Different Temporal and Spatial Gene Expression Patterns Occur during Anther
Development
Author(s): Anna M. Koltunow, Jessie Truettner, Kathleen H. Cox, Marco Wallroth and
Robert B. Goldberg
Source: The Plant Cell, Vol. 2, No. 12 (Dec., 1990), pp. 1201-1224
Published by: Oxford University Press
Stable URL: https://www.jstor.org/stable/3869340
Accessed: 06-11-2023 14:52 +00:00
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The Plant Cell, Vol. 2, 1201-1224, December 1990 ? 1990 American Society of Plant Physiologists
Different Temporal and Spatial Gene Expression
Patterns Occur during Anther Development
Anna M. Koltunow, Jessie Truettner,1 Kathleen H. Cox,2 Marco Wallroth,3 and Robert B. Goldberg4
Department of Biology, University of California, Los Angeles, California 90024-1606
We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at
elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum
degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is
localized within the stomium and connective, and also decays as these cell types degenerate during anther
maturation. The third mRNA sequence set persists throughout anther development and is localized within most
anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runo
transcription studies and experiments with chimeric /?-glucuronidase and diphtheria toxin A-chain genes showed
that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5' region can progra
the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the
differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plant
Together, our studies show that several independent gene expression programs occur during anther development
and that these programs correlate with the differentiated state of specific anther cell types.
INTRODUCTION
Reproductive processes in higher plants take place within
two specialized floral organ systems, the stamen and the
pistil (Esau, 1977; Raven et al., 1986; Goldberg, 1988;
Drews and Goldberg, 1989). The anther compartment of
the stamen contains diploid cells that undergo meiosis to
form haploid microspores that differentiate into the pollen
grains or male gametophyte (Vasil, 1967; Esau, 1977;
Raven et al., 1986). By contrast, sporogenous cells within
the ovary of the pistil ultimately lead to the production of
the female gametophyte, or embryo sac, that contains the
egg cell (Esau, 1977; Raven et al., 1986). Pollination is
required to transfer pollen grains containing the sperm
cells to the stigma of the pistil so that fertilization can
occur in the embryo sac (Esau, 1977; Raven et al., 1986).
Stamens are differentiated from primordia that are spec?
ified within the floral meristem following the transition from
a vegetative to a flowering pathway (Esau, 1977; Raven
et al., 1986). Stamen primordia specification depends upon
the action of several genes (Bowman et al., 1989; Meye?
rowitz et al., 1989; Carpenter and Coen, 1990), some of
1 Current address: Department of Cell Biology, University of Miami,
Miami, FL33101.
2 Current address: Department of Microbiology, University of Tennessee,
Memphis, TN 38163.
3 Current address: Plant Cell Research Institute, San Carlos, CA
94070.
4 To whom correspondence should be addressed.
which may encode transcriptional activator proteins (Som?
mer et al., 1990; Yanofsky et al., 1990). Following stamen
primordia initiation, several highly specialized anther tis?
sues are differentiated from cell lineages present in the
floral meristem (Satina and Blakeslee, 1941) and are re?
sponsible for carrying out nonreproductive functions (e.g.,
support, dehiscence) and reproductive functions (e.g.,
spore and gamete formation).
The molecular processes that are responsible for specifying
functionally distinct anther cell types after primordia
initiation are not well understood. Previously, we used
RNA-excess DNA/RNA hybridization experiments to show
that approximately 25,000 diverse genes are expressed in
the tobacco anther (Kamalay and Goldberg, 1980). Comparisons
of tobacco organ system mRNA and nuclear RNA
populations showed that about 10,000 mRNAs are anther
specific and are undetectable in the cytoplasm and nucleus
of cells in other vegetative and floral organ systems (Ka?
malay and Goldberg, 1980,1984). These findings suggest
that the transcriptional activation of specific gene sets is
required for the establishment and maintenance of dif?
ferentiated cell types and functions during anther
development.
In this paper, we describe the temporal and spatial
expression patterns of several different anther-specific
genes. Our results show that distinct gene expression
programs occur during anther development and that these
programs correspond with the differentiation of functionally
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(B)
DBHBHIH
1 3 5 7 9 11
STAGE
13 5 7 9
STAGE
3 5 7 9 11
STAGE
Figure 1. Tobacco Flower Development.
(A) Flower development from 8-mm bud to opening. Flower buds were divided
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distinct cell types. Transformation studies with one antherspecific
gene, designated as TA29 (Mariani et al., 1990;
Seurinck et al., 1990), demonstrated that sequences re?
sponsible for the transcriptional activation of this gene in
the tapetum are localized within a 122-bp 5' region. De?
struction of the tapetum by a chimeric TA29 diphtheria
toxin A-chain gene indicated that the tapetum is not re?
quired for the differentiation and/or function of other specialized
cell types later in anther development.
RESULTS
Anther Development Has Two Distinct Phases
Previously, we divided tobacco flower development into
12 stages to provide reference points for the expression
of genes in different floral organ systems (Goldberg, 1988).
Figure 1A shows these stages, and the floral bud lengths
and morphological markers used to describe each stage
are summarized in Table 1. Figures 1B and 1C correlate
each stage with the developmental state of the pistil and
anthers. Measurements presented in Figure 1D indicate
that both the flower bud (calyx plus corolla) and pistil
increase in fresh weight and length continuously from
stages 1 to 12. By contrast, the anthers increase in weight
and length only until stage 5. These parameters remain
relatively constant until stage 11 and then decrease precipitously
when flower opening and anther dehiscence
occur at stage 12 (Figures 1A and 1C).
We prepared transverse anther sections at each stage
to correlate changes in external morphology (Figure 1C)
with the presence of specific sporophytic tissues and cell
types and with the presence of microspores and pollen
grains. Bright-field photographs of representative anther
Anther Gene Expression Patterns 1203
sections are shown in Figure 2A, and the major tissue
present and developmental events that occurred from
stages 1 to 12 are summarized in Table 1. At stage 1,
major anther tissues have been specified. These include
the epidermis, endothecium, vascular bundle, connectiv
stomium, and tapetum. In addition, meiosis was finish
and the microspores were bound together as tetrads
the pollen sac. Following stage 1, the pollen grains diffe
entiated and the anther underwent a series of events
leading to dehiscence and pollen release (Figure 2A an
Table 1). For example, by stage 8, the tapetum wa
degenerated, the connective separating the pollen sac
had begun to degrade, and the anther wall showed sig
of splitting in the stomium region (Figure 2A and Table
At stage 11, the connective was absent, the anther w
bilocular, and pollen grains filled the locules (Table 1).
We characterized anthers at earlier development
stages to determine when the major histodifferentiat
events occurred. Figure 1E shows pictures of flower b
development before stage 1. We divided these buds in
seven stages on the basis of both length (Table 1) and
morphological markers observed in the scanning electr
microscope (Figure 1F). Early flower bud stages w
designated with a minus sign to indicate that they occurr
before the completion of meiosis within the anth
(Table 1).
The scanning electron micrographs shown in Figure 1F
and the transverse sections shown in Figure 2A indicate
that anther primordia were present in stage -7 flower
buds and that the differentiation of specific anther tissues
had begun. For example, archesporial cells, connective
tissue, prevascular tissue, and epidermis were already
visible at this developmental stage (Table 1). Between
stages -7 and -1, the anther acquired its characteristic
external shape (Figure 1F), all major anther tissues were
differentiated (Table 1), and the microspore mother cells
Figure 1. (continued).
criteria (Table 1). Stage 1 was designated as the period when tetrads are present within the anther and corresponds to an 8-mm flower
bud. Stage 12 was designated as the time of flower opening and anther dehiscence. The transition from stage 1 to stage 12 occurred
over a 1 -week period during the summer.
(B) Pistil development from stage 1 to stage 12. Pistil lengths and weights are presented in (D).
(C) Anther development from stage 1 to stage 12. Anther lengths and weights are presented in (D).
(D) Changes in organ system weight and length during flower development. Five flower buds were picked at each developmental stage.
and their lengths and fresh weights were measured individually. Similar measurements were made for the anthers and pistil of each flower
bud. Data points represent the mean of these five measurements. Anther fresh weights represent the collective weight of all five anthers
within the flower bud.
(E) Flower development from 0.75-mm bud to 7-mm bud. Young flower buds were divided into seven stages on the basis of size and
developmental events occurring within the anther (Table 1). Stage -7 was designated as the period when tissue differentiation begins in
the anther primordium [(F)] and stage -1 was designated as the period just before the completion of meiosis within the anther (Figure 2
and Table 1). Stage -7, -6, -5, -4, -3, -2, and -1 flower buds averaged 0.75 mm, 1.5 mm, 3 mm, 4 mm, 5 mm, 6 mm, and 7 mm in
length, respectively. The transition from stage -7 to stage -1 occurred over approximately 5 days. Photographs were taken using a
dissecting microscope with a magnification factor of x75.
(F) Scanning electron micrographs of anthers during the early stages of development. Flower buds at the designated stages were
harvested, and their sepals and petals were removed to expose the anthers. The dissected flower buds were then photographed in the
scanning electron microscope as outlined in Methods. Magnification factors for the stage -7, -6, -5, -4, and 1 anthers were X170,
X120, X120, x125, and x60, respectively.
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1204 The Plant Cell
Table 1. Major Events during Tobacco Anther Development
Flower Anther
Bud
Stage Length8 Morphological Markersb Tissues Present0 Major Events and Morphological Markersd
-7
-6
-5
-2
12
0.75 Petal and stamen primordia present; E, V, A, C
carpels forming; calyx almost closed.
Calyx closed; carpels not fused. E, V, Sp, P, C1.5
3
46
Carpels fused; stamen filaments elongated; E, En, T, V, MMC, C
petals equal in length with anthers.
Anthers yellowish; petals enclose anthers; E, En, T, V, MMC, C
stigma forming.
Style elongating; stamen filament extension E, En, T, V, MMC, C
continues; anthers below stigma.
Style clearly elongated; ovary expanded;
anthers below stigma.
Petals approaching top of sepals.
Anthers and pistil fully differentiated and
green.
Calyx opens slightly at top of bud.
Corolla emerges from calyx.
Sepals completely separated at top of
calyx.
Corolla tube bulge just inside calyx.
Corolla tube bulge at tip of calyx.
E, En, T, V, MMC, C, S
E, En, T, V, MMC, C, S
E, En, T, V, TDS, C, S
E, En, T, V, Msp, C, S
E, En, T, V, Msp, C, S
E, En, T, V, Msp, C, S
E, En, T, V, Msp, C, S
E, En, T, V, Msp, C, S
E, En, V, Msp, C, S
E, En, V, Msp, C, S
E, En, V, Msp, C, S
Corolla tube bulge above calyx; petals
closed.
Corolla elongating; petals green and
slightly open.
