Stamen Structure and Function
R. J. Scott,a,1 M. Spielman,a and H. G. Dickinsonb
a Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom
b Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom
INTRODUCTION
Stamens are the male reproductive organs of flowering plants.
They consist of an anther, the site of pollen development, and in
most species a stalk-like filament, which transmits water and
nutrients to the anther and positions it to aid pollen dispersal.
Within the anther, male sporogenous cells differentiate and undergo
meiosis to produce microspores, which give rise to pollen
grains, whereas other cell types contribute to pollen maturation,
protection, or release. Pollen development involves an array of
extraordinary events, including cell division and differentiation
independent of a conventional meristem, the transition from
sporophytic to gametophytic generation, and modifications of
cell division to produce structures that are unusual in plant development,
including coenocytic tissues (the tapetum and the
microsporocyte mass), and subsequently free cells (microspores)
that give rise to self-contained units for genome dispersal
(pollen grains). Reviews of stamen development and gene
expression include articles by Bhandari (1984), Scott et al.
(1991a), Goldberg et al. (1993), and Irish (1999).
A change in emphasis will be apparent since anther development
was reviewed in the first Plant Reproduction special issue of
The Plant Cell (Goldberg et al., 1993). The earlier review drew
largely on studies of tobacco, but this article focuses more tightly
on Arabidopsis, which has surpassed other species in the genetic
tools publically available, including a sequenced genome,
microarrays, well-characterized mutants associated with cloned
genes, and knockout collections for reverse genetics. The advances
of the last 10 years include an increased understanding
of stamen specification, stamen-specific gene expression, internal
patterning of the anther, regulation of male meiosis, and
anther dehiscence.
Stamen and pollen development are intimately linked. We
discuss those aspects of pollen genesis that are essentially
controlled by the sporophytic genome (differentiation of the male
germline, meiosis, and pollen wall formation), but the biology of
the male gametophyte, pollen tube growth, and fertilization are
covered by other reviews in this volume (Edlund et al., 2004;
Hanson and Bentolila, 2004; McCormick, 2004).
SPECIFICATION OF STAMENS
Flowers of eudicots are organized into four concentric whorls
of organs (sepals, petals, stamens, and carpels) that arise
sequentially from the floral meristem (Figure 1). The third whorl in
Arabidopsis flowers contains six stamens, four medial (long) and
two lateral (short). Stamen primordia in Arabidopsis appear
during floral stage 5 (defined by Smyth et al., 1990)/anther stage
1 (Sanders et al., 1999), with the long stamens arising first.
The stamen primordia differentiate a stalked basal region, which
gives rise to the filament, and a wider upper region, which
becomes the anther. The anther locules, in which pollen
develops, appear as convex protrusions on the adaxial surface
(facing the carpels) at floral stage 8/anther stage 4. During this
stage, the sporogenous cells, which give rise to pollen, are
visible within locules of sectioned anthers. Concentric rings of
other cell types associated with pollen development and release
differentiate around the sporogenous cells; collectively, these
constitute the microsporangium. All anther cell types are
present by floral stage 9/anther stage 5. Sporogenous cells
develop into microsporocytes (also known as pollen mother
cells or male meiocytes), which undergo meiosis to form tetrads
of haploid microspores. These are released into the anther
locule to begin male gametophyte development. By floral/anther
stage 12, the anthers are nearly at their final length and contain
tricellular pollen, and the filaments begin to elongate rapidly. At
floral/anther stage 13, the flower opens and anther dehiscence
occurs to release the pollen. The filaments continue to extend
so that the anthers brush past the receptive stigma at floral
stage 14.
There are many recent reviews of floral organ specification
(Jack, 2001, 2004; Theissen, 2001; Lohmann and Weigel, 2002);
therefore, the regulation of stamen identity will be treated only
briefly here. The identity of floral organ primordia is controlled by
three classes (termed A, B, and C) of homeotic selector genes
with overlapping areas of activity; stamens develop in the third
whorl, where both B and C genes are expressed. It has been
discovered that additional transcription factors, encoded by
SEPALLATA (SEP) genes in Arabidopsis, are required to confer
full activity on the homeotic genes in stamens (as well as
petals and carpels). The ABC model was based on genetic
studies in Arabidopsis thaliana and Antirrhinum majus (Coen
and Meyerowitz, 1991). The B class genes are APETALA3 (AP3)
and PISTILLATA (PI) in Arabidopsis and DEFICIENS (DEF ) and
GLOBOSA (GLO) in Antirrhinum; the C genes are AGAMOUS
(AG) and PLENA, respectively. Mutations in any one of the B or C
genes result in homeotic conversion of the third-whorl organs to
a different type. Loss of B function causes transformation of
stamens to carpels, loss of C function converts stamens to
petals, and loss of both transforms stamens to sepals. Singlegene
mutations of the SEP genes have very subtle phenotypes,
but in sep1 sep2 sep3 triple mutants, all floral organs resemble
1 To whom correspondence should be addressed. E-mail bssrjs@
bath.ac.uk.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.017012.
The Plant Cell, Vol. 16, S46–S60, Supplement 2004, www.plantcell.org ª 2004 American Society of Plant Biologists
sepals, suggesting that B and C function have been abolished
(Pelaz et al., 2000).
The signals that determine the number of floral organ primordia
in each whorl are unknown (Irish, 1999). However, the number
of stamens can be perturbed by various mechanisms: extra
stamens develop in clavata1 mutants, which form enlarged
meristems (Clark et al., 1993), and in superman mutants, which
havean expanded BCdomain (Bowman et al., 1992).By contrast,
arrest of stamen primordia is a feature of normal development in
some species. In the radially asymmetric flowers of Antirrhinum,
five stamens initiate, but the dorsal stamen arrests early in development
in response to the activity of the CYCLOIDEA gene
(Luo et al., 1995).Insome speciesthat produce unisexual flowers,
such as maize and white campion, stamens in female flowers
initiate but later arrest or abort (Tanurdzik and Banks, 2004).
The B class, C class, and SEP proteins all belong to the MADS
family of transcription factors, which bind a target DNA sequence
(the CArG box) as homodimers or heterodimers. In vitro, the
B class proteins DEF and GLO (Antirrhinum) or AP3 and PI
(Arabidopsis) bind CArG box sequences only as heterodimers
(Jack, 2001; Thiessen, 2001). Yeast two-hybrid assays show that
PI/AP3 interacts directly with SEP3 but not AG, and it has been
proposed that SEP3 mediates interactions between PI/AP3 and
AG dimers so that PI/AP3, AG, and SEP bind DNA in quaternary
complexes; this effect would explain the combinatorial action of
B and C class genes (along with SEP genes) in stamens (Honma
and Goto, 2001).
