RESEARCH ARTICLE 2409
Development 137, 2409-2416 (2010) doi:10.1242/dev.049320
© 2010. Published by The Company of Biologists Ltd
Tapetal cell fate, lineage and proliferation in the Arabidopsis
anther
Xiaoqi Feng and Hugh G. Dickinson*
SUMMARY
The four microsporangia of the flowering plant anther develop from archesporial cells in the L2 of the primordium. Within each
microsporangium, developing microsporocytes are surrounded by concentric monolayers of tapetal, middle layer and endothecial
cells. How this intricate array of tissues, each containing relatively few cells, is established in an organ possessing no formal
meristems is poorly understood. We describe here the pivotal role of the LRR receptor kinase EXCESS MICROSPOROCYTES 1
(EMS1) in forming the monolayer of tapetal nurse cells in Arabidopsis. Unusually for plants, tapetal cells are specified very early in
development, and are subsequently stimulated to proliferate by a receptor-like kinase (RLK) complex that includes EMS1.
Mutations in members of this EMS1 signalling complex and its putative ligand result in male-sterile plants in which tapetal initials
fail to proliferate. Surprisingly, these cells continue to develop, isolated at the locular periphery. Mutant and wild-type
microsporangia expand at similar rates and the ‘tapetal’ space at the periphery of mutant locules becomes occupied by
microsporocytes. However, induction of late expression of EMS1 in the few tapetal initials in ems1 plants results in their
proliferation to generate a functional tapetum, and this proliferation suppresses microsporocyte number. Our experiments also
show that integrity of the tapetal monolayer is crucial for the maintenance of the polarity of divisions within it. This unexpected
autonomy of the tapetal ‘lineage’ is discussed in the context of tissue development in complex plant organs, where constancy in
size, shape and cell number is crucial.
INTRODUCTION
Unlike animals, which can initiate germlines as early as
embryogenesis, plant reproductive cells form late in development.
All male reproductive cells of Arabidopsis develop within
concentrically organised microsporangia derived from archesporial
cells in the L2 of the stamen primordium (Fig. 1A,E). These
archesporial cells divide periclinally to form an inner primary
sporogenous cell and an outer primary parietal cell (Fig. 1B,E).
Primary sporogenous cells continue division to give rise to a central
sporogenous mass, and consecutive divisions of neighbouring cells
result in the formation of three concentric parietal layers – the
tapetum adjacent to the sporogenous cells, a middle layer, and the
endothecium subjacent to the epidermis (Fig. 1C-E) (Canales et al.,
2002; Feng and Dickinson, 2007; Scott et al., 2004; Sorensen et al.,
2002). In common with the development of other plant
reproductive organs, where consistency in size and shape is
essential for effective function, microsporangial formation does not
involve ‘classical’ plant meristems, but rather finite numbers of
divisions within apparent cell lineages – reminiscent of
development in some animal tissues.
Patterning of the concentric microsporangia involves the
interplay of many genes (Feng and Dickinson, 2007; Ma, 2005;
Wilson and Yang, 2004). The transcription factor
SPOROCYTELESS (SPL; also known as NOZZLE, NZZ)
(Schiefthaler et al., 1999; Yang et al., 1999) acts directly
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1
3RB, UK.
*Author for correspondence ([email protected])
Accepted 14 May 2010
downstream of the floral ‘C’ gene AGAMOUS (AG) (Ito et al.,
2004) to promote sporogenous cell identity (Schiefthaler et al.,
1999; Yang et al., 1999) and is reported to interact with the leucinerich repeat receptor-like kinases (LRR-RLKs) BARELY ANY
MERISTEM 1 and 2 (BAM1 and BAM2) (Hord et al., 2006).
BAM1 and BAM2 are held to promote somatic cell fates, first in
the outward-facing products of the archesporial cell division, and
subsequently during microsporangial expansion by restricting the
expression domain of SPL to the locular centre (Hord et al., 2006).
In turn, SPL appears to promote BAM1 expression, possibly
creating a regulatory loop similar to that involving CLAVATA 1
(CLV1) and WUSCHEL (WUS) in shoot apical and floral meristems
(Hord et al., 2006).
