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- Materials and Methods
- Supporting Information
The genetic basis of organ identity in standard floral organs – the sepals, petals, stamens, and carpels – has been well studied in multiple angiosperm models (reviewed in Causier et al., 2010; Litt & Kramer, 2010; Rijpkema et al., 2010). This program is summarized in the well-known ABC model, which holds that over-lapping gene activities create combinatorial codes for each of the four organ identities: A alone encoding sepals; A + B, petals; B + C, stamens; and C alone, carpels (Coen & Meyerowitz, 1991). Although the conservation, and perhaps even existence, of A function has not been supported, it generally appears that the B and C class functions are broadly conserved, albeit with interesting exceptions (reviewed in Causier et al., 2010; Litt & Kramer, 2010; Rijpkema et al., 2010). Beyond the conservation of standard organ identity is the question of how new floral organ identities arise. The best understood example of this phenomenon is probably the lodicules of grasses, which represent a modification of the pre-existing petal identity program (Whipple et al., 2007 and references therein). Another striking case is the de novo derivation of petaloid organs from outer stamens in the Caryophyllales (Brockington et al., 2012). Both of these instances, however, still result in a total of four organ types, so an outstanding issue is how floral organ identity is established in taxa that produce more than four types of floral organs. Most commonly, these fifth organ types are thought to be evolutionarily derived from pre-existing stamens, therefore termed staminodia, and range enormously in morphology and function (Walker-Larsen & Harder, 2000). Currently, the only functionally tractable model for the genetic study of such novel organs is the lower eudicot Aquilegia (columbine), a member of the Ranunculaceae with numerous available genetic and genomic tools (Kramer, 2009). These flowers produce five petaloid sepals in the first whorl, five spurred petals in the second whorl, four to seven whorls of ten stamens, a single whorl of ten sterile staminodia, and four to six unfused carpels (Fig. 1a–f). Compared with the stamens, the staminodia lack anthers and are laterally expanded with ruffled margins (Fig. 1e). Although their ecological function remains unknown, it is clear that the staminodia represent a completely distinct, fifth organ type (Tucker & Hodges, 2005; Kramer, 2009).
In a first effort to characterize the genetic basis of staminodium identity, homologs of the B class MADS box transcription factors APETALA3 (AP3) and PISTILLATA (PI) were characterized in Aquilegia (Kramer et al., 2007). The first discovery of note was that there are, in fact, four copies of the AP3 lineage, termed AqAP3-1, AqAP3-2, AqAP3-3 and AqAP3-3b, although expression of AqAP3-3b is so low that we will not consider it further (Kramer et al., 2007; Sharma et al., 2011). AqAP3-1 and AqPI are broadly expressed in the presumptive petals, stamens and staminodia before these primordia even initiate. At the same time, AqAP3-3 is detected only in the petal whorl but AqAP3-2 expression in the stamens and staminodia does not become apparent until somewhat later, after the primordia begin to form. These patterns remain constant until roughly the point at which carpel primordia initiate. This marks a clear transition when AqAP3-2 begins to decline in the staminodia but remains on in the stamens, while AqAP3-1 expression declines in the petals and stamens but not the staminodia, leading to a largely mutually exclusive expression pattern that persists through anthesis. AqAP3-2 expression also increases in the petals during later stages after the stamens have clearly differentiated locules. Throughout this period, AqAP3-3 remains constant in the petals while AqPI is expressed in petals, stamens and staminodia. The broad overlap of AqPI expression relative to the more restricted domains of the AP3 paralogs is consistent with the general finding that AP3 and PI homologs act as obligate heterodimers (reviewed in Immink et al., 2010) and, more specifically, that the Aquilegia B gene homologs similarly form heterodimers (Kramer et al., 2007).
These complex expression patterns suggest that both subfunctionalization and neofunctionalization (sensu Force et al., 1999) have occurred among the Aquilegia AP3 paralogs. The ancestral expression domain of AP3 homologs in petals and stamens appears to have been subfunctionalized such that AqAP3-3 is petal-specific and AqAP3-2 is largely stamen-specific, while AqAP3-1 may have experienced neofunctionalization in assuming a role in the novel staminodium identity program. In this regard, it is important to note that studies in other ranunculids show that orthologs of AqAP3-1 and AqAP3-2 are typically broadly expressed in petals and stamens, indicating that their differential expression in Aquilegia must be recently derived. The first test of this hypothesis was RNAi-based downregulation of AqPI using the virus-induced gene silencing (VIGS) (Kramer et al., 2007) technique. This confirmed that petal, stamen and staminodium identity is completely dependent on AqPI, which supports the conclusion that the protein is required for each AP3-containing dimer (Kramer et al., 2007). Silencing of AqPI also resulted in downregulation of AqAP3-2 and AqAP3-3 but not AqAP3-1 (Kramer et al., 2007), which we speculated could indicate a dependency of AqAP3-2 and AqAP3-3 expression on functional B protein heterodimers (Kramer et al., 2007). Such autoregulation and cross-regulation is common among B gene homologs across the core eudicots (reviewed in Rijpkema et al., 2010). Further silencing of AqAP3-3 confirmed that this locus is required only for petal identity and does not play a significant role in any other organ identity program (Sharma et al., 2011).