IDENTIFICATION OF GENES INVOLVED IN STAMEN
DEVELOPMENT AND FUNCTION
Ten years ago, one of the major unanswered questions about
stamen development was the nature of the genes downstream of
the B and C class transcription factors (Goldberg et al., 1993).
After stamen specification, the B and C class genes as well as
SEP genes continue to be expressed during stamen development
(Bowman et al., 1991; Pelaz et al., 2000; Jack, 2001), so they
could be directly responsible for activating many of the genes
involved in stamen morphogenesis and function. Among the
targets of B class genes in Arabidopsis are AP3 and PI themselves,
because both genes are required for the continued expression
of each in the developing flower (Lohmann and Weigel,
2002). Two main approaches have been used to identify additional
targets of B and C class genes. These involve either transcriptional
profiling methods, including subtractive hybridization,
Figure 1. Anther Development.
(A) Scheme of a transverse section through an Arabidopsis floral bud
showing the number, position, and orientation of the floral organs (after
Hill and Lord, 1989).
(B) Schemes of transverse sections through Arabidopsis anthers at
different stages (after Sanders et al., 1999). Floral stages are as
described by Smyth et al. (1990); anther stages are as described by
Sanders et al. (1999). C, connective; E, epidermis; En, endothecium; ML,
middle layer; S, septum; St, stomium; StR, stomium region; T, tapetum;
Td, tetrads; TPG, tricellular pollen grains; V, vascular bundle.
Stamen Development S47
differential display of RNAs, and differential screening of cDNA
libraries or arrays to find genes with stamen-specific or stamenpreferred
expression, or screening for mutations that affect
stamens. Both approaches have yielded interesting results, but
there is not yet enough information to reconstruct the developmental
pathways that start with homeotic gene expression.
Expression Studies
Stamen expression studies have been conducted in many crop
and model species, including tomato (Ursin et al., 1989;
Chmelnitsky et al., 2003), tobacco (Koltunow et al., 1990),
Antirrhinum (Nacken et al., 1991), oilseed rape (Scott et al.,
1991b; Shen and Hsu, 1992), maize (Wright et al., 1993), rice
(Tsuchiya et al., 1994), lily (Crossley et al., 1995), white campion
(Barbacar et al., 1997), Lotus japonicus (Endo et al., 2002), and
Arabidopsis (Rubinelli et al., 1998; Sablowski and Meyerowitz,
1998; Amagai et al., 2003; Zik and Irish, 2003). Some of these
studies have manipulated B and/or C class genes themselves to
help identify their targets; these experiments are discussed in
more detail below. Proteomics analysis also has been used to
compare and identify proteins extracted from rice anthers at
different developmental stages (Kerim et al., 2003). The expression
studies have identified genes involved in protein, starch, and
sucrose metabolism, osmoregulation, cell wall biosynthesis and
expansion, sugar transport, lipid transfer, flavonoid synthesis,
and cytoskeleton structure. These are consistent with features of
stamen and/or pollen development, such as rapid growth, water
movements associated with desiccation and dehiscence, stress
response, and the accumulation of storage compounds. Many
genes of unknown function also were recovered. Few of the
genes have obvious developmental or signaling roles. Exceptions
to this include several candidate transcription factors in
white campion and Arabidopsis (Barbacar et al., 1997; Zik and
Irish, 2003) and a subunit of the COP9 signalosome (a repressor
of photomorphogenesis in plants) found to be upregulated in rice
anthers at the panicle heading stage, suggesting a role in
mediating light signals to coordinate flowering with anther dehiscence
(Kerim et al., 2003).
In situ hybridization shows that genes identified in the expression
studies are specific to a variety of stamen regions and cell
types, including microsporocytes, microspores, tapetum, endothecium,
connectives, epidermis, and filaments. A common
theme is the predominance of tapetum-specific transcripts in
early anthers, reflecting the high metabolic activity of this tissue
(Scott et al., 1991a; see below).
In some of the experiments referred to above, transcriptional
profiling techniques were used in conjunction with mutant or
transgenic plants with altered function or expression of B and C
class genes to aid the identification of their targets. For example,
differential hybridization of an inflorescence-enriched cDNA
library was used to isolate mRNAs expressed in wild-type
flowers but not def mutants of Antirrhinum, which lack B function.
This experiment yielded 12 differentially expressed genes,
including tap1, expressed in the tapetum and encoding a putative
secreted protein, and filamentous flower1 (fil1), expressed
mainly in stamen filaments and petal bases and encoding
a candidate cell wall protein (Nacken et al., 1991). Rubinelli et al.
(1998) compared wild-type and ag mutant flowers of Arabidopsis,
which lack C function, using subtractive hybridization, and
after a further screen making use of RNA from ap3 mutants
(which have pistils but not stamens) identified 13 genes that are
expressed differentially in stamens. These included genes
predicted to encode hydrolytic enzymes and a lipid transferase.
Sablowski and Meyerowitz (1998) analyzed gene expression in
flowers with no C function and inducible B function, which also
were treated with a protein synthesis inhibitor, to discover direct
targets of PI/AP3. Three mRNAs were upregulated consistently
by induction of B function in ap3-3 ag3 double mutant flowers in
the absence of protein synthesis, one of which, NAC-LIKE,
ACTIVATED BY AP3/PI (NAP), was studied in detail. In situ
hybridizations to wild-type flowers showed expression in stamens
and petals, both sites of B function, as well as in some
organs outside the domain of PI/AP3 activity. Based on ectopic
and antisense expression studies, the authors proposed that
NAP functions in the transition between cell division and
expansion, for instance in elongating stamen filaments.
Zik and Irish (2003) used microarray analysis to identify genes
affected by the misexpression of AP3 and PI. Gene expression
was compared in flowers from wild-type plants and from mutant
and transgenic plants with altered B function. This study
identified 47 genes likely to be regulated directly or indirectly by
PI/AP3 in petal and/or stamen development. Because the
microarray used represented 25% of the Arabidopsis genome,
some 200 genes may be affected by changes in PI/AP3 activity.
PI/AP3-responsive genes found to be expressed in stamens, or in
stamens and petals, included many genes implicated in the rapid
cell expansion that is a feature of petal and stamen growth. Only
two candidate transcription factors were identified, suggesting
that AP3 and PI act relatively directly in regulating gene
expression. Twenty-eight of the 47 genes had one or more candidate
CArG boxes in a 1-kb upstream region, indicating that they
could be activated directly by PI/AP3, although these sequences
do not provide specific targets for particular MADS proteins.