Also playing an important part in microsporangial patterning, the
LRR-RLK EXCESS MICROSPOROCYTES 1 (EMS1; also
known as EXTRA SPOROGENOUS CELLS, EXS) (Canales et
al., 2002; Zhao et al., 2002) is reported to form a receptor complex
with two further LRR-RLKs – SOMATIC EMBRYOGENESIS
RECEPTOR-LIKE KINASE1 and 2 (SERK1 and SERK2) – in the
plasma membrane of tapetal cells (Albrecht et al., 2005; Colcombet
et al., 2005). This complex is held to bind TAPETUM
DETERMINANT 1 (TPD1) (Yang et al., 2005; Yang et al., 2003),
a putative ligand synthesised by the microsporocyte mass, and is
reported to specify tapetal cell fate (Albrecht et al., 2005; Feng and
Dickinson, 2007; Jia et al., 2008; Ma, 2005; Yang et al., 2005).
ems1 microsporangia have no tapetal layer but excess
microsporocytes (Canales et al., 2002; Zhao et al., 2002), a
phenotype shared with serk1 serk2 double mutant lines (Albrecht
et al., 2005; Colcombet et al., 2005) and tpd1 plants (Yang et al.,
2003). How this EMS1 complex mediates the formation of a
functional tapetal layer is unknown; models range from a role in
regulating the number of functional archesporial cells (Canales et
DEVELOPMENT
KEY WORDS: Anther tapetum, Arabidopsis, Cell fate establishment, EMS1, Reproductive cell lineage
2410 RESEARCH ARTICLE
MATERIALS AND METHODS
Plant materials and growth conditions
Wild-type (wt) Arabidopsis thaliana (Linneaus) Landsberg erecta (Ler)
plants were used unless otherwise specified. exs1, exs2, exs3 seeds were
available in the laboratory (Canales et al., 2002). ems1 seeds were kindly
provided by Hong Ma (Pennsylvania State University, USA) (Zhao et al.,
2002); tpd1 seeds by De Ye (China Agricultural University, China) (Yang
et al., 2003); and serk1 serk2 seeds by Sacco de Vries (Wageningen
University, Holland) (Albrecht et al., 2005). Rod Scott (Bath University,
UK) provided pA9::Barnase lines in both Ler and Columbia-0
backgrounds (Dilkes et al., 2008). The ems1 line was genotyped using
XF301, XF504 and XF3R primers, and tpd1 (Yang et al., 2003) and serk1
serk2 (Albrecht et al., 2005) mutants were genotyped as previously
described. Plants were grown at 21°C on a 16 hour light/8 hour dark cycle.
RT-PCR analysis
Biological replicates comprised floral buds at flower stages (Smyth et al.,
1990) 1-14, collected from three plants, and three independent replicates
were used from each line. Quantitative RT-PCR (qRT-PCR) was performed
with an Applied Biosystems 7300 Real-Time PCR system, using
XG102F/XG102R primers for AtA9 and At/AhEF1a primers (Becher et al.,
2004) as a control. Thirty-five cycles of RT-PCR were carried out
(PyrobestDNA polymerase, TaKaRa) using XG1206F/XG1206R primers
for EMS1 and GAPDH primers (Hay et al., 2002) as a control.
Transgenic plants
4005 bp of EMS1 genomic DNA and 979 bp of the A9 promoter (pA9)
were cloned using primers XF201/XF202, and XF203/XF204, respectively.
The pA9::EMS1 fragment was amplified through interlaced PCR using
primers XF201/XF204. This fragment was cloned into pMLBART (Gleave,
1992) using NotI digestion. The same fragment was also cloned into
Gateway pDONR207 vector (Invitrogen) after amplification using primers
XL101/XL102, and finally recombined into Gateway vector pGWB16
(Invitrogen). Both constructs were then introduced into ems1/+
heterozygous plants using floral dip transformation (Zhang et al., 2006).
The pMLBART-pA9::EMS1 and pGWB16-pA9::EMS1-6xcMYC T1 plants
resulting were screened for BASTA, and for Kanamycin and Hygromycin
resistance, respectively, and were genotyped for the transgene using
XH201/XH203 primers and for the genomic EMS1 allele using
XF301/XF504/XF3R primers. T4 plants from each independent transgenic
line were genotyped and examined for phenotype.
Determination of stage specificity of the AtA9 promoter
Interpretation of our experiments required precise knowledge of the timing
of pA9 expression in our plant lines; previous reports had only described
pA9 driven reporter constructs as being tapetal specific (Sorensen et al.,
2002). We thus investigated tapetal ablation in pA9::Barnase expressing
lines of Arabidopsis (Dilkes et al., 2008; Paul et al., 1992). These plants
form a full complement of microsporocytes and tapetal cells, but the latter
become highly vacuolated during the proliferation phase (S5; see Fig. S1
in the supplementary material), expand rapidly and degenerate after S5,
confirming pA9 to be tissue specific and active during the proliferation of
the tapetum.
