Original Paper
|
|
|
file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 747-753
doi:10.1111/j.1745-7270.2008.00446.x
Cloning and characterization
of a flowering time gene from Thellungiella halophila
Qiaoyun Fang1,2, Jun Liu2, Zhengkai Xu2,3, and Rentao Song2,3*
1 The Key Laboratory of Gene Resource Utilization
for Severe Diseases, Ministry of Education, Anhui Medical University, Hefei
230032, China
2 School of Life Sciences, Shanghai
University, Shanghai 200444, China
3
Shanghai Key Laboratory
of Bio-energy Crop, Shanghai University, Shanghai 200444, China
Received: April 9,
2008
Accepted: June 3,
2008
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30471119)
*corresponding
author: Tel, 86-21-66133225; Fax, 86-21-66135163; E-mail, [email protected]
Thellungiella
halophila (T. halophila) (salt cress) is a close
relative of Arabidopsis and a model plant for salt tolerance research.
However, the nature of its later flowering causes some difficulties in genetic
analysis. The FRIGIDA (FRI) gene plays a key role in the Arabidopsis
vernalization flowering pathway, whose homolog in T. halophila may also
be a key factor in controlling flowering time. In order to study the molecular
mechanism of vernalization responses in T. halophila, a full length cDNA
named ThFRI (Thellungiella halophila FRIGIDA) was isolated from
the young seedlings of T. halophila by RT-PCR and RACE. The ThFRI
cDNA was 2017 bp in length and contained an open reading frame encoding a
putative protein of 605 amino acids. The ThFRI showed significant
homology to AtFRI (74.5% at the nucleotide level and 63.9% at the amino
acid level). To study its function, ThFRI cDNA was transformed into Arabidopsis
thaliana, driven by CaMV 35S promoter. Transgenic plants expressing
ThFRI exhibited late-flowering phenotype, which suggests that ThFRI
is the funtional FRI homolog in T. halophila. The cloning and
funtional characterization of the FRI homolog of T. halophila
will faciliate further study of flowering time control in T. halophila.
Keywords Thellungiella halophila; vernalization; FRIGIDA; ThFRI;
Arabidopsis thaliana
In plant development, the transition from vegetative to reproductive
phase is critical for seasonal changes. The timing of reproductive transition is
determined by developmental status and environmental conditions. This
combination promotes flowering at the appropriate time by coupling the
accumulation of sufficient nutrients to favorable environmental conditions [1–4]. Genetic
research has revealed that multiple pathways have evolved to regulate flowering
time in many plant species. These pathways monitor both a plant’s developmental
state and environmental cues, such as photoperiod and temperature. Previous studies on genes controlling flowering time have been
conducted predominantly in Arabidopsis. The FRI, FLC, CO,
FT and FCA have been isolated by generating mutants with altered
flowering times [1,5,6]. FRI and FLC were found to be the two key
loci determining flowering time in Arabidopsis; they act synergistically
to cause late flowering [7–11]. The FRI gene encodes a novel protein with two
predicted coiled-coil domains [12,13]. Functional FRI alleles accelerate
FLC messenger RNA accumulation, which in turn inhibits flowering [9],
unless down-regulated by vernalization. The FRI alleles are thought to
promote early flowering in the absence of vernalization [8]. Thus, Arabidopsis
mutants with non-functional or weak FRI alleles have been widely used as
research materials because of their early flowering [14]. For example, Columbia
(Col) carries a dominant FLC but a recessive FRI allele, and Landsberg
erecta possesses a weak FLC and a recessive FRI.T. halphila (salt cress), a typical
halophyte, is an extremophile that is native to harsh environments [15]. It can
grow in a medium containing 500 mM NaCl and can survive at –15 ?C. T. halophila has many features that make it a useful
model system, such as its relatively small genome (twice the size of Arabidopsis),
small size, copious seed production, self-pollination and genetic
transformation by the floral dip procedure. Therefore, T. halophila has
also been used as a research model to study plant salinity tolerance [16,17].
However, as a late flowering plant, T. halophila has a major drawback as
a genetic model because of the prolonged period of vernalization treatment to
induce early flowering. Given that T. halophila is closely related to Arabidopsis
and that most of their genes are similar (>90% similarity in cDNA
sequences) [16,17], it is possible to clone and characterize genes in the
vernalization pathway of T. halophila based on genetic information from Arabidopsis
[18]. In this study, the cDNA of the FRI homolog from T.
halophila was cloned and functionally characterized in Arabidopsis.
