Plant CellPhysiol. 41(3): 377-382 (2000)
JSPP © 2000
Short Communication
Zinc-Finger Genes That Specifically Express in Pistil Secretory Tissues of
Petunia
Ken-ichi Kubo lf 2, Yoshiaki Kanno lf 3, Tokuzo Nishino 2 and Hiroshi Takatsuji1' 4
1
2
3
Laboratory of Developmental Biology, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602
Japan
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 07, Sendai, 980-8579 Japan
Aomori Green BioCenter, 221-10 Yamaguchi, Nogi, Aomori, 030-0142 Japan
Tissue-specific expression patterns of petunia zincfinger genes, ZPT2-10 and ZPT3-3, were analyzed by using
GUS reporter system. The GUS expression directed by
ZPT2-10 promoter was specifically found in the stylar
transmitting tissue of pistil, and that by ZPT3-3 promoter
in stigmatic and stylar transmitting tissues. These tissues
play important roles in reproductive process. We discuss
possible roles of the zinc-finger proteins in these specialized
tissues.
Key words: Petunia — Pistil — Transmitting tissue —
Transcription factor — Zinc finger.
The pistil is composed of three parts; stigma, style and
ovary, from the top. Upon pollination, pollen grains adhere to the surface of stigma, get hydrated, and germinate
pollen tubes, which penetrate into the stigmatic tissue. The
pollen tubes grow through transmitting tissue of the style,
reach the ovary that contains female gametophytes, then
enter the micropyles of ovules and finally release sperm
cells to complete fertilization.
The stylar transmitting tissue consists of elongated
sausage-like cells. Intercellular spaces between the cells are
filled with secretion (Bell and Hicks 1976), through which
the pollen tubes elongate towards the ovary. Small molecules such as glucose and galactose are among the components of the secretion in the transmitting tissue and they are
thought to serve as energy sources for the growing pollen
tubes (Labarca and Loewus 1973). Many kinds of proteins
are known to express specifically in the transmitting tissue
and they are implicated in the pollen-pistil interaction
and/or maintenance of this specialized tissue. These proteins include self-incompatibility-related S-RNases in Solanaceae (Cornish et al. 1987) and transmitting-tissue-specific
Abbreviations: PR, Pathogenesis related; ZPTx-y, PEThy;
ZPTx-y (x; number of zinc finger, y; serial number).
The nucleotide sequences reported in this paper have been
submitted to the DDBJ, GenBank and EMBL under accession
numbers ABO35132 (ZPT2-10) and ABO35133 (ZPT3-3).
4
Corresponding author.
377
glycoprotein in Nicotiana tabacum that has been implicated in attracting the pollen tubes (Cheung et al. 1995).
Abundantly present in the stylar transmitting tissue are the
PR-like proteins (Gasser and Robinson-Beers 1993, Kuboyama 1998) that have structural similarity to PR proteins,
such as (l-3)-/?-glucanase, endochitinase, extensin and
thaumatin.
We previously described a family of TFIIIA-(Cys2/
His2-) type zinc-finger transcription factors in petunia (EPF
family) (Takatsuji 1998, 1999). Members of this family,
ZPT2-1, ZPT2-2 and ZPT2-3 (renamed from EPF1,
EPF2-5 and EPF2-7, respectively) showed floral-organspecific expression patterns (Takatsuji et al. 1994), suggesting their roles in floral-organ-specific transcriptional regulation. Subsequently, we reported seven EPF-family
genes that express transiently and sequentially during anther development as if they form a regulatory cascade
(Kobayashi et al. 1998). We have recently found that some
of these anther-specific zinc-finger proteins play a role in
pollen development (our unpublished results). Besides, we
have reported on several new genes encoding EPF-type
zinc-finger motifs, which remain to be characterized (Kubo
et al. 1998).