Corolla tube bulge enlarging; petal tips
becoming pink.
Corolla limb beginning to open; petal tips E, En, V, Msp, S
pink.
Corolla limb halfway open; stigma and E, En, V, Msp, S
anthers visible.
Flower open; anthers dehisced; corolla limb E, En, V, PG
fully expanded and deep pink.
Rounded primordium; tissue
differentiation begun.
Intense mitotic activity in four corners;
invagination of inner side.
Wall layers including endothecium and
tapetum being formed; connective
established.
Tapetum and pollen sacs distinct; inner
and outer tapetum morphologically
different; middle layer crushed.
Meiosis begins; callose deposition
between microspore mother cells
evident.
Meiosis in progress; tapetum large and
multinucleate; stomium differentiation
begins; thick callose walls between
microspore mother cells.
Meiosis in progress; continued stomium
differentiation.
Meiosis complete; microspores in tetrad;
stomium differentiated; all sporophytic
tissues formed.
Microspores separate.
Tapetum shrunken; secondary
thickening in outer wall layers; pollen
grains begin to form.
Cells adjacent to stomium degenerated;
tapetum degenerating.
Secondary thickening in outer wall
layers intensified.
Remnants of tapetum present;
microspore nucleus dividing.
Degradation of connective tissue in
stomium region.
Disruption of connective tissue
separating pollen sac.
Continued connective degradation.
Connective tissue almost fully degraded;
pollen binucleate.
Anthers bilocular; connective absent;
locules filled with mature pollen
grains.
Anthers dehisce along stomium; pollen
released.
a Mean of five individual determinations expressed in millimeters. Data from stages 1 to 12 taken from those presented in Figure 1
b Major markers that can be used with bud length measurements to identify stage. Tube refers to the white elongated portion
corolla; limb refers to the top pink corolla region where the petal tips are separated from each other (Figure 1A). Markers for
to -1 taken from the scanning electron micrographs (Figure 1F); markers for stages 1 to 12 were taken from visual inspection
(Figure 1 A).
c A, archesporial cells; C, connective; E, epidermis; En, endothecium; MMC, microspore mother cells; Msp, microspores; P, parietal layer;
PG, pollen grains; S, stomium; Sp, sporogenous cells; T, tapetum; TDS, tetrads; V, vascular tissue. Taken from bright-field photographs
of transverse anther sections (Figures 2 and 14).
d Taken from bright-field photographs of transverse anther sections (Figures 2 and 14).
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were undergoing meiosis (Figure 2A). Taken together,
these data indicate that anther developmental stages can
be correlated with floral bud lengths and that there are two
major phases of anther development: phase 1?tissue
differentiation, microspore mother cell specification, and
meiosis (stages -7 to -1), and phase 2?growth, pollen
grain differentiation, and tissue degeneration and dehiscence
(stages 1 to 12).
Anther-Specific Clones Were Identified in an Anther
cDNA Library
We constructed a stage 6 (Figures 1A and 1C) anther
cDNA library and then screened this library for cDNA
clones that represented mRNAs present exclusively, or at
elevated levels, in the anther (see Methods). We used
stage 6 anther mRNA to construct the library because our
earlier solution DNA/RNA hybridization studies indicated
that stage 6 anthers contain a large number of antherspecific
mRNAs (Kamalay and Goldberg, 1980,1984). We
obtained 58 anther-specific cDNA clones out of a screen
of 768 plasmids and then sorted 26 of these clones into
groups by RNA gel blot and cross-hybridization analyses.
Sixteen cDNA clones failed to cross-hybridize at a moderately
stringent criterion (42?C, 50% formamide, 1 M Na+)
and were designated as unique. The remainder were found
to belong to four different cross-hybridizing groups.
Table 2 lists representative cDNA clones from three of
the groups, as well as others that sorted into the unique
category. DNA sequencing studies (Seurinck et al., 1990;
J. Seurinck, J. Leemans, and R.B. Goldberg, unpublished
results) showed that the TA32/36 and TA56/57 groups
represented mRNAs encoding lipid transfer proteins (Bouillon
et al., 1987; Takishima et al., 1988) and thiol endopeptidase
proteins (Miller and Huffaker, 1982; Mitsuhashi and
Minamikawa, 1989), respectively. In addition, the TA13/
TA29 group represented mRNAs that encoded glycinerich
proteins with properties of cell wall proteins (Condit
and Meagher, 1986; Keller et al., 1988; Varner and Cassab,
1988). By contrast, the unique anther cDNA clones sequenced
to date (e.g., TA20, TA25, and TA26) failed to
show relatedness to any known mRNA or protein.
We hybridized representative anther cDNA probes with
gel blots containing floral and vegetative organ system
mRNAs. As shown in Figure 3, all probes produced strong
hybridization signals with anther mRNA. Prevalence estimates
indicated that these mRNAs represented from
0.05% to 0.5% of the anther mRNA, depending upon the
message (Table 2). In most cases, the cDNA probes failed
to produce a detectable signal with heterologous organ
mRNAs, even after the gel blots were exposed 50 times
longer than that required to produce a weak anther signal.
By contrast, three cDNA clones (TA20, TA26, TA4) pro?
duced signals with other mRNAs in addition to anther
mRNA; however, in each case, the heterologous hybridiAnther
Gene Expression Patterns 1205
zation signal was at least 10-fold lower than that observed
in the anther. In addition, each of these cDNA clones
produced a distinct hybridization pattern with the heterol?
ogous mRNAs (Figure 3). For example, the TA20 probe
hybridized with pistil and petal mRNAs, whereas the TA26
probe hybridized only with leaf mRNA. Taken together,
these data indicate that we have identified several different
anther-specific cDNA clones, that these clones represent
relatively prevalent anther mRNAs, and that most of these
mRNAs are either undetectable in other organ system
mRNAs at our sensitivity level (e.g., TA13, TA29, TA36)
or are present at a level at least 10-fold lower than that
observed in the anther (e.g., TA4, TA20, TA26).
Gene Expression Is Temporally Regulated during
Anther Development
We hybridized representative anther cDNA plasmids with
mRNAs from different anther stages to determine whether
their corresponding genes were regulated temporally dur?
ing anther development. These plasmids included the eight
listed in Table 2 in addition to 11 others. Figures 4A and
4B present the results of RNA dot blot studies, and Figure
4C shows duplicate RNA gel blot experiments with some
of the same plasmids.
Three distinct gene expression patterns were observed.
First, Figures 4A and 4C show that most anther mRNAs
investigated accumulated coordinately between stages 1
and 4 and then their levels declined significantly after stage
6. These mRNAs included those encoding the glycine-rich
proteins (TA13, TA29) and the lipid transfer proteins (TA32,
TA36). Second, Figure 4B shows that the TA20 and TA25
mRNAs were present at relatively similar levels in all stages
studied. Closer inspection of the data, however, indicated
that these mRNAs increased slightly in prevalence be?
tween stages 2 and 3 (Figure 4B). Finally, Figures 4B and
4C show that the thiol endopeptidase mRNAs (TA56,
TA57) accumulated between stages 1 and 6, remained
relatively constant until stage 8, and then declined to low
levels before dehiscence at stage 11. Together, these data
indicate that many anther-specific genes are regulated
temporally and that there are timing differences in anther
gene expression patterns.
Gene Expression Is Spatially Regulated during Anther
Development
We hybridized single-stranded 35S-RNA probes with anther
sections in situ (see Methods) to correlate the temporal
gene expression patterns observed during anther devel?
opment (Figure 4) with the presence of specific anther
tissues and cell types (Figure 2 and Table 1).
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1206 The Plant Cell
(B) TA29/DTA
Figure 2. Bright-Field Photographs of Tobacco Anther Development in Untransformed Plants and Plants Transformed with a C
TA29/DTA Gene.
Anthers at the designated stages were fixed, embedded with paraffin, and sliced into 10-/xm transverse sections as described in Me
The fixed sections were stained with toluidine blue and photographed with bright-field illumination. A, archesporial cells; C, connec
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8 Taken from Figure 3.
b Percentage of stage 3 anther polysomal poly(A) mRNA. Esti?
mated relative to calibrated RNA standards. The TA13 and TA29
mRNA prevalences were estimated using mRNA-specific oligo?
nucleotide probes.
c Translation of DNA sequence (Seurinck et al., 1990), or highest
identity score with proteins in the GenBank.
d Different members of the same family. TA13 and TA29 mRNAs
are 90% similar at the nucleotide level (J. Seurinck, J. Leemans,
and R.B. Goldberg, unpublished data).
6 Not significantly similar at the nucleotide level to each other or
to the other mRNAs listed in this table (J. Seurinck, J. Leemans,
and R.B. Goldberg, unpublished data).
f Different members of the same gene family. TA32 and TA36
mRNAs are 63% similar at the nucleotide level (J. Seurinck, J.
Leemans, and R.B. Goldberg, unpublished data), and their cDNA
plasmids only weakly hybridize at a moderately stringent criterion
(42?C, 50% formamide, 1.2 M Na+).
9 The TA56 and TA57 cDNA plasmids (Figure 4B) cross-hybridize
at a moderately stringent criterion.
Several Different Anther mRNAs Are Localized within
the Tapetum
Figure 5 shows the localization patterns of the TA26,
TA29, and TA32 mRNAs (Table 2). As seen in Figures 5A
to 5C, no hybridization grains above background were
observed with the TA29 mRNA control probe. By contrast,
Figures 5D to 5L show that the TA26, TA29, and TA32
anti-mRNA probes produced identical, tapetal-specific lo?
calization patterns, as did TA13 and TA36 anti-mRNA
probes (data not shown). For example, the TA29 antimRNA
probe produced an intense hybridization signal over
the tapetum at stage 3 (Figure 5H) and did not produce
any hybridization grains above background over any other
Anther Gene Expression Patterns 1207
anther regions. Nor were there any detectable hybridiza?
tion signals produced at stage 1 (Figure 5G) and stage 6
(Figure 51). The hybridization signals at each stage correlated
directly with the mRNA levels observed in the RNA
dot blot and gel blot experiments (Figures 4A and 4C). The
absence of a detectable signal at stage 6 (Figures 5F, 51,
and 5L) coincided with the degeneration process occurring
in the tapetum (Figure 2 and Table 1).
We hybridized 3H-poly(U) with the anther sections to
localize the distribution of total poly(A) RNA molecules. In
contrast with the results obtained with the TA13, TA26,
TA29, TA32, and TA36 anti-mRNA probes (Figure 5), the
distribution of grains was uniform throughout the anther
at each stage investigated and grains were present over
all tissues (data not shown). Together, these results indi?
cate that several different mRNAs that accumulate and
decay coordinately during early phase 2 of anther devel?
opment are localized within the tapetum and that these
mRNAs include those encoding the glycine-rich proteins
(TA13, TA29) and the lipid transfer proteins (TA32, TA36).