Light microscopy and measurements
Material was prepared as previously described (Sorensen et al., 2002),
observed under an Olympus BX50 microscope and imaged using ImagePro 6.2 software. The longitudinal mid-point of abaxial microsporangia
from the medial anthers of anther stage 6 was chosen for cell number
measurement. Mean value and s.d. were calculated from 150 independent
locules of each line.
Transmission electron microscopy
Material was prepared as previously described (Sorensen et al., 2002).
In situ hybridisation
A 1157-bp fragment for EMS1 detection was amplified using primers
XH402F/XH402R (Zhao et al., 2002) using wild-type cDNA as template;
similarly, a 305-bp A9 fragment was amplified using primers
XH401F/XH401R, and a fragment for A7 detection was made using DNA
from vector pMC1577 kindly provided by Hong Ma (Zhao et al., 2002;
Rubinelli et al., 1998). Fragments were cloned into the pGEM T-Easy
vector (Promega) and checked by sequencing. In situ hybridisation was
carried out on 8-mm paraffin wax-embedded sections using antisense and
sense probes for each gene as previously described (Lincoln et al., 1994).
Primers
See Table S1 in the supplementary material for a list of primers used.
RESULTS
Tapetal initials in the Arabidopsis anther are
specified early, and require the EMS1 receptor
kinase to proliferate
To investigate the role of the EMS1 complex in tapetal
development, we first examined events immediately following the
division of the archesporial cell in wild-type Arabidopsis (Fig. 1AI). At this stage, and only in the region under the radius of the
anther lobe (distant from the connective), the primary parietal cell
and its adjacent two or three L2 cells (in transverse section)
surrounding the primary sporogenous cell divide to form outer, and
inner, secondary parietal cells (Fig. 1C). The primary sporogenous
cells commence division at this point and, simultaneously, the inner
secondary partietal cells adjacent to them divide periclinally to give
rise to tapetal and middle cell layer initials, which we term primary
tapetal cells (PTCs) and primary middle layer cells, respectively.
This results in the sporogenous cells becoming surrounded by a
small number of PTCs (Fig. 1F). Dramatic growth of the
microsporangium then follows, achieved principally by cell
expansion and anticlinal division of the investing layers, and one
or two further, apparently unpolarised, divisions of the sporogenous
cells. The PTCs participate actively in this proliferation through
successive anticlinal divisions, maintaining a monolayer of cells
between the microsporocyte mass and the dividing middle layer
(Fig. 1G). The microsporocytes then enter meiosis and produce
tetrads (Fig. 1H) from which individual microspores are released
(Fig. 1I) to develop, via pollen mitosis 1 and 2, into pollen grains.
ems1 microsporangia develop similarly to wild type prior to
anther stage 5 [S5, anther stages refer to Sanders et al. (Sanders et
al., 1999) (Fig. 1J)]; however, the PTCs in these lines fail to
proliferate, and as both sporogenous cells (on the inside), and the
outer layers of the microsporangium (on the outside) continue to
divide and expand, individual PTCs either remain grouped (Fig.
DEVELOPMENT
al., 2002) to the specification of tapetal fate in the inner division
products of the inner secondary parietal cells, cells which, in ems1
mutant lines, are held to revert to a default microsporocyte fate –
implying a switch in fate within a cell lineage (Zhao et al., 2002).
In an attempt to define further the role of the EMS1 complex in
the specification of the Arabidopsis tapetum and the mechanism by
which it proliferates as a monolayer, we expressed EMS1
ectopically in ems1 plants after its normal period of expression.
These experiments show that a small population of founder cells is
committed to a tapetal fate very early in development, but that the
EMS1 complex is not involved in this early specification.
However, the EMS1 complex is required for the subsequent
proliferation of these cells to form the tapetal monolayer, but not
divisions in any other cell layer. We also show competition for
space to exist between the products of the microsporocyte and
tapetal cell lineages. This early independence of the tapetal lineage
and its ability to interact with neighboring cell layers provide the
first clues as to how these small but complex plant organs develop
– apparently in the absence of functional meristems.