We showed that ThFRI is highly homologous to FRI and that
heterologous expression of ThFRI in Arabidopsis (Col) could
restore the late-flowering phenotype. These data suggest that ThFRI is
the functional homolog of FRI in T. halophila. The cloning of ThFRI
will facilitate future studies of flowering time control in T. halophila and
the genetic engineering involved in early flowering T. halophila for
plant salt tolerance.
Materials and Methods
Plant material and treatment
Arabidopsis thaliana (Col) and T. halophila
(stock number: CS22504) were cultivated in a growth chamber at 23 ?C under a 16
h light and 8 h dark cycle. For aseptic growth, seeds were surface-sterilized
and plated on a growth medium of 1/2 Murashige Skoog (MS), 0.3% sucrose, 0.9%
agar with pH 5.8, and stratified for 3 d at 4 ?C in the dark before germination
in the growth chamber. Five independent transgenic lines were grown under the
same conditions and used for flowering time measurement.
Total RNA extraction and cDNA
synthesis
Total RNA extraction and cDNA synthesis were performed [18]. Total
RNA was extracted from the seedlings of plants using the Trizol reagent
(Tiangen, Shanghai, China) according to Arabidopsis laboratory manual.
Genomic DNA contamination was removed by RNase-free DNase I (Invitrogen,
Carlsbad, USA) treatment at 37 ?C for 30 min. First-strand cDNAs were
synthesized from 4.0 mg of total RNA with the 3‘-RACE kit (Invitrogen) according to
the manufacturer’s instruction.
Cloning full-length cDNA of ThFRI
Based on the high sequence similarity between Arabidopsis and
T. halophila cDNA [16,17], ThFRI/P1 and ThFRI/P2 primers
were designed according to Arabidopsis FRI (Table 1). The cDNA
fragment from T. halophila was amplified by RT-PCR, and cloned into
pSK-T vector (GENE-tech,
Shanghai, China) for sequencing analysis. To obtain the 5‘ missing portion of the cDNA, two specific
primers, ThFRI/P4 and ThFRI/P5, were designed based on sequence
information acquired from the partial cDNA fragment. The 5‘-RACE was
performed using the FirstChoiceTM RLM-RACE kit (Ambion Inc.,
Austin, USA) according to the manufacturer’s instructions. Random-primed RT and nested PCR reactions were performed to amplify
the 5‘-end of the ThFRI cDNA (Fig. 1). The 3‘-RACE
was performed by the anchored primer and internal gene specific primer to
obtain the 3‘ missing portion of ThFRI cDNA (Table 1). PCR
products were purified from a 1% agarose gel and cloned into pSK-T vector for
further sequence identification. Full length ThFRI cDNA was then
amplified by gene specific primers (Table 1) with nested PCR. Random-primed RT and nested PCR reactions were performed to amplify
the 5‘-end of the ThFRI cDNA (Fig. 1). The 3‘-RACE
was performed by the anchored primer and internal gene specific primer to
obtain the 3‘ missing portion of ThFRI cDNA (Table 1). PCR
products were purified from a 1% agarose gel and cloned into pSK-T vector for
further sequence identification. Full length ThFRI cDNA was then
amplified by gene specific primers (Table 1) with nested PCR. Three independent clones were sequenced to minimize errors
introduced during cloning. The full-length ThFRI sequence was submitted
to GenBank (accession No. DQ089808).
Sequence analyses of ThFRI
A homology search was performed with the BLAST program (http://www.ncbi.nlm.nih.gov/blast.cgi).
Alignment analysis was performed with VECTOR NTI 8.0 software (http://www.informaxinc.com). The
deduced protein sequences were analyzed using the ExPASy Proteomics Server (http://us.expasy.org).Construction of heterologous expression vector for heterologous
expression in Arabidopsis, the ORF of ThFRI cDNA was amplified by
ThFRI/P8 and ThFRI/P9 (Table 1). NcoI and XbaI
sites were introduced at their respective 5‘-ends. PCR product was
digested with NcoI and XbaI, and cloned into corresponding sites
of the vector pAVA321 [19]. The vector contained dual 35S promoter from CaMV, a
translational enhancer sequence of tobacco etch virus and a 35S transcriptional
terminator from CaMV. The expression cassette from the resulting construct was
released by BamHI and KpnI and sub-cloned into corresponding
sites of pPZP211 [20], making the heterologous expression construct pPZP211-ThFRI
(Fig. 2).
