The Plant Cell, Vol. 5, 1217-1229, October 1993 O 1993 American Society of Plant Physiologists
Anther Development: Basic Principles and
Practical Applications
Robert B. Goldberg,’ Thomas P. Beals, and Paul M. Sanders
Department of Biology, University of California, Los Angeles, California 90024-1606
INTRODUCTION
Male reproductiveprocessesin flowering plants take place in
the stamen (Esau, 1977).This sporophyticorgan system contains
diploid cells that undergo meiosis and produce haploid
male spores, or microspores. Microspores divide mitotically
and differentiate intomulticellularmalegametophytes, or pollengrains,
that contain the sperm cells. Figures 1and 2 show
that the stamenconsistsof two morphologicallydistinct partsthe
anther and the filament. The filament is a tube of vascular
tissue that anchors the stamen to the flower and serves as
a conduit for water and nutrients. By contrast, the anther contains
the reproductive and nonreproductivetissues that are
responsible for producingand releasing pollengrains so that
pollination and fertilization processes can occur within the
flower.
Figure 1showsthat anther development can be divided into
two general phases. During phase 1, the morphology of the
anther isestablished,cell and tissue differentiationoccur, and
microspore mother cells undergo meiosis.At the endof phase
1,the anther contains most of itsspecializedcellsandtissues,
and tetrads of microsporesare presentwithin the pollen sacs
(Figure 1). Duringphase2, pollengrains differentiate, the anther
enlarges and is pushed upward in the flower by filament
extension, and tissue degeneration, dehiscence, and pollen
grain releaseoccur (Figure 1). The cellular processesthat regulate
anther cell differentiation, establish anther tissue patterns,
and cause the anther to switch from a histospecification program
(phase l)to acelldegenerationand dehiscence program
(phase 2) are not known.
The developmentalevents leadingto anther formation and
pollen release are exquisitely timed and choreographed
(Koltunow et al., 1990; Scott et al., 1991). Cell differentiation
and dehiscence events occur in a precise chronological order
that correlates with floral bud size (Koltunow et al., 1990;
Scott et al., 1991). This permitsthe mechanisms responsible
for cell-type differentiation, tissue degeneration, and cellspecific
gene activation within the anther to be explored with
relative Base. During the past few years, there has been an
explosive burst of interest in anther biology, both as a system
to dissect plant developmentalprocessesat the molecular and
genetic levels(Koltunowet al., 1990;Gasser, 1991; McCormick,
To whom correspondenceshould be addressed.
1991; Scott et al., 1991; Chaudhury et al., 1992; Dawe and
Freeling, 1992; Spena et al., 1992; van der Meer et al., 1992;
Worrall et al., 1992; Aarts et al., 1993; Preusset al., 1993)and
for practicalgenetic engineering studies to improvecrop plants
(Mariani et al., 1990, 1992; Denis et al., 1993; Schmülling et
al., 1993). In this review,we outlinethe major events that occur
during anther development, using our studies of the
tobacco anther as a focal point (Koltunow et al., 1990). We
concentrate primarily on processes concerned with anther
cell-type differentiation and dehiscence, because others
in this volume have concentratedon the male gametophyte
and have reviewed pollen grain biology and development
(Mascarenhas, 1993,this issue; McCormick, 1993,this issue).
STAMEN INlTlATlON IS CONTROLLED BY HOMEOTIC
GENE INTERACTIONS
As shown in Figure 2 and represented schematically in Figure
1, stamen primordiaare initiatedas awhorl of small bumps
at specific locations on the floral meristemsurface. The number
of stamen primordiavaries in different plant species, but
their locationon the floral meristemisfixed in what is referred
to as the thirdwhorl (Coen, 1991; Coenand Meyerowitz, 1991).
Intobacco, for example, five stamen primordiaare initiatedin
the third whorl (Figures 1 and 2) over a 1- to 2-day period
(Koltunowet al., 1990).Stamen primordiaappear after the sepal
and petalprimordiahave initiatedbut priorto the initiation
of carpel primordia(Figure2). A short time after stamenprimordia
initiation occurs, the anther and filament compartments
differentiate, two bilaterallysymmetrical theca are established
within the anther, major anther cell types and tissues form,
and the anther takes on its characteristic paddlelike shape
(Figures 1 and 2).
Stamen primordia initiation does not depend uponthe presente
of sepal or petal primordia, because surgical removal
of these primordiadoes not preventthird-whorlprimordiafrom
becoming stamens (Hicks and Sussex, 1971). In addition,
homeoticmutationsthat affect the identity of petal and sepal
primordiado not affect stamen primordiainitiationand differentiation
(Coen, 1991; Coen and Meyerowitz, 1991). Thus,
1218 The Plant Cell
Sepal Petal Carpel
Sepal
Stamen
Petal Phase 1|
Stamen
Cell Specification
Tissue Differentiation
Meiosis and Spore Formation
Stages -6 to -1
'/
-Filament
Stage -7 Stage +1
Phase 2|
Pollen Grain and Sperm Cell Differentiation
Anther Enlargement and Filament Extension
Tissue Degeneration
Dehiscence and Pollen Release
Stages
+ 2 to+10
CCC
PG
Stage +1 Stage +11 Stage +12
Figure 1. A Generalized Overview of Anther Development.
Schematic representations of anther developmental stages and cross-sections are based on scanning electron and light microscopy studies of
tobacco anther development (Koltunow et al., 1990; Drewset al.,1992). The dashed line through the stage 1anther drawn in the phase 1portion
of the figure represents the cross-section plane for anthers drawn schematicallyin phase 2. The verticallines drawn through the endothecium
at stages 11and 12 represent fibrous cell wall bands. C, connective; CCC, circular cell cluster; E, epidermis; En, endothecium; PG, pollen grain;
PS, pollen sac; St, stomium; T, tapetum; Td, tetrads; Th, theca; V, vascular bundle.
stamen primordia specification occurs independently of the
presence of other floral organ primordia and vice versa. What
processes control stamen morphogenesis and the differentiation
of diverse stamen cell and tissue types are not known.