Development 137 (14)
Tapetal development in Arabidopsis
RESEARCH ARTICLE 2411
1K,L), or become separated from each other, isolated at the locular
periphery (Fig. 1M,N). As these cells do not develop into
functional tapeta in ems1 locules, we have termed them locular
peripheral cells (LPCs) from this stage onwards. On average, each
ems1 microsporangium contains 3.15±0.34 LPCs in transverse
section at the mid-point of the abaxial microsporangium of medial
anthers at anther stage 6. The locular content of ems1 plants
degenerates shortly following the second meiotic nuclear division
(Canales et al., 2002; Zhao et al., 2002), with LPCs showing
dramatic hypertrophy (Fig. 1L,N).
The locular peripheral cells of ems1
microsporangia express tapetal cell markers
To determine what ‘tapetal’ features are possessed by the LPCs in
the ems1 mutants, we examined these cells using transmission
electron microscopy (TEM) and investigated the expression of two
tapetal markers, At5g07230 (A9) expressed early in development
(Paul et al., 1992) and At4g28395 (A7) expressed post meiosis
(Rubinelli et al., 1998). TEM analysis showed the LPCs to feature
a high density of ribosomes, and active populations of Golgi bodies
and mitochondria (see Fig. S2 in the supplementary material) – all
features of tapetal cells. Quantitative RT-PCR (qRT-PCR) shows
A9 to be expressed in wild-type and ems1 plants – but at a lower
level than in the mutant (see Fig. S3 in the supplementary
material). In situ hybridisation confirms the presence of A9
transcript in the developing tapetal cells of wild type (Fig. 2A-D),
as well as solely in the LPCs of ems1 anthers (Fig. 2E-H). A7 is
highly expressed in the active, late tapetal cells of wild-type anthers
(see Fig. S4A in the supplementary material) and, although the
loculi of ems1 plants degenerate after meiosis 2 (Canales et al.,
2002), the A7 transcript can nevertheless be detected in the
hypertrophic LPCs of these anthers using in situ hybridisation (see
Fig. S4C,D in the supplementary material). A combination of
position and marker gene expression thus clearly identifies the
LPCs of ems1 locules as partially developed tapetal cells.
We investigated the possibility that the LPCs in ems1 plants
result from low levels of normal or aberrant EMS1 expression by
searching for functional EMS1 transcripts in ems1 plants using RTPCR (see Fig. S5 in the supplementary material). No transcripts
were detected, and further evidence that the LPCs do not result
from incomplete penetrance of the ems1 mutation is provided by
the presence of identical numbers of morphologically similar LPCs
in exs-1, and exs-2 and exs-3 mutants in the C24 ecotype (data not
shown).
Ectopic expression of the EMS1 receptor kinase in
the locular peripheral cells of ems1 plants
stimulates their proliferation and restores tapetal
function
To discover whether the few LPCs present in ems1 microsporangia
can be ‘reactivated’ to generate functional tapetal cells (mimicking
wild-type development), we expressed EMS1 in ems1 plants under
DEVELOPMENT
Fig. 1. Development of Arabidopsis
microsporangia from L2 archesporial initials.
Cross sections of anthers at different developmental
stages (anther stages S2-S9). (A-D)Wild-type (WT)
microsporangial pattern formation through cell
divisions. (E) Cell division events. The cell lineage
giving rise to reproductive cells is highlighted in pink.
(F-N)Microsporangial development in WT (F-I) and
ems1 mutant (J-N). ems1 and WT development is
similar (F,J) until S4 when ISP cell division gives rise to
PTCs and PMLCs. In WT, these proliferate and
develop into tapetum and middle cell layer (G). In
ems1 mutants, PTCs fail to divide and differentiate.
Instead these cells (hereafter termed LPCs) remain at
the periphery of the locule (red arrowheads), while
MSCs continue to divide (K,M). At S6, WT MSCs
enter meiosis to produce tetrads (H); these release
microspores (I). Tetrads degenerate (L,N) in ems1 lines
and LPCs (red arrowheads) become enlarged and
vacuolate. In all figures: Ar, archesporial cell; En,
endothecium; ISP, inner secondary parietal cell; LPC,
locular peripheral cell; M, middle cell layer; MSC,
microsporocyte; Msp, microspore; OSP, outer
secondary parietal cell; PMLC, primary middle layer
cell; PP, primary parietal cell; PS, primary sporogenous
cell; PTC, primary tapetal cell; Sp, sporogenous cell; T,
tapetum; Td, tetrad; WT, wild type. S denotes anther
stages (Sanders et al., 1999). Scale bars: 20mm.
