Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK
* Author for correspondence (e-mail: enrico.coen{at}bbsrc.ac.uk)
Accepted 9 September 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cycloidea, Radialis, Dorsoventral, Gene networks, Atavism, Arabidopsis, Antirrhinum
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several genes controlling floral asymmetry have been identified in
Antirrhinum majus. Antirrhinum flowers are asymmetric along their
dorsoventral axis, having distinct dorsal, lateral and ventral organ types.
Asymmetry is most evident in the petal and stamen whorls and depends on the
action of the duplicate genes CYCLOIDEA (CYC) and
DICHOTOMA (DICH). CYC and DICH are both
expressed in the dorsal domain of the flower meristem and continue to be
expressed at later stages in dorsal floral organs, although expression of
CYC occurs in a wider region than that of DICH
(Almeida et al., 1997;
Luo et al., 1999
;
Luo et al., 1996
). cyc
dich double mutants have radially symmetric ventralised flowers, while
single cyc or dich mutants have partially ventralised
flowers, consistent with sub-functionalisation of paralogs following the
CYC-DICH duplication (Gubitz et
al., 2003
; Hileman and Baum,
2003
). Developmental analysis of single and double mutants has
shown that CYC and DICH can enhance or repress organ growth,
depending on developmental stage and organ type, with CYC having a
stronger phenotypic effect than DICH
(Almeida et al., 1997
;
Luo et al., 1999
;
Luo et al., 1996
).
CYC and DICH encode transcription factors belonging to
the TCP family, many members of which influence patterns of plant cell growth
and proliferation (Almeida et al.,
1997; Crawford et al.,
2004
; Kosugi and Ohashi,
1997
; Kosugi and Ohashi,
2002
; Kosugi et al.,
1995
; Luo et al.,
1999
; Luo et al.,
1996
; Nath et al.,
2003
; Palatnik et al.,
2003
; Tremousaygue et al.,
2003
). Members of this family can be grouped into two classes, I
and II, based on sequence similarity in the TCP domain and the consensus
DNA-binding sequence. A good candidate for a direct target of CYC and
DICH is RADIALIS (RAD). RAD is expressed
in the dorsal domain of floral meristems, in a manner that depends on
CYC and DICH. Plants mutant for RAD have almost
fully ventralised flowers, retaining only slight dorsal identity in their
uppermost regions, suggesting that many of the effects of CYC and
DICH are mediated through RAD. RAD acts antagonistically to
DIVARICATA (DIV), which promotes ventral identity
(Almeida et al., 1997
;
Corley et al., 2005
;
Galego and Almeida, 2002
).
RAD and DIV encode related MYB-like proteins that are
thought to compete for common protein or DNA targets.
Unlike Antirrhinum, Arabidopsis has radially symmetrical flowers.
The TCP1 gene of Arabidopsis is the closest homologue to
CYC/DICH and is expressed asymmetrically in the dorsal domain of
young flower meristems and axillary meristems
(Cubas et al., 2001). This
indicates that the common ancestor of Antirrhinum and
Arabidopsis had a CYC/TCP1-related gene that was
asymmetrically expressed even though the flowers were presumably radially
symmetric. In contrast to CYC and DICH, TCP1 is only
transiently expressed at very early stages of flower development.