The current study builds on this previous work by examining the phenotypes of flowers silenced for AqAP3-1 or AqAP3-2 alone as well as simultaneous silencing of AqAP3-1/2. Our results suggest that AqAP3-1 is, in fact, essential to staminodium identity while silencing of AqAP3-2 has no effect on these organs. By contrast, AqAP3-2 is required for normal stamen development but AqAP3-1 also contributes to the establishment of stamen identity; complete stamen to carpel transformation is only observed when both loci are silenced. Although these data seem straightforward enough, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of the expression of the other B gene homologs in each case reveals evidence for complex regulatory feedback interactions. Overall, our current picture of floral organ identity in Aquilegia reveals an intricate evolutionary history by which ancient gene duplications laid the foundation for a stepwise series of subfunctionalization and neofunctionalization that facilitated the evolution of a completely new floral organ identity.
- Top of page
- Materials and Methods
- Supporting Information
Each of the constructs tested resulted in highly distinct phenotypes. Targeting of AqAP3-1 primarily affected staminodia, which were transformed towards carpel identity, with some perturbation of inner stamen identity as well. Silencing of AqAP3-2 strictly affected stamens, causing sterility because of late anther necrosis, severe reduction of anther development, or partial transformation toward carpel identity. By contrast, dual silencing produced a very dramatic phenotype in which all of the stamens and staminodia were strongly transformed towards carpel identity. There was no effect on petal development in either of the singly silenced cohorts but double silencing resulted in stunted petal spurs that failed to completely elongate. Analyses of gene expression patterns within the context of these phenotypes yielded complex results. Our targeted silencing of AqAP3-1 also produced downregulation of AqAP3-2, but had only weak effects on AqAP3-3 and none on AqPI. By contrast, targeted silencing of AqAP3-2 resulted in upregulation of AqAP3-1 in staminodia and moderate downregulation of AqAP3-3 in petals, but no effect on AqPI. Similarly, dual silencing of AqAP3-1/2 resulted in strong reduction in AqAP3-3 expression but not in AqPI expression.
The primary issue in interpreting these results is determining whether the reduced expression of AqAP3-2 in the TRV2-AqAP3-1-treated plants is most likely caused by off-target silencing or, alternatively, by genetic interactions among the paralogs. We favor the latter explanation for a number of reasons. First, both the decline in AqAP3-2 expression in TRV2–AqAP3-1-treated plants and that of AqAP3-3 in TRV2–AqAP3-2-treated plants are consistent with the previous findings in AqPI-silenced plants, where AqAP3-2 and AqAP3-3 expression decreased (Kramer et al., 2007). In this case, off-target silencing between AqPI and the distantly related AP3 paralogs is highly unlikely but autoregulation and cross-regulation is common among B gene homologs (reviewed in Goto & Meyerowitz, 1994; Liu & Mara, 2010). Second, if the issue was off-target silencing owing to siRNA amplification, one would expect that it would be a general response and affect both TRV2–AqAP3-1 and TRV2–AqAP3-2. This is not the case because AqAP3-1 expression appears to increase in the AqAP3-2-silenced plants. Third, if AqAP3-2 was being targeted by VIGS in TRV2–AqAP3-1-treated plants, we might expect the phenotypes to be equivalent to those of TRV2–AqAP3-1/2. Instead, these phenotypes differ dramatically with the former primarily affecting staminodia while the latter affects all of the stamens and staminodia. Alternatively, if the loss of AqAP3-2 was caused by regulatory interactions, the different phenotypes could be explained by differences in the relative timing of repression. Lastly, while it is theoretically possible that signal amplification could lead to siRNA production from regions outside the original target fragment, several studies have revealed high levels of fidelity for VIGS (reviewed in Burch-Smith et al., 2004), including when working with the closely related MADS box genes (Liu et al., 2004; Drea et al., 2007; Sharma et al., 2011). In light of all of these facts, we believe that the simplest explanation for the collective phenotypic and expression data is that the AP3 paralogs exhibit both positive and negative cross-regulation.
