Plant Cell Physiol. 41(6): 757-766 (2000)
JSPP © 2000
Expression of Bra r 1 Gene in Transgenic Tobacco and Bra r 1 Promoter
Activity in Pollen of Various Plant Species
Takashi Okada, Yoko Sasaki, Rieko Ohta, Noriyuki Onozuka and Kinya Toriyama'
Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural Science, Tohoku University, 1-1 TsutsumidoriAmamiyamachi, Aoba-ku, Sendai, 981-8555 Japan
Bra r 1 encodes a Ca2+-binding protein specifically
expressed in anthers of Brassica rapa. In this study, we
isolated a genomic clone of Bra r 1 and found sequences
similar to Pollen Box core motifs and LAT56/59 box,
pollen-specific cw-acting element, in the 5' upstream region
of Bra r 1. Reporter gene fusion revealed that the Bra r 1
promoter directs male gametophytic expression in Nicotiana tabacum, Arabidopsis thaliana and B. napus, showing strong expression in mature pollen grains similar to that
of endogenous Bra r 1. Genomic DNA of Bra r 1 was introduced into tobacco plants and the highest accumulation
of Bra r 1 protein was observed in mature pollen in the
same manner as reporter gene expression. Using in vitrogerminated pollen tubes of transgenic tobacco, we firstly
demonstrated the subcellular localization of Bra r 1 in
pollen tubes. Bra r 1 protein was distributed throughout
the pollen tube of transgenic tobacco and slightly intense
signals of Bra r 1 were observed in the tip region. In
long-germinated pollen tubes, Bra r 1 was detected only in
the cytoplasmic compartments while no signals were observed in the empty part of the pollen tube, indicating that
cytoplasmic movement toward the tube tip is accompanied
by Bra r 1. Hence, we suggest that Bra r 1 is involved in
pollen germination and pollen tube growth.
phases of specific gene activity are involved in the production of pollen (Mascarenhas 1990). The first phase is during
microspore development, with mRNA levels reaching a
peak around microspore mitosis and then decreasing during pollen maturation. A second phase of gene expression
follows microspore mitosis and is characterized by the
synthesis and accumulation of transcripts which are translated at the time of pollen germination. Genes expressed
during this second phase have been reported and the regulation of gene expression during male gametophyte development has been investigated by using promoter and
reporter gene fusion of Zea mays Zml3 (Guerrero et al.
1990), tomato LAT52 and LAT59 (Twell et al. 1990, 1991)
and Brassica Bcpl (Xu et al. 1993). Furthermore, pollenspecific cis regulatory elements have been characterized in
tomato LA T52, LA T56 and LA T59 promoters (Twell et al.
1990, 1991).
Since transcripts are accumulated and translation is
very active at the mature pollen stage, it seems that these
late expressed gene products would be prepared for successful fertilization. When pollen grains set on the stigma,
they germinate and the pollen tubes grow through the style
to deliver sperm cells to the embyro sac. In this process,
the existence of a tip-focused gradient of cytosolic free
calcium ([Ca2+]c) is essential for the growth of pollen tubes
(Rathore et al. 1991, Malho et al. 1994, Pierson et al.
1994). This tip-focused Ca 2+ gradient is coupled with an
influx of extracellular Ca 2+ (Pierson et al. 1994, 1996), and
changes in the distribution of [Ca2+]c in the tube apex
controls pollen tube orientation (Malho and Trewavas
1996). These Ca2+-mediated processes such as pollen maturation and pollen tube growth should be correlated
with Ca2+-binding proteins, intracellular Ca 2+ receptors.
Calmodulin (CaM) is well characterized as a Ca2+-binding protein ubiquitously expressed in eukaryotic cells and
known to have many functions mediated by cytosolic free
Ca 2+ (Cheung 1982, Rasmussen 1989, Roberts and Harmon 1992). The distribution of CaM in pollen tubes, which
was fluorimetrically visualized by the fluorescent CaM
inhibitor fluphenazine, has been compared with that of
[Ca2+]c and it is thought to be correlated with tip-based
Ca 2+ gradient and pollen tube growth of Lilium longiflorum (Hausser et al. 1984), Nicotiana tabacum (Tirlapur
and Cresti 1992) and Gasteria verrucosa (Tirlapur and
Willemse 1992).
Key words: Bra r 1 — Ca2+-binding protein — Beta-glucuronidase — Pollen allergen — Pollen tube — Promoter
activity.
Pollen, the male gametophyte of flowering plants, develops in anthers and plays a central role in sexual reproduction, i.e., in the production and transmission of the
sperm cells to the embryo sac for double fertilization.
Gametophytic gene expression indicates that two distinct
Abbreviations: CaM, calmodulin; [Ca2+]c, cytosolic free
calcium; DIG, digoxygenin; FDA, fluorescein diacetate; FITC,
fluorescein isothiocyanate; GUS, beta-glucuronidase; IEF, isoelectric focusing; PBS, phosphate-buffered saline; PBST, PBS
containing Tween 20; PEP, pollen extracellular protein; PIP,
pollen intracellular protein; TBS, tris-buffered saline.
The nucleotide sequence reported in this paper has been
submitted to DDBJ under accession number AB032260.
1
Corresponding author.
757
758
Expression of Bra r 1 in transgenic tobacco
We previously reported that allergenic proteins of
B. rapa encoded by Bra r 1 showed significant sequence
similarities to Ca2+-binding proteins (Toriyama et al.