Plant transformation
expression
constructs pPZP211-ThFRI and pPZP211, the latter as a negative control,
were transformed into Agrobacterium tumefaciens GV3101 via
electroporation. Agrobacterium containing these constructs were used to
transform Arabidopsis thaliana by floral dip method [21]. Arabidopsis
transformants were selected on agar plates containing 1/2 MS medium and 50 mg/ml kanamycin.
semi-quantitative RT-PCR
analyses
Total RNA was extracted from Arabidopsis plants and treated
with DNase I (RNase-free) to remove genomic DNA contamination [18].
First-strand cDNA was synthesized by Superscript II (Invitrogen). RT-PCR was
performed on first-strand cDNA using gene-specific primer sets (Table 1).
To normalize the RT mixtures, Arabidopsis UBQ10 was used as
internal control. The following conditions were used for PCR: 40 s at 94 ?C, 40
s at 56 ?C, and 1 min at 72 ?C for 30 cycles. For each primer set, three
independent biological repeats were performed.
Results
Isolation of full-length FRI
cDNA from T. halophila
It has been shown that the flowering behavior of T. halophila mimics
the winter annual Arabidopsis in which AtFRI acts as the major flowering
inhibitor [22]. This suggests a FRI homolog could exist in T.
halophila. Because most genes in T. halophila have an approximately
90% sequence similarity to Arabidopsis counterparts at the cDNA sequence
level [16,17], specific primers were designed based on Arabidopsis AtFRI
cDNA (GenBank accession No. AF228499) to obtain a partial cDNA fragment from T.
halophila by RT-PCR. Sequence analysis of this partial fragment revealed a
sequence with 80% similarity to AtFRI, suggesting it could be the FRI
homolog in T. halophila. The 5‘-RACE and 3‘-RACE were
carried out to obtain the 5‘ and 3‘ missing portions of the cDNA,
respectively. Subsequently, full-length cDNA was obtained and designated ThFRI
(GenBank accession No. DQ089808).
Sequence analysis of ThFRI cDNA
The full-length sequence of ThFRI cDNA is 2017 bp and
contains a 1818 bp ORF that encodes a protein of 605 amino acids. The
full-length nucleotide sequence and the deduced amino acid sequence are shown
in Fig. 3(A). The cDNA contains a 46 nucleotides 5‘ untranslated
region (UTR) and a longer 3‘ UTR of 153 nucleotides, including the
polyA-tail. The deduced protein has a molecular weight of 67.86 kDa and a
theoretical isoelectric point of 7.42.ThFRI? 605 amino acids were found to be significantly homologous only to AtFRI
in the GenBank database, with 74.5% identity at nucleotide level and 63.9%
identity at amino acid level [Fig. 3(B)]. However, a comparison between ThFRI
and AtFRI protein sequences revealed some differences; for example, the
region between amino acids 108 and 157 showed much lower identity (24.1%), and
14 amino acids were deleted in ThFRI at amino acid 548 [Fig. 3(B)].
In addition, extra amino acids were found in ThFRI at the N-terminal and
C-terminal (seven and three amino acids respectively). Overall, significant
sequence homology was found across the entire gene, suggesting that the cloned ThFRI
was an ortholog of AtFRI in T. halophila.
Heterologous expression of ThFRI
in Arabidopsis restored late-flowering phenotype
Arabidopsis thaliana (ecotype Col)
flowers early due to a recessive FRI-Col allele. If a functional FRI allele
were introduced into Arabidopsis (Col), FRI protein would
accelerate the FLC messenger RNA accumulation, which, in turn, would
inhibit flowering and cause late-flowering phenotype [9]. To assess its
biological function and determine whether ThFRI is functional, a
complementary test was carried out in the Arabidopsis (Col) with
recessive FRI-Col. We constructed pPZP211-ThFRI, the heterologous
expression construct of ThFRI (see “Materials and Methods”) (Fig.
2).Expression constructs were transformed into FRI-Col Arabidopsis
by the floral dip method [21]. Transgenic lines were selected by kanamycin
resistance and detected by PCR with specific primers according to recombinant
plasmid (data not shown). Five independent transgenic lines (designated
OV1-OV5) were selected for further analysis (Fig. 4). The expression of ThFRI
in transgenic Arabidopsis was verified by RT-PCR using gene-specific
primers ThFRI/P11 and ThFRI/P12 (Table 1). All positive
transgenic lines had ThFRI transcripts except the OV4 line, which was
proved to be a false-positive transgenic line; no such transcript was detected
in negative control plants that transformed with empty pPZP211 [Fig. 4(E)].