Genetic and molecular studies have shown that stamen
primordia specification is controlled by the interaction of several
homeotic genes (Figure 1), including APETALA3 (APS),
PISTILLATA (PI),and AGAMOUS(AG) in Arabidopsis and their
counterparts DEFICIENS (DEF), GLOBOSA (GLO), and PLENA
(PLE) in snapdragon (Schwarz-Sommer et al., 1990; Coen,
1991, 1992; Coen and Carpenter, 1993, this issue; Okamuro
et al., 1993, this issue). Mutations in any one of these genes
result in the loss of stamen primordia and the conversion of
third-whorl primordia to adifferent floral organ type (SchwarzSommer
etal., 1990; Coen, 1991,1992; Coen and Meyerowitz,
1991). These homeotic genes also play a role in the specification
of other floral primordia and are discussed in detail by
Okamuro etal. (1993, thisissue) and Coen andCarpenter (1993,
this issue). Related homeotic genes have also been identified
in tobacco (Hansen et al., 1993), petunia (Angenent et al., 1992;
van der Krol and Chua, 1993, this issue), tomato (Pnueli et
al., 1991), and corn (Schmidt et al., 1993; Veil et al., 1993, this
issue), indicating that the same genes control stamen primordia
specification in distantly related plants.
Homeotic genes controlling stamen specification encode
transcription factors related to those in yeast and mammals
and contain a conserved MADS box region that probably
represents the DMAbinding domain (Coen and Meyerowitz,
1991). DEF (AP3) and GLO (PI) encode transcription factors
that interact as heterodimers with their target DMAsequence
motifs and, with the PLE (AG) gene product, activate genes
required for stamen specification inthe third whorl (Coen, 1992;
Schwarz-Sommer et al., 1992; Trobneret al., 1992). In situ hybridization
studies have shown that these homeotic genes are
expressed in the predicted pre-third-whorlterritories ofthe floral
meristemjust prior tostamen primordia initiation andthat they
Anther Development 1219
continue to be expressed throughout stamen development in
both the anther and filament (Schwarz-Sommer et al., 1990,
1992; Coen and Meyerowitz, 1991; Coen, 1992). Thus, the floral
homeotic genes DEF (AP3), GLO (PI), and PLE (AG) set
off a cascade of events that cause third-whorl primordia to follow
a stamen differentiation pathway (Figure 1).
Although progress has been made in identifying genes required
for stamen primordia specification, several crucial
questions remain unanswered. First, which genes control the
stamen primordia identity genes, and howdo these upstream
genes activate DEF(APS), GLO(PI), and PLE(AG) in the proper
third-whorl territories? That is, what processes regulate the position
and number of stamen primordia on the floral meristem?
Several homeotic genes have been identified in Arabidopsis
and snapdragon that play a role in regulating floral meristem
identity (Schwarz-Sommeret al., 1990; Coen, 1991,1992; Coen
and Meyerowitz, 1991; Coen and Carpenter, 1993, this issue),
including SOUAMOSA and FLORICAULA in snapdragon (Coen
et al., 1990; Schwarz-Sommer et al., 1990), and their counterparts
APETALA1 (Mandel et al., 1992) and LEAFY (Weigel et
al., 1992) in Arabidopsis (Okamuro et al., 1993, this issue). It
is not yet known, however, which genes and processes
function in the interval between the activation offloral meristem
identity genes and those involved in stamen primordia specification.
Second, and most important, it is not known what target,
or downstream, genes are activated transcriptionally by the
DEF (AP3), GLO (PI), and PLE (AG) proteins within the stamen
primordia. That is, what are the genes whose products
are required to carry out stamen morphogenesis and tissue
differentiation programs after the stamen primordia have been
specified? Among these downstream genes should be those
that (1) regulate the differentiation of the stamen into anther
and filament compartments, (2) control the shape of the anther
and its size, (3) initiate cell and tissue specification
programs required forthe development of a mature anther, and
(4) activate anther cell-specific gene sets. Clearly, the identification
of the downstream genes and their products should
provide insight into the detailed mechanismsresponsible for
anther morphogenesis and differentiation.
STAMEN DEVELOPMENT IS INFLUENCED BY
NUCLEAR AND MITOCHONDRIAL
GENOME INTERACTIONS
Figure 2. Scanning Electron Micrographs of Tobacco Stamen Primordia
Development.
Floral buds and anthers harvested at the designated stages were photographed
in the scanning electron microscope(Koltunowet al.,1990;
Drews et al.,1992). Magnification factors and stage numbers are marked
on each micrograph. Numbers have not been assigned to stagesprior
to -7, which are collectively referred to as pre -7 (Figure 1). A, anther;
C, carpel; F, filament; P, petal; S, sepal; T, theca.
Several homeotic-like mutants have been identified in tobacco
that affect stamendevelopment (Rosenberg and Bonnett, 1983;
Kofer et al., 1990,1991,1992). These mutants were identified
originally as male-sterile progeny of crosses between different
tobacco species (Sand, 1968; Hicks et al., 1977) and, more
recently, in plants regenerated from tissue culture after fusion
of protoplasts from different male-sterile cultivars (Kofer et al.,
1990, 1991, 1992). Unlike male-sterile homeotic mutants in
Arabidopsis and snapdragon that arecaused by nuclear gene
lesions, tobacco homeotic-like mutants are inherited cytoplasmically
and are associated with alterations in specific mitochondrial
DNAfragments (Kofer et al., 1991,1992). This implies
that mitochondrial genes play a role in the stamen differentiation
pathway.