1995). Bra r 1 is distinct from other Ca2+-binding proteins
with a half size of CaM, and contains two Ca2+-binding
domains (EF-hand motif) and both domains bind Ca 2+
(Okada et al. 1998). Bra r 1 was found to be expressed in
anthers and pollen grains at the mature stage, a peak
around second mitosis, but not in stigmas and leaves,
and was characterized as an anther-specific Ca2+-binding
protein. Bra r 1 protein is localized at the pollen coat
(tryphine) and cytoplasm of tapetum and pollen (Okada et
al. 1999). Recently, cDNAs encoding such a two EF-hand
Ca2+-binding protein which is highly homologous to Bra r
1 were isolated from pollen cDNA libraries of birch (Bet v
4), alder (Aln g 4), olive (Ole e 3), Bermuda grass (Cyn d 7),
and timothy grass (Phi p 7) and they were characterized as
Ca2+-binding pollen allergens (Batanero et al. 1996, Engel
et al. 1997, Suphioglu et al. 1997, Hayek et al. 1998,
Niederberger et al. 1999). These Bra r 1-like Ca2+-binding
proteins have been characterized as (1) containing two
EF-hand motifs for Ca2+-binding; (2) being small (ca. 9
kDa), water soluble, acidic proteins; and (3) being expressed in pollen grains. Calcium-dependent conformational changes such as that of CaM have been reported for
the other members of two EF-hands Ca2+-binding proteins
(Batanero et al. 1996, Engel et al. 1997, Hayek et al. 1998).
However, there have been no reports on the genomic sequences and the promoter activity of these genes which
regulate expression in mature pollen. The biological function of Bra r 1-like Ca2+-binding proteins is still unknown.
In this study, we isolated a genomic clone of Bra r 1
and nucleotide sequence of the 5' upstream region was determined. Reporter gene fusion was introduced into tobacco, Arabidopsis thaliana and B. napus, because these
plant species were easier to produce transgenic plants than
B. rapa. Bra r 1 promoter directs expression in the male
gametophyte of transgenic plants, and expression pattern
of reporter gene was compared in each plant species. We
previously detected Bra r 1 protein in the pollen tubes
elongating in the transmitting tissue of the pollinated pistils
of B. rapa, suggesting functional relationship with pollen
tube growth (Okada et al. 1999). However, detailed subcellular localization of Bra r 1 could not be carried out
because in vitro germination of Brassica pollen is rather
difficult. In this sense tobacco is an ideal plant for the study
of pollen tubes, because techniques for in vitro and semi
vivo germination of pollen have been well established
(Mulcahy and Mulcahy 1985) and we can easily obtain
long-elongated pollen tubes. We, therefore, decided to
produce transgenic tobacco carrying Bra r 1 genomic DNA
and to analyze the subcellular localization of Bra r 1 in
pollen tubes of transgenic tobacco.
Materials and Methods
Genomic Southern blot analysis—Genomic DNA from four
members of the family Brassicaceae (Brassica rapa L.,B. napus L.
cv. Westar, B. oleracea L. and Arabidopsis thaliana ecotype
Columbia) was digested with Hindlll, and Southern blot analysis
was performed as described by Suzuki et al. (1995). The blots were
hybridized with a digoxigenin (DIG)-labeled cDNA probe of Bra
r 1 and washed at high or low stringency (0.1 x SSC or 0.5 x SSC,
0.1% SDSat65°C).
Screening of genomic library and sequence determination—A
genomic library of Brassica rapa (Suzuki et al. 1995) was screened
by DIG-labeled cDNA of Bra r 1 according to the manufacturer's
instructions. Out of 120,000 plaques, we identified 3 clones that
hybridized to Bra r 1 cDNA. After restriction mapping and
Southern hybridization analysis, a 3.2 kb Hindlll fragment was
found to contain sequences corresponding to Bra r 1 cDNA; this
fragment was subcloned into the Hindlll site of the pUC18 vector, and the entire nucleotide sequence was determined.
Construction of plasmids—The genomic DNA fragment was
used as a template and the promoter region of Bra r 1 was amplified by PCR using Pfu DNA polymerase (Stratagene, La Jolla,
CA, U.S.A). A primer was designed with a Sail site 16 bp upstream of the ATG codon of Bra r 1 (GTGTTTTGTCGACGTGTTG; Sail site is underlined, see Fig. 2A). A 2.6 kb promoter
region was amplified using Ml3 reverse primer and the designed
primer, and subcloned into the Sail site of the pGEM vector
(Promega, Madison, Wisconsin, U.S.A). The Bra r 1 promoter
fragment extending from this introduced Sail site to an endogenous Hindlll site, 2.6 kb upstream of the ATG codon, was then
ligated into a pBI 101-derived plasmid containing a hygromycinresistant gene (Yokoi et al. 1998) as a Hindlll/Sail fragment to
form pBI-BG (see Fig. 2C).
The 3.2 kb Hindlll genomic DNA fragment of Bra r 1 was
ligated into the same site of the SLJ1006 vector (Jones et al. 1992)
to generate pSLJ-Bra (see Fig.4A).
Transient GUS expression in pollen grains after DNA delivery by particle bombardment—To confirm the Bra r 1 promoter
activity in pollen grains and to check the reliability of the constructed vector, pBI-BG was introduced into pollen grains of
B. napus by particle bombardment as described by Nishihara et al.
(1993). Transient beta-glucuronidase (GUS) expression in pollen
grains was assayed as described in the following section.
Plant transformation—The pBI-BG was transferred to
Agrobacterium tumefaciens strain A136 that contained helper
plasmid pCIB542 (Moore and Nasrallah 1990) and this strain
was used to transform Nicotiana tabacum cv. Petit Havana SRI
(tobacco), A. thaliana ecotype Columbia and B. napus cv. Westar
(rape). The leaf disk method for tobacco (Horsch et al. 1998), the
in planta transformation method for A. thaliana (Bechtold et al.
1993), and the flower stem method for Brassica napus cv. Westar
(Fry et al. 1987) were used for transformation. pSLJ-Bra was also
introduced into tobacco as mentioned above. The primary generation of transgenic plants was defined as Ti and transgenic plants
transformed with pBI-BG or pSLJ-Bra were named BI-BG or
SLJ-Bra plants, respectively.