Among the undercharacterized zinc-finger genes of
petunia, two of them (ZPT2-10 and ZPT3-3) were found to
express preferentially in the pistil, which was suggestive of
their involvement in transcriptional regulation leading to
the development of this reproductive organ. To characterize the roles of these zinc-finger proteins in post-organogenesis tissue-specific transcriptional regulation, we isolated 5' upstream regions of these genes and analyzed their
promoter activity using the GUS reporter system (Jefferson
et al. 1987). We show herein that the promoter activities of
these two genes were localized in similar, but not identical,
specialized tissues of pistil; the stigmatic secretory zone,
the transmitting tissues in style and the ovary. Based on
their specific expression patterns, we discuss possible roles
of these transcription factors.
ZPT2-10 and ZPT3-3 contain two and three zincfinger motifs, respectively, as putative DNA-binding domains (Kubo et al. 1998). The ZPT2-10 is relatively similar
to the ZPT3-3 among EPF family proteins, particularly in
Pistil-specific zinc-finger genes
378
ZPT2-10
ZPT3-3
Ethidium
Bromide
staining
Fig. 1 Organ specificities of ZPT2-10 and ZPT3-3 expression. Total RNAs were extracted with a RNeasy plant mini kit
(QIAGEN, Hilden, Germany) from floral organs of 50-60 mm
buds and vegitative organs. Five-microgram each of total RNA
was separated on 1% agarose gels that contained 2.2 M formaldehyde, transferred to GeneScreen Plus filters (Du Pont-New
England Nuclear, Boston, MA) and hybridized with digoxigeninlabeled antisense RNA probes for ZPT2-10 and ZPT3-3 full
length cDNA fragments. The temperatures for the hybridizations
were 68 and 60°C for the ZPT2-10 and ZPT3-3 probes, respectively. In these conditions, the both probes specifically hybridize
only their homologous transcripts as examined by separate experiments.
the C-terminal region that includes two zinc-finger motifs.
Northern blot analysis indicated that ZPT2-10 expressed
specifically in the pistil. ZPT3-3 was also found to express
in the pistil, with low level of expression being in petals
as well (Fig.l). Then, we isolated corresponding genomic
ZPT2-10
ZPT3-3
-326
-295
ZPT2-10
ZPT3-3
-285
-250
ZPT2-10
ZPT3-3
-235
-205
ZPT2-10
ZPT3-3
-192
-159
AGA GATGG 3ATTG
GA GATGG
-286
-251
AAAG:TCGCA TGTTACATGT rGTTCCC itTt TACTCTCTTT
CACTC
AAAGGTCGCA TGTTACATGT CCTTCCC
-236
-206
CCTTTATGCA TACGdTTAA TTGAG -TTT TCTTT
CATGCA TACGCTTTAA TTGAG :TTTT TCTTTTCCCT
-193
-160
GGG
TAMGGG
IAAATGC
tfTAAATGC
ATTAATT
ATTAATT
clones of these two proteins and subcloned 5' upstream
regions for the analysis of respective promoter activities.
Sequence analysis revealed putative TATA boxes for both
the genes at proximal upstream of the predicted transcription initiation sites which we described previously (Kubo
et al. 1998). Comparison of the two upstream DNA sequences revealed high similarity (72% identity) stretches of
about 170-bp (Fig. 2) starting at 155- and 123-bp upstream
from the predicted transcription initiation sites of ZPT2-10
and ZPT3-3, respectively, indicating a close relationship of
these two genes. To characterize their tissue-specific expression patterns, the upstream DNA fragments of ZPT210 (2.4 kb) and ZPT3-3 (2.3 kb) were joined to the coding
sequence of the GUS reporter gene (Jefferson et al. 1987)
and introduced into petunia by using Agrobacterium-mQdiated transformation procedure (Jorgensen et al. 1996).
In the upper part of petunia pistil, a high cell-densified
stigmatic secretory zone is connected with stylar transmitting tissue that runs along the axis of the style (Fig. 3A, B).