The phenotypes of tobacco homeotic-like mutants vary,but
they correlate with the presence of a specific cytoplasm and
mitochondrial genome (Kofer et al., 1991,1992). For example,
in some mutants, the third-whorl primordia emerge and then
abort just prior to carpel primordia initiation (Rosenberg and
Bonnett, 1983). In other mutants, the third-whorl primordia develop
into petal-like structures or antherless stamens whose
filaments have stigmatoid structures at their tips (Hicks et al.,
1977; Rosenbergand Bonnett, 1983). The varied effects of the
mitochondrial genome on stamen development suggest that
the nuclear/mitochondrial gene interactions occur after stamen
primordia specification (Hicks etal., 1977; Rosenberg and
Bonnett, 1983). If so, this suggests that the altered cytoplasms
interfere with the activation and/or function of the downstream
genes required for stamen differentiation and morphogenesis.
The question of how mitochondrial and nuclear gene
interactions affect the stamen differentiation pathway remains
unresolved.
1220 The Plant Cell
ANTHER CELL TYPES CAN BE TRACED TO SPECIFIC
FLORAL MERISTEM LAYERS
At the end of phase 1 (Figure 1), a differentiated anther has
several highly specialized cells and tissues that are responsible
for carrying out nonreproductive functions (e.g., support
and dehiscence) and reproductive functions (e.g., spore and
pollen formation). Schematic representations of differentiated
stage 1 tobacco anther cross-sections are shown in Figures
1 and 3, actual bright-field photographs are shown in Figure
4, and anther cell types and their functions are listed in Table
1. The nonreproductive tissues include the epidermis, endothecium,
tapetum, circular cell cluster, connective, stomium,
and vascular bundle (Table 1). Each of these tissues and cell
types carries out specialized tasks (Esau, 1977). For example,
the stomium and circular cell cluster are involved in dehiscence
(Bonner and Dickenson, 1989), the tapetum plays a role in pollen
wall formation (Esau, 1977; Pacini et al., 1985), and the
connective is responsible for anchoring the four pollen sacs
together into a single anther structure (Weberling, 1989). In
addition to these diploid sporophytic tissues, the anther also
contains haploid microspores that fill the pollen sacs and
differentiate into pollen grains (McCormick, 1991, 1993, this
issue; Scott et al., 1991; Mascarenhas, 1993, this issue).
Cytochimera cell lineage studies performed with the Jimson
weed (Datura stramonium), a solanaceous relative of a
tobacco, showed that the floral meristem consists of three
"germ" layers, designated L1, L2, and L3 (Satina et al., 1940),
which give rise to different anther tissues following stamen
primordia initiation (Satina and Blakeslee, 1941). Thus, once
specified, the developmental fate of L1, L2, and L3 layer
STAGE
10
Phase 1
Anther Primordium
Stage -7
Phase 2
Inner
Tapetum
Microspores
Connective
Vascular
Bundle
Outer
Tapetum
Endothecium
Stomium
Epidermis
Anther Stage +1
Circular Cell
Cluster
Dehiscence
Figure 3. Cell Lineages and Major Events That Occur during Anther Differentiation and Dehiscence.
Stages, timing of events, and anther schematic representations were taken from Koltunow et al. (1990) for tobacco anther development. Similar
events take place during anther development in other plants (Esau, 1977). Cell lineages derived from the L1, L2, and L3 primordia layers were
inferred from the histological observations of Satina and Blakeslee (1941), Esau (1977), and Koltunow et al. (1990).
Anther Development 1221
0)
V)
(0
CM
0)
(A
CO
-6
-2
-1
8
11
st En
Figure 4. Bright-Field Photographs of TobaccoAnther Development.
Anthers at the designated stages were fixed, embedded in paraffin,
and sliced into 10-nm transverse sections through the plane drawn
schematically in Figure 1. The sections were stained with toluidine
blue and photographed with bright-field illumination. Pictures aretaken
from Koltunow et al. (1990), except for stage 11. A, archesporial cells;
C, connective; CCC,circular cell cluster; E,epidermis; En,endothecium;
MMC, microspore mother cells; Msp, microspores; P, parietal cells;
PS, pollen sac; Sp, sporogenous cells; St, stomium; T,tapetum; TDS,
tetrads; V, vascular bundle.
derivatives is fixed. Figure 3 shows a schematic representation
of a tobacco stamen primordium and the anther cell
lineages that are inferred from histological analysis to be derived
from the L1, L2, and L3 layers (Satina and Blakeslee,
1941; Esau, 1977; Koltunow et al., 1990). In most cases, individual
tissues and cell types arederived from a single germ
layer. For example, the L1 layer gives riseto the epidermis and
the stomium; the L2 layer gives rise to the archesporial cells,
microspore mother cells, endothecium, and middle-wall layers
that lie between the epidermis and tapetum; and the L3 layer
gives riseto the connective, vascular bundle, and circular cell
cluster adjacent to the stomium (Figure 3). By contrast, both
the L2 and L3 layers contribute to tapetum formation (Figure
3). Tapetal cells along the upper (inner) portion of the pollen
sacs are specified from the LS-derived connective tissue,
whereas those that line the lower (outer) portion of the pollen
sacs are specified from the L2-derived archesporial lineage
(Figure 3; Esau, 1977). The underlying mechanisms responsible
for the differentiation of specific anther cell types after
stamen primordia initiation occurs are not known.