The TA56 Thiol Endopeptidase mRNA Is Localized
within the Connective and Stomium
The TA56 mRNA localization pattern is shown in Figure 6.
Figures 6A to 6D show that the TA56 mRNA control probe
produced no hybridization grains above background levels
with anther sections from stage 1 (Figure 6A), stage 3
(Figure 6B), stage 8 (Figure 6C), and stage 11 (Figure 6D).
By contrast, Figures 6E to 6H show that the TA56 antimRNA
probe produced a localized hybridization signal at
all stages investigated and that both the location and
intensity of the hybridization grains changed with the de?
velopmental state of the anther. Bright-field photographs
of the anther regions containing the TA56 mRNA are
shown in Figures 61 to 6L, and Figures 6M to 6P show
high-magnification dark-field photographs of the hybridi?
zation grains at each stage.
As predicted from the RNA accumulation studies (Fig?
ures 4B and 4C), a relatively weak TA56 hybridization
signal was observed at stage 1 of anther development
(Figures 6E and 6M). This signal was localized on both
sides of the anther over a circular cluster of cells between
the stomium and the connective (Figures 6E, 61, and 6M).
At stage 3, the TA56 mRNA hybridization signal intensified
Figure 2. (continued).
epidermis; En, endothecium; MMC, microspore mother cells; Msp, microspores; PS, pollen sac; S, stomium; Sp, sporogenous cells; T
tapetum; TDS, tetrads; V, vascular bundle. Magnification factors for stage -7, -6, -2, 1, 3, and 8 anthers were x400, x200, x10
X100, X100, and X100, respectively.
(A) Anther development in wild-type untransformed plants.
(B) Anther development in plants transformed with a chimeric TA29/DTA gene. Tobacco plants were transformed with a TA29/DTA gene
as described in Methods. The TA29/DTA gene contained a TA29 5' DNA fragment containing nucleotides -1477 to +51 (Mariani et al
1990; Seurinck et al., 1990) fused with the DTA gene coding sequence (Greenfield et al., 1983; Maxwell et al., 1986; Palmiter et
1987).
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ORGAN SYSTEM
cDNA A Pi P L S R kb
TA13
TA29
TA20
TA25
TA26
TA36
TA39
TA51
TA4
Figure 3. Representation of Anther-Specific mRNAs in Floral and
Vegetative Organ Systems.
Tobacco anther (A), pistil (Pi), petal (P), leaf (L), stem (S), and root
(R) polysomal poly(A) mRNAs were fractionated on denaturing
agarose gels, transferred to nitrocellulose paper, and hybridized
with labeled plasmid DNA probes as outlined in Methods. Anther
and pistil mRNAs were isolated from stage 6 flower buds (Figure
1), whereas petal mRNA was isolated from stage 12 flowers
(Figure 1). Leaf, stem, and root mRNAs were isolated from plants
described in Methods. One microgram of each mRNA was used
for the TA29 gel blot, and 0.5 ng of each mRNA was used for the
TA13, TA20, and TA25 gel blots. By contrast, the TA26, TA35,
TA51, and TA4 gel blots contained 0.1 pg of anther mRNA and 1
Mg of all other mRNAs. Film exposure times varied for each gel
blot but, with the exception of the TA20 gel blot, were the same
for all RNA lanes. Exposure times for the TA20 petal, leaf, stem,
and root RNA lanes were approximately 30 times longer than that
used for the anther and pistil RNA lanes.
but remained localized over the same circular cell cluster
(Figures 6F, 6J, and 6N). No hybridization grains above
background levels were observed over any other anther
tissues at this stage, including the epidermis, connective,
endothecium, tapetum, and wall. By stage 8, however, the
TA56 mRNA localization pattern changed. Figures 6G, 6K,
and 60 show that the TA56 mRNA was now localized
over both stomium regions and was uniformly distributed
over the connective separating the pollen sacs. By con?
trast, TA56 mRNA was undetectable in the circular cell
cluster between the stomium and connective because
these cells had degenerated (Figure 6K). Finally, at s
11 after the connective degenerated (Table 1), the T
mRNA hybridization signal was reduced significantly
was still localized over the stomial cells (Figures 6H
and 6P). Together, these data indicate that the TA56 t
endopeptidase mRNA is present in specific anther c
types and that the appearance of this mRNA occurs
just before the degeneration of these cells in anther
development.
The TA20 mRNA Is Distributed throughout the Anther
and Is Localized in Specific Pistil Regions
Figures 3 and 4B show that the TA20 mRNA is present
throughout phase 2 of anther development and is also
present in the pistil at a level 10-fold lower than that
observed in anthers of the same stage flower bud. We
hybridized a TA20 anti-mRNA probe with anther and pistil
sections to localize TA20 molecules within both of these
floral organ systems. Figure 7A shows that this probe
produced an intense hybridization signal over most anther
tissues at stage 2, including the connective, stomium, and
wall layers. Highest concentrations of grains occurred over
the connective and over cells immediately surrounding the
tapetum. By contrast, the TA20 anti-mRNA probe did not
produce any hybridization grains above background levels
(Figure 7E) over the vascular bundle, the tapetum, or the
circular cell cluster between the stomium and connective
that contained the TA56 thiol endopeptidase mRNA (Fig?
ures 6M and 6N). Figures 7B to 7D show that at later
developmental stages the TA20 mRNA remained localized
within the same tissues, but beginning at stage 6 became
progressively concentrated in connective regions adjacent
to the vascular bundle (Figure 7C), and by stage 8 became
less prevalent throughout the anther (Figure 7D).
Figures 8A to 8C show bright-field photographs of ovary
cross-sections from a stage 4 pistil (Figure 8A) and a stage
6 pistil (Figure 8B), as well as a stigma/style longitudinal
section from a pistil at stage 6 (Figure 8C). Two carpels
can be visualized within the ovary of each pistil, along with
locules, placenta, vascular bundle, ovules, and the ovary
wall (Figures 8A and 8B). A prominent stigma and a style
with transmitting tissue were also visualized within the
pistil (Figure 8C). Figures 8D and 8E show that the TA20
mRNA was localized within the ovary wall and a narrow
cell layer that connects the ovules to the placenta. No
TA20 hybridization grains above background levels (data
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(A) STAGE
cDNA 123456789 10 11 Control
TA13 _
TA29 ^^BIIliiiplBiiM
TA32
TA26
TA33
TA36
TA39
TA45
TA46
TA5i -^MWM-MM
(B) STAGE
cDNA 123 45 6789 10 11 Control
TA20
TA25
TAX
TA54
TA56
TA57
TA30
(C) STAGE
cDNA 12345678 10 kb
?- -p?^ j^^ip^*^
TA26
TA29
TA32
TA56
V?f*f?
j-0.7
(-1.5
Figure 4. Accumulation of Anther-Specific mRNAs during Anther
Development.
Polysomal poly(A) mRNAs were isolated from anthers at different
developmental stages (Figure 1) and either spotted onto nitrocel?
lulose paper [(A) and (B)] or fractionated on denaturing agarose
gels and transferred to nytran [(C)]. Dot blots and gel blots were
hybridized with labeled plasmid DNAs as outlined in Methods.
Film exposure times differed for each dot blot or gel blot devel?
opmental series but were the same for each RNA hybridized with
a given DNA probe.
(A) Hybridization of anther cDNA plasmids with anther mRNAs at
different developmental stages. The TA13 and TA29 dots con?
tained 0.25 pg of mRNA; the other dots contained 0.05 pg of
mRNA. Control dots contained an equivalent amount of soybean
embryo polysomal poly(A) mRNA.
(B) Hybridization of anther cDNA plasmids with anther mRNAs at
different developmental stages. The TA20 and TA25 dots con?
tained 0.25 Mg of mRNA; the other dots contained 0.05 ^g of
mRNA. Control dots contained an equivalent amount of soybean
embryo polysomal poly(A) mRNA.
Aniner uene txpression rauerns
not shown) were observed over the ovules, vascular
dles, and most of the placenta. Figure 8F shows that
hybridization grains were also absent from the stigma
the transmitting tissue of the style. By contrast,
mRNA molecules were localized within parenchyma t
that extended from beneath the stigma surface thr
the style to the ovary (Figure 8F). Together, these
show that the TA20 mRNA is present in the majorit
anther cells throughout phase 2 of development an
concentrated within specific regions of the pistil. We
clude from these data and those obtained with the other
mRNAs (Figures 5 and 6) that temporal differences in
anther gene expression programs correlate with the differ?
entiation and degeneration of specific anther cell types.
Most Anther Genes Are Represented only a Few
Times in the Tobacco Genome
We hybridized stage 6 anther 32P-cDNA with an exces
tobacco DNA in solution to estimate the copy number
genes expressed in the anther. We utilized an internal
single-copy DNA standard to calibrate the hybridiza
kinetics (Goldberg et al., 1978, 1981). Figure 9A show
that 70% of the 32P-cDNA hybridized with a rate cons
identical to that of the 3H-single-copy DNA (0.00045
see"1). By contrast, 30% of the 32P-cDNA hybridized w
a rate constant of 0.047 M~1 sec~\ suggesting that t
sequences are reiterated approximately 100 times (0.0
0.0045) in the tobacco genome. These studies, and th
presented previously (Kamalay and Goldberg, 1980), i
cate that mRNAs comprising most of the stage 6 ant
mRNA mass and sequence complexity are encoded
single-copy genes, or genes present only a few times
per tobacco genome.
We hybridized anther cDNA plasmids with DNA gel bl
to estimate the copy number of individual anther-spe
genes. We utilized several tapetal-specific cDNA plasm
for these experiments (Figure 5) and included those r
resenting the glycine-rich protein mRNAs (TA29, TA
and the lipid transfer protein mRNAs (TA32, TA36) (T
2). Figure 9B shows that each of these probes hybrid
with only three or four EcoRI fragments in the toba
genome at the hybridization criterion employed (42
50% formamide, 1 M Na+). For example, the TA29 plas
probe hybridized with two similar-sized EcoRI fragm
6.5 kb to 7.0 kb in length and two additional Ec
fragments 8.6 kb and 10.5 kb in length, respectively.
restriction map of the TA29 gene region, shown in Fi
10A, and sequencing studies with TA13 and TA29 cD
(Seurinck et al., 1990; J. Seurinck, J. Leemans, and
(C) Hybridization of the TA26, TA29, TA32, and TA56 cD
plasmids with anther mRNAs at different developmental st
Each RNA gel blot contained 0.7 ^g of mRNA per lane. The T
TA29, TA32, and TA56 RNA gel blots were exposed for 1.5
1.5 hr, 5 hr, and 96 hr, respectively.
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Figure 5. Localization of the TA26, TA29, and TA32 mRNAs during Tobacco Anther Development.