Genomic DNA was isolated from kanamycin or hygromycin-resistant plants, and digested with Hindlll. Southern blot
analysis was performed as mentioned above to confirm the
integration of the introduced genes and to estimate their copy
numbers. The blots were hybridized with DIG-labeled Bra r 1
promoter probe (a 2.6 kb fragment) and washed at high stringency.
Expression of Bra r 1 in transgenic tobacco
Histochemical GUS assay—Histochemical and fluorometric
GUS assays were performed essentially as described by Jefferson
(1987). Various tissues and different developmental stages of anthers were stained with 2 mM 5-bromo-4-chloro-3-indolyl-beta-Dglucuronide in GUS assay buffer containing 100 mM Tris-HCl
(pH 7.0), 50 mM NaCl and 2 mM potassium ferricyanide at 37°C
for 8 h (De Block and Debrouwer 1992). Developmental stages
of pollen grains were assessed by 4,6-diamidino-2-phenylindole
staining as previously described (Singh et al. 1985). In vitro and
semi vivo-germinated pollen tubes in growth medium (0.01%
H2BO3, 0.01% KH2PO4) 0.1 % CaCl2, 15% sucrose) were prepared as described by Mulcahy and Mulcahy (1985) and incubated
in GUS assay buffer for 2 h.
Protein extraction and immunoblot analysis—Anthers at
four different developmental stages as well as stigmas, styles,
sepals, petals and leaves from transgenic tobacco plants carrying
Bra r 1 genomic DNA were homogenized with extraction buffer
containing 50 mM Tris-HCl (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, and 2 fig ml'1 aprotinin. After centrifugation
(12,000 xg, 15 min, 4°C), the supernatant was used for immunoblot analysis as a total protein. The amounts of proteins were estimated by the method of Bradford (1976) using protein assay
(Bio-Rad, Hercules, California, U.S.A).
An extracellular fraction of pollen grains was also extracted
by an aqueous buffer as described by Evans et al. (1991). Extracellular proteins were extracted by vigorously vortexing tobacco pollen grains for 5 min in 15% sucrose, which was the same
concentration as that in the pollen tube growth medium so as to
avoid osmotic damage of pollen grains during extraction. The
suspension was centrifuged for 15 min at 12,000xg and the supernatant was removed as a pollen extracellular fraction (PEP).
The remaining pollen grains were washed with the same buffer
twice, and then pollen intracellular proteins (PIP) were extracted
by homogenization. Pollen viability was examined using fluorescein diacetate (FDA) after extraction of pollen extracellular
protein as previously reported (Heslop-Harrison and HeslopHarrison 1970); more than 95% of the pollen grains were intact.
Examination of pollen homogenates showed that more than 95%
of the pollen grains were ruptured.
The protein extracts were subjected to thin-layer polyacrylamide gel isoelectric focusing (IEF; pi 3.5-9.5, Amersham-Pharmacia, Upsala, Sweden) or 15% SDS-polyacrylamide gel, and the
separated proteins were electroblotted onto polyvinylidene difluoride membrane (Millipore, Bedford, MA, U.S.A). Protein blots
were incubated with anti-Bra r 1 antibody (Okada et al. 1998)
or anti-a-tubulin monoclonal antibody (Amersham-Pharmacia,
Upsala, Sweden). The blots were washed in tris-buffered saline
(TBS), and incubated with goat anti-mouse IgG conjugated with
alkaline phosphatase (Promega, Madison, Wisconsin, U.S.A).
After TBS wash, the bound antibody was visualized by incubation
with 5-bromo-4-chloro-3-indolyl-phosphate /?-toluidine salt and
nitroblue tetrazolium chloride solution.
Indirect immunofluorescence microscopy—Indirect immunolocalization of Bra r 1 in the pollen tubes of transgenic tobacco
carrying Bra r 1 genomic DNA was determined as described by
Lin et al. (1996). Pollen grains from the SLJ-Bra tobacco line were
germinated in a liquid medium. Germinated pollen tubes were
incubated in a fixation buffer (4% paraformaldehyde, 50 mM
PIPES buffer, pH 6.9, 2 mM MgSO4). After being washed with
PBS, pollen tubes were treated with 2% (w/v) cellulase R-10
(Yakult) and 1% (w/v) Macerozyme R-10 (Yakult) in a buffer
containing 15 mM MES, pH 5.5, 400 mM mannitol, 5 mM CaCl2
and 1 fig ml ' aprotinin. After being washed with PBS containing
759
Tween-20 (PBST), the pollen tubes were blocked with 3% (w/v)
nonfat dry milk in PBS, and incubated with the anti-Bra r 1
antibody. After being washed in PBST, the pollen tubes were
incubated with a secondary antibody (fiuorescein anti-mouse
IgG, Vector Laboratories, Burlingame, CA, U.S.A). Slides were
mounted with Perma Fluor Aqueous Mountant (Shandon, Pittsburgh, PA, U.S.A). Observations and photography were conducted on a Zeiss Axioskop.
Results
Southern blot analysis of Bra r 1—Bra r 1 has been
shown to have significant sequence similarities to Ca 2+ binding proteins, and homologous genes have been reported to exist in several plant species (Batanero et al. 1996,
Engel et al. 1997, Suphioglu et al. 1997, Hayek et al. 1998,
Niederberger et al. 1999). To investigate the genomic organization of Bra r i m the Brassicaceae, Southern blot
analysis was performed using genomic DNA from B. rapa,
B. napus, B. oleracea, and Arabidopsis thaliana.
A single intense band corresponding to Bra r 1 was
observed at 3.2 kb for the Brassica species and a weak band
at 4.7 kb for A. thaliana (Fig. 1A). These results suggest
that Bra r 1 gene is present as a single copy gene in B. rapa,
B. oleracea and A. thaliana. When the blots were washed
at lower stringency, a few weak bands were also detected
(Fig. IB), indicating existence of related genes of Bra r 1
in the Brassica genome. Since B. napus is an amphidiploid between B. rapa and B. oleracea, restriction fragment
length polymorphism revealed that B. napus has genes derived from both B. rapa and B. oleracea (Fig. IB). In fact,
A
1 2
B
3 4
1 2
3 4
23.1 —
9.4 —
6.6 —
4.4 —
******
2.3 —
2.0 —
kb
Fig. 1 DNA gel blot analysis of Bra r 1 in the Brassicaceae.