2412 RESEARCH ARTICLE
Development 137 (14)
Fig. 2. A9 is expressed in ems1 LPCs, and the A9
promoter drives high levels of EMS1 expression
in ems1 pA9::EMS1 tapeta. (A-T)In situ
hybridisations showing anthers at different
developmental stages. (A-H)A9 transcript in WT
(A-D) and ems1 mutant (E-H). A9 expression
commences from S5: exclusively within the tapetum
(C and D; green arrows) in WT, or in the LPCs (G and
H; red arrows) of the ems1 mutant. (I-P)EMS1
transcript in WT (I-L) and ems1 pA9::EMS1 plants
(M-P; ems1 pA9::EMS1 line #5). In WT, EMS1 is
expressed in all cells of the microsporangium before
S5 (I), and accumulates strongly in the tapetum
(green arrows) from S5 onwards (J-L). In ems1
pA9::EMS1 plants, EMS1 is only induced in the
tapetum (N-P; green arrows), but at a greater level
than when driven by its own promoter in WT.
(Q-T)Control hybridisations with sense probes on WT
(Q,R) and ems1 pA9::EMS1 (S,T) anthers at stages 4
(Q,S) and 11 (R,T). The background signal generated
by developing microspores is evident. Scale bars:
20mm.
Although pA9 is activated after EMS1 transcription in wild-type
plants (Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002)
(Fig. 2A-D,I-L), it must direct expression sufficiently early for the
transgenic tapeta to follow closely the ‘wild-type’ developmental
pathway. pA9::EMS1 rescues the ems1 mutant phenotype, but it
fails to do so in either ems1 tpd1 double or ems1 serk1 serk2 triple
mutant backgrounds, although EMS1 is strongly expressed in the
LPCs of the mutant anthers (see Fig. S6 in the supplementary
material). However, these transgenic plants have phenotypes very
similar to those of ems1, tpd1 and serk1 serk2 mutants (Fig. 4),
providing further evidence that these four genes are members of the
same signalling pathway (Albrecht et al., 2005; Colcombet et al.,
2005).
Inducing primary tapetal cells to proliferate in
transgenic plants restricts the microsporocyte
population
As the reduction in tapetal cell number in ems1 lines is
accompanied by an increase in the microsporocyte population, we
investigated the relationship between microsporocyte number and
the level of tapetal proliferation in ems1 pA9::EMS1 transgenic
plants. In wild-type plants, mid-point transverse sections of
medial anthers with microsporangia at stage 6 transect 7.17±0.17
microsporocytes, whereas similar ems1 locules contain
14.38±0.32 microsporocytes. The number of microsporocytes in
ems1 pA9::EMS1 microsporangia varies markedly between lines,
but they are always in excess of those in wild-type plants, and
less than those found in ems1 anthers. For example, ems1
DEVELOPMENT
the A9 promoter (pA9), which drives tapetal-specific expression
during the proliferation stage of the tapetum (see Materials and
methods) and which, in ems1 locules, is expressed only in the
LPCs. In all 42 ems1 pA9::EMS1 and pA9::EMS1-cMYC lines
generated, LPCs both proliferated and differentiated into apparently
normal tapetal cells (see Fig. 3) – to which the EMS1 protein was
restricted (data not shown). All lines produced fertile pollen,
despite the comparatively late expression of EMS1 when driven by
pA9 [EMS1 expression precedes that of A9 in the wild-type anther
(Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002)] (Fig. 2AD,I-L), but each transgenic line possessed a different proportion of
sterile locules. The tapetum was also restored in different patterns
in different lines, varying from a normal monolayer (e.g. in ems1
pA9::EMS1 line #10; Fig. 3A-C) to clumps of multilayered
tapetum (e.g. in ems1 pA9::EMS1 line #5; Fig. 3D-F).
pA9::EMS1 expression in ems1 microsporangia restores tapetal
function sufficiently to produce viable pollen and, because only the
LPCs express A9 in these locules, it follows that the functional
tapeta of the ‘restored’ plants must be derived from this cell type.