Based on this, we can expand our hypotheses of regulatory interactions among the Aquilegia B gene homologs (Fig. 4). It seems that maintained expression of AqAP3-2 is particularly dependent on full function of AqAP3-1. As AqAP3-1 expression in the stamens is normally weak at late stages, we would propose that either this low level of AqAP3-1 expression is required for persistent AqAP3-2 activation or the strong, early AqAP3-1 expression potentiates long-term expression of AqAP3-2. Given the distinct phenotypes of AqAP3-1-, AqAP3-2- and AqAP3-1/2-silenced flowers, we assume that AqAP3-1 is not essential to AqAP3-2 initiation but rather its maintenance. Conversely, AqAP3-2 appears to feed back negatively onto AqAP3-1, certainly in the stamens and possibly also in the other floral organs. It is important to note that even if there is a degree of off-target silencing in these experiments, the data still suggest negative regulation of AqAP3-1 by AqAP3-2. At the same time, late AqAP3-2 expression in petals is important for strong AqAP3-3 expression, likely with contributions from AqAP3-1 (double AqAP3-1/2 silencing affects AqAP3-3 expression more than silencing of AqAP3-2 alone). Alternatively, it may simply be that AqAP3-2 silencing was more effective in the doubly silenced plants, leading to stronger reduction in AqAP3-3. As AqPI would be expected to participate in all of these interactions through heterodimerization with the AP3 proteins, the loss of AqAP3-2 and AqAP3-3 in AqPI-silenced flowers (Kramer et al., 2007) is consistent with the model, although we cannot rule out additional autoregulatory feedback for AqAP3-2 and AqAP3-3. Interestingly, neither AqAP3-1 nor AqAP3-2 expression was affected when AqAP3-3 was silenced (Sharma et al., 2011) and, at least so far, no positive feedback onto AqPI has been observed in any of the AP3 silencing experiments (this study, Sharma et al., 2011).
Figure 4. A modified ABC model for Aquilegia petals, stamens and staminodia based on the expression and apparent functional roles of each Aquilegia AP3 paralog as well as AqPI. Dashed orange lines indicate regulatory feedback interactions hypothesized based on changes of paralog expression in different silenced backgrounds. Note that AqPI is inferred to participate in all of the AP3 paralog interactions because of their demonstrated heterodimerization (Kramer et al., 2007). PET, petals; STA, stamens; STD, staminodia.
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Beyond these potential regulatory interactions, it appears clear that the novel staminodium identity program is dependent on AqAP3-1 function but does not especially require AqAP3-2. Conversely, in stamens, AqAP3-1 expression seems to be sufficient to prevent the development of carpel identity but is not capable of promoting proper stamen development. It may even be that the effect in inner stamens in AqAP3-1-silenced flowers results more from the loss of AqAP3-2 than from AqAP3-1 itself. Thus AqAP3-1 and AqAP3-2 contribute differentially to staminodium and stamen identity, respectively, but what is the basis of the contrast? Is it simply because of their differential expression or are they biochemically differentiated as well? Obviously, B gene homologs typically control both petal and stamen identity but the separate programs result from the presence or absence of other factors, such as C gene homologs (Coen & Meyerowitz, 1991). In Aquilegia, the C gene homologs AqAG1 and AqAG2 appear to be expressed in both stamens and staminodia, although there may be reduced expression in the latter (Kramer et al., 2007), which bears further investigation. There are cases where duplications in the B lineage, particularly AP3, have led to subfunctionalization between petal and stamen identity, a situation best characterized in petunia. In that case, although there are distinct expression patterns and dimerization preferences for the two AP3 paralogs, they are both biochemically capable of promoting petal identity (Vandenbussche et al., 2004; Rijpkema et al., 2006). Conversely, paralogs of the C gene AGAMOUS (AG) in Antirrhinum are subfunctionalized both in terms of their expression and biochemical ability to promote stamen vs carpel identity (Causier et al., 2005; Airoldi et al., 2010). In Aquilegia AqAP3-2-silenced flowers, we see that even though AqAP3-1 is upregulated, it is still incapable of rescuing normal stamen development. It may be that the degree of upregulation is not enough to rescue the absence of AqAP3-2, but we must consider the possibility that the loci are not biochemically equivalent. This being said, it is also true that AqAP3-1 expression alone in AqAP3-2-silenced stamens does not appear to be sufficient to convert these organs to full staminodium identity, indicating that other factors specific to the staminodium whorl are required (or even higher AqAP3-1 expression). Protein interaction studies to date have found that both AqAP3-1 and AqAP3-2 interact with AqPI, although the AqAP3-1/AqPI dimerization is particularly strong (Kramer et al., 2007). Addressing the question of biochemical differentiation, as well as testing our hypotheses of regulatory interactions, will require the use of transgenic studies in Aquilegia. In addition, a thorough investigation of AqAG1 and AqAG2 may shed light on the full genetic identity program that distinguishes staminodia from stamens.