To understand the evolution of asymmetry, we analysed how CYC acts in Antirrhinum and Arabidopsis. We show that CYC binds to DNA, and use random binding site selection to define the consensus binding site. Sequences matching this site are found in the RAD promoter and intron, and are bound by CYC, indicating that RAD may be a direct target of CYC. CYC is also able to activate the RAD gene of Antirrhinum in the context of Arabidopsis but is unable to activate endogenous RAD-like genes of Arabidopsis, which lack sequences identical to the consensus binding site. CYC is nevertheless able to act in Arabidopsis, reducing leaf and increasing petal size, through changes in cell proliferation and expansion. By activating CYC protein at specific times, we show that CYC can act at various stages of Arabidopsis organ growth. Taken together, our results suggest that ancestral TCP1/CYC-like genes played a role in regulating a network of target genes involved in growth and development. Some of these interactions could have been retained in both Antirrhinum and Arabidopsis, accounting for the common developmental effects of CYC expression in both species. In addition, an interaction between CYC and RAD was established or preserved specifically in the Antirrhinum lineage.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of recombinant CYC protein and electrophretic mobility shift assays (EMSAs)
The pRSET-b vector (Invitrogen) was used for production of recombinant CYC
protein in E. coli (plasmid pJAM2093). BL21(DE3) SBET pLysE
(Schenk et al., 1995;
Studier and Moffatt, 1986
)
E. coli cells were transformed with the construct and the recombinant
His-CYC fusion protein was expressed under the control of the T7 promoter and
purified from the soluble fraction or from inclusion bodies produced in
bacteria, as described previously (Kosugi
and Ohashi, 1997
; Kosugi and
Ohashi, 2002
). The conditions for the DNA-binding reaction and
electrophoresis, and the DNA probes used were as described
(Kosugi and Ohashi, 2002
). For
the EMSA with the RAD promoter and intron, the DNA probes were
obtained by PCR using the primer combinations 5733/5734 and 5735/5736 with the
plasmid pJAM2283, respectively (5733,
5'-ACCGTAGAACATTATAGACAACA-3'; 5734,
5'-CACCAACAAAACCTTCCACATAG-3'; 5735,
5'-GCTATAACGTCGATGTGTCTC-3'; 5736,
5'-ATTCTAAAAACCACGAGAGTCC-3'). For the EMSA with the RAD
promoter and intron, the primer combinations 5733-5734 and 5735-5736 were
used, respectively.
Random binding site selection
Random binding site selection for the His-CYC protein was performed using
the oligonucleotide BS18N, as described previously
(Kosugi and Ohashi, 1997;
Kosugi and Ohashi, 2002
). The
DNA-protein complex was separated by polyacrylamide gel electrophoresis and
the DNA recovered from the gel was amplified by PCR. After the fifth round of
selection, the DNA amplified by PCR was cloned into pGEMT-easy vector
(Promega) for sequencing.
Plasmid construction
The binary plasmid coding for the CYC protein fused to the rat
glucocorticoid receptor (GR) (Lloyd et
al., 1994) under the control of cauliflower mosaic virus (CaMV)
35S promoter was obtained as follows. The CYC open reading frame was
amplified by PCR using the primers 5'-CYC
(5'-CGGGATCCATGGTTGGGAAG-3') and 3'-CYC
(5'-GAAGATCTTTGATGAACTTGTGCT-3') from plasmid pJAM2095. The
forward primer introduced a Kozac sequence and a BamHI site before
the start codon and the reverse primer removed the CYC stop codon and
created a BglII restriction site at the C terminus. This PCR fragment
was digested with BamHI and BglII and cloned into the
BamHI restriction site of the plasmid GR-pBluescript (pRS020)
(Sablowski and Meyerowitz,
1998
) resulting in an in-frame translational fusion at the C
terminus of CYC with the rat glucocorticoid hormone-binding domain (pJAM2387).
The CYC cDNA was sequenced to check for PCR errors and to check if
the protein was in frame with the GR sequence. This fusion was called
CYC:GR. The double CaMV 35S promoter and 35S polyadenylation signal from CaMV
were isolated as a 1.56 kb KpnI/EcoRV fragment from pJIT60
(Guerineau, 1993) and cloned into a SacI filled in/KpnI
sites in pGreenII 0029 (plasmid pJAM2388). A 1.7 kb
XbaI/BamHI fragment containing the CYC:GR fusion was
isolated from plasmid pJAM2387 and the ends filled in. This fragment was
cloned into the SmaI dephosphorylated site of plasmid pJAM2388,
adjacent to the double 35S promoter and the orientation was checked by
restriction mapping. This construct was called 35S::CYC:GR (plasmid
pJAM2389).
Transformation of Arabidopsis
Arabidopsis plants were transformed with the 35S::CYC:GR construct
by floral dipping (Clough and Bent,
1998). The Agrobacterium strain used was C58C1 pGV101
pMP90. Kan-resistant transformants (T1 generation) were selected on GM plates.