Genomic DNA of B. rapa (lane 1), B. napus (lane 2), B. oleracea
(lane 3), and A. thaliana (lane 4) was digested with Hindlll. The
blots were hybridized with DIG-labeled Bra r 1 cDNA probe, and
washed at high (A) and low (B) stringencies. Molecular markers
are indicated in kb on the left.
760
Expression of Bra r 1 in transgenic tobacco
we obtained cDNA clones from B. napus which has exactly
the same sequences as that of Bra r 1 (Toriyama et al.
1995). It is noteworthy that Bra r 1 was also found to be
conserved in Arabidopsis, another genus of Brassicaceae.
Genomic structure of Bra r 1 gene—We isolated genomic clones of Bra r 1 and subcloned a 3.2 kb HindlW
fragment corresponding to the band detected by Southern
blot analysis in Figure 1A. Determination of the entire
DNA sequence revealed the fragment to be comprised of
3,168 bp nucleotides (DDBJ Accession no. AB032260).
Figure 2A shows a part of the nucleotide sequence of Bra
r 1, including the coding region and 510 bp of the 5' upstream and 3' downstream regions. Bra r 1 was shown to
have no introns. The promoter region contains several sequence motifs similar to pollen specific cis-elements, for
example the PB core motif and the LAT56/59 box (Twell
et al. 1991). These elements have been reported to be pollen-specific enhancer elements identified in tomato pollen-specific genes. Within the 500 bp 5' upstream region
of the initiation codon, four repeats of sequence motifs
TTTGGTT, in which six out of seven nucleotides matched
those of the PB core motifs (TGTGGTT), were present at
— 278, —284, —433 and —480, when nucleotides were
numbered with the first nucleotide of initiation codon
marked as + 1 . (Fig. 2A, B). A sequence motif GAAATAGTGA, in which nine out of ten nucleotides matched
those of the LAT56/59 box (GAAATTGTGA), was
present at —360. We also found four repeats of a unique
sequence TTTTG at - 2 4 2 to - 2 6 1 , although the significance of this sequence repeat is unknown.
The Bra r 1 promoter activity during pollen development—To investigate the spartial and temporal regulation
of the Bra r 1 promoter, we fused a 2.6 kb promoter of
Bra r 1 with the GUS reporter gene to construct pBIBG (Fig. 2C). We introduced this construct into tobacco,
A. thaliana and B. napus to compare the expression pattern in different plant species. Prior to transformation of
the plants, pBI-BG was introduced into pollen grains of
B. napus by particle bombardment, and we confirmed
the transient expression of GUS gene in mature pollen of
B. napus (data not shown).
We obtained 12 transgenic plants with pBI-BG in
tobacco, 20 in A. thaliana and 3 in B. napus, respectively.
Pollen grains were histochemically stained for GUS in all
transformants. GUS expression in BI-BG tobacco was
initially observed in uninucleate microspores just before
pollen mitosis at the late uninucleate microspore stage and
increased during pollen development; the highest expression was detected in mature pollen (Fig. 3A-C). The BI-BG
A. thaliana and B. napus plants showed similar expression
patterns in which GUS activity was initially detected in bicellular pollen and dramatically increased in mature pollen
(Fig. 3D-I). The expression pattern was essentially the
same among all the transformants in each plant species.
-510
-459
-408
-357
-306
-255
-204
-153
-102
-51
+1
+ 52
+ 103
+ 154
+205
+256
+ 307
+ 358
+ 409
+ 460
+ 511
ATATTCTGTTATAATTTAGTATTAATATTaTTTGGTTtTATAATAGGTAAGA
TGGTGATTGTTTGTATTATTTTTCTTlTTTGGTTtrAAGGATTGTTTGTTTCT
AACTGTGGAATTTACACGACATATAATATAGTTTTGTTCGCATGAACTim
S^jjflliTTTATAGAAGCTATTGCTTTAGGGACCGGTTTACGTTTGTTAAA
AAACTTGAAACAAATTGAAAT^fTTTGGTTTTGGTTtrTCTAGTTAATTTTGT
TTTCTjrTTGTTTTGTTTTTTTTTAAATTACAGTTAATTCGTGAAAGACCAA
GTCACATCCTAATTCTGGTAAATTAATTAGTTTTAACTCGGACCATTTGAA
TTCAATGGTTGTTCAGTGTTATTCCTCCAAAAGTTCCTAATTAAAAGCACA
TCCTCAACTTTTTTGTCACTCAGATTTOiTAfATAAtTTGATGATTCTTTGT
TTTCTCAACCAAAAAGGAAAATCATCTTTCAACACATAGACAAAACACACT
3' GTTGTGQAQCTGTTTTGTG 5'
ATGGCTGATGCTGAGCACGAACGTATATTCAAGAAATTTGACACTGACGGC
M A D A E H E R I F K K F D T D G
GATGGTAAAATATCAGCAGCCGAACTTGAAGAAGCTCTTAAGAAACTTGGC
D G K I
S A A E L E E A L K K L G
TCGGTGACCCCTGATGACGTGACTCGTATGATGGCTAAAATCGATACTGAT
S V T P D D V T R M M A K I D T D
GGTGATGGAAACATATCGTTTCAAGAATTCACCGAGTTTGCATCTGCCAAT
G D G N I S F Q E F T E F A S A N
CCTGGACTCATGAAGGATGTTGCCAAAGTTTTCTAGAATGGTAGTATTTGC
P G L M K D V A K V F
CATCGTCTTATTTTTGAATTTTTCTAATATTTTTTTTCAATATGATTTGGT
TCTAAGAAACTATCCATATTATAATTCATGTAACTGATCATTGGAGTTGTA
TATCTCTCAATGAATACTCTCGAGTATTTTTAATTTTTGTTTATTTCCCTA.