Anthers were fixed, embedded in paraffin, sliced into 10-Mm sections, and hybridized with single-stranded 35S-RNA probes as outlined in
Methods. Photographs were taken by dark-field microscopy, and all film emulsion exposures were for 3.5 days. Corresponding brightfield
photographs are shown in Figure 2. Magnification factors for the stage 1, stage 3, and stage 6 anthers were x100, X100, and x66,
respectively. C, connective; E, epidermis; T, tapetum; V, vascular bundle.
(A) to (C) In situ hybridization of a TA29 mRNA control probe with anthers at stage 1 [(A)], stage 3 [(B)], and stage 6 [(C)]. White grains
represent background hybridization levels; white patchy areas in the anther wall represent light-scattering effects due to dark-field
illumination through stained sections.
(D) to (F) In situ hybridization of a TA26 anti-mRNA probe with anthers at stage 1 [(D)], stage 3 [(E)], and stage 6 [(F)]. White grains in
the stage 3 anther represent regions containing RNA/RNA hybrids.
(G) to (I) In situ hybridization of a TA29 anti-mRNA probe with anthers at stage 1 [(G)], stage 3 [(H)], and stage 6 [(I)]. White grains in
the stage 3 anther represent regions containing RNA/RNA hybrids.
(J) to (L) In situ hybridization of a TA32 anti-mRNA probe with anthers at stage 1 [(J)], stage 3 [(K)], and stage 6 [(L)]. White grains in
the stage 3 anther represent regions containing RNA/RNA hybrids.
Goldberg, unpublished data) indicated that these EcoRI
fragments were derived from cleavage of the TA13 and
TA29 genes. DNA sequencing studies (J. Seurinck, J.
Leemans, and R.B. Goldberg, unpublished results) also
suggested that each TA32 and TA36 EcoRI fragment
represented a different gene (Figure 9B). Together, these
data indicate that each tapetal-specific mRNA investigated
is encoded by a small gene family.
We hybridized the TA29 cDNA plasmid with gel blots
containing tobacco DNA (Nicotiana tabacum) and the
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Anther Gene Expression Patterns 1211
Figure 6. Localization of the TA56 mRNA during Tobacco Anther Development.
Anthers were fixed, embedded in paraffin, sliced into 10-/im sections, and hybridized with single-stranded ^S-RNA probes as outlined in
Methods. C, connective; E, epidermis; En, endothecium; S, stomium; T, tapetum; TDS, tetrads.
(A) to (D) In situ hybridization of a TA56 mRNA control probe with anthers at stage 1 [(A)], stage 3 [(B)], stage 8 [(C)], and stage 11
[(D)]. Photographs taken with dark-field microscopy, and film emulsion exposure times were for 13 days. White grains represent
background hybridization levels; white patchy areas represent light-scattering effects due to dark-field illumination through stained anther
sections. The magnification factor for anthers at each stage was x100.
(E) to (H) In situ hybridization of a TA56 anti-mRNA probe with anther sections at stage 1 [(E)], stage 3 [(F)], stage 8 [(G)], and stage
11 [(H)]. Photographs were taken with dark-field microscopy, and film emulsion exposure times were for 13 days. White grains represent
regions containing RNA/RNA hybrids. The magnification factor for anthers at each stage was x100.
(I) to (L) Bright-field photographs of anther stomium region at stage 1 [(I)], stage 3 [(J)], stage 8 [(K)], and stage 11 [(L)]. Anther regions
correspond to those showing intense hybridization in (E) through (H) and (M) through (P). The magnification factor for anthers at each
stage was x400.
(M) to (P) In situ hybridization of a TA56 anti-mRNA probe with anther stomium region at stage 1 [(M)], stage 3 [(N)], stage 8 [(O)], and
stage 11 [(P)]. Dark-field photographs represent the stomium region of anthers shown in (E) to (H) at a magnification factor of x350.
DNAs of its diploid progenitor species (N. tomentosiformis
and N. sylvestris; Goodspeed, 1954) to determine whether
genes encoding this tapetal-specific mRNA were derived
from one or both tobacco parental lines. As seen in Figure
9C, the gel blot hybridization patterns obtained with all
three tobacco DNAs were similar with the exception of
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1212 The Plant Cell
one or two polymorphisms. Polymorphic DNA frag
were not unexpected because our previous experim
showed that N. tomentosiformis and N. sylvestris
copy DNAs had diverged considerably from each o
(Okamuro and Goldberg, 1985). Together, these re
indicate that members of the TA13/TA29 glycine-rich
tein gene family are present in both parental species
tabacum and that these genes were probably prese
the genus before the divergence of N. sylvestris a
tomentosiformis from a common ancestor (Goods
1954).
The TA29 Gene Does not Contain Introns and Is
Contiguous to a Constitutively Expressed Gene
We isolated a genomic clone containing the TA29 gene
(Figure 10A) to begin to dissect processes that regulate
the coordinate expression of the tapetal-specific gene set
(Figures 3 to 5). We hybridized XTA29 phage DNA with
anther mRNA under conditions that form R-loops to visualize
TA29 gene structure in the electron microscope (see
Methods). Figure 10A shows that the TA29 R-loops were
simple in structure and lacked detectable introns, and that
their average length was approximately the size of the
TA29 mRNAs. The TA29 gene' sequence confirmed the
absence of introns and revealed that this gene has a
continuous open reading frame of 963 bp (Seurinck et al.,
1990). Translation of the reading frame indicated that the
TA29 gene encoded a 33-kD glycine-rich protein that has
several potential glycosylation sites and is organized into
two similar 120-amino acid modules that are highly hydro?
phobic (Seurinck et al., 1990). A schematic representation
of the TA29 gene, including the positions of relevant
consensus sequences, is shown in Figure 10A.
We hybridized the XTA29 phage DNA with gel blots
containing floral and vegetative organ system mRNAs to
determine whether other genes were present in the TA29
gene region. Figure 10B shows that the phage probe
produced an intense hybridization signal with the 1.1-kb
and 1.2-kb TA29 anther mRNAs. In addition, this probe
Figure 7. Localization of the TA20 mRNA during Tobacco Anther
Development.
Anthers were fixed, embedded in paraffin, sliced into lO-^m
sections, and hybridized with single-stranded 35S-RNA probes as
outlined in Methods. Photographs were taken by dark-field mi?
croscopy, film emulsion exposure times were 6 days, and the
magnification factor for anthers at each stage was X100. Corre?
sponding bright-field photographs are shown in Figure 2. C,
connective; S, stomium; T, tapetum; V, vascular bundle; W, wall.
(A) to (D) In situ hybridization of a TA20 anti-mRNA probe with
anthers at stage 2 [(A)], stage 4 [(B)], stage 6 [(C)], and stage 8
[(D)]. White grains represent regions of RNA/RNA hybridization.
(E) In situ hybridization of a TA20 mRNA control probe with a
stage 4 anther. White grains represent background hybridization
levels.
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Figure 8.
Pistil.
Localization of the TA20 mRNA within the Tobacco
Pistils were fixed, embedded in paraffin, sliced into lO-^m sec?
tions, and hybridized with a TA20 anti-mRNA probe as outlined
in Methods. The film emulsion exposure times and magnification
factors were 6 days and x40, respectively. L, locule; O, ovule; P,
placenta; TTi, transmitting tissue; Sti, stigma; Sty, style; V, vas?
cular bundle; W, wall.
(A) and (B) Bright-field photographs of ovary cross-sections from
pistils at stage 4 [(A)] and stage 6 [(B)].
(C) Bright-field photograph of a stigma and style longitudinal
section from a stage 6 pistil.
(D) and (E) In situ hybridization of a TA20 anti-mRNA probe with
ovaries from pistils at stage 4 [(D)] and stage 6 [(E)]. Photographs
were taken by dark-field microscopy. White grains represent
regions of RNA/RNA hybridization.
(F) In situ hybridization of a TA20 anti-mRNA probe with the
stigma and style of a stage 6 pistil. Photograph was taken by
dark-field microscopy. White grains represent regions of RNA/
RNA hybridization.
hybridized weakly with a 0.9-kb mRNA in all other organs.
The prevalence of the 0.9-kb mRNA in the leaf, root, pistil,
and petal was at least 2 orders of magnitude lower than
Anther Gene Expression Patterns 1213
that observed for the TA29 anther mRNAs. By contrast,
the 0.9-kb mRNA was approximately 10-fold more prevalent
in the stem than in the other organs (Figure 10B).
We localized DNA sequences responsible for producing
the 0.9-kb mRNA signal to a region just 3' to the TA29
gene (Figure 10A). R-loop studies with stem mRNA (Figure
10A) and the DNA sequence of this region (Seurinck et al.,
1990) revealed the presence of a gene with two introns
that was transcribed in the opposite direction as the TA29
gene. We designated this gene as TSJT1 (Seurinck et al.,
1990), and a schematic representation of its structure and
position in the TA29 gene region is shown in Figure 10A.
The TSJT1 coding sequence was not related to any known
gene or protein in the GenBank.
We hybridized a DNA fragment containing a portion of
the TSJT1 gene with gel blots containing floral and vege?
tative organ system mRNAs to determine whether it was
also active in the anther. Figure 10C shows that a 0.9-kb
TSJT1 mRNA signal was produced with anther mRNA at
a level equivalent to that observed with all organ system
mRNAs except the stem. Together, these data show that
the TA29 tapetal-specific gene has a relatively simple
structure and that it is contiguous to a convergently tran?
scribed gene that is differentially regulated and expressed
in all organ systems.
The TA29 Gene Is Regulated at the Transcriptional
Level
We hybridized DNA gel blots containing TA29 gene se?
quences with 32P-RNAs synthesized in isolated anther and
leaf nuclei (see Methods) to determine whether the organ
specificity of the TA29 gene was controlled by transcrip?
tional or post-transcriptional processes. We included
TSJT1 DNA sequences on these blots as a control be?
cause this gene was active in both the anther and the leaf
(Figure 10C). The results are shown in Figures 11A and
11B and are summarized schematically in Figure 11C.
Figure 11 shows that both the TA29 gene (Figure 11 A)
and the TSJT1 gene (Figure 11B) hybridized with anther
32P-nuclear RNA. The TSJT1 hybridization signal was
slightly more intense than that obtained with the TA29
gene, even though the 0.9-kb TSJT1 mRNA was approx?
imately 100-fold less prevalent than the TA29 mRNAs in
the anther (Figures 10B and 10C). By contrast, no detect?
able TA29 gene hybridization signal was observed with
leaf 32P-nuclear RNA (Figure 11 A), even though this probe
produced a signal with DNA fragments representing the
TSJT1 gene (Figure 11B). Together, these data indicate
that the TA29 gene is regulated primarily at the transcrip?
tional level and that post-transcriptional processes contribute
to establishing TA29 and TSJT1 mRNA levels in the
anther.