Mutant Analysis
Mutant screens conducted over many decades have revealed
a large number of sporophytic genes required for male fertility
(Kaul, 1988; Chaudhury, 1993; Dawson et al., 1993; Chaudhury
et al., 1994; Taylor et al., 1998; Sanders et al., 1999; Bhatt et al.,
2001; Sorensen et al., 2002; Caryl et al., 2003). Pollen development
also is affected by male gametophytic mutations
(McCormick, 2004) and indirectly by mutations in the mitochondrial
genome that result in degeneration of the tapetum (Hanson
and Bentolila, 2004). The reported sporophytic mutations disturb
avarietyofprocessesinthestamen,suchaschromosomepairing
or segregation in meiosis, viability of the tapetum, pollen wall
formation, filament elongation, and anther dehiscence. Genes
required for pollen development that were first identified through
mutant analysis include DETERMINATE, INFERTILE1 (DIF1)/
SYN1, encoding a cohesin required for chromosome segregation
(Bai et al., 1999; Bhatt et al., 1999); MALE STERILITY 2 (MS2),
encoding a predicted fatty acyl reductase with a potential role in
pollen wall formation (Aarts et al., 1997); and MS1 and ABORTED
MICROSPORES, both candidate transcription factors expressed
S48 The Plant Cell
in the tapetum and possibly in microspores (Wilson et al., 2001;
Sorensen et al., 2003).
General screens for male sterility yield surprisingly few mutants
that affect the differentiation of anther cell types (Sanders et al.,
1999). Therefore, we conducted an extinction screen for the loss
of expression of a b-glucuronidase reporter gene fused to early
tapetum-specific promoters, with the aim of specifically detecting
mutations that disrupt microsporangium development
(Sorensen et al., 2002). This screen identified three loci
involved in patterning of the anther locule. Of these, EXTRA
SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1
(EMS1) has been studied most thoroughly (Canales
et al., 2002; Zhao et al., 2002). Because this appears to be a key
gene in patterning the stamen, it is discussed in more detail
below.
GENETIC CONTROL OF ANTHER DEVELOPMENT
Most plant organs derive from meristems, undifferentiated
populations of cells that divide to replenish themselves and also
provide founder cells for organ primordia. Anther development is
unusual in that the microsporangia arise from single archesporial
cells rather than from meristems. Key stages in this process are
the establishment of adaxial-abaxial polarity, the specification of
cell types, and the formation of the radially symmetrical micro-
sporangia.
Asymmetry first becomes evident in the anther primordium
with the formation of the microsporangia, for the two abaxial
locules (facing the petals) are larger than the adaxial pair and are
further separated by the connective tissue (Figure 1). In Arabidopsis
leaves, asymmetry is established by cadastral interactions
among genes that promote adaxial or abaxial identity, with
the former suppressing the latter (Bowman et al., 2002). Mutations
in either set of genes result in either ‘‘adaxialized’’ or
‘‘abaxialized’’ filamentous leaves, and the study of flowers in
these mutant lines suggests that anther polarity is regulated
similarly (Sawa et al., 1999; Siegfried et al., 1999). For example,
double mutants for fil1 and yabby1 or kanadi1 (kan1) and kan2
produce radially symmetrical filamentous structures, the internal
structure of which has yet to be determined. As with leaves, FIL
is expressed abaxially in the connective tissue of the anther,
supporting the view that stamens evolved from leaves bearing
microsporangia on their upper surfaces.
The floral meristem of Arabidopsis, like the shoot apical
meristem, is composed of three ‘‘histogenic layers’’ of cells with
separate lineages: L1 (epidermis), L2 (subepidermis), and L3
(core). Stamen primordia are initiated by periclinal divisions in the
floral meristem, usually within the L2 (Jenik and Irish, 2000). The
L2 gives rise to most of the cell types of the anther, including the
sporogenous cells (discussed in detail below). The L3 contributes
to the vasculature and sometimes to the connectives. With
the growth of the anther primordium, cells of the L2 undergo
a complex series of divisions leading to the formation of the four
radially symmetrical microsporangia (Figure 2) and conducting
tissue that will eventually become linked to the filament. The
founder cells of the four microsporangia are single L2 archesporial
cells, each of which divides periclinally to form a primary
parietal cell (PPC) subjacent to the L1 and a primary sporogenous
cell (PSC) facing inward (Canales et al., 2002).
There has been significant progress in the last few years
toward identifying genes involved in the first steps of microsporangium
differentiation. In Arabidopsis, the candidate transcription
factor NOZZLE/SPOROCYTELESS (NZZ/SPL) (Schiefthaler
et al., 1999; Yang et al., 1999) is required for archesporial specification,
because nzz/spl mutant anthers fail to form archesporial
cells. In maize, male sterile converted anther1 (msca1) mutants
produce archesporial cells, but these do not divide into a PSC
and a PPC, and no microsporangia are formed (Chaubal et al.,
2003). Intriguingly, the msca1 mutation also appears to convert
the entire anther to another structure, because stomata form
ectopically on the epidermis, although the filament maintains its
identity.
The number of cells that can acquire archesporial fate in the
anther appears to be regulated by a similar mechanism to that
used in the shoot apical meristem to control numbers of
pluripotent cells. In the shoot apical meristem, cell number is
determined by the Leu-rich repeat (LRR) receptor kinase
CLAVATA1 (CLV1), its signaling partner CLV2, and its ligand
CLV3 (Clark, 2001). In the anther, the number of cells acquiring
archesporial fate is restricted to one per premicrosporangial
domain by EXS/EMS1, a putative Leu-rich repeat receptor
kinase (Canales et al., 2002; Zhao et al., 2002). In exs/ems1
mutants, which are affected only in male development, multiple
L2 cells in the anther enter archesporial development (Figure 2A),
with the result that an excess of PSCs is formed after the first
periclinal division. The ligand for EXS/EMS1 is unknown, but
mutants in the TAPETAL DETERMINANT1 (TPD1) gene have
phenotypes similar to those of exs/ems1 lines (Yang et al.,
2003b), suggesting that TPD1, a novel putative secreted protein,
is involved at some point in this signaling pathway, whether or not
as a ligand for EXS/EMS1. However, TPD1 expression differs
from that of EXS/EMS1, and although still consonant with a role
in archesporial cell fate determination, this finding suggests that
TPD1 plays a wider role in microsporangial development. An
EXS/EMS1 ortholog, MULTIPLE SPOROCYTE1 (MSP1), has
been reported in rice (Nonomura et al., 2003), and in common
with multiple archesporial cells1 (mac1) mutants in maize (which
also share phenotypic features with exs/ems1 mutants [Sheridan
et al., 1999]), msp1 lines show defects in both male and female
development.