CELL TERRITORIES MAY PLAY A ROLE IN THE
ANTHER DIFFERENTIATION PROCESS
Figures 3 and 4 show that cells destined to become major anther
tissues and cell types form at precise times and positions
during anther development. For example, archesporial cells
destined to differentiate into microsporangia (pollen sacs) and
surrounding tapetum and endothecium tissues arise simultaneously
in each corner of the anther primordium (Figures
3 and 4). By contrast, vascular tissues differentiate within the
Table 1. Functions of Anther Cell and Tissue Types
Cell or Tissue Type8
Major Function6
Connective
Circular Cell Cluster0
Endotheciumd
Epidermis
Microspore
Middle Layerd
Stomium
Tapetum
Vascular Bundle
Join anther theca together; connect
anther to filament; structure, support,
and morphology
Dehiscence
Structure and support; dehiscence
Structure and support; prevent water
loss; gas exchange; dehiscence
Pollen grain and sperm cell
development
Structure and support; dehiscence
Dehiscence
Pollen wall components; nutrients for
pollen development; enzymes for
microspore release from tetrads
Connection between anther, filament,
and flower; nutrient and water supply
8
Cells present in a stage 1 tobacco anther (Figures 1 to 4).
"Taken from Esau (1977), Weberling (1989), and Koltunow et al.
(1990).
c
Also referred to as the crystal-containing idioblasts (Trull et al.,
1991), intersporangial septum (Bonner and Dickenson, 1989), and
hypodermal stomium (Horner and Wagner, 1992).
d
Collectively referred to as the anther wall.
1222 The Plant Cell
center of the anther primordium and establish a connection
with the filament (Figures3 and 4). Other tissues, such as the
circular cell cluster and stomium, form at the boundaries between
each microsporangialpair later indevelopment (Figures
3 and 4). Thus, specific regions,or territories, are established
early in anther development within which unique histodifferentiation
events occur in a precise chronological order.
No information exists about whether the differentiation of
anther cells and tissues is induced (or inhibited) by signals
originating from contiguous regions of a territory, or is controlledautonomouslyby
sequesteringmorphogeneticfactors
during divisions of the L1, L2, and L3 layers, or both. The
territory-baseddifferentiationof four microsporangiawith identicaltissue
patternsand the differentiationof tapetalcellswithin
each territory from two different cell lineages (Figure 3) suggest
that cell-cell communication processes may play an
important role in the anther histospecificationprocess. For example,
it is conceivable that a gradient of morphogens,
synthesizedby archesporial cells and interpreted by contiguous
L2 and L3 cells, triggers the differentiationof tapetal and
endothecial cells (Figures 3 and 4). Other morphogens appearing
later at the boundaries of the microsporangia could
be perceived by neighboring L1-derived epidermal cells and
L3-derived connective cells and cause them to differentiate
intothe stomium and circular cellcluster, respectively (Figures
3 and 4). lnterpretation of putative intercellular signalswould
lead ultimatelyto the transcriptionalactivationofgenes responsible
for establishing the differentiated fate of each cell type
within the territory. Although speculative, this model leads to
testable predictions.
Severa1lines of evidence suggest that intercellular signals
play an important role in determiningthe fate of cells within
each floral meristem germ cell layer. These studies are reviewed
by Sussex (1989)and by Hualaand Sussex (1993,this
issue). Genetic chimera experimentshave indicatedthat the
position of L1, L2, and L3 cells within the floral meristem is
the primary factor in specifyingtheir fate rather than priorcell
ancestry (Sussex, 1989; Hualaand Sussex, 1993,this issue).
For example, rare divisions that enable L2 cells to penetrate
or invade the L1 meristem layer cause these cells to follow
an epidermalpathway.Conversely, penetrationof L1cells into
the L2 layer causes these cells to have a fate identical to that
of surroundingL2cells. In addition, recent studies usinggene
markersspecific for each germ layer have shown that the L3
layer influencesthe developmentof cells derivedfrom the L1
and L2 layers (Szymkowiak and Sussex, 1992). These experimentssuggestthat
cellswithin each meristemgerm layer are
conditionallyspecified(Davidson, 1991) and that the ultimate
fate of these cells depends upon their position in the floral
meristem. That is, position-dependent communication processes
probablyoccur between adjacent meristematiccells,
and these signaling events influencefloral organ cell specification
pathways. The extent to which cell-autonomous and
position-dependent cell interaction processes play a role in
the differentiationof specialized cell types derivedfrom each
anther primordiumgerm layer (Figure 3) remains a major unanswered
question.
ANTHER DEHISCENCE INVOLVES THE PROGRAMMED
DESTRUCTION OF SPEClFlC CELL TYPES
One of the major mysteriesof anther development is how a
differentiated anther switchesfrom a histodifferentiation program
(phasel)to a cell degeneration and dehiscence program
(phase 2) that leads ultimately to pollen release and stamen
senescenceat flower opening. The dehiscence program begins
after the formation of tetrads, results in the sequential
destruction of specific anther cell types, and is coordinated
temporally with the pollen differentiationprocess (Figure 1).
In tobacco, the entire dehiscence and cell degeneration process
occurs over a l-week period (Koltunow et al., 1990) and
involves three major cell types that are formed during phase
1from different anther primordiumcell layers(Figure 3).These
include the stomium, the endothecium, and the circular cell
cluster (Table 1; Figures 1 and 4). The latter tissue, which is
common in solanaceous anthers, is also referred to as the
hypodermal stomium (Horner and Wagner, 1992), the intersporangialseptum
(Keijzer, 1987; Bonner and Dickenson, 1989)
and, becauseit containscalcium oxalatecrystals, the crystalcontaining
idioblasts (Trull et al., 1991; Horner and Wagner,
1992). The onset of the dehiscence program sets off an ordered
series of events within the anther, including (1) the
formation of fibrous band thickeningson the endothecial cell
walls, (2) degenerationof the circularcellcluster and merging
of the two pollen sacs in each theca into a single locule, (3)
breakdownof the tapetum and connective, and (4) rupture of
the anther at the stomium and pollen release (Figures 1and
4; Bonner and Dickenson, 1989). Male-steriletobacco anthers
that lack pollengrains and tapetal cells undergoa normaldehiscenceprocess(Koltunow
et al., 1990; Marianiet al., 1990),
indicating that dehiscence is not set into motion by signals
derived from either the tapetum or differentiatingpollengrains.