Approximately, two thirds of T2 plants (segregating 3:1 on Kan) showed a
phenotype when also germinated on DEX. T3 progeny of plants that showed a
phenotype on DEX-containing media were hemizygous, whereas the progeny of
plants that did not show a phenotype were homozygous. 35S::CYC:GR homozygous
plants were crossed to other homozygous independent lines and to wild type.
Double hemizygous plants never showed a phenotype when grown in DEX-containing
media, whereas the hemizygous lines obtained from the cross with wild type
showed the phenotype.
Analysis of expression by northern blot and by RT-PCR
For Northern-blot analysis, total RNA was extracted with TRI-Reagent
(Sigma), according to the manufacturer's instructions, from 35S::CYC:GR T2
seedlings grown on Kan and DEX, with and without a phenotype. The RNA was
blotted onto Hybond N+ (Amersham) according to the manufacturer's instructions
and probed with CYC or NPTII (neomycin phosphotransferase
II) probe.
For RT-PCR analysis, RNA was extracted using RNeasy Plant mini kit (Qiagen), from duplicate samples of tissue from 21-day-old 35S::CYC:GR RAD::RAD plants grown on GM media, 6, 18 and 48 hours after transfer to 10 µM DEX plates. cDNA was prepared from 2 µg of total RNA using Thermoscript RT-PCR system (Invitrogen). Aliquots of the cDNA were used as template for PCR with gene-specific primers.
Phenotypic analysis
Scanning electron microscopy (SEM) analysis was carried out on `2-Ton'
epoxy (Devcon) replicas of Arabidopsis petals and leaves as
previously described (Carpenter et al.,
1995; Green and Linstead,
1990
). These replicas were sputter coated with gold palladium,
analysed and photographed with a Philips XL 30 FEG SEM. Petal and leaf areas
were calculated using a program written in MatLab called CellFinder. Medians
were obtained by analysis of variance. The analyses were carried out using the
statistical package GenStat for Windows 7th Edition (VSN International,
Oxford, UK).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
CYC affects growth of Arabidopsis
To investigate whether CYC expression could affect growth in
Arabidopsis, the coding region of CYC was fused to that of the rat
glucocorticoid receptor and expressed under the control of the 35S promoter
(35S::CYC:GR). Sixteen transformants were obtained, of which 10 had a clear
developmental phenotype when grown on the glucocorticoid inducer dexamethasone
(DEX). Northern-blot analysis on three independent transformants segregating
for the construct showed that the severity of the phenotype correlated with
the level of CYC mRNA accumulation
(Fig. 3). Curiously, only
plants hemizygous for the construct showed strong expression, indicating that
expression was silenced in homozygotes. To check whether this was a
consequence of transgene dosage or allelic suppression, 35S::CYC:GR
homozygotes were crossed to other independent homozygous lines and to wild
type. Double hemizygous plants did not show a phenotype when grown in DEX
media, whereas the hemizygous lines obtained from the cross with wild type
showed the phenotype. These results indicate that two doses of the transgene
result in gene silencing, a phenomenon that has been recorded previously for
some transgenes (de Carvalho et al.,
1992).
|
|
|
|
To determine the developmental stage at which CYC exerts its effects on petal development in Arabidopsis, 35S::CYC:GR plants were transferred to DEX after bolting, when the first two flowers had opened in the main inflorescence. The inflorescence was then allowed to develop further. All flowers showed delayed senescence and abscission, indicating that CYC can have an effect late in development even after the flowers have opened.
CYC represses growth of Arabidopsis leaves
35S::CYC:GR plants grown in DEX-containing media, were dwarfed and
exhibited small oval-shaped leaves (Figs
6,
7,
8). Mature leaves of
35S::CYC:GR plants were on average eight times smaller in surface area than
those of wild type (Fig. 7A,C).
To investigate whether the difference in leaf size was due to a difference in
cell size or in cell number, the adaxial epidermal layer of leaf 4 was
analysed by SEM at different stages of development
(Fig. 7B,D). At maturity, cells
of 35S::CYC:GR leaves were about four times smaller than those in wild-type
plants, suggesting that contrary to what was observed in the petals, CYC has a
role in repressing cell expansion in the leaves. However, if reduced cell size
was solely responsible for the reduction in overall leaf size, the leaves of
35S::CYC:GR plants should be four times smaller rather than observed value of
eight times. This indicates that 35S::CYC:GR leaves also had about half the
number of cells of wild type. To determine whether this reflected an early
arrest of cell proliferation, epidermal cells of younger leaves were analysed.