The stylar transmitting tissue spreads into two directions at
the top of ovary and merges with uppermost cell layer of
placenta. Two thick vascular bundles run through parenchymatous cortex tissue in parallel with the stylar transmitting tract and merge with vascular systems of ovary
walls. A microscopic view of a pollinated pistil after staining with aniline blue showed that pollen tubes penetrated
into the surface of stigma, grew passed the stigmatic secretory zone and stylar transmitting tract, then finally
reached the ovary (Fig. 3C, D). In the ovary, pollen tubes
continue to elongate on the surface of placenta and penetrate into micropyles of ovules.
Histochemical analysis revealed that ZPT2-10-promoter-driven GUS activity was localized in the stylar
TTTAG GAA
TTTAGHGAAF
CCGTT TTAA\G|AAAA TTAC|r|TCC
CGTT TTAA ]TAAAA TTAC TCC
-155
-123
Fig. 2 Conserved sequences in the 5' upstream region of ZPT2-10 and ZPT3-3 genes. Nucleotide sequences were aligned for conserved
5' upstream regions of the ZPT2-10 and ZPT3-3 genes. Boxes indicate identical bases. The sequences are numbered with regard to the
putative transcription start sites. To clone the upstream regions of the ZPT2-10 and ZPT3-3 genes, a genomic library of petunia {Petunia
hybrida var. Mitchell) in EMBL3 vector (Stratagene, La Jolla, CA) were screened with 32P-labeled cDNAs as probes (Sambrook et al.
1989). The DNA fragments containing the upstream regions of ZPT2-10 (3.0 kb) and ZPT3-3 (2.5 kb) were subcloned into an
EcoRV-Xbal and an KpnI-EcoRI sites of the pBluescript SK+ vector (Stratagene), respectively. Then, they were sequenced with an
automatic DNA sequencer (model 373, ABI, Foster City, CA) and analyzed with the GENEWORKES software version 2.5 (InteliGenetics, Mountain View, CA).
Pistil-specific zinc-finger genes
379
Fig. 3 Tissues in the pistils of wild-type petunia and CaMV 35S-promoter-driven GUS activities. (A-D), Microscopic views of wildtype pistils. (A), A longitudinal section of a stigma and the upper part of a style. (B), A transverse section of a style. (C), A longitudinal
section of a stigma and the upper part of a style at six hours after pollination. (D), A longitudinal section of an ovary at 24 h after
pollination. Pistils were fixed and stained according to a standard procedure (Ruzin 1999), then rehydrated, embedded in 5% agar and
sectioned to the thickness of 100 //m with a microslicer (Dosaka EM, Kyoto, Japan). The sections were stained with safranin (A and B)
or aniline blue (C and D). (E-G), GUS staining of 35S::GUS transgenic plants. (E), A longitudinal section of a stigma and the upper
part of a style. (F), A transverse section of a style. (G), A longitudinal section of an ovary. Transformation of petunia (Petunia hybrida
var. Mitchell) was performed as described by Jorgensen et al. (1996). To analyze the tissue specificities of promoter activity,
histochemical staining for GUS activities was performed as described by Gallagher (1992). Bars indicate the lengths of 500 /urn in panel
A, C, D, E and G, SO^um in the panel B, and 100 jum in the panel F. Abbreviations are: co, cortex; ep, epidermis; ov, ovule; ow, ovary
wall; pg, pollen grain; pi, placenta; pt, pollen tube; re, receptacle; sz, stigmatic secretory zone; tt, transmitting tissue; and vb, vascular
bundle.
transmitting tissue (Fig. 4A, C). At the top of the ovary,
the zone of GUS activity separated into two directions and
spread on the surface of placenta (Fig. 4E). The level of
GUS activity in the placenta declined towards the bottom
of the ovary. Under higher magnification, the GUS activity
was found to be confined to the uppermost cell layer of the
placenta (Fig. 4F, G). In no other tissue of the plant was
the GUS activity detected.