The developmental fates of the two products of archesporial
cell division in Arabidopsis are very different. The PSC undergoes
a small number of divisions to generate the meiocytes, whereas
the PPC divides periclinally to form an endothecial cell subjacent
to the L1 and a secondary parietal cell (SPC). The SPC again
divides periclinally to generate a middle layer cell next to the
endothecium and a tapetal cell adjacent to the sporogenous
cells. Mutants have been described with defects in anther wall
layer development—for example, ms23 and ms32 in maize, in
which the prospective tapetal layer undergoes an extra periclinal
division but neither layer differentiates as tapetum (Chaubal et al.,
2000)—but no clear picture is emerging regarding how cell fates
are specified. Interestingly, this process is affected in exs/ems1
anthers, for the tapetal and, frequently, middle layer cells are
absent (Canales et al., 2002; Zhao et al., 2002). Whether EXS/
Stamen Development S49
EMS1 is itself required for tapetal specification, or whether the
mass of extra sporogenous cells formed disrupts tapetal and
middle layer differentiation, is unclear.
Periclinal divisions of the single archesporial cell thus give rise
to a linear array of different cell types (Figure 2B). However, this
process alone—even accompanied by anticlinal divisions—cannot
generate the radially symmetrical microsporangium, for cells
of the microsporangium adjacent to the connective have been
shown to have a different origin from those on the outer face in
a number of species (Nanda and Gupta, 1978; Goldberg et al.,
1993). The model for microsporangial development most consistent
with the data confers a key organizational role on the
sporogenous cells (Figure 2). In this scheme, the PSC, after its
formation from the archesporial cell, sets up a radial field of
signals around itself. This field, which continues to be generated
by the subsequent division products of the PSC, induces
periclinal division and development in adjacent cells. Thus, the
PPC, sister to the PSC, is induced to divide to form an
endothecial cell and, adjacent to the source of signals, the
meristematic SPC. The SPC then executes the final division of
the program, forming tapetal and middle layer cells. However,
these signals also are received and interpreted by other cells
adjacent to the sporogenous cells, regardless of their origin,
which then are recruited into the developmental pathway
described above. This model may seem inconsistent with some
observations—for example, the cell lineage described here for
Arabidopsis is not accepted universally (Yang et al., 1999)—and
certainly does not hold for all other species (Davis, 1966).
However, whatever the division sequence followed by these
cells, the sporogenous cells or their immediate antecedents are
specified at an early stage in all plants and the wall layers
subsequently develop radially around them. There are many
examples in plants in which tissue organization is established in
the absence of a coherent pattern of cell division (Torres-Ruiz
and Ju¨ rgens, 1994). A second problem is a time disparity
between the development of the inner- and outer-facing domains
of the microsporangium (Nanda and Gupta, 1978). However,
data from exs/ems1 mutant anthers suggest that cells of the
Figure 2. Microsporangium Development.
(A) Light micrographs of wild-type and exs anther primordia with archesporial cells marked by asterisks; extra archesporial cells arise in exs mutants
(Canales et al., 2002).
(B) Model for the differentiation of the microsporangial cell layers in Arabidopsis.
S50 The Plant Cell
subepidermal L2 are ‘‘primed’’ for development and, together
with the PPCs, may be able to respond to signals generated from
the PSC earlier than other L2 cells and those of the connective
tissue. The nature of the hypothetical radial signal is unknown,
but mutants would have phenotypes resembling those of exs/
ems1 lines. The fact that transcripts of TPD1, a putative partner
for the EXS/EMS1 receptor kinase, are concentrated in the spore
mass (Yang et al., 2003b) provides a first indication of a radial
disposition of signals centered on the developing microspor-
ocytes.
MICROSPORANGIAL DEVELOPMENT
Development of the different cell types proceeds at varying
speeds: although the micosporocytes and tapetal cells enlarge
rapidly and display differing patterns of gene expression, the
endothecial layer remains undifferentiated through meiosis and
much of microsporogenesis. The middle layer, of which little is
known, apparently is crushed by the expansion of the microsporocytes
and the tapetum. After the initial phase of cell
expansion, cytoplasmic contact is lost between the microsporocytes
and the tapetum as plasmodesmata are ruptured,
seemingly by the rapid synthesis of the microsporocyte callose
wall (see below). Both the microsporocytes and the tapetum
eventually develop into coenocytes. As the microsporocytes
enter meiosis, plasmodesmata between them enlarge to form
‘‘cytomictic channels’’ up to 0.5 mm in diameter, through which
cytoplasmic exchange must occur. These channels are believed
to promote synchrony within the microsporocyte mass (HeslopHarrison,
1966). Later in tapetal development, particularly in
plants such as Arabidopsis with secretory tapeta (see below),
walls between the tapetal protoplasts become gelatinous and
interconnecting plasmodesmata enlarge to form irregular channels.
Postmeiotic development thus involves crosstalk between
two coenocytes, sealed within the microsporangium by a lipid/
sporopollenin peritapetal membrane (Dickinson, 1970). Surprisingly,
this interaction does not become essential until after
meiosis, for exs/ems1 microsporocytes progress to the tetrad
stage in the absence of any visible tapetum, and it is believed to
be the lack of tapetally derived b-1,3-glucanase, which releases
the microspores from the tetrad callose walls (see below), that
causes developmental arrest (Canales et al., 2002).
Meiosis in the Sporogenous Cells
The principal events of meiosis—chromosome pairing, recombination,
and segregation—are common to all eukaryotes, and
the genes involved have been the subject of a number of reviews
(Vershon and Pierce, 2000; Rabitsch et al., 2001) and are the
focus of an interkingdom database (www.germonline.unibas.ch).
However, unlike in animals, meiosis in plants stands at the
threshold not of gametogenesis but of the alternation of generations.
The microspores formed as a product of male meiosis
develop into pollen, an independent microgametophyte. Perhaps
for this reason, developmental events surrounding plant
meiosis, and some details of the process itself, differ from those
in other kingdoms.
In plant microsporocytes, cytoplasmic reorganization accompanies
nuclear events, including the dedifferentiation and division
of plastids and mitochondria and dramatic decreases in
rRNA and mRNA (Dickinson, 1987). Little DNA synthesis occurs
during this period, except in the organelles, and prophase cDNA
libraries reveal a steep decline in the numbers of nuclear genes
expressed (Crossley et al., 1995). This de facto purging of sporophytic
information from the microsporocyte cytoplasm has
been interpreted either as facilitating gametophytic development
after meiosis or freeing the germline of detrimental RNA species,
including viruses and silencing elements (Dickinson, 1987).
Organellar dedifferentiation and replication also may be caused
by this decrease in information from the nucleus, but microsporogenesis
is unusual in that it is highly sensitive to mitochondrial
mutation. This is believed to result from a need for extreme
efficiency in energy production by the tapetum, and mutations in
mitochondrial DNA, and nuclear restorer genes that ‘‘correct’’
the defect, have been exploited in the development of cytoplasmic
male sterility systems used in plant breeding (Kaul, 1988;
Hanson and Bentolila, 2004).