The dehiscence program probably requiresthe activation
of manygenes, includingthose thatencode hydrolyticenzymes
requiredfor cell death, such as RNases, proteases, and cellulases(Keijzer,
1987; Bonner and Dickenson, 1989;de1Campillo
and Lewis, 1992). One marker for the dehiscence process in
tobacco is the TA56 thiol endopeptidase gene (Koltunow et
ai., 1990).Asshown in Figure5, TA56mRNAaccumulatesfirst
in the circular cell cluster prior to its destruction, then in the
stomium, and finally in the connective. Although the role of
the TA56thiol endopeptidasegene inthe dehiscenceprocess
is not known, it marks the cell-specific degeneration events
that occur prior to stomium rupture and pollen release from
the anther (Figures 1 and 4; Koltunow et al., 1990). Recent
experimentswith chimeric genes containingTA56 5’sequences
showed that the sequential accumulation of TA56 mRNA in
specific anther cell types is controlled primarily at the transcriptionalleve1(T.
F! Beals and R. B. Goldberg, unpublished
results). Thus, the onset of the dehiscence program results
in the transcriptionalactivationof genes that are inactive duringthe
histodifferentiationphaseof anther development(Figure
1). Which cellular events trigger the dehiscence program,
activatedehiscencespecificgenes, andcoordinatepollengrain
Anther Development 1223
DEVELOPMENTAL STAGE
m R N A - I I Z 3 4 5 6 7 8 9 10 II 12
TA13 TA36
TA29 TA32
TA26
TA20
TA39
TA55
TA56
CCC
W MS
Figure 5. Temporaland Spatial Regulation of mRNAs during Tobacco
Anther Development.
Data represent a summary of RNA gel blot and in situ hybridization
studies performed in our laboratory with anther-specific cDNAs
(Koltunow et al., 1990; T.P. Seals and R.B.Goldberg, unpublished
results). Proteins encoded by these mRNAs and their cell specificities
are summarized in Table 2. The colors present in each bar
correspond with the cell and tissue types in which each mRNAis localized
at that period of anther development. The tapering in each bar
shows periods when the mRNA accumulates or decays, and the bar
thickness approximates the relative mRNA prevalences. C, connective;
CCC,circular cell cluster; E, epidermis; MS, microspore; S,
stomium; T, tapetum; V,vascular bundle; W, wall. The wall includes
the endothecium and middlelayer (see Table 1). Figure adapted from
Koltunow et al. (1990).
differentiation with the sequential destruction of specific anther
tissues are not known.
GENE EXPRESSION IS TEMPORALLY AND SPATIALLY
REGULATED DURING ANTHER DEVELOPMENT
RNA-excess DNA/RNA hybridization experiments with singlecopy
DNA indicated that there are ~25,000 diverse genes
expressed in a stage 6tobacco anther (Figure 4; Kamalay and
Goldberg, 1980). The vast majorityof these genes encode rare
mRNAs that constitute less than 0.001% of the mRNA mass
when averaged over the entire anther mRNA population. Comparison
of floral and vegetative organ system RNApopulations
showed that ~10,000 diverse anther mRNAsare undetectable
in the nuclear RNA and mRNA populations of other organs
and are anther specific (Kamalay and Goldberg, 1980,1984).
Although it is not known how many additional anther-specific
genes are expressed during phase 1,when major histospecification
events occur (Figure 4), these results indicate that the
differential regulation of a large number of genes is required
to specify and maintain the differentiated state of cells and tissues
within the anther.
Experiments conducted in several laboratories have identified
cDNA clones that represent prevalent mRNAs that are
present exclusively, or at elevated levels, in the anther (Koltunow
et al., 1990; Smith etal., 1990; Evrard etal., 1991; McCormick,
1991,1993, this issue; Nacken et al., 1991; Scott et al., 1991;
Shen and Hsu, 1992). Table 2 lists several tobacco antherspecific
mRNAs studied in our laboratory. Anther-specific
mRNAs have been shown to encode lipid transfer proteins,
protease inhibitors, thiol endopeptidases, glycine-rich and
proline-rich polypeptides with properties of cell wall proteins,
pectate lyases, polygalacturonases, and chalcone synthase
isoforms (Table2; Koltunow et al., 1990; McCormick, 1991; Scott
et al., 1991). Many anther-specific mRNAs, such as TA20,are
found exclusively in sporophytic tissues (Table 2; Koltunow et
al., 1990). Others are pollen grain specific or, like TA39 (Table
2), are present in both sporophytic and gametophytic cells
(McCormick, 1991; Scott et al., 1991).
Experiments conducted with anther-specific cDNAs have
demonstrated the striking degree to which different temporaland
cell-specific gene expression programs occur during
anther development (Koltunow etal., 1990; McCormick, 1991,
1993, this issue; Scott et al., 1991). Figure 5 presents a
Table 2.
mRNA8
'"
TA13
TA20
TA26
TA29
TA32
TA36
TA39
TA55
TA56
Gene Markers for Tobacco
Protein Encoded
Glycine-rich
Unknown
Unknown
Glycine-rich
Lipid transfer
Lipid transfer
Unknown
Unknown
Thiol endopeptidase
Anther Cell Types
Cell Specificity
Tapetum
Connective, stomium,
endothecium, middle layer
Tapetum
Tapetum
Tapetum
Tapetum
Tapetum, microspore
Tapetum, connective,
endothecium, middle layer
Connective, circular cell
cluster, stomium
a
Taken from Koltunow et al. (1990) and T.P. Beals and R.B.