ATAAATTCAAGTTTTGCTACGTTTACACATTCTTAATATTGCTTTCAATAT
AATAAATACATTTAATCCATACATAGCCAATCTAAATTGGTAACTTGCACA
TAATCTTAGCTACTATAAGCTT
Hindlll
B
1
II
H
BX
s
1LAT 56/59 box
DPB core motif
1TATA box
c
pBI-BG
H
RB
4H
NPTII
LB
HPT
H
1kb
Fig. 2 (A) A part of the nucleotide sequence of Bra r 1 gene.
The deduced amino acid sequence is shown below the nucleotide
sequence. Nucleotides are numbered with the first nucleotide of
the initiation codon marked as + 1 . Shaded, open and dotted
boxes indicate significant sequence similarity to LAT 56/59 box,
PB core motif and TATA box, respectively. The dotted underline
indicates four of TTTTG sequence repeats. The putative polyadenylation signal is underlined. Hindlll site at the 3' end is also
underlined. The nucleotides shown in italics below the genomic
sequence indicate the primer used for amplifying the promoter
region and the substituted nucleotides are underlined. (B) Restriction map of a 3.2 kb Hindlll fragment containing Bra r 1 gene.
Gray, white and black boxes indicate the LAT 56/59 box, PB core
motif and TATA box, respectively. The restriction sites indicated
are as follows: B, BamHl; E, EcoRl; H, ifwidlll; S, Sphl; X,
Xbal; Xh, Xhol. (C) Construction of transformation vector
pBI-BG. Abbreviations: NPTII, gene for kanamycin resistance;
HPT, gene for hygromycin resistance; Bra pro, promoter sequence of Bra r 1; RB and LB, right and left borders of T-DNA;
H and S, Hindlll and Sad sites, respectively, used for inserting
the Bra r 1 promoter.
Expression of Bra r 1 in transgenic tobacco
761
Fig. 3 Histochemical GUS staining of pollen grains from BI-BG transformed N. tabacum (A-C), A. thaliana (D-E) and B. napus
(G-I). Upper panel (A, D, G), middle panel (B, E, H) and lower panel (C, F, I) represent uninucleate microspores, mid-bicellular pollen,
and mature pollen, respectively. (J) In vitro-germinated pollen tubes of BI-BG tobacco, transgenic No. 12. (K) Semi vivo-germinated
pollen tube of BI-BG tobacco, No. 33. Bars in (A) to (I) = 20//m, (J) = 50//m (K) = 200//m.
Histochemical GUS staining of the in vitro and semi
vivo-germinated pollen tubes of BI-BG tobacco was examined. The in vitro-germinated pollen tubes showed uniform staining (Fig. 3J). With the semi vivo technique,
pollen tubes grow from the bases of cut styles into the
growth medium (Mulcahy and Mulcahy 1985). Semi vivogerminated pollen tubes were sharp and germinated
straightly, and blue staining was stronger in the tip region
(Fig.3K).
Expression of Bra r 1 in tapetum cells in B. rapa has
been reported and Bra r 1 protein in tapetum cells is considered to be deposited on the pollen coat during the degeneration of tapetum cells (Okada et al. 1999). None of
the BI-BG transformants showed GUS activity in tapetum
cells by histochemical GUS staining (data not shown). This
may be due to the lack of some factors in Bra r 1 promoter
needed to activate the expression in tapetum cells. Low
GUS activity was observed in the leaves and roots for all
species, in stigmas, sepals, petals and ovaries of tobacco,
and in stigmas and sepals of A. thaliana (data not shown).
The expression level, however, was much lower than that
of mature pollen.
Segregation of GUS expression in pollen grains—Segregation of GUS-positive and GUS-negative pollen of Ti
BI-BG plants was observed in all species, indicating that
the Bra r 1 promoter was gametophytically active. The
stained pollen was counted and scored. According to
Southern blot analysis and GUS expression in pollen
grains, 7 plants of tobacco, 3 of A. thaliana and 3 of
B. napus plants were selected for segregation analysis of
GUS pollen. Segregation of GUS pollen of tobacco was
consistent with the T-DNA copy number determined by
Southern blot, except for transformant No. 5 (Table 1).
Transgenic tobacco containing one copy of T-DNA revealed 1 : 1 segregation of GUS positive and negative pollen
grains, and two and three copies showed 3 : 1 and 7 : 1
segregation, respectively. Although two B. napus transformants revealed 1 : 1 segregation, they had more than two
copies of T-DNA, indicating the existence of several copies
in one locus or only one active gene. We could not determine a significant segregation ratio in A. thaliana; however, transformant No. 1 showed close to a 3 : 1 ratio and
No. 2 and 3 showed a ratio of nearly 1 : 1 .
Expression of Bra r 1 in transgenic tobacco—We have
previously demonstrated that Bra r 1 protein was detected
in the pollen tubes elongating in the transmitting tissue
of the pollinated pistils of B. rapa (Okada et al. 1999).
Tobacco is a more ideal plant for the study of pollen tube
growth than Brassica species, because in vitro and semi
vivo pollen tube germination methods are well established
(Mulcahy and Mulcahy 1985). To analyze the subcellular
localization of Bra r 1 in pollen tubes, the vector pSLJ-Bra
containing Bra r 1 genomic fragment was introduced into
tobacco to express Bra r 1 in tobacco pollen (Fig. 4A). Ten
Expression of Bra r 1 in transgenic tobacco
762
Table 1 Segregation of GUS expression in pollen grains of BI-BG transformants
Pollen a
GUS +
GUS-
Plant line
Segregation
ratio b
T-DNA copy
number c
1
2
3
4
5
6
7
502
227
158
66
108
158
234
213
164
78
0
49
33
7:
1
1
1
1
3
7
1
1
1
1
0
1
1
3
1
1
1
2
2
3
A. thaliana
1
2
3
341
307
235
156
230
318
N.D.