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1214 The Plant Cell
(A) (B) (O
TA29
Copy*
ix sx
1.0
-oSfngttcopy
? AnttwrcONA
J_I_L
TA26 TA32 TA36
-Ul ? Jfe.
I
l* Copy*
RI H3 RIH3 RIH3 IX 5X
KT1 KP 101 10* I03 K>4 ?5 W6
EQUIVALENT COT
Figure 9. Representation of Anther mRNA Sequences in the Tobacco Genome.
(A) Hybridization of stage 6 anther cDNA with an excess of tobacco DNA. Trace amounts of ^P-anther cDNA and 3H-single-copy
were hybridized together with an excess (>25,000/1 mass ratio) of unlabeled total genomic DNA, and the extent of hybridizatio
measured by hydroxyapatite chromatography (see Methods). The single-stranded fragment size of driver and tracer DNAs was 0.
The reassociation kinetics of the driver genomic DNA were described elsewhere (Zimmerman and Goldberg, 1977; Okamuro and Goldbe
1985). Curves through the data points represent the best least-squares solution for either one second-order component (single-c
DNA) or two second-order components (anther cDNA). For these solutions, the rate constant of the slowest hybridizing (singlecomponent
was fixed at 0.00045 M~1 see-1, a value obtained previously from several independent reassociation experiments with t
single-copy DNA (Goldberg et al., 1978; Kamalay and Goldberg, 1980; Okamuro and Goldberg, 1985). Other solutions to thes
points produced higher root mean square errors. The fraction of DNA fragments, second-order rate constant, and average copy n
for each component were: 3H-cDNA; component 1, 0.29, 0.047 M~1 see-1, 100; component 2, 0.71, 0.00045 M~1 see-1, 1; 32P-s
copy DNA; component 1, 1.0, 0.00045 M"1 see"1,1. The fraction of DNA fragments in each component was normalized to 100% t
reactivity (Kamalay and Goldberg, 1980; Okamuro and Goldberg, 1985).
(B) Hybridization of tobacco DNA gel blots with labeled TA26, TA29, TA32, and TA36 plasmid DNAs. Tobacco DNA was digested w
EcoRI, fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized with each labeled plasmid D
outlined in Methods. The reconstruction lanes contained one copy (1 x) and five copy (5x) equivalents of EcoRI-digested XTA29 p
DNA (Figure 10) and were calculated using a XTA29 phage DNA length of 43 kb (Figure 10) and an N. tabacum genome size of 2.4
kb [(A) and Okamuro and Goldberg, 1985]. The TA29 probe hybridized with TA13 sequences at the criterion employed, whereas
TA32 and TA36 probes did not hybridize significantly to each other (Table 2).
(C) Hybridization of labeled TA29 plasmid DNA with gel blots containing DNAs of different tobacco species. Tobacco DNAs were di
with the designated restriction endonucleases, fractionated by electrophoresis on agarose gels, and hybridized with labeled TA29 p
DNA as described in Methods. The one copy (1 x) and five copy (5x) reconstruction lanes were calculated on the basis of the N. tab
genome size as described in (B).
A 122-bp 5' Region Programs TA29 Gene TapetalSpecific
Expression
We showed elsewhere (Mariani et al., 1990) that the TA29
gene 5' region can direct Escherichia coli 0-glucuronidase
(GUS) gene expression within the tapetum, and that the
chimeric TA29/GUS gene is regulated exactly like the
endogenous TA29 gene (Figures 3 to 5). We generated
deletions of the TA29 gene 5' region to localize sequences
required to activate TA29 gene transcription within tapetal
cells. These deletions were fused with the GUS gene and
then the chimeric TA29/GUS genes were transferred to
tobacco plants (see Methods). A schematic representation
of some of the chimeric TA29/GUS genes is shown in
Figure 12A. We obtained 170 transformants representing
20 different 5' deletions ranging from -5670 to +18
nucleotides relative to the TA29 transcription start site
(Figures 10A and 12A). The number of individual transform?
ants per deletion varied from one (-2070) to 19 (-1990),
but averaged approximately nine. DNA gel blot studies
and genetic analysis of kanamycin-resistant (Knr) gene
segregation patterns showed that most transformants had
one unrearranged copy of the TA29/GUS gene (data not
shown).
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TA29 Anther TSJTI Stem
Figure 10. Organization of the TA29 Anther Gene Region.
(A) Structures of the TA29 anther gene and contiguous TSJT1
stem gene. The TA29 cDNA plasmid was used to isolate a 13.2kb
genomic clone from an EcoRI XCharon 32 tobacco DNA library
(Seurinck et al., 1990). DNA gel blot experiments (Figures 9B and
9C) showed that this clone represents an unrearranged copy of
the TA29 genomic region. The 6.2-kb Clal/Aval region containing
the anther and stem genes was sequenced by Seurinck et al.
(1990). Large arrows represent gene locations and transcriptional
orientations. Solid and open regions represent exons and introns,
respectively. TA29 nucleotides +1, +1141, and +1238 indicate
the transcription start site and 3' gene ends as determined by
both S1 nuclease and primer extension analyses. The TA29 cDNA
plasmid represents nucleotides +452 to +821. The TSJT1 stem
gene is numbered relative to the ATG start codon because the
transcription start site has not yet been determined. Nucleotide
coordinates for each exon, as well as the start and stop codons,
are boxed. Fusion of the three stem gene exons results in an
open reading frame of 498 nucleotides, which is approximately
60% of the mRNA length [(B) and (C)]. The TA29 gene 3' end is
only 412 nucleotides from the TSJT1 gene stop codon (Seurinck
et al., 1990). Only relevant XTA29 restriction endonuclease sites
are shown. Sites contained within the 6.2-kb Clal/Aval region can
be obtained from the DNA sequence (Seurinck et al., 1990). XL
and XR refer to the XCharon 32 left and right arms, respectively.
The R-loops were formed by hybridizing a sequence excess of
either anther mRNA or stem mRNA with XTA29 DNA as described
by Fischer and Goldberg (1982). The bar in the electron micro?
graphs represents 1 kb. E and I refer to exons and introns,
respectively. The number-average anther gene R-loop size was
0.84 ? 0.2 kb (n = 26). The R-loop and intron sizes for the stem
gene were: 0.31 kb (E1), 0.22 kb (11), 0.29 kb (E2), 0.42 kb (12),
and 0.35 kb (E3) (n = 1). The anther and stem gene R-loops were
oriented relative to the XTA29 restriction endonuclease map and
the XCharon 32 left and right arms.
(B) Representation of XTA29 sequences in vegetative and floral
Anther Gene Expression Patterns 1215
We used fluorimetric analysis to measure GUS enzyme
activity in stage 4 anthers from 148 individual transform?
ants. Although the GUS enzyme levels varied between
transformants with a given deletion, the results showed
that TA29/GUS gene transcriptional activity remained rel?
atively constant as the 5' region was deleted from -5670
to -279 nucleotides (data not shown). By contrast, no
detectable GUS enzyme activity was observed in anthers
containing TA29/GUS genes with only 150 nucleotides of
TA29 5' region (data not shown). We hybridized a GUS
probe with gel blots containing pooled stage 4 anther
mRNAs from five independent transformants per deletion
(Figure 12A) to show directly that transcriptional control
sequences were localized between -279 and -150 nu?
cleotides from the TA29 gene transcriptional start site
(Figures 10A and 12A). Figure 12B shows that GUS mRNA
levels were similar in transformants containing TA29/GUS
genes with 5270 to 279 nucleotides of the TA29 5' region.
Relative to the RNA standards shown in Figure 12C, the
GUS mRNA prevalence in these transformants averaged
0.1%, a value similar to that obtained for the endogenous
TA29 mRNA gene at the same developmental stage (Table
2). In situ hybridization studies showed directly that tapetal
cell GUS and TA29 mRNA levels were similar in TA29/
GUS transformants containing either 5270 or 1477 nucleo?
tides of the TA29 5' region (Mariani et al., 1990; K.H. Cox
and R.B. Goldberg, unpublished results). By contrast, no
detectable GUS mRNA was observed in transformants
containing either 150 nucleotides of the TA29 5' region or
no TA29 5' region, even though endogenous TA26 mRNA
levels were similar to those obtained in all of the other
transformants (Figure 12B). This result indicated that the
TA29/GUS gene transcriptional level dropped to below our
detection limit (>100-fold) when the TA29 5' region was
deleted from -279 to -150 nucleotides. Together, these
organ system mRNAs. Anther (A), pistil (Pi), petal (P), leaf (L),
stem (S), and root (R) mRNAs were fractionated by electropho?
resis on denaturing agarose gels, transferred to nitrocellulose,
and hybridized with labeled XTA29 phage DNA as outlined in
Methods. Anther and pistil mRNAs were isolated from stage 6
flowers; petal mRNA was isolated from stage 12 flowers (Figure
1). Each lane contained 0.5 ng of mRNA, but the anther mRNA
gel blot (1x) was exposed for only 1/100 of the time used to
expose the other gel blots (100x).
(C) Representation of the TSJT1 gene sequence in vegetative
and floral organ system mRNAs. Gel blot conditions and mRNAs
were the same as those used in (B). For these gel blots, a DNA
fragment containing portions of the TSJT1 stem gene exon 2 and
intron 2 [0.7-kb Hindlll fragment, (A)] was used as a probe. The
reconstruction lanes contained 0.5 ng (0.7%), 0.05 pg (0.07%),
and 0.005 fig (0.007%) of soybean midmaturation stage mRNA,
and were hybridized with the soybean L9 lectin cDNA plasmid
(Goldberg et al., 1983). Experiments published previously estab?
lished that the 1-kb lectin mRNA represents approximately 0.7%
of the midmaturation stage embryo mRNA (Goldberg et al., 1983).
All gel blot RNA lanes were exposed for 3 days.
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(A)
(C) TA29 TSJTI
xL n i ^p3^prc j, j. jfoSo 5 5 S 5 ? j| 5
u o cl C ? u
*_A A+S S ?
* A+S S ?
Leaf rzzzzzzzzzzzzzzm wss^mmmm^m
Figure 11. Transcriptional Regulation of the Tapetal-Specific
TA29 Gene.
(A) Hybridization of the TA29 gene with anther and leaf ^Pnuclear
RNAs. A plasmid (TA29Eco6.6L) representing the left half
of the XTA29 phage DNA (Figure 10A) was digested with both
Clal and EcoRI, transferred to nytran, and hybridized with MPnuclear
RNAs synthesized in vitro from either stage 3 anther
nuclei or leaf nuclei (see Methods). The band labeled with an
asterisk represents an RNA polymerase II (a-amanatin sensitive)
transcript that hybridized with the 4.2-kb Clal DNA fragment [(C)].