As in all eukaryotes, homologous chromosomes pair during
plant meiosis. Although there have been many reports of presynaptic
alignment of homologs, little unequivocal data exist,
save for work with polyploid cereals, in which homeologous
pairing clearly takes place in floral tissue before meiosis
(Martinez-Perez et al., 1999). The balance between homologous
and homeologous pairing in cereals is regulated by the Ph locus
(Vega and Feldman, 1998), and this premeiotic alignment of
homeologs has been cited as evidence of an ancestral system of
presynaptic alignment of homologs (Martinez-Perez et al., 1999).
Evidence is now emerging of homologous pairing during interphase
in somatic cells, as occurs in animals and microorganisms
(Fransz et al., 2002), but any mechanistic relationship between
these two types of pairing has yet to be established.
After pairing, DNA processing in the male meiocytes is identical
to that of other organisms, including the formation of doublestrand
breaks, resection, strand invasion, ligation, and resolution
of the complexes formed via Holliday junctions. As in most
eukaryotes, these events are accompanied by the formation of
synaptonemal complexes and recombination nodules at zygotene/pachytene.
Generally, the genes involved in meiotic processing
in plants are orthologous with animal sequences, but
differences in gene number and expression profile do occur
(Bhatt et al., 2001; Caryl et al., 2003). Evidence from a range of
meiotic mutants suggests that meiotic checkpoints also may
differ between plants and other eukaryotes (Bhatt et al., 2001),
with at least one checkpoint absent from male development
in Arabidopsis (Bhatt et al., 1999). However, mutations in the
homeodomain protein MALE MEIOCYTE DEATH, which cause
meiocytes to arrest at diplotene/diakinesis (Yang et al., 2003c),
suggest that some level of checkpoint control is present in
anthers.
The Cell Wall in Male Meiosis
Cell wall formation (and dissolution) during and after meiotic
divisions is another unique feature of male meiosis in plants.
During prophase I of meiosis, the microsporocytes of most
Stamen Development S51
angiosperm species secrete a wall of callose, a b-1,3-glucan,
between the plasmalemma and the original cellulosic wall. At the
end of meiosis, the walls separating the microspores also are
composed of callose. Most monocot species undergo successive
cytokinesis during male meiosis, in which a callose wall is
formed between the dyad cells after meiosis I and the microspores
of the nascent tetrad after meiosis II. As in mitotically
dividing cells, these walls form centrifugally. By contrast, most
eudicots, including Arabidopsis, undergo simultaneous microsporocyte
cytokinesis, meaning that no walls are formed until the
end of meiosis II. In simultaneous cytokinesis, intersporal walls
first appear as ingrowths of callose from the parent wall surrounding
the microsporocyte and expand centripetally until the
microspores are separated (Brown and Lemmon, 1988, 2001;
Owen and Makaroff, 1995).
Control of the division plane also occurs differently in sporophytic
mitosis and in male meiosis. In meiotic divisions, the
future site of cell separation is not marked by a preprophase
band. Instead, after each meiotic division in species with
successive cytokinesis, or after meiosis II in species with
simultaneous cytokinesis, each microspore nucleus becomes
surrounded by a radial array of microtubules that partition the
surrounding cytoplasm into ‘‘spore domains’’ (Dickinson and
Sheldon, 1984; Brown and Lemmon, 1988, 2001). Cytokinesis
proceeds along the planes defined by the intersection of the
arrays as vesicles contributing membranes and wall components
coalesce at the spore domain interfaces. In tetraspore/
stud (tes/std) mutants of Arabidopsis, there is partial or
complete failure of male meiotic cytokinesis, so that all four
microspore nuclei begin development in an undivided cytoplasm
(Hu¨ lskamp et al., 1997; Spielman et al., 1997). The TES
gene encodes a putative kinesin that appears to be required for
the establishment of the radial microtubule arrays surrounding
the microspore nuclei at the end of meiosis, because these are
lacking in tes mutants (Yang et al., 2003a). The tes/std
mutations are recessive, and TES is expressed throughout
the anther before meiosis, indicating that male meiotic
cytokinesis is under sporophytic control.
In wounded plant cells, callose is synthesized by a plasma
membrane–localized glycosyl transferase UDP-glucose b-1,3glucan
synthase (callose synthase) (Delmer, 1987). Recently,
Østergaard et al. (2002) described an Arabidopsis gene, glucan
synthase-like5 (AtGsl5), that encodes a plasma membrane
protein homologous with yeast b-1,3-glucan synthase and that
partially complements a yeast b-1,3-glucan synthase mutant.
The gene is highly expressed in flowers and may represent the
enzyme responsible for microsporocyte callose synthesis.
Several explanations of the biological functions of the callose
wall have been advanced. Callose has been proposed to act as
a barrier, or ‘‘molecular filter,’’ between the sporogenous cells
and the rest of the sporophyte that is necessary for meiosis
(Heslop-Harrison, 1964; Heslop-Harrison and Mackenzie, 1967).
It also has been suggested that callose functions as a temporary
wall that first isolates the products of meiosis to prevent cell
cohesion and fusion and then releases free microspores upon its
dissolution (Waterkeyn, 1962) or as a template for the formation
of the species-specific exine-sculpting patterns seen on mature
pollen grains (Waterkeyn and Bienfait, 1970). These hypotheses
are not mutually exclusive. This last potential function is considered
in more detail below.
Since these hypotheses were proposed, natural exceptions,
mutants, and plants engineered to degrade the callose wall
prematurely have provided more insight into the role of the
callose wall. Several lines of evidence suggest that the callose
wall is not required for male meiosis: both Pandanus odoratissimus,
which naturally lacks a callose wall (Periasamy and
Amalathas, 1991), and transgenic tobacco plants in which the
callose wall is absent throughout meiosis as a result of theectopic
expression of an endo-b-1,3-glucanase (Worrall et al., 1992)
successfully initiate and complete meiosis to produce a tetrad of
microspores. The engineered absence of the callose wall in
lettuce (Curtis et al., 1996), oilseed rape, tomato, and maize (R.J.
Scott, unpublished data) also failed to disrupt meiosis.
At the end of meiosis, the external and intersporal walls of the
tetrad are dissolved to release individual microsporocytes by
a mixture of enzymes (‘‘callase’’) containing endoglucanases
and exoglucanases secreted by the tapetum. In the anthers of
petunia and lily, the expression and secretion of callase activity is
under strict developmental control (Frankel et al., 1969; Stieglitz
and Stern, 1973). Alterations in the timing of b-1,3-glucanase
expression, or failure to express b-1,3-glucanase, leads to abnormal
dissolution of the tetrad callose walls, which has been
shown to be a primary cause of male sterility in cytoplasmic
male-sterile lines of several species, including petunia (Izhar and
Frankel, 1971). Callase also must contain cellulases to degrade
the original cellulosic wall of the microsporocyte, because premature
removal of the callose component of the wall does not
release microspores into the locule until the normal time of
callase production (Worrall et al., 1992).