Furthermore, as the tapetum of some of the transgenic lines (e.g.
ems1 pA9::EMS1 line #10) so closely resembles that of wild type,
and because the LPCs themselves resemble tapetal cells both in
position and in expression of the A9 (early) and A7 (late) tapetal
markers, we conclude that the wild-type tapetal layer must also be
derived from the few PTCs formed around the early sporogenous
cells. The extra tapetal cells that are frequently generated in ems1
pA9::EMS1 lines are likely to be the result of the higher activity of
pA9 compared with pEMS1 (Fig. 2I-P).
Tapetal development in Arabidopsis
RESEARCH ARTICLE 2413
Fig. 3. pA9::EMS1 restores functional tapetum to ems1 mutant
anthers. Light micrographs of sectioned anthers of ems1 pA9::EMS1
plants. Rescued tapetal cells form a continuous layer (A-C), or multiple
layers (D-F) that are sometimes discontinuous (E,F). Monolayered tapeta
with near-normal MSC numbers are fertile (e.g. ems1 pA9::EMS1 line #10;
A-C); excess tapetal cells result in degeneration (e.g. ems1 pA9::EMS1 line
#5, D-F). Green arrows indicate tapetum. Scale bars: 20mm.
Fig. 4. pA9::EMS1 does not restore functional tapetum to ems1
tpd1 or ems1 serk1 serk2 mutant lines. (A-L)Light micrographs of
sectioned anthers. (A-C)tpd1 mutant microsporangia. (D-F)ems1 tpd1
pA9::EMS1 microsporangia. (G-I)serk1 serk2 microsporangia. (J-L)ems1
serk1 serk2 pA9::EMS1 microsporangia. pA9::EMS1 rescues tapetum in
ems1 but not in tpd1 or serk1 serk2 mutant backgrounds; these show
phenotypes identical to the those of the original mutant lines (tpd1,
A-C; serk1 serk2, G-I), where PTCs (red arrows) are present either as a
group (A,D,G,J) or individual cells (B,E,H,K). Scale bars: 20mm.
Tapetum derived from isolated locular peripheral
cells proliferates via periclinal and anticlinal
divisions in transgenic plants
Our screen of microsporangia from different transgenic lines
revealed a wide range of phenotypes, including plants containing
clumped or multilayered tapetum (Fig. 3D-F). We believe this, like
the higher numbers of microsporocytes (see above), to result from
differences in the timing of EMS1 function (Fig. 5). Thus, in
different ems1 pA9::EMS1 lines, the promoter might drive EMS1
transcription to attain an effective threshold either before or after
the fragmentation of the monolayer of LPCs around the
sporogenous cells: if before, exclusively anticlinal division takes
place; if afterwards, a mixture of anti- and periclinal division
occurs to generate tapetal layering (Fig. 5). The association
between the integrity of the tapetal monolayer and division polarity
is striking, and points to the polarity of division (anticlinal in this
case) being maintained by coordinating signals from neighbouring
cells. Such signals might be transmitted through plasmodesmata,
or generated by mechanical stress set up in the tapetal monolayer
itself. Alternatively, division polarity could be regulated by the
directionality of the TPD1 signal from the microsporocytes. In
wild-type locules, this signal would be received on the inward
DEVELOPMENT
pA9::EMS1 line #5 sporangia contain 10.38±0.18
microsporocytes in transverse section (see Fig. S7 in the
supplementary material). Similar comparisons cannot be carried
out for tapetal cells as the number in ems1 pA9::EMS1
microsporangia cannot be measured accurately owing to their
multiple division planes and irregularity in size and shape.
However, the fact that ems1 anthers (which contain excess
microsporocytes) contain few LPCs (about three per section),
transgenic ems1 pA9::EMS1 lines (which contain fewer
microsporocytes than ems1 plants) have significant numbers of
tapetal cells, and wild-type anthers (which possess fewer
microsporocytes than ems1 pA9::EMS1 lines) feature a complete
tapetal monolayer, points to a clear inverse relationship between
tapetal cell and microsporocyte numbers.
However, even when a functional monolayer of tapetal cells is
formed in ems1 pA9::EMS1 lines (e.g. as in ems1 pA9::EMS1 line
#10), the number of microsporocytes always exceeds that of wildtype anthers. pA9 is activated later in anther development than
pEMS1, and this interval between expression stages may provide an
opportunity for the microsporocytes to commence proliferation in the
absence of a developing tapetal layer. Significantly, in all the ems1
and pA9::EMS1 ems1 lines screened, the numbers of cells
composing the middle, endothecial and epidermal layers were
identical to those of wild-type plants, showing that the proliferation
of these cell layers is unaffected by the presence or absence of both
EMS1 expression and a functional tapetal layer.