An RNA complementary to DNA sequences in this region was not
detected in the gel blot experiments with organ system mRNAs
shown in Figure 10B. The band labeled A represents the TA29
nuclear RNA that hybridized with the 2.3-kb Clal/EcoRI DNA
fragment containing the TA29 gene [(C)].
(B) Hybridization of the TSJT1 gene with anther and leaf ^Pnuclear
RNAs. A plasmid (TA29Eco6.6R) representing the right
half of the XTA29 phage DNA (Figure 10A) was digested with Pstl
and EcoRI, transferred to nytran, and hybridized with ^P-nuclear
RNA synthesized in vitro from either stage 3 anther nuclei or leaf
nuclei (see Methods). The band labeled with a circled asterisk
represents an RNA polymerase II transcript that hybridized with
the 2.2-kb Pstl/EcoRI DNA fragment [(C)], and that was unde?
tectable in the organ system mRNA gel blots shown in Figure
10B. The band labeled with an S represents the TSJT1 nuclear
RNA that hybridized with the 3.3-kb Pstl fragment containing the
TSJT1 gene [(C)]. The band labeled A + S represents TA29 and
TSJT1 nuclear RNAs that hybridized with the 1.1-kb EcoRI/Pstl
fragment that contained sequences from both genes [(C)].
(C) Transcribed TA29 genomic regions. Restriction map and
schematic representations of the TA29 and TSJT1 genes were
taken from Figure 10A. Bars with diagonal lines and dots represent
restriction fragments contained within plasmids TA29Eco6.6L and
TA29Eco6.6R, respectively. These bars show regions that hybrid?
ized with anther and leaf 32P-nuclear RNAs. A, S, asterisk and
circled asterisk represent TA29, TSJT1, and two unidentified RNA
polymerase II transcripts, respectively.
data demonstrate that TA29 5' sequences between -279
and -150 are required to program high levels of transcrip?
tion within the tapetum.
We delineated the TA29 gene transcriptional control
region further by investigating GUS enzyme activity in
TA29/GUS transformants containing only 178 nucleotides
of the TA29 5' region. The chimeric TA29/GUS genes in
these transformants and in control transformants with 279
nucleotides of the TA29 5' region are schematically rep?
resented in Figure 13A. Figure 13B shows that, as pre?
dicted, intense blue color resulting from GUS enzyme
activity was visualized within the tapetal cells of stage 4
anthers containing a TA29/GUS gene with 279 nucleotides
of TA29 5' sequences. No color was observed in the
tapetal cells of stage 1 and stage 6 anthers of the same
transformants (Figure 13B). By contrast, no detectable
enzyme activity was visualized at any developmental stage
within the tapetum of transformants containing a chimeric
TA29/GUS gene with only 178 nucleotides of the TA29 5'
region (data not shown). Nor was there any GUS enzyme
activity visualized within the roots, stems, leaves, and
cotyledons of germinated seedlings containing either the
-279 or the -178 TA29/GUS genes (data not shown).
Together, these data indicate that sequences between
-279 and -178 relative to the TA29 gene transcription
start site are required for both temporal specificity (Figures
4A and 4C) and tapetal-cell specificity (Figures 5G to 51)
during anther development.
We fused selected portions of the TA29 gene -279 5'
region with a GUS gene that contained a minimal cauliflower
mosaic virus (CaMV) promoter (Figure 13A) and
then transferred these chimeric TA29/CaMV/GUS genes
to tobacco plants to show directly that sequences in this
region were sufficient to transcriptionally activate a heter?
ologous promoter in the tapetum. Figures 13C and 13D
show that a high level of GUS enzyme activity was visu?
alized within stage 4 anther tapetal cells of TA29/CaMV/
GUS gene transformants containing TA29 5' nucleotides
-279 to -85 (Figure 13C) and -207 to -85 (Figure 13D).
By contrast, Figure 13E shows that no GUS activity was
visualized within tapetal cells of control transformants con?
taining a GUS gene with the minimal CaMV promoter
(Figure 13A). Nor was GUS activity visualized in the tape?
tum of TA29/CaMV/GUS transformants with TA29 5' nu?
cleotides -178 to -85 (Figure 13A) (data not shown).
Surprisingly, TA29/CaMV/GUS transformants containing
the -279 to -178 TA29 5' region (Figure 13A) did not
have detectable GUS activity above control levels (Figure
13E) in their tapetal cells (data not shown). We conclude
from these data that a 122-bp TA29 5' region between
nucleotides -207 and -85 (Figures 10A and 13A) contains
sequences sufficient to program TA29 gene tapetal-specific
transcription during the tobacco life cycle. These
transcriptional control sequences either exist as separate
elements within the -207 to -178 and -178 to -85
regions, or extend as one domain across nucleoti
into each region, or both.
The Tapetum Functions Autonomously during Ph
of Anther Development
We fused a 1.5-kb TA29 5' region containing the
cell transcriptional control sequences (Figure 10A;
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(A)
{B) ####* * $$ + (cv/i i 1 i i i i i i i i
TA29/GUS iiiiiBi^HHH
TA26
Figure 12. Localization of the Tapetal-Specific TA29 Gene Tran?
scriptional Control Region.
Chimeric TA29/GUS genes containing different amounts of the
TA29 gene 5' region (Figure 10A) were transferred to tobacco
plants as described in Methods. Approximately 150 stage 3
anthers (0.4 g) from each of five different transformants per
deletion were pooled, and their polysomal poly(A) mRNAs were
isolated as outlined in Methods. One microgram of mRNA from
each deletion pool was fractionated on denaturing agarose gels,
transferred to nytran, and hybridized with a mixture of 32P-antimRNAs
synthesized from TA26 and GUS plasmids.
(A) Schematic representation of the 5' TA29 region in each
chimeric TA29/GUS gene. End points of the +18, -150, -279,
-412, and -717 deletions were determined directly by DNA
sequence analysis. The remainder were estimated by agarose gel
electrophoresis relative to known marker DNAs.
(B) GUS and TA26 mRNA levels in anthers of pooled transform?
ants containing a TA29/GUS gene.
(C) Reconstruction lanes calibrating the TA29/GUS deletion
mRNA gel blots. GUS and TA26 mRNAs were synthesized in
vitro. Equal mass amounts of each mRNA were mixed, fraction?
ated on denaturing agarose gels, transferred to nytran, and hy?
bridized with TA26 and GUS 32P-anti-mRNAs as outlined in Meth?
ods. The 0.1% and 0.01% reconstruction lanes contained 10~3
Mg and 10~4 pg each of GUS and TA26 mRNAs, respectively. All
TA29/GUS and reconstruction RNA gel blots were exposed for
the same time period (4 hr).
et al., 1990) with the cytotoxic diphtheria toxin A-chain
(DTA) gene (see Methods) to (1) determine whether tapetai
cell specification occurred before TA29 gene transcrip?
tional activation, and (2) to determine whether destruction
of the tapetum had any effect on the differentiation and/or
function of other anther cells. We transferred the chimeric
TA29/DTA gene to tobacco cells and regenerated seven
independent transformants (see Methods). DNA gel blot
studies and Knr gene segregation patterns demonstrated
that each transformant had a single, unrearranged copy of
the TA29/DTA gene (data not shown).
Anther Gene Expression Patterns 1217
Figure 2 shows that anther development in plants with
the chimeric TA29/DTA gene (Figure 2B) was identical to
untransformed wild-type plants (Figure 2A) from stages
-7 to -2. The bright-field photographs shown in Figure
14 indicate that stage -2 anthers of control plants (Figure
14A) and TA29/DTA plants (Figure 14D) were indistinguishable
and that major anther tissues such as the epi?
dermis, endothecium, connective, and tapetum had differ?
entiated, as well as the microspore mother cells. By con?
trast, Figures 14B, 14C, 14E, and 14F show that both
tapetal and microspore mother cells were abnormal in
appearance in stage -1 TA29/DTA anthers (Figure 14E)
and that complete tapetum destruction and pollen sac
collapse occurred by stage 1 (Figure 14F). Figure 2B
shows that subsequent stages of anther development in
plants with the TA29/DTA gene were identical to those of
the control plants (Figure 2A). The anthers enlarged, the
connective and stomium degenerated, and the anthers
dehisced. The only difference between plants with the
TA29/DTA gene and wild-type plants was the absence of
pollen grains at dehiscence; that is, the TA29/DTA plants
were male sterile. We conclude from these data that the
TA29 gene is activated after tapetal cell differentiation has
occurred during a period when meiosis is taking place
(Table 1) and that destruction of the tapetum has no
effect on anther differentiation and/or function later in
development.
DISCUSSION
Gene Expression Is Temporally and Spatially
Regulated during Anther Development
We have identified several different prevalent mRNAs
are present either exclusively (e.g., TA13, TA29) or at
vated levels (e.g., TA20, TA26) in the anther (Figure 3). T
experiments complement our previous results (Kama
and Goldberg, 1980, 1984) and those of others (Stins
et al., 1987; Gasser et al., 1988; Ursin et al., 1989; Twe
et al., 1990; van Tunen et al., 1990) and indicate that
differentiated state of the anther is associated with the
expression of unique genes. A major aspect of the results
presented here is the finding that different temporal and
spatial gene expression programs occur during anther
development and that these programs correlate with the
differentiation of specific tissues and cell types.
A schematic representation of the gene expression pro?
grams that we have identified during anther development
is presented in Figure 15. First, the TA13, TA26, TA29,
TA32, and TA36 mRNAs accumulate coordinately during
early phase 2 of anther development, decay before the
stage when microspore nuclei divide in the pollen sac
(Figure 4 and Table 1), and are localized exclusively within
the tapetum (Figure 5). Second, the TA56 mRNA accumulates
later than the tapetal-specific mRNAs, persists
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1218 The Plant Cell
tt w
Figure 13. Identification of Transcriptional Control Sequences
within the TA29 Gene -279 5' Region.
DNA fragments representing different segments of the TA29 gene
-279 5' region (Figure 10A) were fused with either the GUS gene
coding region or a chimeric GUS gene containing a CaMV minimal
promoter (see Methods) and transferred to tobacco plants as
outlined in Methods. Stage 1, 4, and 6 anthers from each trans?
formant were harvested and assayed for GUS enzyme activity as
outlined in Methods. We examined eight individual transformants
per chimeric TA29 gene construction. Although GUS levels varied,
the qualitative pattern was the same in each transformant.
(A) Schematic representation of the TA29 gene -279 5' region
in each chimeric TA29/GUS gene. Blue 5' regions represent
chimeric genes that produced the tapetal-specific GUS expression
patterns shown in (B) to (D).