The GUS activity driven by the ZPT3-3 promoter
showed a pattern similar to that by the ZPT2-10 promoter
in the stigmatic and stylar transmitting tissues (Fig. 4B, D),
except that the activity in the ovary did not decline towards
the bottom (Fig. 4H). The GUS activity was detected in
the stigmatic secretory zone that serves as a pathway for
pollen-tube growth. In addition, a minor GUS activity was
also detected in receptacle and nectary at the bottom of the
ovary (Fig.4H) and in anthers (data not shown).
In a control experiment, the GUS expression pattern
driven by cauliflower mosaic virus (CaMV) 35S promoter
was found to be quite different from those by the ZPT2-10
and ZPT3-3 promoters. A strong GUS activitiy was detected in the two vascular bundles, whereas the stylar cortex showed a rather weak GUS expression (Fig. 3E, F). The
35S::GUS activity was barely detectable in the stigmatic
secretory zone and the transmitting tissue, unlike those by
the ZPT2-10 and ZPT3-3 promoters. In the ovary, all the
tissues except for the uppermost layer of placenta showed
GUS staining, it being particularly strong in vascular system (Fig. 3G). It is noteworthy that the staining pattern by
35S::GUS appeared to be just the reverse image of that by
ZPT3-3::GUS. The observed 55S-promoter-driven GUS
expression in petunia was inconsistent with that reported
for Nicotiana tabacum (Sessa and Fluhr 1995), where it
was shown to be constitutive in the whole pistil, indicating
interspecies differences in the tissue specificity of the 35Spromoter activities.
Temporal expression patterns of ZPT2-10 and ZPT33 during the maturation of pistil were characterized by
northern blot analysis. The transcripts of ZPT2-10 were
detected at a high level in the style, whereas only at a low
level in the ovary (Fig. 5). The expression level in style increased during pistil elongation, peaking just before anthesis (Fig. 5). The transcripts of ZPT3-3 were detected
in the stigma, style, ovary (Fig. 5) and receptacle (data
not shown), with their levels remaining nearly constant
throughout the maturation process of flowers. These re-
380
Pistil-specific zinc-finger genes
Fig. 4 GUS expression patterns driven by the ZPT2-10 and ZPT3-3 promoters. GUS expression patterns in a ZPT2-10::GUS (A, C,
E, F and G) and a ZPT3-3::GUS transformants (B, D, and H) are shown. (A and B), Longitudinal sections of the stigmas and the upper
parts of the styles in ZPT2-10::GUS (A) and ZPT3-3::GUS (B) transformants. (C and D), Transverse sections of the styles of the
ZPT2-10::GUS (C) and ZPT3-3::GUS (D) transformants. (E and G), Longitudinal sections of the ovaries in the ZPT2-10::GUS
transformant. Shown in the panel G is a higher-magnification view of the boxed area in the panel E. (F), A transverse section of the
ovary of the ZPT2-10::GUS transformant. The position of the section is shown by a line in the panel E. (H), A transverse section of
the ovary of the ZPT3-3::GUS transformant. To construct promoter::GUS chimeric genes, BamHI sites were introduced +21 bp
downstream from the translation initiation sites of the both genes in the plasmids containing the 5'upstream regions by polymerase chain
reaction (PCR) using the primers containing a BamHI site. The DNAs containing the 5' upstream regions were then excised and inserted
into a Sail/BamHI site in a binary vector pBINPLUS (van Engelen et al. 1995) to generate in-flame fusions with the GUS coding sequences. Other experimental procedures are as described in the legend to the Figure 3. Bars indicate the lengths of 500 jum in the panels
A, B, E and H, and 100 ^m in the panels C, D, F and G. Abbreviations are: ne, nectary and se, septum. Other abbreviations are the
same as those in the Figure 3.
suits are consistent with the promoter activities of the both
genes revealed by histochemical analyses using the GUS
reporter gene. The pollination did not affect the levels of
transcripts in any tissues (data not shown).