Many ems1 pA9::EMS1 microsporangia degenerate after S9
(Fig. 3F), and we found a clear relationship between
microsporocyte number, the extent of tapetal disruption and locular
fertility. Lines with a large number of microsporocytes were likely
to possess more fragmented and multilayered tapetum, and a higher
proportion of sterile locules. For example, ems1 pA9::EMS1 line
#5, which contains 10.38±0.18 microsporocytes in transverse
section (the highest number among all 24 transgenic lines), has the
most irregular tapetum and the largest number of sterile locules
(see Fig. S7 in the supplementary material).
2414 RESEARCH ARTICLE
Development 137 (14)
Fig. 5. Model for wild-type, ems1 and ems1 pA9::EMS1 microsporangial development. Division of ISPs occurs between S3 and S4 to
generate PTCs and PMLCs. In WT PTCs, the EMS1 signalling complex becomes active, stimulating proliferation. In ems1, the complex is inactive and
PTCs fail to divide, instead they differentiate further as LPCs. The MSCs and PMLCs divide as the locule expands in ems1, fragmenting the LPC layer.
Expression of the pA9::EMS1 transgene activates the EMS1 signalling complex later in development: if this occurs before fragmentation of the LPC
layer, a tapetal monolayer is formed (as in ems1 pA9::EMS1 line #10); if afterwards, clumps of cells resulting from peri- and anticlinal division are
formed (as in ems1 pA9::EMS1 line #5). The later the EMS1 functional threshold is achieved, the more time MSCs have to proliferate before
inhibition by tapetal cells – either through physical competition for locular space or by signalling.
DISCUSSION
The sequence of cell divisions leading to tapetal formation is well
understood in Arabidopsis (Canales et al., 2002; Feng and
Dickinson, 2007; Scott et al., 2004), but how cells generated by the
periclinal division of the inner secondary parietal (ISP) cell layer
acquire and maintain their tapetal fate is unclear. The similarity in
cell numbers between wild type and ems1 mutants (where no
tapetum is formed) has reasonably been interpreted as evidence that
these ISP cells revert to a default microsporocyte fate in the
absence of the EMS1 receptor kinase (Zhao et al., 2002). However,
our findings that a small number of cells with tapetal features
(PTCs/LPCs) are formed early in development in wild-type and
ems1 plants, and that ectopically expressed EMS1 stimulates
proliferation of only these cells in the latter indicate that an
alternative interpretation of these events might be required.
The specification, proliferation and maintenance
of the Arabidopsis tapetal cell lineage
The new data point to the small number of cells formed adjacent
to the sporogenous cells immediately becoming committed to a
‘pre-tapetal’ fate, and the EMS1 signalling pathway being
responsible for the subsequent proliferation of these cells, allowing
the tapetal monolayer to keep pace with the rapid expansion of the
other layers of the loculus, which, because they are unaffected by
the absence of functional EMS1, must be regulated by different
signals. This role for EMS1 in proliferation implies that the EMS1
complex cannot be involved in the initial establishment of tapetal
fate in the PTCs; the genes responsible for this are unknown, but
other LRR-RLKs, such as BAM1/2, remain possible candidates.
Arguably, the LPCs of ems1 anthers could represent a small
number of partially developed tapetal cells resulting from the
incomplete penetration of the ems1 mutation, with the remainder
of the inner division products of the inner secondary parietal cell
being converted into microsporocytes. This is unlikely because: (1)
EMS1 transcripts are absent from ems1 mutant plants; (2)
DEVELOPMENT
facing surfaces of the tapetal cells, whereas in ems1 pA9::EMS1
lines, where LPCs are isolated, the TPD1 signal could be perceived
on their radial (and perhaps outward) surfaces. Such a system has
been reported for stomatal development, where the LRR-RLKs
ERECTA (ER), ERECTA LIKE 1 (ERL1) (Shpak et al., 2005) and
TOO MANY MOUTHS (TMM) (Geisler et al., 2000; Nadeau and
Sack, 2002) form a heterodimer, which orients the cell division
plate depending upon from which direction it receives the ligand
EPIDERMAL PATTERNING FACTOR 1 (EPF1) (De Smet et al.,
2009; Hara et al., 2007).