At day 14, cells of 35S::CYC:GR leaves were larger than those of wild type,
suggesting that cell division had arrested early and the cells were starting
to differentiate (Fig. 7B,D).
Moreover, evidence of recent cell divisions could be seen in the epidermal
cells of wild-type leaves (formation of new stomata), whereas in 35S::CYC:GR
leaves no such events could be seen. Thus, CYC reduces both cell proliferation
(by promoting early arrest of cell division) and cell expansion in leaves.
|
This showed that removal of CYC could restore leaf growth at early stages of development, before they were about 0.5 mm wide. Moreover, the effect was greater the earlier that CYC was removed.
Conversely, when plants were grown without DEX and moved onto DEX at day 10 (Fig. 8C), growth repression was first detected for leaves 3-4 and became progressively more pronounced in later leaves (compare Fig. 8C with 8D). These results suggest that activation of CYC can influence leaf development at early stages, before leaves are 0.5 mm wide, leading to a reduction in final leaf size.
|
To test whether CYC could activate the Antirrhinum RAD gene in the context of Arabidopsis, 35S::CYC:GR Arabidopsis plants were crossed to transgenic plants containing RAD under the control of its own promoter (RAD::RAD) (C.B., M.M.R.C. and E.C.). When plants with both transgenes were grown in DEX, they showed the same phenotype as 35S::CYC:GR plants. The double transgenics were also grown without DEX and transferred, at 21 days after germination, to media containing DEX. Fig. 9 shows that RAD expression was upregulated 6 hours after the induction of CYC, confirming that CYC can bind to the RAD promoter in vivo, and showing that this interaction can be reconstituted in Arabidopsis.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In considering the evolutionary changes in a regulatory network, it is convenient to distinguish between two types of target process influenced by a transcription factor. One is the set of potential targets that could be influenced by expression of the transcription factor at any developmental stage and in any part of the plant. This class of targets can be revealed through constitutive expression of the transcription factor. Evolutionary changes in potential targets may occur, for example, through changes in the structure of the transcription factor (i.e. binding site) or changes in the promoters of target genes. The other type of target process is the set of actual targets that are influenced only where and when the transcription factor is normally active. Given a set of potential targets, changes in the expression pattern of the transcription factor can lead to modifications to the defined subset of actual targets.
The ability of CYC to influence both Antirrhinum and Arabidopsis development suggests that a range of potential developmental targets were present in their common ancestor. A subset of these would have been the ancestral actual targets in the dorsal regions of axillary meristems. Although these actual targets are unknown, they are unlikely to have involved morphological asymmetry of the flower as the ancestral condition is thought to have been radial symmetry.
|
In addition to co-option of RAD, other alterations in the regulatory interactions may also have occurred in the Antirrhinum lineage. For example, CYC has developmental effects on stamen arrest as well as a slight effect on petal lobe asymmetry independent of RAD. These effects may have arisen through divergence in some of the target processes influenced by CYC (Fig. 10, step b). It is also possible that the target sequence recognised by CYC diverged to some extent (the CYC consensus binding site is not typical of type II TCP proteins).
Another evolutionary change may have involved persistent expression of
CYC in the dorsal domain of Antirrhinum floral meristems [in
contrast to transient expression of TCP1 in Arabidopsis
(Cubas et al., 2001)], leading
to a change in expression or range of actual targets. Given the phenotypic
effects of CYC on petal size in Arabidopsis, it is possible that such
persistence could create an asymmetric Arabidopsis flower with larger
dorsal petals. Persistence in the Antirrhinum lineage may have arisen
through evolution of an autoregulatory loop
(Fig. 10, step c), as
CYC contains promoter sequences matching the consensus CYC-binding
site (the TCP1 promoter does not contain such sites). However,
persistent expression could also have arisen through interaction with other
factors that are not directly dependent on CYC. In addition, we
cannot rule out the possibility that the ancestral CYC/TCP1 gene was
persistently expressed in dorsal floral meristems and that expression became
transient in the lineage leading to Arabidopsis.