The transmitting tissue and the uppermost cell layer of
placenta, in which both ZPT2-10::GUS and ZPT3-3::GUS
activities were localized, and the stigmatic secretory zone,
in which only ZPT3-3::GUS activity was observed, are
specialized secretory tissues in pistil that serve as a pathway
for pollen-tube growth (Fig. 3C, D) (Cheung 1996). Both
of these tissues are derived from the same cell layer in
carpel primordia (Satina 1944) and are presumed to play
similar functions in the attraction and support of pollentube growth. Several genes that express in these tissues have
been reported to date. The gene for S-RNase, which is involved in the arrest of tube growth from incompatible
pollen, specifically expresses in the stylar transmitting tissue and the epidermal cell layer of placenta in Nicotiana
alata (Cornish et al. 1987). The genes for extensins and
arabinogalactan-proline-rich protein are also known to express in these secretory tissues of pistil (Cheung 1996).
Transmitting-tissue-specific glycoprotein of Nicotiana tabacum, which has been implicated in the attraction of pollen
tubes (Cheung et al. 1995), specifically expresses in the
transmitting tissues (Cheung et al. 1993). Some genes
whose products are related to PR proteins in their structures have been reported to express in the secretary tissues;
e.g. the genes of /?-glucanase (Sessa and Fluhr 1995), endochitinase (Harikrishna et al. 1996, Ficker et al. 1997) and
thaumatin-like protein (Kuboyama 1998) specifically express in stylar transmitting tissue, and the gene for proteinase inhibitor (Atkinson et al. 1993) expresses in stig-
Pistil-specific zinc-finger genes
style
stigma
upper
lower
ovary
ZPT2-10
ZPT3-3
Ethidium
Bromide
staining
Fig. 5 Temporal expression patterns of ZPT2-10 and ZPT3-3
during the maturation of pistil. Total RNAs from stigmatic, stylar
and ovary fractions at different developmental stages were analyzed by northern hybridization as described in the legend to the
Figure 1. The developmental stages were standardized by the
length of floral buds. The stigmatic fraction includes the top part
of the style as well. The styles were separated into upper and lower
parts at later developmental stages.
matic secretory zone. These pistil PR-like genes are not
inducible by wound treatments, whereas the PR genes are
wound-inducible (Brederode et al. 1991), suggesting the
presence of a pistil-specific defense system. Considering the
similarity in the spatial expression patterns, ZPT2-10 and
ZPT3-3 could be involved in the transcriptional regulation of some of the pistil-specific genes mentioned above,
thereby playing a role in self-incompatibility system, development and maintenance of the secretory tissues, pollen
tube guidance, and/or defense system. Their possible involvement in the pistil-specific defense system is of particular interest, considering the expression patterns of other
EPF-family members. We have recently found that some
of the EPF genes are inducible in leaves by wound treatments (our unpublished data), implying their involvement
in the wound-inducible expression of the PR-family genes.
By extending this speculation, ZPT2-10 and ZPT3-3 could
be the tissue-specific transcriptional regulators of the noninducible PR-like gene family in pistil.
The tissue specificities of ZPT2-10 and ZPT3-3 promoter activities are common in the stylar transmitting
tissue and the uppermost cell layer of placenta, although
the ZPT3-3 promoter showed some extra activities in stigmatic secretory zone, nectary and anthers. The similarity in
the tissue specificity suggested the presence of common cis
element(s) that direct tissue specificity of promoter activity.