Fig. 6. Relationships between the microsporocytes and the
tapetal and middle layer cells during anther development in
Arabidopsis. (A,B)Following the division of the inner secondary
parietal cell (A), the cell adjacent to the microsporocyte acquires a
tapetal fate and is converted into a PTC (B). (C)The EMS1 complex is
expressed in the PTC cell membrane, and the MSC synthesises TPD1.
(D)TPD1 activates the EMS1 complex and the PTC is induced to
proliferate. Other signals induce division in the middle layer cells and
the MSCs. (E)The proliferation of the tapetal layer represses further
MSC divisions. (F)Later the tapetal cells become fully functional,
perhaps as a result of later signalling events. MSC, microsporocytes;
PTC, primary tapetal cell; MLC, middle layer cell; T, functional tapetal
cell.
reactivation of these few LPCs in ems1 lines can generate a
complete and functional tapetum that supports the production of
viable pollen; and (3) LPCs are present in all plants carrying ems1
alleles in both Ler and C24 backgrounds (some encoding nonfunctional receptor kinases), as well as in tpd1 and serk1 serk2
plants.
Although the EMS1 complex is required for the proliferation of
PTCs, its role in their final differentiation into functional tapetal
cells is unclear. LPCs certainly complete some aspects of tapetal
development in ems1 lines as they express A7, a gene encoding a
lipid transfer protein that is normally expressed post meiosis
(Rubinelli et al., 1998), but they nevertheless fail to develop into
functional tapetal cells. The EMS1 complex and its putative ligand
TPD1 thus emerge as a component of a larger gene network
regulating tapetal development, with other network members being
responsible for specifying initial tapetal fate in the PTCs and,
perhaps, aspects of subsequent development. Although an inverse
relationship exists between microsporocyte and tapetal cell
numbers in our transgenic plants, our data do not show whether this
is the result of intercellular signalling, or simply the proliferation
of reproductive cells to fill space that would otherwise be occupied
by the tapetal monolayer.
The formation of the tapetal monolayer during
microsporangial development
Our experiments begin to explain how complex tissue arrays can
develop within small plant organs in the absence of organised
meristems. Like the microsporocytes, tapetal cells are specified
very early, and once specified their fate cannot be altered. These
tapetal ‘founders’ are then stimulated to proliferate (via the EMS1
complex) resulting in the formation of a monolayer investing the
microsporocyte population. This signal for proliferation is unique
to the tapetum, and differs from that regulating division of the
microsporocytes and the other cell layers of the microsporangium.
As TPD1 binds to the EMS1 complex (Yang et al., 2005), and is
principally synthesised by the microsporocytes (Yang et al., 2003),
its presence must serve as a developmental checkpoint confirming
the presence of functional microsporocytes (Fig. 6). Maintenance
RESEARCH ARTICLE 2415
of the developing monolayer requires its continued integrity,
revealing that anticlinal divisions within it are either controlled by
internal signals, or by microsporocyte-generated TPD1 acting as a
directional cue. Cells of the tapetum and the microsporocytes thus
develop as ‘communicating’ neighbours, but as independent
lineages occupying defined domains within the expanding
microsporangium. Whether the proliferation of microsporocytes
into ‘tapetal territory’ in ems1 lines results from the lack of signals
from the tapetum, or simply from available space, remains to be
determined.
Perhaps the most significant difference between development of
these complex microsporangial tissues and development of those
derived from the apical and root meristems is their early and
continued independence; certainly they can receive both directional
and ‘checkpoint’ signals and perhaps alter the space they occupy
but, once formed, their founder cells generate independently
proliferating lineages committed to single developmental fates.
Without this degree of independence and level of ‘internal’ control
it is difficult to see how small numbers of cells of different fates
can assemble into such a range of intricate patterns within a small,
terminally differentiated organ such as the anther.
Acknowledgements
We thank the following for providing mutant lines and reagents: Hong Ma, De
Ye, Sacco De Vries, and Rod Scott for providing the pA9::Barnase lines and
information on A9 expression patterns. Carla Galinha and Paolo Piazza gave
valuable help with in situ hybridisation and qRT-PCR, respectively, and we
acknowledge Qing Zhang, Helen Prescott and Matthew Dicks for providing
excellent technical assistance. We are indebted to Miltos Tsiantis and Angela
Hay for helpful discussion, and the research was funded by Oxford University
through a Clarendon Scholarship to X.F., with additional financial support from
Magdalen College (Oxford).
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.049320/-/DC1
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