(B) to (E) Localization of GUS enzyme activity in transformants
with TA29/GUS genes 1 [(B)], 3 [(C)], 4 [(D)], and 5, 6, and 7
[(E)] as schematically diagrammed in (A). 1, 4, and 6 refer to
stages of anther development.
throughout phase 2 of anther development, and decays
just before dehiscence and flower opening (Figures 4 and
6). The TA56 mRNA is localized within the stomium, the
connective, and the circular cell cluster between th
mium and connective (Figure 6). TA56 mRNA locali
within these anther regions, however, occurs in a p
temporal cascade?first circular cell cluster, next sto
and then connective (Figure 15). Finally, the TA20
accumulates to a relatively high level before stage 1 (F
4) and is localized continuously in the epidermis, sto
wall layers, and the connective (Figure 7).
Several of the mRNAs investigated here encode pr
that correlate well with the known functions of the cell
which they are localized (Table 2). For example, the
tum is essential for the differentiation of the pollen
and produces both proteins and lipids that are sec
into the pollen sac and form part of the pollen grain
wall (Vasil, 1967; Heslop-Harrison, 1975). The TA
and TA32/36 tapetal-specific mRNAs encode putativ
cine-rich cell wall proteins and lipid transfer protei
spectively (Table 2). These proteins are excellent c
dates for molecules that play a critical role in tap
physiological processes. By contrast, the TA56 m
encodes a protein similar to the thiol endopeptidase
are present in germinating cotyledons (Mitsuhash
Minamikawa, 1989) and senescing leaves (Miller and
faker, 1982). The appearance of the TA56 mRNA i
connective, stomium, and circular cell cluster just
their degeneration (Figure 6) suggests that the TA5
endopeptidase may aid in the degenerative proc
either activating hydrolytic enzymes that degrade c
macromolecules, or by functioning as a hydrolytic en
or both.
A Mosaic of Regulatory Programs Operate in the
Anther
Our results suggest that genes expressed in the anther
are regulated, in part, by a mosaic of control programs
that are partitioned with respect to both cell type and
developmental time within the anther. For example, the
TA20, TA29, and TA56 genes respond differentially to
regulatory signals present within the wall layers, tapetum,
and circular cell cluster, respectively (Figure 15). By con?
trast, the TA20 and TA56 genes both respond to signals
present within the stomium and connective, but are coexpressed
within these tissues only during specific develop?
mental periods (Figure 15). We showed directly using
runoff transcription studies (Figure 11) and transformation
experiments with chimeric genes (Figures 12 to 14) that
the tapetal-specific expression of the TA29 gene is controlled
at the transcriptional level. Assuming that the other
anther-specific genes that we have investigated are also
regulated by transcriptional processes (Kamalay and Gold?
berg, 1984), our results suggest that the mRNA localiza?
tion patterns that we have observed reflect the distribution
of transcription factors capable of activating these genes
during anther development.
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Figure 14. Bright-Field Photographs of Anthers from Untransformed
Plants and Plants Containing a Chimeric TA29/DTA Gene.
Anthers were fixed, embedded in paraffin, and sliced into 10-^m
sections as outlined in the legend to Figure 2 and in Methods. C,
connective; E, epidermis; En, endothecium; MMC, microspore
mother cells; T, tapetum; TDS, tetrads.
(A) to (C) Bright-field photographs of untransformed anthers at
stage -2 [(A)], -1 [(B)], and 1 [(C)]. Magnification factor was
X160.
(D) to (F) Bright-field photographs of anthers containing the
TA29/DTA gene at stages -2 [(D)], -1 [(E)], and 1 [(F)]. Mag?
nification factor was x160.
The gene expression programs that we have identified
take place within sporophytic tissues and probably repre?
sent only a fraction of the regulatory programs that occur
during anther development. Other anther-specific gene
expression programs have been identified that occur within
the developing male gametophyte or pollen grain (Stinson
et al., 1987; Mascarenhas, 1988; Hanson et al., 1989;
Ursin et al., 1989; Brown and Crouch, 1990; Twell et al.,
1990). In each case, these programs deal with the prevalent
mRNA sets that are regulated during phase 2 of anther
development and represent only a small fraction of the
thousands of anther-specific mRNAs known to occur (Ka?
malay and Goldberg, 1980,1984). For example, the TA29
mRNA constitutes 0.2% of the stage 3 anther mRNA mass
as a whole (Table 2) and on a cell basis represents a much
larger percentage of the tapetai mRNA mass (Figure 5).
By contrast, a typical rare class anther mRNA constitutes
only 10_3% of the anther mRNA mass (Kamalay and
Anther Gene Expression Patterns 1219
Goldberg, 1980), or approximately the level of the TSJT1
mRNA in the anther (Figure 10C). No information exists at
the present time regarding the regulation of genes encod?
ing rare mRNAs during anther development. Nor is there
any information regarding gene expression programs that
operate during phase 1 of anther development when major
histodifferentiation events occur (Figure 2). Nevertheless,
the cell-specific mRNAs identified by us and by others
(Ursin et al., 1989) provide useful markers for dissecting
the regulatory pathways that control the specification of
different cell types during anther development.
Several Coordinately Regulated Tapetal-Specific
Genes Have Been Identified
One of the most striking aspects of our results is t
identification of several genes that are coordinately
pressed with respect to both time and cell type dur
anther development. Figures 4 and 5 show that the TA
TA26, TA29, TA32, and TA36 mRNAs accumulate and
decay in the same temporal sequence during anther de?
velopment and are localized collectively within the tape?
tum. Other anther-specific mRNAs (e.g., TA1, TA51) have
identical accumulation and decay programs and are prob?
ably localized within the tapetum as well (Figure 4).
At least one tapetal-specific gene, that encoding the
TA29 mRNA, does not appear to be expressed detectably
at any other time of the sporophytic life cycle. No detect?
able TA29 mRNA is present at the level of RNA gel blots
in heterologous vegetative and floral organ systems (Figure
3). Transformed plants containing cytotoxic genes controlled
by the TA29 5' gene region are indistinguishable
from control plants except that they are male sterile as a
consequence of tapetai cell destruction (Figure 14; Mariani
et al., 1990). Finally, we have not detected these tran?
scripts or their TA13 relatives in any other organ system
using the polymerase chain reaction (PCR) (C. Mariani, M.
De Bueckeleer, and R.B. Goldberg, unpublished results).
By contrast, other tapetal-specific mRNAs are present in
heterologous organs. For example, the RNA gel blot stud?
ies shown in Figure 3 indicate that the TA26 mRNA is
present in the leaf, and PCR studies have shown that the
TA32 and TA36 mRNAs are present at low levels in the
leaf, stem, and root (C. Mariani, M. De Bueckeleer, and
R.B. Goldberg, unpublished results). We also cannot exclude
the possibility that tapetal-specific genes, including
those encoding the TA13 and TA29 mRNAs, are ex?
pressed during the gametophytic phase of the life cycle,
although in situ hybridization experiments have not de?
tected these mRNAs within developing pollen grains (data
not shown). We conclude that several different genes
investigated form part of a regulatory network that is
activated in the tapetum and that some of these tapetalspecific
genes are linked to additional regulatory circuits
that are active at other times of the life cycle.
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1220 The Plant Cell
DEVELOPMENTAL STAGE
mRNA i ! 2 3 4 5 6 7 8 9 10 11 12-1 i ?.
TA26 T
TA56
TA20
Figure 15. Temporal and Spatial Regulation of mRNAs during
Anther Development.
Data represent a summary of the RNA blot and in situ hybridization
experiments shown in Figures 4 to 7. The tapering in each colored
bar shows periods when the mRNA accumulates or decays, and
the bar thickness approximates the prevalence of each set aver?
aged over the entire anther mRNA population (Table 2). The color
of each bar corresponds with the anther region in which each
mRNA is localized. Three colors were used for TA56 because this
mRNA is localized in several anther regions at different develop?
mental periods. C, connective; S, stomium; T, tapetum; V, vas?
cular bundle; W, wall.
Experiments with the chimeric TA29/DTA gene (Figure
14) indicated that the TA29 gene is activated transcriptionally
after the tapetum has differentiated during a period
when meiosis occurs in the microsporangia (Figure 14 and
Table 1). We do not know how many other tapetal-specific
genes are also activated at this time. However, the tran?
scriptional activation of the TA29 gene occurs close to the
developmental stage when tapetal cell specification takes
place within the anther (Figure 2). Identification of tran?
scription factors that regulate this gene and others in the
tapetal-specific regulatory network should provide entry
into the tapetum differentiation pathway close to the period
when critical specification decisions occur.
A Tapetal-Specific Regulatory Domain Has Been
Identified
Dissection of the TA29 5' region by both deletion experi?
ments (Figures 12 and 13) and gain of function experi?
ments with a heterologous promoter (Figure 13) has iden?
tified a 122-bp 5' region (-207 to -85) that is both
necessary and sufficient to program tapetal-specific
expression within the anther. Sequences within this do?
main are required to activate TA29 gene transcription
within the correct organ system and cell type and at the
appropriate time of the tobacco life cycle. Activation of the
TA29 gene probably requires transcription factors that are
either present exclusively in the tapetum or are present in
an active state only in the tapetum. Tapetal-specific tran?
scription factors must be highly conserved because the
TA29 gene 5' region can program tapetal-specific expres?
sion of both GUS and RNase genes in distantly related
oilseed rape plants (Mariani et al., 1990). Comparisons of
the TA29 -207 to -85 regulatory domain with the TA36
tapetal-specific 5' gene region did not reveal any significant
DNA sequence homologies (J. Seurinck, A.M. Koltunow,
and R.B. Goldberg, unpublished results). What the exact
tapetal-specific cis elements are and how factors interact
with these elements to activate the transcription of tapetalspecific
genes remain to be determined.
Chimeric Cytotoxic Genes Can Be Used To Study Cell
Interaction Processes during Anther Development
The mechanisms by which different cells and tissues form
during anther development are not known. As shown in
Figures 2 and 14, cells that serve as precursors to major
tissues appear at specific positions within the anther. For
example, archesporial cells arise simultaneously in each
corner of the anther, and these archesporial cells differ?
entiate into the microsporangia and their surrounding tis?
sues such as the tapetum and endothecium (Vasil, 1967;
Esau, 1977; Mascarenhas, 1988). By contrast, the vas?
cular tissues differentiate within the center of the anther
(Figure 2). Thus, specific regions, or territories, are estab?
lished early in anther development within which unique
histodifferentiation events occur. In some cases, other
tissues (e.g., stomium) form at the boundaries of these
territories later in anther development (Figures 2 and 6).
Cell lineage studies (Satina and Blakeslee, 1941)
showed that after the stamen primordia are specified, the
epidermis is derived from the L1 floral meristem layer.