Late-flowering phenotype analysis was carried out with those confirmed
transgenic lines. They all displayed well-characterized late-flowering
phenotype: significantly more rosette leaves were found before flowering [Fig.
4(A,B)]. The first 10 rosette leaves formed on transgenic lines, as
represented by OV1, were apparently larger than those of control plants [Fig.
4(C)]. Within the same transgenic line, there was no significant difference
in ThFRI expression level or flowering time among individual plants [Fig.
4(D,E)]. These results indicate that heterologously expressed ThFRI
could functionally complement the FRI-Col in Arabidopsis and
cause late-flowering phenotype in transgenic Arabidopsis. Therefore, we
demonstrated that ThFRI is a functional FRI homolog in T.
halophila.
Discussion
AtFRI has been shown to be a key
regulator in the flowering time pathway of Arabidopsis. In this study, ThFRI
was isolated from T. halophila, and characterized in Arabidopsis
(Col) with recessive FRI-Col allele. By RT-PCR with primers designed on
AtFRI and the RACE method (Fig. 1), the full-length cDNA ThFRI
was cloned from T. halophila (Fig. 3). Sequence analysis
indicated that ThFRI and AtFRI shared significantly high
similarity. Heterologous expression of ThFRI in Arabidopsis with FRI-Col
resulted with late-flowering phenotype (Fig. 4), which indicated that ThFRI
had functions similarly to AtFRI. These results suggested that ThFRI and
AtFRI are functionally conserved during evolution, and that the heterologous
expression of ThFRI could result in flowering delay in closely related
species, such as Arabidopsis. As a result, the vegetative growth was
extended, which could be valuable in some crop and vegetative plants [24]. By controlling
the expression of FRI, vegetative growth could be rationally
manipulated.Our data showed that the heterologous expression of ThFRI in Arabidopsis
could give rise to the late-flowering phenotype. However, the heterologous
expression of ThFRI could only extend the vegetative growth period, but
not fully complement AtFRI-deficiency in Arabidopsis (Col). Arabidopsis
ecotype with wild-type AtFRI had about twice the vegetative growth
period before flowering (about 4 months) as compared with ThFRI heterologous
expression lines from this study [25]. Such differences suggested that there
might already be some functional divergence between AtFRI and ThFRI,
although differences due to expression pattern changes could not be ruled out.
The results showed that the heterogeneous expression of ThFRI could
partially restore the late-flowering phenotype in Arabidopsis. Due to
its excellent salinity tolerance, T. halophila is considered as a model
system for salinity tolerance studies. However, its long life cycle presents a
major limitation for lab research. Genetic manipulation at FRI locus
would create early flowering T. halophila. FRI takes effect
synergistically during the formation of late flowering behavior in Arabidopsis;
loss of its function would promote early flowering. With ethane methyl
sulfonate mutagenesis or RNA interference technique, loss-of-function ThFRI
mutants could be obtained, resulting in early flowering T. halophila. A
previous study of another key gene involved in the vernalization pathway in T.
halophila, ThFLC, had demonstrated the feasibility of genetic
engineering of short life cycle T. halophila through the vernalization
pathway [18]. The cloning of the functional FRI homolog in T.
halophila was a critical step towards better understanding of both
flowering time regulation in T. halophila and the engineering of early
flowering T. halophila as a model plant for salt tolerance study.
Acknowledgements
We would like to thank Dr. Xiongfei Ju for
his help in preparing this manuscript.
References
1 Levy YY, Dean C. The
transition to flowering. Plant Cell 1998, 10: 1973–1990
2 Reeves PH, Coupland G.
Response of plant development to environment: control of flowering by daylength
and temperature. Curr Opin Plant Biol 2000, 3: 37–42
3 Mouradov A, Cremer F,
Coupland G. Control of flowering time: interacting pathways as a basis for
diversity. Plant Cell 2002, 14: S111–S130
4 Simpson GG, Dean C. Arabidopsis,
the Rosetta stone of flowering time? Science 2002, 296: 285–289
5 Koornneef M, Alonso-Blanco
C, Peeters AJ, Soppe W. Genetic control of flowering time in Arabidopsis.