The 170-bp sequences conserved between the two promoters (Fig. 2) might as well include such cis element(s). In this
regard, several sequences have been proposed as candidates
for the cis elements that direct the secretory-tissue-specific
gene expression in pistil. Among such sequences are boxes
I-V (Dzelzkalns et al. 1993, Stein et al. 1996) and RK-boxes
381
1 and 2 (Suzuki et al. 1997) that are conserved in the 5'
upstream regions of SLG (S-locus glycoprotein) and SRK
(S-receptor kinase) genes in Brassica. Bipartite-structured
sequences conserved in the 5' upstream regions of the SRNase genes are also regarded as the candidates for such
cis elements (Ficker et al. 1998). The sequences similar to
the ones mentioned above were not found either within the
170-bp sequences or the rest of the 5' upstream regions
of ZPT2-10 and ZPT3-3 promoters. Moreover, no other
conserved sequence was recognized between the upstream
regions of the zinc-finger genes and the structural genes
mentioned above. However, the absence of a common cis
element is not unexpected, considering a possibility that the
structural genes could be under the control of transcription
factors like ZPT2-10 and/or ZPT3-3. If this is the case,
ZPT2-10 and/or ZPT3-3 should interact with the promoter
regions of the structural genes. One of the approaches
towards characterization of the regulatory functions of
ZPT2-10 and ZPT3-3 will be to examine the interaction of
these proteins with the promoter regions of the structural
genes that show similar tissue-specific expression patterns
to those of ZPT2-10 and ZPT3-3 themselves.
The authors are grateful to Dr. Sanjay Kapoor for critical
reading of the manuscript. This work was supported by a COEpromotion fund from the Science and Technology Agency of
Japan and by a PROBRAIN grant from the Bio-Oriented Technology Research Advancement Institution (BRAIN) of Japan.
References
Atkinson, A.H., Heath, R.L., Simpson, R.J., Clarke, A.E. and Anderson, M.A. (1993) Proteinase inhibitors in Nicotiana alata stigmas are
derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5: 203-213.
Bell, J. and Hicks, G. (1976) Transmitting tissue in the pistil of tobacco:
light and electron microscopic observations. Planta 131: 187-200.
Brederode, F.T., Linthorst, H.J.M. and Bol, J.F. (1991) Differential induction of acquired resistance and PR gene expression in tobacco by
virus infection, ethephon treatment, UV light and wounding. Plant Mol.
Biol. 17: 1117-1125.
Cheung, A.Y. (1996) The pollen tube growth pathway: its molecular and
biochemical contributions and responses to pollination. Sex. Plant
Reprod. 9: 330-336.
Cheung, A.Y., May, B., Kawata, E.E., Gu, Q. and Wu, H.-m. (1993)
Characterization of cDNAs for stylar transmitting tissue-specific
proline-rich proteins in tobacco. Plant J. 3: 151-160.
Cheung, A.Y., Wang, H. and Wu, H.-m. (1995) A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their
growth. Cell 82: 383-393.
Cornish, E.C., Pettitt, J.M., Bonig, I. and Clarke, A.E. (1987) Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata. Nature 326: 99-102.
Dzelzkalns, V.A., Thorsness, M.K., Dwyer, K.G., Baxter, J.S., Balent,
M.A., Nasrallah, M.E. and Nasrallah, J.B. (1993) Distinct ris-acting
elements direct pistil-specific and pollen-specific activity of the Brassica
5 locus glycoprotein gene promoter. Plant Cell 5: 855-863.
Ficker, M., Kirch, H.-H., Eijlander, R., Jacobsen, E. and Thompson,
R.D. (1998) Multiple elements of the S2-RNase promoter from potato
(Solanum tuberosum L.) are required for cell type-specific expression in
transgenic potato and tobacco. Mol. Gen. Genet. 257: 132-142.
Ficker, M., Wemmer, T. and Thompson, R.D. (1997) A promoter direct-
382
Pistil-specific zinc-finger genes
ing high level expression in pistils of transgenic plants. Plant Mol. Biol.
35: 425-431.
Gallagher, S.R. (1992) GUS protocols: using the GUS gene as a reporter of
gene expression. Academic Press, Inc., San Diego.