Goldberg (unpublished results).
b
Gene markers for anther cells in other plants have been reviewed
recently (Gasser, 1991; McCormick, 1991; Scott et al., 1991).
1224 The Plant Cell
schematic representationof several different tobacco antherspecific
gene expressionprogramsthat have been identified
recently inour laboratory(Koltunowet al., 1990;T.P. Beals and
R.B. Goldberg, unpublishedresults). One strikingexample is
the coordinated accumulation and decay of the tapetal-specific
TA13, TA26, TA29, TA32, and TA36 mRNAs during early phase
2 (Figure 5; Koltunow et al., 1990). This pattern, and others
shown in Figure5, probably represent only a few of the gene
expression programs that occur during anther development
(Evrardet al., 1991;McCormick, 1991; Scott et al., 1991). However,
these patterns illustrate that (1) genes expressed in the
anther are regulated by many differentcontrol programsthat
are partitionedwith respectto bothcell type and developmental
time, (2) anther-specific gene expressionprogramscorrelate
with the differentiation and degeneration of specific anther tissues
andcelltypes, and (3)anther cell-specific mRNAsprovide
useful markersfor dissectingthe regulatory pathwaysthat control
the specification of different cell types during anther
development. The mechanisms responsible for establishing
cell- and temporal-specific gene expression patterns during
anther development are not known.
TRANSCRIPTIONAL PROCESSES CONTROL
ANTHER-SPECIFIC GENE EXPRESSION PROGRAMS
Solution hybridization experiments carried out with anther
mRNA and nuclear RNA populations suggested that most
anther-specific genes are under transcriptional control
(Kamalay and Goldberg, 1984). Run-offtranscriptionstudies
andtransformation experimentswith chimericgenes containing
anther-specificgene promoters demonstrated directly that
the temporal and spatialgene expression programsthat operate
in the anther are controlled primarily by transcriptional
processes (Koltunowet al., 1990; McCormick, 1991,1993, this
issue; Scott et al., 1991).For example,the tapetal-specificTA29
gene (Table 2 and Figure 5) is not transcribed detectably in
other plant organs, and chimeric genes with TA29 S’sequences
are active only in the tapetum (Koltunow et al., 1990; Mariani
et al., 1990, 1992). Chimeric genes driven by other antherspecific
gene promoters, such as TA56 (Table 2 and Figure
5),producetranscription patternsthat also reflectthe cell-and
temporal-specific expression programscharacteristic of their
parental, or endogenous, genes (McCormick, 1991; Scott et
al., 1991; T.P. Beals and R.B. Goldberg, unpublishedresults).
These results suggest that the mosaicof gene expression programs
that function in the anther (Figure 5) reflects the
distributionof transcriptionfactors capable of activatingunique
gene sets in each specialized cell type. If so, this impliesthat
anther cell differentiationevents lead directly or indirectly to
the expressionof regulatory genesthat are necessaryto activate
each cell-specific gene set. The identificationof anther
transcriptionfactors and their correspondinggenes should provide
usefulentry points intoanther cell-specific differentiation
pathways(Figure 3). How genes encodinganther transcription
factors are regulatedwith respectto cell type and time during
anther development, activate batteries of cell-specificgene
sets, and connectwith regulatory circuitsthat ultimately trace
backto homeoticgenes controllingstamen primordiaspecification
(Figure 1) remain major unanswered questions.
CELL ABLATION STUDIES WlTH CYTOTOXIC GENES
CAN BE USED TO STUDY ANTHER CELL
DlFFERENTlATlONPROCESSES
One powerful use of the anther cell-specific promotersidentified
in the gene expression studies (Table 2 and Figure 5)
isto fusethem to cytotoxic structuralgenes, such as the diphtheria
toxin A-chain gene (Palmiter et al., 1987), the RNase
T1 gene (Mariani et al., 1990), or the barnase gene (Mariani
et al., 1990),to selectively destroy specific cell types during
anther development. This strategy, illustratedin Figure 6,is
an extension of the microbeam laser cell ablation approach
that (1)providedthe first insightsintothe roleof cell-cell interactions
in Caenorhabditiselegans cell-typespecificationand
(2) laid the foundation for molecular and genetic approaches
that identified genes encoding components of different cell
inductionpathways (Sulstonet al., 1983;Horvitzand Sternberg,
1991). 60th of these strategies rely on the selective destruction
of a specific cell type and an identificationof the effect,
if any, that the loss of this cell type has on the differentiation
of contiguous cells (Palmiter et al., 1987). The microbeam laser
approachis not readily applicableto studies of anther cell
differentiation,becausethe anther consists of many cell layers
and is contained within a closed flower bud (Figures 1 to 4).
Thus, specific cell targets are refractory to identificationand
physical destruction by a laser beam. By contrast, ablation
studies using“genetic lasers” in transgenic plants can, in theory,
be applied to an unlimited number of cell types. All that
is required is a set of cell-specificpromotersthat is active in
a variety of anther cell types (Table 2).