N.D.
N.D.
2
4
3
B. napus
1
2
3
121
201
752
130
191
182
1: 1
1: 1
N.D.
2
3
5
N. tabacum
74
a
Number of GUS positive ( + ) or negative (—) pollen grains from Tj transformant.
* Segregation ratio of GUS expression which showed significant difference. N.D., non-significant
difference is determined. Probability >0.05.
c
T-DNA copy number was determined by Southern blot analysis detected by Bra r 1 promoter
probe.
kanamycin-resistant plants were generated, and three SLJBra plants showing Bra r 1 expression were selected to
generate T2 progeny. The SLJ-Bra tobacco line 1 to 3 each
showed a strong expression of Bra r 1 in anthers which was
detected as a band at pi 4.3 with the same pi value of Bra
r 1 from B. napus (Fig.4B). Weak signals were also detected in the non-transformant and SLJ-Bra line 4. These
signals would be derived from the expression of endogenous Bra r 1 homologue of tobacco and indicate that the
anti-Bra r 1 antibody could be cross-reactive, since the expression of Bra r 1-like Ca2+-binding protein in pollen of
several plant species have been reported and considered to
be conserved in various plant pollen (Batanero et al. 1996,
Engel et al. 1997, Suphioglu et al. 1997, Okada et al. 1998).
The expression of Bra r 1 in tobacco has no effect on pollen
development and pollen tube growth. SLJ-Bra tobacco and
non-transformants showed no differences in pollen viability, in vitro pollen tube growth, seed sets and transmission
of the transgene into T2 progeny (data not shown).
Koltunow et al. (1990) denned 12 developmental stages
of tobacco anthers after meiosis. Bra r 1 was initially detected at stages 7 to 9 containing mid-bicellular pollen and
was mostly detected at stages 10 to 12 containing mature
pollen from SLJ-Bra tobacco No. 18 (Fig. 4C). The results
for the other transformant, SLJ-Bra No. 22, were similar
(data not shown). The temporal expression pattern of Bra
r 1 in anthers of SLJ-Bra tobacco was similar to GUS expression of BI-BG tobacco, with highest expression in
mature pollen. BI-BG tobacco showed weak GUS expres-
sion at stages 1 to 6, however, Bra r 1 protein was not detected in SLJ-Bra tobacco at these stages. These difference
is probably due to the difference in method used for detection of gene expression, i.e., GUS assay and immunoblot analysis, or stability of proteins. Although endogenous
Bra r 1 was expressed in pollen grains and tapetum cells in
Brassica (Okada et al. 1999), no Bra r 1 was detected at
stages 1 to 3 when the tapetum is very active, indicating no
expression in tapetum cells of SLJ-Bra tobacco. This result
is consistent with the expression of reporter genes in BI-BG
plants which showed no GUS expression in tapetum. Bra r
1 protein was not detected in vegetative tissue and other
floral organs (leaves, ovaries, stigmas, styles, petals and
sepals) of transformant SLJ-Bra No. 22 (Fig. 4D) and No.
34 (data not shown).
Distribution of Bra r 1 in pollen of transgenic tobacco—We have previously demonstrated that Bra r 1 protein
is localized in the pollen coat as well as in the pollen
cytoplasm, and released from pollen grains into an aqueous buffer (Okada et al. 1999). It is not clear, however,
whether or not Bra r 1 protein released into an aqueous
buffer comes from inside the pollen grains or pollen coat
which is derived from tapetum. Such a tapetum-derived
Bra r 1 was not included in the transgenic tobacco carrying
Bra r 1 genomic DNA, because the transgene was not active
in the tapetum as described above.
We prepared pollen extracellular proteins by vigorously vortexing pollen grains in 15% sucrose solution for
5 min. This procedure hardly damage the pollen grains
Expression of Bra r 1 in transgenic tobacco
based on the FDA staining. Figure 4E shows that a significant amount of Bra r 1 protein was detected in the
pollen extracellular fraction (PEP), as well as in the pollen
intracellular fraction (PIP), although the amount of protein loaded in the PEP lane was one-sixth of that in the PIP
A
pSLJ-Bra
RB
2'35S
i—1
Brar1genomi~H
LB
NPTll
B
1
B.n wt
2
3
4
8.65 —
8.45 —
8.15 —
7.35 —
6.85 —
6.55 —
5.85 —
5.20 —
4.55 —
m
m
3.75 —
a-tubulin
B.n
1-3
4-6
B.n
Le
Wt
7-9 10-12 1-3
No. 18
4-6
7-9 10-12
Brari
a-tubulin
D
Ov
St
Pe
Brar 1
PEP
Brar 1
a-tubulin
PIP
m
—*.
Se
An
763
lane (upper panel). As an intracellular control, anti-atubulin monoclonal antibody was reacted with the protein
gel blot: signals were mainly obtained in the PIP fraction
and slight amount of protein was detected in the PEP
fraction (Fig.4E, lower panel), indicating that pollen
grains were hardly damaged during the extraction procedure. These results indicate that Bra r 1 protein was
released from inside the pollen grains upon hydration.
Localization of Bra r 1 in the pollen tube—To study
the localization of Bra r 1 in pollen tubes in transgenic
tobacco, an indirect immunofluorescence experiment was
performed (Fig. 5). In vitro-germinated pollen tubes of
SLJ-Bra tobacco were stained using anti-Bra r 1 antibody
and FITC-conjugated secondary antibody. The signal,
green fluorescence, was detected evenly throughout the
cytoplasm of pollen tubes and pollen grains; however,
slightly intense fluorescence was observed in the tip region (Fig. 5C, F). In contrast, no fluorescent signal was
observed in the pollen tubes of the non-transformant
(Fig. 5A). The morphology of pollen tubes was checked by
light microscopy (Fig. 5B, D) and we confirmed normal
pollen tube growth in SLJ-Bra tobacco.