Gasser, C.S. and Robinson-Beers, K. (1993) Pistil development. Plant Cell
5: 1231-1239.
Harikrishna, K., Jampates-Beale, R., Milligan, S.B. and Gasser, C.S.
(1996) An endochitinase gene expressed at high levels in the stylar
transmitting tissue of tomatoes. Plant Mol. Biol. 30: 899-911.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions:
yff-glucuronidase as a sensitive and versatile gene fusion marker in higher
plants. EMBO J. 6: 3901-3907.
Jorgensen, R.A., Cluster, P.D., English, J., Que, Q. and Napoli, C.A.
(1996) Chalcone synthase cosuppression phenotypes in petunia flowers:
comparison of sense vs. antisense constructs and single-copy vs. complex
T-DNA sequences. Plant Mol. Biol. 31: 957-973.
Kobayashi, A., Sakamoto, A., Kubo, K., Rybka, Z., Kanno, Y. and
Takatsuji, H. (1998) Seven zinc-finger transcription factors are expressed
sequentially during the development of anthers in petunia. Plant J. 13:
571-576.
Kubo, K., Sakamoto, A., Kobayashi, A., Rybka, Z., Kanno, Y.,
Nakagawa, H., Nishino, T. and Takatsuji, H (1998) Cys2/His2 zincfinger protein family of petunia: evolution and general mechanism of
target-sequence recognition. Nucl. Acids Res. 26: 608-616.
Kuboyama, T. (1998) A novel thauatin-like protein gene of tobacco is
specifically expressed in the transmitting tissue of stigma and style. Sex.
Plant Reprod. 11: 251-256.
Labarca, C. and Loewus, F. (1973) The nutritional role of pistil exudate in
pollen tube wall formation in Lilium longiflorum. II. Production and
utilization of exdate from stigma and stylar canal. Plant Physiol. 52:
87-92.
Ruzin, S.E. (1999) Plant Microtechnique and Microscopy. Oxford University Press, Oxford.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a
Laboratory Manual. 2nd Edition Cold Spring Harbor Laboratory, New
York.
Satina, S. (1944) Periclinal chimeras in datura in relation to development
and structure (A) of the style and stigma (B) of calyx and corolla. Amer.
J. Bot. 31: 493-502.
Sessa, G. and Fluhr, R. (1995) The expression of an abundant transmitting
tract-specific endoglucanase (Sp41) is promoter-dependent and not essential for the reproductive physiology of tobacco. Plant Mol. Biol. 29:
969-982.
Stein, J.C., Dixit, R., Nasrallah, M.E. and Nasrallah, J.B. (1996) SRK,
the stigma-specific S locus receptor kinase of Brassica, is targeted to the
plasma membrane in transgenic tobacco. Plant Cell 8: 429-445.
Suzuki, G., Watanabe, M., Kai, N., Matsuda, N., Toriyama, K.,
Takayama, S., Isogai, A. and Hinata, K. (1997) Three members of the
S multigene family are linked to the S locus of Brassica. Mol. Gen.
Genet. 256: 257-264.
Takatsuji, H. (1998) Zinc-finger transcription factors in plants. Cell. Mol.
Life Sci. 54: 582-596.
Takatsuji, H. (1999) Zinc-finger proteins: the classical zinc finger emerges
in contemporary plant science. Plant Mol. Biol. 39: 1073-1078.
Takatsuji, H., Nakamura, N. and Katsumoto, Y. (1994) A new family of
zinc finger proteins in Petunia: structure, DNA sequence recognition,
and floral organ-specific expression. Plant Cell 6: 947-958.
van Engelen, F.A., Molthoff, J.W., Conner, A.J., Nap, J.P., Pereira, A.
and Stiekema, W.J. (1995) pBINPLUS: an improved plant transformation vector based on pBIN19. Trans. Res. 4: 288-290.
(Received October 6, 1999; Accepted January 6, 2000)