We fused the tobaccotapetal-specific TA29 gene promoter
(Table2 and Figure5)to several cytotoxic genes to selectively
destroy the tapetum during anther development (Figure 6;
Koltunowet al., 1990;Mariani et al., 1990).These experiments
showedthat the tapetum is critical for pollen developmentbut
that it is not necessary for the function and/or degeneration
of other anther cell types during phase 2 of anther development
(Koltunow et al., 1990; Marianiet al., 1990). That is, the
tapetum functions in a cell-autonomousmanner following its
formation during phase 1 of anther development (Figures 3
and4), and tapetal cells are not requiredfor the inductionand/or
completion of the dehiscence program(Figure 1). Of particular
interest, of course, is how the loss of cells formed during
the early stages of anther development would affect the differentiation
of adjacent cell types (Figures 3 and 4). For
example, how would the loss of primary parietal cells affect
the differentiationof contiguoussporogenouscells within developing
microsporangialterritories? These ablation studies,
Anther Development 1225
Male Sterile Plants Restored Male Fertile Plants
MS NOT No MS MS
Normal Tapetum
Development
[TA29J BARNASE)' |TA29| BARNASE) |TA29| BARSTAR)
Tapetal-
specific
Barnase
Destruction
of RNA in
Tapetum Tapetal-
specific
Barnase
\
Tapetal-
specific
Barstar
Tapetum Destroyed
Barnase
INACTIVE
Barnase/Barstar
Complex
Normal Tapetum
Development
Figure 6. Genetic Engineering for Male Fertility Control.
A schematic representation of the male sterility and male fertility restoration experiments described by Mariani et al. (1990, 1992). MS, microspores;
T, tapetum.
however, will require promoters that are active in specific cell
types of the developing anther primordium, such as the archesporial
and parietal cells (Figures 3 and 4). The availability
of anther primordium-specific promoters should permit athorough
investigation of the lineage relationships, cell functions,
and cell-cell interaction processes that occur during anther
development (Figure 3).
MALE-STERILE MUTANTS CAN BE USED TO
GENETICALLY DISSECT ANTHER DEVELOPMENT
Cell ablation studies can provide important conceptual information
about the processes that control the differentiation of
anther cell types. They cannot, however, identify genes that
are responsible for directing cell specification events. To accomplish
this will require, in part, a genetic approach analogous
to that used in Drosophila and C. elegans to identify genes
involved in both position-dependent and cell-autonomous
differentiation pathways(Horvitz and Sternberg, 1991; Kramer
et al., 1991).
Male-sterile mutants have been identified in many plants,
including Arabidopsis (Kaul, 1988). Although most male sterility
genes and their wild-type alleles have not been cloned and
studied (Aarts et al., 1993), phenotypic and histological analyses
suggest that some male sterility mutations affect the
differentiation and/or function of many anther cell types, including
those in the stomium, tapetum, endothecium, and the
archesporial and sporogenous layers of the anther primordium
(Kaul, 1988; Chaudhury, 1993, this issue). Arabidopsis malesterile
mutants are easy to identify because they flower for
a longer time period, grow taller, and remain in a green or
nonsenesced state longer than their male-fertile, wild-type
counterparts (Feldmann, 1991; Chaudhury et al., 1992;
Forsthoefel et al., 1992; Aarts et al., 1993; Preuss et al, 1993).
Because large numbers of mutagenized plants can be
screened readily (Feldmann, 1991; Chaudhury et al., 1992;
Forsthoefel et al., 1992), genetic studies in Arabidopsis have
the potential to dissect gene pathways that control both the
1226 The Plant Cell
histodifferentiation program (phase 1) and the dehiscence and
cell degeneration program (phase 2) of anther development
(Figure 1).
Recently, we screened a large number of Arabidopsis lines
that were treated with either T-DNA (Feldmann, 1991;
Forsthoefel et al., 1992) or ethyl methanesulfonate (EMS)
(Koornneef et al., 1982)for male-sterile mutants. Wewere particularly
interested in anther mutants that failed to dehisce and
those that had defects in the anther differentiation pathway.
Figure 7 shows two male-sterile mutants that were identified
in our EMS screen. One mutant, designated as undeveloped
anther, has stamens with normal filaments but anthers that lack
pollen and have an undeveloped, club-shaped appearance
(Figure 7A). The selective effect of this mutation on anther development
suggests that distinct genes control anther and
filament differentiation following stamen primordia specification.
By contrast, the other mutant, designated as late
dehiscence, has stamens that undergo a normal differentiation
pathway and contain anthers that are morphologically
similar to their wild-type counterparts (Figure 7B). Late dehiscence
anthers, however, fail to dehisce on time (Figure 7B)
and release their pollen grains after the stigma is no longer
receptive to pollination. These male-sterile mutants and others
with analogous phenotypes should permit the isolation of
genes controlling anther developmental processes in the near
future.
ANTHER GENE EXPRESSION STUDIES HAVE LED TO
THE GENETIC ENGINEERING OF CROP PLANTS
FOR MALE FERTILITY CONTROL
The anther plays a prominent role in crop production because
it is responsible for carrying out male reproductive processes
necessary for generating seeds that will produce the next plant
Figure 7. Arabidopsis Male-Sterile Mutants That Are Defective in Anther
Development.
(A) undeveloped anther.
(B) late dehiscence.
A, anther; F,filament; OV,ovary; P,petal; S, sepal; SL, style; ST,stigma.
generation (Figure 1). One of the major factors contributing
to increases in crop productivity over the past few years has
been the breeding of hybrid varieties in crops such as corn
and oilseed rape (Brewbaker, 1964; Feistritzer and Kelly, 1987).
Crosses between inbred plant lines give rise to hybrid progeny
that, at times, exhibit heterosis or hybrid vigor (Brewbaker,
1964). Although the molecular basis of hybrid vigor is not
known, hybrids with a heterotic phenotype are more resistant
to disease, less susceptible to environmental stress, and generate
higher yields than their inbred parents (Brewbaker, 1964;
Feistritzer and Kelly, 1987). Increased crop yields are particularly
important to keep the world'sfood supply sufficient to meet
the needs of an ever-expanding population (Klein, 1987).