The elongated pollen tubes after overnight culture
were divided by callose plug and cytoplasmic components
had moved toward the tip, leaving the pollen tubes empty
in the base region. The fluorescent signal was observed only
in the cytoplasmic compartments of long pollen tubes,
while no signals were detected in the empty parts of the
Fig. 4 Expression of Bra r 1 in T2 progeny of transgenic
tobacco. (A) Construction of transformation vector pSLJ-Bra.
Abbreviations: 2'35S, divergently transcribed 2' and 35S promoter
in pSLJ1006 (Jones et al. 1992); Bra r 1 gene, 3.2 kb Hindlll
fragment containing Bra r 1; others, see legend to Fig. 2. (B) Immunoblot analysis of tobacco carrying Bra r 1 genome. Protein
extracts were prepared from anthers of B. napus (B.n), nontransformant (wt) and 4 independent transformants (lanes 1 to 4).
Thirty micrograms of B.n and 60 /ug of tobacco extracts were
separated by IEF for the detection of Bra r 1 or SDS-PAGE for
the detection of a-tubulin as a control. The protein blots were
incubated with anti-Bra r 1 (upper panel) or anti-a-tubulin antibodies (lower panel), pi markers are indicated on the left. (C)
Expression of Bra r 1 during anther development. Protein extracts
were prepared from tobacco anthers at 4 different developmental
stages (1-3, 4-6, 7-9, 10-12), according to Koltunow et al. (1990).
Protein blot of non-transformant (wt) and transgenic plant No. 18
was prepared and immunoblot was performed as in (B). (D) Detection of Bra r 1 in various tissue of SLJ-Bra tobacco No. 22.
Forty micrograms (B.n) or 100^/g (others) of protein extracts were
separated by IEF as in (B). B.n, anthers of B. napus; Le, leaves;
Ov, ovaries; St, stigmas and styles; Pe, petals; Se, sepals; An,
anthers. (E) Distribution of Bra r 1 protein in pollen extracellular
fraction (PEP) and intracellular fraction (PIP) of mature pollen
of SLJ-Bra tobacco. The pollen extracellular proteins (7.5 fig) and
intracellular proteins (50 jug) were separated by IEF for the detection of Bra r 1 or SDS-PAGE for a-tubulin as a control of intracellular proteins. The protein blots were incubated with antiBra r 1 (upper panel) and anti-a-tubulin antibodies (lower panel).
Expression of Bra r 1 in transgenic tobacco
764
Fig. 5 Subcellular localization of Bra r 1 in growing pollen tubes
of SLJ-Bra tobacco transformant. In vitro-germinated pollen
tubes were fixed, incubated with anti-i?ra r 1 antibody followed by
FITC-conjugated secondary antibody. (A and B) FITC and light
microscopic images of short pollen tube of non-transformant. (C
and D) Same image of SLJ-Bra transformant. (E to G) Long
pollen tube divided by a callose plug or pollen wall. Cytoplasmic
compartments after staining with FITC are shown at lower magnification; the pollen tube was germinating left to right (E), higher
magnification (F) and that of light microscopic image (G).
Bars = 50 jum.
pollen tubes or in the callose plugs (Fig. 5E-G). These
results indicate that Bra r 1 is distributed in the cytoplasm
of pollen tubes and that the cytoplasm of pollen tubes
moves toward the tip which is accompanied by Bra r 1.
Discussion
We have previously described anther-specific expression of the gene Bra r 1 in B. rapa (Toriyama et al. 1995,
Okada et al. 1999). In the present study, we isolated a
genomic clone of Bra r 1 and demonstrated its promoter
activity in pollen grains of TV. tabacum, A. thaliana and
B. napus. The GUS gene controlled by the Bra r 1 promoter
revealed strong expression in mature pollen of all species
similar to that of the endogenous Bra r 1 in B. rapa.
Pollen-specific cis-elements have been well characterized in tomato LAT52, LAT56 and LAT59 promoters
(Twell et al. 1990, 1991). Twell et al. (1991) reported that
the PB core motif activated pollen-specific gene expression in transgenic tobacco. Sequence analysis of Bra r 1
demonstrated that six out of seven nucleotides of the four
regions in the 5' upstream region of Bra r 1 matched those
of the PB core motif (Fig. 2). The LAT56/59 box has been
also reported to enhance the basal promoter activity in
tobacco pollen (Twell et al. 1991) and the GTGA sequence
has been determined for the core sequence for activation.
There is one sequence showing nine of ten matches to the
LAT56/59 box and GTGA motif is also conserved in the
Bra r 1 promoter (Fig. 2). The Bra r 1 promoter activity was
increased during pollen development and showed the
highest expression in mature pollen of transgenic tobacco,
A. thaliana and B. napus. These results were consistent
with our previous finding that Bra r 1 transcripts and protein were accumulated during pollen maturation (Toriyama
et al. 1995, Okada et al. 1999). Hence, these putative cisregulatory elements may act as a pollen-specific gene expression of Bra r 1. Further promoter deletion analysis will
elucidate the contribution of such putative cis-elements to
activate expression in pollen.