Annu Rev Plant Physiol Plant Mol Biol 1998, 49: 345–370
6 Ratcliffe OJ, Nadzan GC,
Reuber TL, Riechmann JL. Regulation of flowering in Arabidopsis by an FLC
homologue. Plant Physiol 2001, 126: 122–132
7 Lee I, Michaels SD,
Masshardt AS, Amasino RM. The late-flowering phenotype of FRIGIDA and
mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta
strain of Arabidopsis. Plant J 1994, 6: 903–909
8 Osborn TC, Kole C, Parkin
IA, Sharpe AG, Kuiper M, Lydiate DJ, Trick M. Comparison of flowering time
genes in Brassica rapa, B. napus and Arabidopsis thaliana.
Genetics 1997, 146: 1123–1129
9 Sheldon CC, Rouse DT,
Finnegan EJ, Peacock WJ, Dennis ES. The molecular basis of vernalization: the
central role of FLOWERING LOCUS C (FLC). Proc Natl Acad Sci USA
2000, 97: 3753–3758
10 Koornneef M, Hanhart CJ, van
der Veen JH. A genetic and physiological analysis of late flowering mutants in Arabidopsis
thaliana. Mol Gen Genet 1991, 229: 57–66
11 Michaels SD, Amasino RM. Loss
of FLOWERING LOCUS C activity eliminates the late-flowering phenotype of
FRIGIDA and autonomous pathway mutations but not responsiveness to
vernalization. Plant Cell 2001, 13: 935–941
12 Clarke JH, Dean C. Mapping FRI,
a locus controlling flowering time and vernalization response in Arabidopsis
thaliana. Mol Gen Genet 1994, 242: 81–89
13 Michaels SD, Bezerra IC,
Amasino RM. FRIGIDA-related genes are required for the winter-annual
habit in Arabidopsis. Proc Natl Acad Sci USA 2004, 101: 3281–3285
14 Lu Q, Xu ZK, Song RT. OsFY,
a homolog of AtFY, encodes a protein that can interact with OsFCA-g
in
rice (Oryza sativa L.). Acta Biochim Biophys Sin 2006, 38: 492–499
15 Inan G, Zhang Q, Li P, Wang Z,
Cao Z, Zhang H, Zhang C et al. Salt cress. A halophyte and cryophyte Arabidopsis
relative model system and its applicability to molecular genetic analyses of
growth and development of extremophiles. Plant Physiol 2004, 135: 1718–1737
16 Bressan RA, Zhang C, Zhang H,
Hasegawa PM, Bohnert HJ, Zhu JK. Learning from the Arabidopsis
experience. The next gene search paradigm. Plant Physiol 2001, 127: 1354–1360
17 Zhu JK. Plant salt tolerance.
Trends Plant Sci 2001, 6: 66–71
18 Fang Q, Xu Z, Song R. Cloning,
characterization and genetic engineering of FLC homolog in Thellungiella
halophila. Biochem Biophys Res Commun 2006, 347: 707–714
19 von Arnim AG, Deng XW, Stacey
MG. Cloning vectors for the expression of green fluorescent protein fusion
proteins in transgenic plants. Gene 1998, 221: 35–43
20 Hajdukiewicz P, Svab Z, Maliga
P. The small, versatile pPZP family of Agrobacterium binary vectors for
plant transformation. Plant Mol Biol 1994, 25: 989–994
21 Clough SJ, Bent AF. Floral dip:
a simplified method for Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J 1998, 16: 735–743
22 Johanson U, West J, Lister C,
Michaels S, Amasino R, Dean C. Molecular analysis of FRIGIDA, a major
determinant of natural variation in Arabidopsis flowering time. Science
2000, 290: 344–347
23 Altschul SF, Gish W, Miller W,
Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990, 215:
403–410
24 Hirai M, Ajisaka H, Kuginuki Y,
Yui M. Utilization of DNA polymorphism in vegetable breeding (utilization of
DNA-marker for breeding and classification in horticultural plants, for further
development of horticulture in East Asia). Journal of the Japanese Society for
Horticultural Science 1998, 67: 1186–1188
25 Stinchcombe JR, Weinig C,
Ungerer M, Olsen KM, Mays C, Halldorsdottir SS, Purugganan MD et al. A
latitudinal cline in flowering time in Arabidopsis thaliana modulated by
the flowering time gene FRIGIDA. Proc Natl Acad Sci USA 2004, 101: 4712–4717