Function
Mutant analysis suggests that male reproductive development of Arabidopsis is more
dependent on GA than female reproductive development, with male fertility being impaired in less severely GA-deficient mutants whilst female fertility remains apparently unaffected (Rieu et al., 2008; Hu et al., 2008). Common Arabidopsis GA-deficient stamen phenotypes include reduced filament elongation and delayed or failed anther dehiscence (Figure 1.8). Arrests in anther and pollen development have been reported in GA-deficient backgrounds of several species (Nester & Zeevaart, 1988; Goto & Pharis, 1999; Aya et al., 2009), indicating that GA functions to promote microgametophyte development. Loss of GA biosynthesis in both
Arabidopsis and rice (as represented by the ga1-3 and oscps1-1 mutants) results in pollen
development arresting once haploid microspores have been released from tetrads, at the young microspore stage in rice (Aya et al., 2009) and further characterised in Arabidopsis as prior to entry into pollen mitosis (Cheng et al., 2004). The immediate cause for developmental arrest at this stage is unknown. Interestingly, the GA-insensitive rice mutant gid1-4 exhibits an earlier block in anther development, prior to the completion of meiosis and the appearance of tetrads (Aya et al., 2009). Stamen development in the Arabidopsis gid1a gid1b gid1c mutant has not yet been studied. This indicates the existence of a potential second checkpoint in anther development dependent on GA signalling. This difference in phenotype between GA- deficient and GA-insensitive mutants may be explained by GA-independent interaction between GID1 and DELLA proteins in GA-deficient plants, resulting in a low basal level of GA signal transduction in these backgrounds (see section 1.3.1). In contrast to Arabidopsis and rice, anther development in the GA-deficient tomato mutant gib-1 is blocked prior to meiosis (Jacobsen & Olszewski, 1991). The pollen mother cells (PMCs) in this mutant were
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Figure 1.8: Floral organ phenotypes associated with Arabidopsis GA-deficient and GA-
insensitive mutants. Adapted from Plackett et al. (2011).
found to be arrested in the G1 stage of premeiotic interphase, suggesting that the GA- dependent developmental block is at least partially enacted through the cell cycle. However, clear developmental differences have been identified between anthers of tomato and other model plant species, as demonstrated by the tapetum, which arises from one L2 tissue layer in
Arabidopsis (Scott et al., 2004, Figure 1.9, see further discussion below), but is derived from
two separate histological sources in tomato (Jacobsen & Olszewski, 1991). There may also be divergence in some plant speciesÕ response to GA signalling during anther development.
Analysis of AtGA3ox::GUS reporter lines (Mitchum et al., 2006, Hu et al., 2008) indicates that bioactive GA is synthesised in both the stamen filament and anthers across much of floral development, from floral stage 9 onwards (Figure 1.10). Weak AtGA3ox expression is seen in anther and microspore tissues from anther stage 6 (Sanders et al., 1999), at the point that PMCs enter meiosis. The four AtGA3ox paralogues display differential expression patterns during stamen development (see section 1.2.1), with two peaks of expression seen: one in the tapetum prior to degeneration (see below) and one in mature pollen prior to anther dehiscence. Tapetal expression of GA-biosynthetic genes has been observed in rice via in situ
hybridisation (OsGA20ox1 and -2, OsGA3ox1 and -2, Kaneko et al. 2003) and in tobacco via GUS reporting (NtGA3ox, Itoh et al., 1999). These results indicate that GA is synthesised in
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Figure 1.9: Development of Arabidopsis anther tissues and tapetum function.
Developmental stages taken from Sanders et al. (1999), showing (a) establishment of anther tissue layers and (b) subsequent meiosis and tapetum-dependent pollen development through to tapetum degeneration. In (a), descendents of the archesporial cell are highlighted in colour. At anther stage 3 the archesporial cell divides into a Primary Sporogenous (PS, red) and Primary Parietal (PP, blue) lineages, the PP subsequently differentiating to establish the endothecium and Secondary Parietal (SP) layers. The SP subsequently differentiates into the tapetum and middle layer. PS cells become pollen mother cells by anther stage 5.
both the tapetum cell layer and developing microspores, though potentially at different developmental stages. The stamen expression patterns of the AtGA20ox family are currently unknown. Recent trancriptomic analyses performed specifically on anther tissues in rice suggest that GA biosynthesis is specifically down-regulated in both the tapetum and meiotic PMCs until after meiosis is complete and unicellular microspores are released from tetrads (Chhun et al., 2007; Hirano et al., 2008; Tang et al., 2010), after which time we see up- regulation of numerous components of GA biosynthesis in both cell types. Conversely, Hirano et al. (2008) showed up-regulation of SLR1 in both the tapetum and PMCs during meiosis, with a concomitant down-regulation from the unicellular stage. This may indicate that GA responses are being tightly regulated during this phase of microspore development.