Other anther tissues are formed from the L2 or L3 layers,
or both (Satina and Blakeslee, 1941). For example, the
tapetum is formed from both the archesporial lineage that
is derived from the L2 layer and the connective lineage
that is established from L3. The specification of the tape?
tum from two lineages within each microsporangia territory
suggests that cell position is more critical than cell ancestry
in the anther histospecification process.
No information exists on whether different anther cells
and tissues are induced by signals from contiguous regions
of a territory, or are specified autonomously by sequestering
morphogenetic factors during divisions of the L1, L2,
and L3 layers, or both. We have shown in this paper
(Figures 2 and 14) and elsewhere (Mariani et al., 1990)
that cytotoxic genes controlled by the TA29 gene 5' region
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(Figure 13) can selectively destroy the tapetum during
anther development. Both the TA29/DTA gene (Figure 14)
and two different TA29/RNase genes (Mariani et al., 1990)
cause tapetai cell breakdown between stages -1 and 1 of
tobacco anther development. Destruction of the tapetum
by either DTA or RNase does not affect the function and/
or differentiation of anther cell types at later developmental
stages (Figures 2 and 14; Mariani et al., 1990). We infer
from these observations that the tapetum functions autonomously
during phase 2 of anther development. We do
not know, of course, what effect tapetai cell loss at an
earlier period would have on differentiation events that
occur within contiguous cells of the same territory. Use of
cell-specific gene regulatory domains that can activate
cytotoxic genes early in anther development should, in
principle, be able to address the role of cell-cell interactions
during the anther differentiation process. Clearly, the
mechanisms that control cell type specification during an?
ther development and how unique gene sets are activated
coordinately within these cell types remain major unanswered
questions.
METHODS
Growth of Plants
Tobacco plants (Nicotiana tabacum cv Samsun) and other Nico?
tiana species (N. tomentosiformis, N. sylvestris) were grown in
the greenhouse. Characteristics of plants and organ systems used
for RNA preparations were described elsewhere (Kamalay and
Goldberg, 1980; Cox and Goldberg, 1988).
Polysomal mRNA Isolation
Polysomal poly(A) mRNAs from tobacco vegetative and floral
organ systems were isolated according to the procedures de?
scribed by Cox and Goldberg (1988).
DNA Isolation and Labeling
Tobacco leaf nuclear DNAs, as well as plasmid and phage DNAs,
were isolated as outlined in Jofuku and Goldberg (1988). Plasmid
and phage DNAs were labeled by nick translation under conditions
specified by Bethesda Research Laboratories. Single-copy DNA
sequences were labeled by gap translation as described previ?
ously (Goldberg et al., 1978; Kamalay and Goldberg, 1980; Oka?
muro and Goldberg, 1985).
Isolation of Single-Copy DNA
Unlabeled single-copy DNA sequences were isolated from total
DNA by two cycles of renaturation and hydroxyapatite fractionation
to Cots 2000 and 200, respectively (Goldberg et al., 1981).
The final nonreassociated single-copy DNA fraction represented
Anther Gene Expression Patterns 1221
4% of the starting DNA, and was reassociated to Cot 7000 to
generate networks for the gap translation labeling reaction (Gold?
berg et al., 1978, 1981; Kamalay and Goldberg, 1980; Okamuro
and Goldberg, 1985).
Solution DNA Hybridization Studies
Procedures used for DNA-excess hybridization experiments with
single-copy DNA and cDNA have been described extensively
elsewhere (Goldberg et al., 1978, 1981; Kamalay and Goldberg,
1980; Okamuro and Goldberg, 1985). Computer analysis of the
hybridization kinetics was carried out as described by Pearson et
al. (1977), using a program that was modified for use on an
IBM personal computer by June Baumer of the UCLA Biology
Department.
Gel Blot Studies
DNA and RNA gel blot studies were carried out according to
previously published procedures (Jofuku and Goldberg, 1988
1989). RNA dot blot experiments were performed according to
Barkeretal. (1988).
Light Microscopy of Anther and Pistil Sections
Anthers and pistils were dissected from flower buds at the relevan
developmental stages and fixed with glutaraldehyde as described
by Cox and Goldberg (1988). The fixed floral organs were dehy
drated, cleared of chlorophyll, embedded in paraffin, and sliced
into 10-A*m sections as outlined previously (Cox and Goldberg,
1988). The sections were stained with 0.05% toluidine blue, and
photographed with Kodacolor Gold100 film with bright-field illu?
mination as described (Cox and Goldberg, 1988; Perez-Grau and
Goldberg, 1989). Color prints were produced by Village Phot
(Westwood, CA) using an automated developing and printing
process. In some cases, these prints were spliced together to
reconstruct the entire organ visualized in the microscopic field.
In Situ Hybridization Studies
In situ hybridization studies with paraffin-embedded organ system
sections were carried out exactly as described by Cox and
Goldberg (1988) and Perez-Grau and Goldberg (1989).
Synthesis of Single-Stranded Probes
Labeled and unlabeled single-stranded RNAs were synthesized
using the pGEM transcription system (Promega Biotec). ^S-RNA
and 32P-RNA probes were used for in situ and gel blot hybridization
studies, respectively.
Runoff Transcription Studies
Synthesis of 32P-RNA in isolated nuclei was carried out as de?
scribed by Walling et al. (1986) and Cox and Goldberg (1988). To
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1222 The Plant Cell
prevent contamination of anther nuclei with developing pollen
grains, the anther homogenates were passed through a 20-miti
nylon mesh screen.
R-Loop Analysis
R-loops were formed between XTA29 phage DNA and organ
system mRNAs as described by Fischer and Goldberg (1982) and
Jofuku and Goldberg (1988). Procedures for visualizing the Rloops
in the transmission electron microscope were described
previously (Fischer and Goldberg, 1982).
Construction and Screening of Anther cDNA Library
A stage 6 anther cDNA library was constructed in pBR329 (Covarrubias
and Bolivar, 1982) according to the RNase H procedure
of Gubler and Hoffman (1983). Complementary DNA clones representing
mRNAs present exclusively or at elevated levels in the
anther were identified by screening replica plates of the anther
library separately with stage 6 anther 32P-cDNA and a mixture of
leaf, root, stem, pistil (stage 6), and petal (stage 12) 32P-cDNAs.
Bacterial colonies were screened by the hybridization procedures
of Grunstein and Hogness (1975) and Hanahan and Meselson
(1980).
Construction and Screening of Tobacco Genome Library
Tobacco leaf DNA sequences were cloned in the XCharon 32
vector (Loenen and Blattner, 1983) according to the procedure of
Jofuku and Goldberg (1988). Approximately 8 x 105 independent
phage isolates were plated and amplified to form a permanent
library, and greater than 99% of these phage were recombinants.
The XTA29 genomic clone was isolated from this library by
the plaque hybridization procedures described by Jofuku and
Goldberg (1988).
Scanning Electron Microscopy
Flower buds and anthers were fixed and prepared for scanning
electron microscopy according to the procedure of Irish and
Sussex (1990). Samples were examined in an ETEC autoscan
scanning electron microscope (ETEC Corporation, Hayward, CA)
with an acceleration voltage of 10 kV.
Histochemical Assay for GUS Enzyme Activity
Histochemical staining for GUS enzyme activity was carried out
according to the procedure of Jefferson et al. (1987), with minor
additions to optimize visual detection of GUS activity in anthers.
In brief, anthers were sliced into 2-mm pieces and fixed by vacuum
infiltration for 15 min in 0.1 M sodium phosphate buffer (pH 7.0)
containing 0.1% formaldehyde, 0.1% Triton X-100, and 0.1%
0-mercaptoethanol. The fixed anthers were then rinsed extensively
with 0.1 M sodium phosphate buffer (pH 7.0) containing
0.1% jS-mercaptoethanol, followed by a rinse with 0.05 M so?
dium phosphate buffer (pH 7.0) plus 0.1% 0-mercaptoethanol.
GUS reactions were carried out at 37?C in 0.05 M sodium ph
phate buffer (pH 7.0) containing 0.1% Triton X-100, 0.1% 0-m
captoethanol, and 0.001 M 5-bromo-4-chloro-3-indolylglucuron
(X-gluc). After a visible histochemical reaction occurred,
anthers were fixed in 5% formaldehyde, 5% acetic acid, and
ethanol for 2 hr, and then cleared of chlorophyll by shakin
50% ethanol for 2 hr and then in 100% ethanol overnight.
thers were photographed with Kodacolor Gold100 film using
Olympus SZH stereo microscope with dark-field illumination.
Gene Fusions Used for Transformation Studies
TA29 gene 5' regions were inserted into a pGV1500-derived Tiplasmid
cointegration vector (Deblaere et al., 1987) that was
modified to contain the Escherichia coli GUS gene (Jefferson et
al., 1987). Deletions of the TA29 gene 5' region were generated
from a 5.7-kb Sall/Hindlll fragment (nucleotides -5670 to +95;
Figure 10A) using the exonuclease III procedure of Henikoff
(1987). The -279 to +1 TA29 transcriptional control region (Figure
13) was inserted directly into the pGV1500 GUS vector. By
contrast, DNA fragments representing portions of this region were
inserted into a similar GUS vector that contained the CaMV 35S
gene minimal promoter (nucleotides -52 to +8; Benfey et al.,
1990a, 1990b). The chimeric TA29/DTA gene was constructed
exactly as described by Mariani et al. (1990) using a 5' TA29
gene fragment containing nucleotides -1477 to +51 (Figure 10A)
and the DTA coding sequence (Greenfield et al., 1983; Maxwell
et al., 1986; Palmiter et al., 1987).
Transformation of Tobacco Plants
Chimeric TA29/GUS genes were transferred from the pGV15
vector to the disarmed Agrobacterium pGV2260 Ti-plasmid (D
blaere et al., 1987), and tobacco leaf discs were transform
according to the procedure of Horsch et al. (1985) using kana
mycin selection.
ACKNOWLEDGMENTS
We express our appreciation to Anthu Bui for spending m
hours harvesting tobacco floral and vegetative organs and
help isolating organ system mRNAs. We thank Elizabeth C
for harvesting and organizing the transformed tobacco anth
and our former colleagues Drs. Tom Sims, Linda Walling, J
Harada, Diane Jofuku, Jack Okamuro, and Gary Drews for ad
and stimulating discussions during the course of this work
also thank Margaret Kowalczyk for preparing the figures for
paper, Dr. Nam-Hai Chua for the CaMV 35S minimal promo
and Drs. Jan Leemans, Titti Mariani, Johan Botterman, and J
Seurinck of Plant Genetic Systems for transformation vect
DNA sequences, and a very productive collaboration. This
search was supported by a National Science Foundation gran
R.B.G.). A.M.K. was supported by a Plant Genetic Syst
Postdoctoral Fellowship. K.H.C. and M.W. were supported
McKnight Foundation Postdoctoral and Predoctoral Fellowsh
respectively.
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Anther Gene Expression Patterns 1223
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