Several candidates for genes encoding the endo-b-1,3glucanase
component of callase have been identified, although
none has been confirmed directly (e.g., by knockout or knockdown
analysis). The Arabidopsis A6 gene encodes a polypeptide
similar in sequence to b-1,3-glucanases, and reporter gene
studies established that A6 gene expression is tapetum specific
and temporally correlated with callase activity (Hird et al., 1993).
Transcripts of the tobacco Tag1 gene, which encodes a polypeptide
related to established tobacco b-1,3-glucanases containing
the conserved pentapeptide motif of the active site of
these enzymes, also show a callase-like pattern of expression
(Bucciaglia and Smith, 1994). Although one or more of the genes
described above may participate in callose wall dissolution,
more effort is required to confirm this and then to understand
how this key stage in pollen production is regulated. The availability
of expression array data and gene knockout lines in
Arabidopsis is likely to assist in this endeavor.
The formation and dissolution of the callose intersporal wall
does appear to be important for the production of individual
microspores. Species that naturally produce permanent tetrads
(four microspores fused together), such as members of the
Juncaceae, Ericaceae, and Oenotheraceae, develop little or
no callose within the intersporal cross-walls of the tetrad
(Blackmore and Crane, 1988). The microspores possess individual
exine walls, but these are fused along the line of the
cross-walls, which presumably prevents their separation. Blackmore
and Crane (1988) proposed that the fusion of exines is
S52 The Plant Cell
related to the extent and timing of callose wall deposition. The
absence of callose in the tetrad cross-walls in some species that
normally produce them also results in permanent tetrads (e.g., in
tomato [R.J. Scott, unpublished data]). However, both P.
odoratissimus (Periasamy and Amalathas, 1991) and engineered
Brassica napus (R.J. Scott, unpublished data) produce individual
microspores in the absence of cross-wall callose, suggesting
that in some species callose is not essential to ensure that
microspores remain separate during exine wall formation within
the tetrad. By contrast, quartet mutants in Arabidopsis produce
permanent tetrads despite apparently normal patterns of callose
wall synthesis and dissolution, whereas pectic components are
unusually persistent in the parent microsporocyte wall surrounding
the tetrads, indicating that pectin dissolution also is
necessary for microspore separation (Preuss et al., 1994; Rhee
and Somerville, 1998).
Tapetal Development and Pollen Coat Formation
Although presumably acting as a nurse tissue to the developing
meiocytes, the tapetum likely only provides nutrition and
materials for the formation of the complex pollen wall. Developmental
signaling, as occurs between nurse cells and the
egg in Drosophila (St. Johnston and Nusslein-Volhard, 1992),
is unlikely to take place because cytoplasmic connections
between these groups of cells are lost at an early stage.
However, microsporocytes and tapetal cells do share many
developmental pathways (Dickinson and Bell, 1976), particularly
during pollen wall formation. Exceptions are intense phases of
protein synthesis early in development and meiosis itself,
although significant nuclear changes do occur in the tapetum,
with endomitosis and endoreduplication common in many
species. In petunia, tapetal nuclei can attain DNA levels of 8C,
requiring an intensity of nucleic acid synthesis that is probably
unique to the tapetum and that, when combined with high levels
of protein synthesis early in development, must create an
exceptionally high demand for energy (Liu and Dickinson, 1989).
Depending on the species, tapetal cells may be either
secretory and remain at the periphery of the microsporangium
throughout development or amoeboid/invasive and move into
the locule and intermingle with the developing microspores
(Pacini, 1990). In other plants, the tapetum fragments at an early
stage, and components of the ruptured protoplast move into the
locule (see below). In addition to its central role in pollen wall
formation (see below), the tapetum also contributes a lipid-rich
exine coating in many species. Thus, the secretory tapetum
of Lilium deposits a blend of carotenins, flavonols, and lipids
(termed pollenkitt) onto the exine surface (Reznickova and
Dickinson, 1982), whereas the invasive tapetal protoplasts of
the Asteraceae penetrate within the cavea of the complex
chambered pollen wall (Howlett et al., 1975). Most is known of
the complex coatings applied to the surface of crucifer pollen,
termed tryphines (Dickinson and Lewis, 1973; Murgia et al.,
1991). These are formed when tapetal fragments become applied
to the pollen exine and contain a range of lipids, glycolipids, and
proteins essential for the successful development of the pollen on
the stigma surface (Ruiter et al., 1997). Arabidopsis mutants
defective in coating components can fail to hydrate after pollination
(Preuss et al., 1993), whereas in Brassica species, small,
Cys-rich pollen coat proteins are involved in interactions with the
stigmas and particularly with the female determinants of the selfincompatibility
system (Doughty et al., 1998). Indeed, the male
self-incompatibility determinants in these species (Schopfer
et al., 1999) belong to the pollen coat protein family of proteins.
The pollen coat and pollination are discussed in this issue by
Edlund et al. (2004).
POLLEN WALL STRUCTURE, SYNTHESIS,
AND PATTERNING
After male meiotic cytokinesis, the individual microspores of the
tetrad initiate development of the pollen wall. The wall of a mature
pollen grain is a multilayered structure consisting of a pectocellulosic
intine surrounded by a sporopollenin-based exine, which
itself contains two layers, the inner nexine and the outer sexine
(terminology as given by Erdtman, 1969). The latter is the most
complex of the layers, providing most of the species-specific
variation in pollen wall patterning through the elaboration of its
columellae and roof-like tectum (Figure 3).
The ephemeral callose wall is the first of several layers
deposited at the microspore surface. This is followed by the
primexine (a precursor of the sexine), the nexine, and finally
the intine (Blackmore and Barnes, 1990). The primexine is
composed largely of polysaccharide and apparently acts as
a template that guides the accumulation of sporopollenin, the
main structural component of the pollen wall. Worrall et al. (1992)
showed that removal of the callose wall early in microsporogenesis
disrupts pollen wall formation in tobacco, deleting the
tectum to leave only exposed columellae; this finding suggests
that the callose wall may provide a solid surface against which
the tectum forms. Because correct columellae positioning
occurs in Brassica napus in the absence of callose (R.J. Scott,
unpublished data), the callose wall probably does not provide
a stencil to guide the positioning of wall elements, as proposed
by Waterkeyn and Bienfait (1970).