|
Expression of CYC also influences petal development in Arabidopsis. All petals are equally affected, reflecting ectopic expression of CYC all around the developing flower. The effect of CYC largely involves cell expansion late in development, in contrast to CYC in Antirrhinum, which also has early developmental effects. Moreover, CYC expression does not affect stamen development in Arabidopsis, unlike the situation in Antirrhinum. Thus, the potential developmental targets in floral organs have probably diverged, consistent with the inability of CYC to switch on RAD-like genes in Arabidopsis.
Taken together, our results indicate that interactions involving
CYC have changed in many ways since the separation of the
Antirrhinum and Arabidopsis lineages. These changes may
themselves represent a subset of the changes that have occurred in the whole
regulatory network in which CYC is embedded. Nevertheless, some
elements of the network have been preserved, such as the initial asymmetric
expression pattern and the modification of developmental target gene activity.
Thus, floral asymmetry has most likely arisen through a process of tinkering
(Jacob, 1977;
Jacob, 2001
) with the
strengths and pattern of connections in a regulatory network, in which some
common elements may still be discernable.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almeida, J., Rocheta, M. and Galego, L. (1997).
Genetic control of flower shape in Antirrhinum majus.
Development 124,1387
-1392.
Babu, M. M., Luscombe, N. M., Aravind, L., Gerstein, M. and Teichmann, S. A. (2004). Structure and evolution of transcriptional regulatory networks. Curr. Opin. Struct. Biol. 14,283 -291.[CrossRef][Medline]
Bleecker, A. B. and Patterson, S. E. (1997).
Last exit: senescence, abscission, and meristem arrest in Arabidopsis.
Plant Cell 9,1169
-1179.
Bleecker, A. B., Estelle, M. A., Somerville, C. and Kende, H. (1988). Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241,1086 -1089.
Butenko, M. A., Patterson, S. E., Grini, P. E., Stenvik, G. E.,
Amundsen, S. S., Mandal, A. and Aalen, R. B. (2003).
Inflorescence deficient in abscission controls floral organ abscission in
Arabidopsis and identifies a novel family of putative ligands in plants.
Plant Cell 15,2296
-2307.
Cantu, J. and Ruiz, C. (1985). On atavisms and atavistic genes. Annu. Genet. 28,141 -142.
Carpenter, R., Copsey, L., Vincent, C., Doyle, S., Magrath, R.
and Coen, E. (1995). Control of flower development and
phyllotaxy by meristem identity genes in antirrhinum. Plant
Cell 7,2001
-2011.
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Corley, S. B., Carpenter, R., Copsey, L. and Coen, E.
(2005). Floral asymmetry involves an interplay between TCP and
MYB transcription factors in Antirrhinum. Proc. Natl. Acad. Sci.
USA 102,5068
-5073.
Crawford, B. C., Nath, U., Carpenter, R. and Coen, E. S.
(2004). CINCINNATA controls both cell differentiation and growth
in petal lobes and leaves of Antirrhinum. Plant
Physiol. 135,244
-253.
Cubas, P., Coen, E. and Zapater, J. M. (2001). Ancient asymmetries in the evolution of flowers. Curr. Biol. 11,1050 -1052.[CrossRef][Medline]
de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inze, D. and Castresana, C. (1992). Suppression of beta-1,3-glucanase transgene expression in homozygous plants. EMBO J. 11,2595 -2602.[Abstract]
Donoghue, M. J., Ree, R. and Baum, D. A. (1998). Phylogeny and the evolution of flower symmetry in the Asteridae. Trends Plant Sci. 3, 311-317.[CrossRef]
Endress, P. K. (1999). Symmetry in flowers: diversity and evolution. Int. J. Plant Sci. 160,S3 -S23.[CrossRef][Medline]
Galego, L. and Almeida, J. (2002). Role of
DIVARICATA in the control of dorsoventral asymmetry in Antirrhinum flowers.
Genes Dev. 16,880
-891.