The reproductive biology of higher plants makes it difficult
to perform directed crosses between inbred lines on a scale
large enough for hybrid seed production. Because stamens
and carpels (Figures 1 and 2) are present within the same
flower (e.g., tobacco, oilseed rape, Arabidopsis) or are contained
within separate flowers on the same plant (e.g., corn),
a high level of self-pollination can occur within individuals of
each parental line. Toovercome this problem and ensure that
crosses occur berween inbred lines, several breeding strategies
have been used, including (1) manual and/or mechanical
removal of stamens from the flowers of one parental line, (2)
use of self-incompatibility alleles that prevent self-pollination,
and (3) employment of dominant cytoplasmic or nuclear male
sterility genes that disrupt pollen development (Feistritzer and
Kelly, 1987). Each strategy has limitations that prevent its use
as a general approach for hybrid seed production in all crop
plants. For example, many crop plants do not have selfincompatibility
alleles, manual removal of stamens is labor intensive
and impractical for crops with small bisexual flowers
(e.g., oilseed rape), and male sterility genes require a restorer
system.
An important practical spin-off of anther cell differentiation
and gene expression studies has been the development of
a genetically engineered system for male fertility control that
is applicable to a wide range of crop plants, including oilseed
rape, tomato, lettuce, and corn (Mariani etal., 1990,1992). This
system, which is illustrated schematically in Figure 6, takes
advantage of the tapetal-specific transcriptional activity of the
tobacco TA29 gene (Table2 and Figure 5; Koltunowet al., 1990)
and an RNase/RNase-inhibitor defense system utilized by the
bacteria Bacillus amyloliquefaciens (Hartley, 1989). The genetically
engineered male fertility control system was developed
as a collaborative effort between our laboratory and Plant
Genetic Systems in Gent, Belgium (Mariani et al., 1990,1992).
In brief, barnase is an extracellular RNase that is produced
by B. amyloliquefaciens to protect itself from microbial predators
(Hartley, 1989). Barstar, on the other hand, is a barnasespecific
RNase inhibitor that is used intracellularly by B.
amyloliquefaciens to protect itself from the cytotoxic effects of
barnase (Hartley, 1989). As illustrated in Figure 6, a cytotoxic
chimeric gene consisting of the TA29 gene promoter and barnase
gene coding sequences leads to the selective ablation
of anther tapetal cells and the production of pollenless, male-
Anther Development 1227
sterile plants (Mariani et al., 1990). By contrast, an anticytotoxic
chimeric gene containingthe same TA29gene promoter
andthe barstar gene coding region does not affect tapetal cell
development and is a dominant suppressor of the TA29/barnase
gene (Figure 6).
When male-sterileplantscontaining the TA29/barnasegene
are crossed with male-fertile plants containing the TA29/barstar
gene, progeny are produced that contain both genes, and
these progeny are malefertile (Marianiet al., 1992). The presente
of barstar within the tapetal cells of F, progeny restores
malefertility by forming acomplex with barnasethat prevents
barnase cytotoxic activity from destroying the tapetal cells
within the anther (Figure 6). Becausethe TA29 gene promoter
is regulated correctly in a wide range of dicot and monocot
crop plants (Mariani et al., 1992),the barnaselbarstar system
illustrated in Figure 6 has the potential for being universally
applicable for hybrid seed production in awide variety of crops
(Mariani et al., 1990, 1992). This system can also be used for
cell ablation studies that investigate anther cell specification
processes because it overcomes the need for cell-specific
promotersto ablate aspecific celltype. That is, acombination
of chimeric barnase and barstar genes containing promoters
with overlapping cell specificities can be used to selectively
ablate single cell types (Table 2; T.P. Beals and R.B. Goldberg,
unpublished results).
Recently, other strategies have been developed for the
productionof male-sterile plants. These include (1) the useof
achimeric tapetal-specific glucanase geneto prematurelydisruptmicrosporedevelopment(Worrallet
al., 1992),(2) antisense
inhibition of flavonoid biosynthesis within tapetal cells to disrupt
pollen development (van der Meer et al., 1992), (3)
tapetal-specific expression of achimeric Agmbacferium rhizogenesm/Bgene
that increasesauxin activity inthe anther and
causes developmental abnormalities (Spenaet al., 1992),and
(4)overexpression of a chimeric cauliflower mosaic virus 35s
atp9 mitochondrial gene that disrupts anther development
(Hernould et al., 1993). Clearly, knowledge gained by studying
anther development at the molecular and genetic levels
will reveal other approaches that can be used to genetically
engineer nove1varieties of hybrid crop plants.
CONCWSION
The anther represents anoutstanding system to study the differentiation
of plant cell types. Excellent progress has been
made in describing many of the gene expression events that
occur during anther development as well as genes controllingstamen
primordia specification. However, much remains
to be learned about the mechanisms that are responsible for
anther cell specification andthose requiredfor the activation
of cell-specific gene sets within specialized anther cells and
tissues. Molecular and genetic tools are now available that
should reveal new insight into the processes controlling anther
development in the near future.
ACKNOWLEDGMENTS
We expressour appreciationtoAnna Koltunow,Titti Mariani,andJan
Leemans for many stimulating discussions on anther development.
We thank Becky Chasan for reviewing a draft of this manuscript,
Margaret Kowalczykfor doingthe art work, and BonniePhanfor typing
this manuscript. Research from our laboratory described in this
paperwas supported by aNationalScienceFoundationgrantto R.B.G.
T.P.B. was supported by a National lnstitutesof Health Postdoctoral
Fellowshipand P.M.S. was supported by a New ZealandCrown Research
lnstitute Predoctoral Fellowship.
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