Until microspore release, sporopollenin is polymerized from
precursors synthesized and secreted by the microspore;
however, the bulk of sporopollenin precursors are secreted by
the tapetum and incorporated into the wall after the dissolution of
the tetrad (reviewed by Scott et al., 1991a). Sporopollenin confers
on the exine an unparalleled combination of physical strength,
chemical inertness, and resistance to biological attack; these
features have greatly hampered progress in understanding both
its chemical composition and details of its biosynthesis. Early
literature frequently cites carotenoids as the main constituents of
sporopollenin (Shaw, 1971; reviewed by Scott, 1994). However,
the demonstration that a potent inhibitor of carotenoid biosynthesis,
norflurazon, failed to prevent sporopollenin biosynthesis
in Cucurbita pepo (Prahl et al., 1985) began a reevaluation
of sporopollenin composition. Subsequently, a large body of
experimental evidence (reviewed by Scott, 1994) established that
sporopollenin consists mainly of long-chain fatty acids and a
minor component of phenolic compounds, perhaps p-coumaric
acid. More recent analyses have confirmed this view (Ahlers et al.,
1999; Domı´nguez et al., 1999). The phenolic monomers are
coupled by ester bonds characteristic of polyphenolics such as
Stamen Development S53
lignin and suberin, which places sporopollenin together with
cutin, suberin, and lignin in a family of functionally (protective) and
compositionally related biopolymers (Scott, 1994).
In addition to layering, pollen wall patterning also involves the
development of a consistent number of precisely positioned
germinal apertures and arrays of columellae (Figure 3). The pollen
wall above the apertures consists of intine only, and consistent
with the proposed role for primexine, this matrix is not deposited
in areas destined to become apertures. Heslop-Harrison (1963)
proposed that primexine synthesis is prevented by the apposition
of a colpal shield (a plate of endoplasmic reticulum) to the
plasma membrane at the position of the future aperture.
Subsequently, Dover (1972) and Sheldon and Dickinson (1986)
implicated the meiotic spindle in aperture positioning, perhaps
by forcing cytoplasmic components against the plasma membrane.
Despite the huge diversity in pollen wall patterning, a basic
design feature shared by many species is the reticulate
arrangement (frequently as hexagons) of the columellae to form
a series of interlocking lacunae (Figure 3).
Several lines of evidence suggest that the genes responsible
for species-specific pollen wall patterning are transcribed
within the diploid nucleus of the premeiotic microsporocyte
and that the pattern information is inherited by the microspores
(Heslop-Harrison, 1963, 1968; Rogers and Harris, 1969). There is
no evidence for the participation of any subcellular organelle in
specifying the columellae position. However, centrifugation
experiments with developing lily microsporocytes suggest that
the agent(s), potentially protein-coated vesicles, responsible for
imposing pattern on the primexine appears in the cytoplasm at
the beginning of meiosis and is inserted into the plasma
membrane as meiosis progresses (Sheldon and Dickinson,
1983; Dickinson and Sheldon, 1986). These vesicles may insert
protein, or another substance, into the plasma membrane that
somehow influences the position of the columellae. HeslopHarrison
(1972) made the following observation: ‘‘Sometimes
when we contemplate biological pattern it is difficult to imagine
how great a part of it could arise from physical causes, and
correspondingly easy to slip into accepting that genetical control
extends down to all detail. Yet this is obviously not so.’’ Sheldon
and Dickinson (1983) suggested that vesicles fuse randomly with
the plasma membrane and the deposited protein undergoes
self-assembly into pattern-specifying units. This would occur
in a manner analogous to the behavior of oil droplets on water,
or the cracks in the drying of a uniform surface such as mud,
or more dramatically in the basalt layers that produced geological
formations such as the Giants Causeway (Ireland), which
naturally generate hexagonal patterns (reviewed by Scott,
1994). In the droplet model, the vesicles insert material into
the membrane that eventually forms hexagonal plates, with
the columellae forming along the interfaces. In the cracking
model, the vesicular material forms a uniform layer, which upon
Figure 3. Pollen Wall.
(A) Scheme of the main architectural features of a generalized mature
pollen wall. The terminology is according to Erdtman (1969).
(B) Scanning electron micrograph of a Lilium pollen wall showing
a reticulate pattern formed by the fused heads of the columellae. The
nexine I is visible through the lacunae. Bar ¼ 5 mm.
(C) Model of pollen wall development based on Lilium (after Scott, 1994).
Events begin before meiosis and proceed left to right until the mature wall
is formed. 1, Transcription of ‘‘pattern’’ genes in the premeiotic
microsporocyte nucleus. 2, Insertion of labile pattern information into
the plasma membrane via endoplasmic reticulum–derived vesicles,
followed by tessellation to produce a negative stencil of plates. 3, First
phase of primexine synthesis over the entire microspore surface except
areas destined to become apertures. 4, Conversion of primexine to
a sporopollenin-receptive state through the activity of a factor(s)
secreted from sites between the plates. 5, Second phase of primexine
synthesis, more rapid than the first, resulting in limited primexine
conversion and specification of columellae. 6, Nascent columellae are
apparent as lamellated strands that lack substantial sporopollenin. 7,
Consolidation of columellae by the appearance of partially polymerized
(proto)sporopollenin on the receptive surfaces. 8, Final phase of
primexine synthesis, during which the pattern stencil dissipates or is
circumvented to produce the solid nexine I. 9, The callose wall is
dissolved. 10, Wall elements are further consolidated by tapetally derived
sporopollenin. 11, Nexine II is synthesized without the participation of the
primexine. 12, Intine synthesis is initiated.
S54 The Plant Cell
shrinkage forms the hexagonal arrays; the columellae are
specified along the fracture lines.
There are very few mutations reported to affect pollen wall
patterning. However, in the recently described defective in exine
formation1 (dex1) mutant of Arabidopsis, primexine deposition is
delayed and reduced significantly and pollen wall patterning is
disrupted (Paxson-Sowders et al., 2001). The DEX1 gene encodes
a predicted membrane-associated calcium binding protein
that may act as a nucleation site for sporopollenin.
ANTHER DEHISCENCE
Mature pollen is released from the anther by dehiscence,
a program of cell destruction culminating in rupture of the
stomium, a furrow separating each pair of anther locules (Figure
1). Sanders et al. (1999) codified the major events that occur
during the dehiscence program in Arabidopsis. These begin with
the degeneration of the middle layer and tapetum, expansion of
the endothecial layer, and deposition of fibrous bands (wall
thickenings) in endothecial and connective cells. Degeneration of
the septum generates a bilocular anther, which is followed by
stomium cell breakage. In tobacco, the dehiscence process is
very similar, differing only in the greater degree of connective
breakdown. An elegant series of cell ablation experiments in
tobacco showed that a functional stomium region is essential
for dehiscence (Beals and Goldberg, 1997). Although events
associated with dehiscence normally are coordinated with pollen
development, male-sterile tobacco anthers that lack pollen and
tapetum undergo a normal dehiscence process (Goldberg et al.,
1993), indicating that dehiscence does not require signals
derived from these cell types.