Gibson, G. and Honeycutt, E. (2002). The evolution of developmental regulatory pathways. Curr. Opin. Genet. Dev. 12,695 -700.[CrossRef][Medline]
Green, P. B. and Linstead, P. (1990). A procedure for SEM of complex shoot structures applied to the inflorescence of snapdragon (Antirrhinum). Protoplasma 158, 33-38.[CrossRef]
Gubitz, T., Caldwell, A. and Hudson, A. (2003).
Rapid molecular evolution of CYCLOIDEA-like genes in antirrhinum and its
relatives. Mol. Biol. Evol.
20,1537
-1544.
Guerineau, F. and Mullineaux, P. M. (1993). Plant transformation and expression vectors. In Plant Molecular Biology Labfax (ed. R. R. D. Croy), pp.121 -148. London: BIOS Scientific Publishers.
Hall, B. (1995). Atavisms and atavistic mutations. Nat. Genet. 10,126 -127.[CrossRef][Medline]
Hileman, L. C. and Baum, D. A. (2003). Why do
paralogs persist? Molecular evolution of CYCLOIDEA and related floral symmetry
genes in Antirrhineae (Veronicaceae). Mol. Biol. Evol.
20,591
-600.
Jacob, F. (1977). Evolution and tinkering. Science 196,1161 -1166.[Medline]
Jacob, F. (2001). Complexity and tinkering.
Annu. New York Acad. Sci.
929, 71-73.
Kosugi, S. and Ohashi, Y. (1997). PCF1 and PCF2
specifically bind to cis elements in the rice proliferating cell nuclear
antigen gene. Plant Cell
9,1607
-1619.
Kosugi, S. and Ohashi, Y. (2002). DNA binding and dimerization specificity and potential targets for the TCP protein family. Plant J. 30,337 -348.[CrossRef][Medline]
Kosugi, S., Suzuka, I. and Ohashi, Y. (1995). Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue-specific expression. Plant J. 7,877 -886.[CrossRef][Medline]
Lloyd, A. M., Schena, M., Walbot, V. and Davis, R. W. (1994). Epidermal cell fate determination in Arabidopsis: patterns defined by a steroid-inducible regulator. Science 266,436 -439.[Medline]
Luo, D., Carpenter, R., Vincent, C., Copsey, L. and Coen, E. (1996). Origin of floral asymmetry in Antirrhinum. Nature 383,794 -799.[CrossRef][Medline]
Luo, D., Carpenter, R., Copsey, L., Vincent, C., Clark, J. and Coen, E. (1999). Control of organ asymmetry in flowers of Antirrhinum. Cell 99,367 -376.[CrossRef][Medline]
Nath, U., Crawford, B. C., Carpenter, R. and Coen, E.
(2003). Genetic control of surface curvature.
Science 299,1404
-1407.
Palatnik, J. F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J. C. and Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature 425,257 -263.[CrossRef][Medline]
Patterson, S. E. (2001). Cutting loose.
Abscission and dehiscence in Arabidopsis. Plant
Physiol. 126,494
-500.
Patterson, S. E. and Bleecker, A. B. (2004).
Ethylene-dependent and -independent processes associated with floral organ
abscission in Arabidopsis. Plant Physiol.
134,194
-203.
Sablowski, R. W. and Meyerowitz, E. M. (1998).
Temperature-sensitive splicing in the floral homeotic mutant apelata3-1.
Plant Cell 10,1453
-1463.
Schenk, P. M., Baumann, S., Mattes, R. and Steinbiss, H. H. (1995). Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19,196 -198, 200.[Medline]
Stebbins, G. L. (1974). Flowering Plants: Evolution Above the Species Level. Cambridge, MA: Harvard University Press.
Studier, F. W. and Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189,113 -130.[CrossRef][Medline]
Tautz, D. (2000). Evolution of transcriptional regulation. Curr. Opin. Genet. Dev. 10,575 -579.[CrossRef][Medline]
Tremousaygue, D., Garnier, L., Bardet, C., Dabos, P., Herve, C. and Lescure, B. (2003). Internal telomeric repeats and `TCP domain' protein-binding sites co-operate to regulate gene expression in Arabidopsis thaliana cycling cells. Plant J. 33,957 -966.[CrossRef][Medline]