OsGAMYB::GUS reporter lines show expression in anthers from the pre-meiotic stage
onward, indicative of GA signalling (Aya et al., 2009, see section 1.3.3). GUS reporter lines indicate that both OsGAMYB and AtMYB33 are expressed in developing microspores and the
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Figure 1.10: Expression of Arabidopsis GA biosynthesis genes during floral and anther
development.
Expression patterns are based on evidence from GUS reporter lines (Silverstone et al., 1997; Mitchum et al., 2006; Hu et al., 2008), demonstrating differential tissue expression patterns between (a) early (AtCPS) and late (AtGA3ox) biosynthetic stages and (b) the four separate
AtGA3ox paralogues. The intensity of colour shown reflects the reported intensity of GUS
staining, and by inference the intensity of gene expression. Floral and anther developmental stages given are as listed by Smyth et al. (1990) and Sanders et al. (1999), respectively. Floral stages 2 and 3are marked as primordia on the inflorescence meristem (IM). Adapted from Plackett et al. (2011).
surrounding tapetal cells (Millar & Gubler, 2005; Aya et al., 2009), suggesting that a GA response is occurring simultaneously in both cell types. However, it is as yet unclear whether the developmental blocks observed during pollen development in GA biosynthesis/signalling
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mutants are due to loss of GA signalling directly in the PMCs, indirectly due to a loss of GA signalling in the tapetum, or both.
The tapetum cell layer is crucial to the successful development of pollen. The innermost sporophytic tissue layer surrounding the anther locule (Figure 1.9), the tapetum acts as a nurse tissue for the developing microspores, secretes callase enzymes necessary to release the microspores from the callose wall surrounding the haploid cells at the end of meiosis (tetrads), and both synthesises and releases key elements of the outer pollen coat into the locule, an action which necessitates the timely degeneration of tapetal cells via programmed cell death (PCD, Scott et al., 2004, see further discussion below). Generally speaking, two broad categories of tapetal types have been classified within angiosperms (Huysmans et al., 1998): amoeboid, in which tapetal protoplasts move into the locule, and secretory, in which tapetal cells remain in situ until they degenerate. Both Arabidopsis and rice develop secretory tapeta, and it is for these two species that most is known about the action of GA signalling on tapetum function.
In Arabidopsis, the development of the tapetum is tightly interlinked with the pollen that it surrounds. Derived from two distinct cell lineages (the tapetum from primary parietal cells and PMCs from primary sporogenous cells, Figure 1.9, Scott et al., 2004), mutant analysis in
Arabidopsis demonstrates that their respective identities and future cell fates are established
by signalling between the two. A tapetally-expressed leucine-rich repeat receptor-like kinase EXTRA SPOROGENOUS CELLS/EXCESS MICROSPORES (EXS/EMS, Canales et al., 2002; Zhao et al., 2002) interacts with the protein ligand TAPETUM DETERMINANT1 (TPD1) secreted by nascent PMCs to establish and maintain tapetum cell fate: loss of either component results in the tapetal precursors forming additional PMCs instead (Yang et al., 2003, Yang et al., 2005, Jia et al., 2008). Development of the PMC lineage is also affected in
exs/ems and tpd1 mutants, with development blocking in meiosis prior to cytokinesis. This
developmental block is reminiscent of the osgid1-4 GA-insensitive phenotype described above, but the presence of a clearly defined tapetum cell layer suggests that GA signalling acts
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downstream of the EXS/EMS-TPD1 signalling module and is not required to specify tapetum cell identity.
A critical function of the tapetum is synthesis of pollen coat components. These processes have been studied most closely in species belonging to the Brassicaceae family, which includes Arabidopsis. In Brassica napus (which also possesses a secretory tapetum,
Huysmans et al., 1998) the tapetum has been demonstrated to be the predominant contributor of sporopollenin (a major structural component of the exine pollen wall layer) and pollen coat lipids (Piffanelli et al., 1997). During formation of the Brassica exine, tapetally-synthesised structural lipids and proteins are secreted into the anther locule, whilst components of the pollen coat are mostly released into the locule later in development, upon tapetum cell death (Piffanelli et al., 1998). These components, which include long-chain fatty acids and large lipid-associated proteins, are sequestered in two tapetal organelles (tapetosomes and
elaioplasts) prior to tapetal cell degeneration (Hern‡ndez-Pinz—n et al., 1999). The importance of these functions to pollen development is clearly demonstrated by Arabidopsis male-sterile mutants with defects in either lipid biosynthesis (male sterility 2, Aarts et al., 1997) or tapetal PCD (Kawanabe et al., 2006), which result in pollen abortion or collapse. It has recently been demonstrated in rice that oscps1-1 displays defects in tapetum secretory function (with corresponding differences to the pollen exine layer) and that GA is necessary for the tapetum to enter PCD (Aya et al., 2009). Developing microspores in this mutant are reported as being morphologically normal at the time that tapetum function is disrupted, suggesting that the action of GA on these two cell types is separable and that the GA signalling controlling tapetum function most likely occurs within the tapetum. Both the GA-deficient tomato mutants gib-2 and sl-2 also demonstrate delayed or abnormal tapetum degeneration (Nester & Zeevaart, 1988; Sawhney, 1992).