The expression of endogenous Bra r 1 has been
characterized and mRNA and protein have been detected
in pollen and tapetum cells, but not in stigmas, leaves
and ovaries (Toriyama et al. 1995, Okada et al. 1999). In
general, proteins produced in the tapetum are reportedly
deposited on the pollen surface as tryphine during the degeneration of the tapetum (Dickinson and Bell 1972, 1976,
Dickinson and Lewis 1973). All transgenic plants showed a
similar GUS expression pattern in microspores and pollen,
but no expression in tapetum. Promoter activity has been
reported to differ among plant species. For example, Bcpl
gene promoter has been found to be active in tapetum in
transgenic A. thaliana, but inactive in transgenic tobacco
(Xu et al. 1993). Twell et al. (1990) also reported that tissue
specificity of LAT59 promoter in transgenic plants was
different from native gene expression in pollen and sporophytic cells in anthers. Sequences required for activation in
the tapetum may not be present in the introduced fragment
of Bra r 1 promoter, as reported for Bcpl gene promoter
(Xu et al. 1993).
Bra r 1 has been characterized as a novel family of
Ca2+-binding protein, and this type of Ca2+-binding protein has been shown to be widespread in plant species and
considered to play a fundamental role in pollen (Toriyama et al. 1995, Batanero et al. 1996, Engel et al. 1997,
Suphioglu et al. 1997, Hayek et al. 1998). Tobacco may
also possess these two EF-hand Ca2+-binding proteins.
Thus, the weak band detected in the non-transformant
tobacco may be the Bra r 1 homologue in tobacco, and the
antibody could be cross-reactive (Fig. 4B). We also isolated
cDNAs homologous to Bra r 1 from tobacco anthers and
they showed high sequence similarity to Bra r 1-like Ca 2+ binding protein (unpublished results).
These Bra r 1-like Ca2+-binding proteins have been
reported to be easily extracted by suspending pollen grains
E x p r e s s i o n o f Bra rim
in an aqueous buffer (Toriyama et al. 1995, Batanero et al.
1996, Engel et al. 1997, Smith et al. 1997, Okada et al.
1999). We demonstrated that Bra r 1 expressed in tobacco
pollen was released into an extracellular buffer (Fig. 4E).
However, Bra r 1 does not have hydrophobic N-terminus,
characteristics of a signal peptide (Okada et al. 1999),
suggesting that the release of Bra r 1 is different from the
general secretion mechanism, this being due to the small
peptide that can pass through the pollen plasma membrane. This release of two EF-hand Ca2+-binding proteins
upon hydration may play a role in such phenomena as
Ca2+-mediated pollen-pistil interaction and change the
concentration and distribution of [Ca2+]c in pollen grains
to trigger pollen germination and tube growth (Okada et al.
1998, 1999).
Effect of extracellular CaM on pollen tube growth has
been reported. Pollen germination and tube growth were
inhibited by anti-CaM serum and CaM antagonist, and
addition of exogenous CaM in medium enhanced pollen
germination and tube growth (Ma and Sun 1997). Ma et al.
(1999) also reported extracellular CaM activated heterotrimeric G protein associated with pollen plasma membrane
that is correlated with pollen tube growth. Hence, we suggest that extracellular Bra r 1 also may have a function like
CaM related with pollen tube growth.
In pollen tubes, the tip-focused gradient of [Ca2+]c is
essential for growth (Rathore et al. 1991, Malho et al. 1994,
Pierson et al. 1994) and this gradient is coupled with an
influx of extracellular Ca 2+ and Ca 2+ channel activity in
the apex of the tube (Pierson et al. 1994, Malho et al.
1995). This calcium mediated pollen tube growth should be
correlated with Ca2+-binding proteins. The typical EFhand Ca2+-binding protein CaM is well characterized and
high levels of CaM have been reported to be present in
germinal apertures, germination bubbles and tube tips using a monoclonal anti-CaM antibody (Tirlapur et al. 1994).
Moutinho et al. (1998) also reported that microinjection of
fluorescently-labeled CaM revealed uniform distribution in
pollen tubes, but that a part of the intense V-shaped signal
was observed behind the apical region, suggesting an interaction with cytoskeletal-bound target proteins.
In this study, the expression of Bra r 1 in transgenic
tobacco revealed that protein showed the greatest accumulation in mature pollen and was distributed throughout
pollen tubes. Slightly intense fluorescence, however, was
observed in the tip region (Fig. 5). Distribution of GUS
protein in pollen tubes also revealed that more protein was
located in the tip region (Fig. 3K). These results are possibly due to the difference of cytoplasmic density in pollen
tubes, which is higher in the tip region. Strong GUS
staining was observed in semi vivo-pollen tubes elongated
more than 3 cm in length after pollination (Fig. 3K). Although it depends on the stability of GUS protein translated in mature pollen, it is reasonable to understand that
765
transgenic tobacco
Bra r 1 protein is newly translated in pollen tubes, which
accounts for strong GUS expression in 3 cm-long pollen
tubes 32 h after pollination. Bra r 1 protein was also detected in the cytoplasmic compartments of long pollen
tubes, indicating cytoplasmic movement toward the tube
tip accompanied by Bra r 1 with translation in the pollen
tube (Fig. 5E, F). Bra r 1 is always localized in cytoplasmic
compartments close to the tube tip, active Ca 2+ entry site.
We therefore suggest that Bra r 1 may control the distribution of [Ca2+]c in pollen tubes during elongation.
In summary, we found that the Bra r 1 gene promoter
is active in mature pollen and that Bra r 1 is distributed
throughout pollen tubes in transgenic tobacco. Release of
Bra r 1 from pollen grains and localization of Bra r 1 in the
pollen tube may be correlated with pollen-pistil interaction,
pollen germination and tube growth. Investigation of promoter sequences and the activity of Bra r 1 in various
plants are expected to yield useful information for further
study of the expression of Bra r 1 and the antisense inhibition of endogenous Bra r 1.
This work was supported in part by Grants-in-Aid for Special
Research on Priority Areas (no. 07281101; Genetic Dissection of
Sexual Differentiation and Pollination Process in Higher Plants)
from the Ministry of Education, Science, Culture and Sports,
Japan. T.O. is a recipient of Research Fellowship from the Japan
Society for the Promotion of Science for Young Scientists.
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