Comparative anatomical studies, microscopic observations,
micromanipulation experiments, and measurements of water
movement in stamens of a range of species suggested a model in
which the endothecium contributes to two steps in the dehiscence
process (Keijzer, 1987; Bonner and Dickinson, 1989).
Just before dehiscence, concomitant with lysis of the cells of the
stomium, the endothecial and epidermal cells become turgid
(Figure 4A). This generates an inwardly directed force in the
anther wall that causes the rupture of the weakened stomium.
Subsequent desiccation of the endothecium causes differential
shrinkage of thickened and unthickened regions of the cell wall,
creating an outward bending force that leads to the retraction of
the anther wall and full opening of the stomium to permit pollen
release (Keijzer, 1987). The ms35 mutant of Arabidopsis (Dawson
et al., 1999) provides supporting evidence for the role of the
endothecium in this second phase of dehiscence. Mature
anthers of ms35 mutants contain viable pollen grains, breakdown
of the septum and stomium occurs normally, and the
pattern of water movement is apparently also normal. However,
the endothecial cells fail to develop lignified secondary wall
thickenings and do not undergo the shrinkage normally associated
with retraction of the anther wall. The MS35 gene
(renamed MYB26) encodes a putative R2R3-type MYB transcription
factor (Steiner-Lange et al., 2003). Because other MYBs
are known to regulate the phenylpropanoid pathway (e.g.,
AtMYBb4 [Jin et al., 2000]), the role of MYB26 may be to activate
this pathway in endothecial cells to provide lignin residues for
wall thickening.
Perhaps the most significant recent advance in understanding
the control of dehiscence has been the discovery of the
involvement of jasmonic acid (JA), a lipid-derived signaling
compound distributed widely in the plant kingdom. All known
mutants in JA biosynthetic or signaling pathways exhibit a similar
phenotype, with (1) reduced elongation of the filaments, (2)
delayed anther dehiscence, and (3) reduced pollen viability
resulting in male sterility. Several mutants in JA biosynthetic
enzymes have been isolated that cause male sterility and can be
rescued by the application of JA (McConn and Browse, 1996;
Sanders et al., 2000; Stintzi and Browse, 2000; Park et al., 2002;
von Malek et al., 2002). The role of JA in male fertility is supported
by the JA signal transduction mutant coronatine-insensitive1,
which is male sterile and insensitive to JA treatment (Feys et al.,
1994; Xie et al., 1998).
The DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) gene
encodes a phospholipase A1 that catalyzes the initial step of JA
biosynthesis (Ishiguro et al., 2001). Desiccation of dad1 anthers
appears delayed so that at the time of flower opening, when
dehydration and shrinkage of the endothecium and connective
cells break the stomium in wild-type anthers, the endothecium
and connective cells of dad1 anthers are fully expanded and the
locules are filled with liquid. Expression studies suggest that
DAD1 activity is restricted to the anther filament immediately
before flower opening; therefore, the filament may represent the
primary source of JA within the flower. Ishiguro et al. (2001)
proposed a model in which JA synthesized in the filaments
regulates water transport in the stamens and petals to bring
about the coordinated opening of flowers, filament elongation,
and anther dehiscence (Figure 4B). In this model, JA is required
for the expression of genes involved in water transport in anthers.
Interestingly, AtSUC1, a plasma membrane H1-sucrose symporter,
which is theoretically capable of transporting sucrose to
increase water uptake, accumulates in some of the connective
cells surrounding the vascular tissue during the final stages of
anther development (Stadler et al., 1999). Filament and petal
Figure 4. Anther Dehiscence.
(A) Brassica oleracea stamens before dehiscence showing fluid-filled
epidermal cells on the abaxial surface of the anther.
(B) Model for the synchronous regulation of pollen maturation, anther
dehiscence, and flower opening by jasmonic acid. The shaded areas
represent regions that actively take up water and elongate in response to
jasmonic acid (adapted from Ishiguro et al., 2001).
Stamen Development S55
elongation also are inhibited in dad1 flowers, suggesting that JA
regulates water transport into these organs. This finding accords
well with the observation that the dehiscence of onion anthers
correlates with the extension rate of the filament (Keijzer, 1987)
and with the suggestions that anther dehiscence is preceded by
dehydration of the locules and that water is exported through
the filaments to the petals (Bonner and Dickinson, 1990). An
alternative model suggests that JA regulates programmed cell
death in the anther as part of the dehiscence process (Zhao and
Ma, 2000).
Rieu et al. (2003) reported evidence for the involvement of
ethylene signaling in dehiscence. Tobacco plants insensitive to
ethylene developed structurally normal anthers, but dehiscence
was late and no longer synchronized with flower opening, in
correlation with delays in the degeneration of stomium cells and
dehydration of the anther. Treatment of nearly mature anthers
with ethylene accelerated dehiscence in wild-type plants.
Ethylene and JA may perform the same role in tobacco and
Arabidopsis, respectively, or act redundantly in both species.
The fact that Arabidopsis mutants such as dde1, which cannot
synthesizeJAwithinthestamens,eventuallyundergodehiscence
might represent evidence in support of redundant pathways.
CONCLUSION
Ten years ago, little was known about the genetic control of
stamen development beyond the specification of stamen
primordia by floral homeotic genes. Now, footholds have been
established in several areas, notably patterning of the microsporangium,
regulation of meiosis, and anther dehiscence. Other
processes, such as the genesis of the pollen wall, have proved
less tractable, perhaps because of a lack of informative mutants.
Important questions remain to be addressed, particularly those
concerning the establishment of cell fates within the microsporangium,
the molecular regulation of the very different nuclear
events in the meiocytes and tapetal cells, the link between
sporophytically controlled callose degradation and male gametophyte
development, the ‘‘molecular mechanics’’ of pollen wall
formation, and bridging the gap between JA levels and water
movement during dehiscence. Understanding stamen development
is key to the successful exploitation of plant breeding
systems. In a world of diminishing resources and an increasing
need for efficient food production, solving these questions
should be accorded a high priority.
ACKNOWLEDGMENTS
We are grateful to all those in the Scott and Dickinson laboratories who
have contributed over the years to our work on anther and pollen
development, particularly Anuj Bhatt, Claudia Canales, Franc¸ ois
Guerineau, Diane Hird, Rachel Hodge, Wyatt Paul, Liz Porter, Ursula
Potter, Mike Roberts, Judith Sheldon, Anna Sorensen, and Dawn
Worrall. This review is dedicated to the memories of Jack HeslopHarrison
and Lynda Bonner.
Received September 12, 2003; accepted January 17, 2004.
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S60 The Plant Cell