We now have a picture of organ identity in Aquilegia that reveals complex subfunctionalization and neofunctionalization, which have evolved along different time-scales. The petal-specific AqAP3-3 paralog is subfunctionalized relative to ancestral AP3 function and appears to be both necessary and sufficient for establishing petal identity (Rasmussen et al., 2009; Sharma et al., 2011). The lack of normal petal development in AqAP3-1/2-silenced plants may result from the reduction in AqAP3-3 expression, but it is also possible that the other AP3 paralogs have some important contribution to petal development. Regardless, all evidence suggests that orthologs of AqAP3-3 are similarly subfunctionalized to a specific role in petal identity across the Ranunculaceae, Berberidaceae and possibly even the Lardizabalaceae (Kramer et al., 2003; Rasmussen et al., 2009; Sharma et al., 2011; Hu et al., 2012), indicating that an ancient subfunctionalization has been broadly maintained. The next key question will be understanding how this expression pattern is controlled. Although a petal-specific element has been characterized in the Arabidopsis AP3 promoter (Hill et al., 1998), it remains to be seen whether the AqAP3-3 subfunctionalization relies on a similar element.
The other two AP3 paralogs have experienced both subfunctionalization and neofunctionalization: neither locus is essential to petal identity, both act to promote stamen identity, with AqAP3-2 being the crucial player, and only AqAP3-1 is essential to staminodium identity. Again, independent of any potential off-target silencing, the phenotypes resulting from the different TRV2 constructs are completely distinct and support this model. Furthermore, there is every reason to believe that the distinct respective roles of AqAP3-1 and AqAP3-2 in staminodium and stamen identity were critical factors in the evolution of this novel organ identity in Aquilegia. It is interesting to note that although the paralogous lineages defined by these two genes are quite ancient, predating the ancestor of the bulk of the Ranunculales (Rasmussen et al., 2009; Sharma et al., 2011), staminodia between the stamens and carpels are recently evolved in the tribe Thalictroideae (Tucker & Hodges, 2005). The only other genus to possess staminodia-like structures is Semiaquilegia, the sister genus to Aquilegia, which has sterile organs in the same position that are more irregular in number and morphology than the broad, elaborated staminodia of Aquilegia (Tucker & Hodges, 2005). In fact, the Semiaquilegia organs bear considerable resemblance to the sterile filaments obtained in strong AqAP3-2-silenced flowers. This suggests a model in which silencing of AqAP3-2 in the innermost whorl of stamens may have been the first step in the evolution of the novel organs. Essentially, the ancestor of Aquilegia + Semiaquilegia could have possessed whorls of fertile stamens that expressed AqAP3-1 and AqAP3-2 together, but in this novel inner whorl, transient AqAP3-2 expression with persistent AqAP3-1 produced sterile organs that ultimately evolved into the full staminodium phenotype of Aquilegia. Even in the fertile stamen whorls of Aquilegia, it appears that the innermost stamens are more sensitive to AqAP3-1 silencing than the outer ones, suggesting a degree of differentiation within the morphologically homogeneous stamen whorls. One possibility is that this partitioning of AqAP3-1 and AqAP3-2 function – with one paralog more responsible for outer stamens while the other primarily controls inner stamens – may have been further exaggerated to yield the novel staminodial whorl. Examination of AqAP3-1 and AqAP3-2 ortholog expression in Semiaquilegia and other related genera will be critical to understanding this evolutionary process, as will exploring the regulatory mechanisms controlling expression of the two paralogs.
What is clear is that the existence of the ancient AP3-1 and AqAP3-2 paralogs, which are otherwise commonly expressed in both petals and stamens (Rasmussen et al., 2009; Sharma et al., 2011), has facilitated the evolution of a whole new organ identity program in the lineage leading to Aquilegia. This involved a series of genetic innovations, including the definition of a new regulatory domain within the previously homogeneous stamen region and the acquisition of negative feedback by AqAP3-2 onto AqAP3-1, which is unusual for B gene homologs. Further studies will be necessary to determine whether gene duplication similarly underlies identity in other taxa that produce more than four types of floral organs, but the common presence of paralogs among the floral MADS box genes (reviewed in Litt & Kramer, 2010; Rijpkema et al., 2010) means that raw material for such neofunctionalization is broadly available. For example, an independent set of AP3 duplicates has been implicated in the evolution of the novel petaloid organ types seen in orchids, but this hypothesis is yet to be functionally tested (Mondragon-Palomino & Theissen, 2011). Of course, the association between gene duplication and morphological or physiological novelty has been well documented in both plants and animals (for review see Flagel & Wendel, 2009; Hall & Kerney, 2012). In Aquilegia, the next goal is to elucidate the mechanistic basis of this process and determine just how many genetic changes were required downstream of the AP3 paralogs to achieve the final elaboration of the staminodia.