In rice, it has been determined through mutant and transcriptomic analysis that GA signalling in anthers acts almost solely through OsGAMYB (Aya et al., 2009, see section 1.3.3). This same study identified a number of immediate downstream targets of OsGAMYB involved directly in lipid biosynthesis, transport, and the signalling cascade that leads to PCD, as well
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Figure 1.11: Regulation of tapetum development and function by GA signalling.
Genetic pathways showing known transcription factors and downstream targets involved in anther/tapetum development in Arabidopsis (a) and rice (b). Putative homologues between the two systems are indicated by matching colours. Dotted arrows indicate transcriptional or indirect regulation. Solid arrows indicate direct enzymatic function or protein-protein interaction (double headed arrow). Black arrows indicate confirmed interactions, grey arrows indicate possible interactions. Adapted from Plackett et al. (2011).
as other transcription factors (Figure 1.11). Recent developments in our understanding of the transcriptional cascade regulating anther development suggest the existence of a conserved genetic framework underpinning anther development in both rice and Arabidopsis (Wilson & Zhang, 2009). Points of integration between this pathway and GA signalling are now being identified. One transcription factor identified as a putative target of OsGAMYB is TAPETUM
DEGENERATION RETARDATION (TDR, Li et al., 2006, Aya et al., 2009), which shares
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(AMS, Figure 1.11, Sorensen et al., 2003). Downstream targets of AtMYB33 and AtMYB65 have not yet been identified in Arabidopsis anther development, and comparisons with the rice system may well provide an important starting point. The expression of AtMYB33 and
AtMYB65 has been shown not to be regulated by the NZZ/SPL-EXS/EMS-DYT1 cascade
(Zhang et al., 2006). Coupled with this, similarities between the anther phenotypes of myb33
myb65 and dyt1 (tapetum hypertrophy and failure of PMC development at meiosis, Millar &
Gubler, 2005; Zhang et al., 2006) have led to speculation that AtGAMYBs and DYT1 interact at the protein level, forming heterodimers to regulate common downstream targets (Zhang et al., 2006). However, as yet no such protein interaction has been demonstrated in vitro or in
planta, and the evidence from rice so far suggests that instead these two signalling pathways
independently regulate common downstream targets. Interestingly, Millar and Gubler (2005) report a low level of sporadic recovery of pollen development in the myb33 myb65 mutant. This recovery is specific to individual locules within an anther, indicative of a highly localised effect. This suggests that the cause may be to do with downstream targets of GA signalling rather than the signalling mechanism itself. The rescue of fertility can be enhanced by altering environmental conditions such as light intensity and temperature (Millar & Gubler, 2005), which suggests interaction between GA signalling and another signalling pathway in anther development. The role of known DELLA binding targets such as PIFs (see section 1.3.3) in reproductive development has not yet been investigated, and these may in time be found to play a role in this phenomenon.
All of the examples given above relate to the effect of the absence of GA signalling on pollen development. In rice and barley, which each possess only one DELLA protein to repress GA downstream responses, loss of DELLA function (therefore leading to constitutive GA signalling) also causes male sterility (Ikeda et al., 2001; Lanahan & Ho, 1988). This phenotype is not well described in published literature, but in the case of barley anthers are recorded as being ÔpollenlessÕ (Lanahan & Ho, 1988), suggesting that inappropriate GA signalling in anther tissues also disrupts pollen development. The fact that Arabidopsis carries five DELLA paralogues has made it difficult to recreate similar constitutive GA signalling in this species, and to date no published combination of DELLA loss-of-function mutant alleles
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has resulted in serious pollen developmental defects (see section 1.3.2). However, a recent combination of RGA and GAI loss-of-function alleles in the Columbia-0 ecotype of
Arabidopsis unexpectedly caused severe defects in male fertility (Thomas, S., personal
communication). If confirmed as having defects in pollen development, this mutant may allow the effects of constitutive GA signalling on tapetum function and pollen development to be investigated more closely.