Global gene expression patterns in Nuphar
We have analyzed spatial and temporal gene expression patterns using custom microarrays targeting transcripts encoding genes involved in Nuphar floral development. Hierarchical clustering of differentially expressed genes revealed two distinct transcriptional programs in the data. One program involves genes that are expressed almost exclusively during the early stages of floral development (pre-microsporangial initiation and/or pre-meiotic), whereas the other harbors a transcriptional program with a later temporal pattern. Within this ‘later’ program, one subset (module 1) appears to be primarily deployed at the latest developmental stage sampled here, seed development, whereas the other is associated with floral organ development. Most of the genes in this subset (the AP3/PI/SEP1 module) are primarily expressed in the perianth organs and stamens, with weaker expression in carpels, and a smaller number (the AGL6 module) is expressed primarily in carpels. Homologs of AG and AP1 are both expressed during the early floral bud stages, and are placed in the ‘early’ transcriptional program, but although AP1 is not readily detectable in mature floral organs by our measurements, the transcript abundance of the AG homolog increases in mature stamens and carpels.
The spatiotemporal expression patterns of the genes noted in the AP3/PI/SEP1 module and the AGL6 module are therefore consistent with the ABCE model. For example, although the AP3/PI/SEP1 module shows the broadest expression areas across all floral organs (expression areas of E-function genes; Figure S1), genes in this module are mainly expressed in the perianth whorls and/or stamens, which agrees with the main expression domain of B-class gene homologs. In the AP1 module, more than half of the genes are strongly expressed in early developmental stages, which is consistent with the expression pattern of A-class gene homologs, but not in tepals (whorls 1 and 2), which represent the expected expression area of A-function genes. Considering that the presence of a ‘true’ A function is currently questionable (Kramer and Hall, 2005; Davies et al., 2006), this result cannot be considered as a deviation from the ABCE model. Genes in the AGL6 module are mainly expressed in carpels and seeds, but Nuphar AGL6 is expressed in all floral organs (see below), and AGL6 homologs are expressed in inflorescence buds, perianth and female reproductive tissues in other angiosperms (Ma et al., 1991; Rounsley et al., 1995; Hsu et al., 2003; Fan et al., 2007; Rijpkema et al., 2009; Schauer et al., 2009). In gymnosperms, however, AGL6 homologs are mainly expressed in the megasporangium and integument (Mouradov et al., 1998; Shindo et al., 1999; Winter et al., 1999; Reinheimer and Kellogg, 2009). It is therefore tempting to speculate that the expression of AGL6 homologs in carpels stems from an ancient role in the female reproductive unit of seed plants, and that they have acquired a different role in the perianth of angiosperms (see also Reinheimer and Kellogg, 2009).
The expression patterns of homologs of MADS-box genes from Nuphar generally agree with those previously reported based on RQ-RT-PCR data (Kim et al., 2005; Zahn et al., 2005; Yoo et al., 2010). This is especially true for homologs of AP3, PI and SEP1. However, there may be differences in the levels of gene expression among floral organs (Figures 3 and S2). For example, the Nuphar AG homolog was detected in stamens and carpels by RQ-RT-PCR techniques (Yoo et al., 2010), and is strongly upregulated in stamens and carpels relative to leaves according to the microarray data, but is also upregulated, albeit at lower expression levels, in outer and inner perianth whorls (Figure 3). In all instances (AP1, AG and AGL6), microarray data suggest broader expression patterns than observed with RQ-RT-PCR. Whether these differences reflect greater sensitivity of microarrays, non-specific binding or experimental variation in cDNA populations remains unclear. RQ-RT-PCR uses sequence-specific primer sets to amplify target cDNA, whereas microarray experiments are based on hybridization between probes and target RNA. Therefore, if duplicate gene copies with sufficient sequence similarity exist in the cDNA population in microarray studies, the probe may bind to both gene copies, and wider gene expression patterns could be detected if duplicates collectively exhibit broader gene expression domains. Similar results would be obtained if microarray probes target conserved motifs present in multiple genes. Also, expression levels measured by RQ-RT-PCR are relative to that of an internal control; therefore, direct comparison between these two sets of experiments may only be partially reliable. When examined without an internal control, RQ-RT-PCR expression of Nuphar AP1 and AGL6 homologs was detected in all floral tissues, even though there is variation in signal intensities among organs, based on the microarray data. However, AG transcripts were not detected in outer and inner perianth whorls by RQ-RT-PCR. Expression levels of AG are relatively high in stamens and carpels (Figures 3 and S2), suggesting that expression levels in outer and inner perianth whorls may lie below the threshold sensitivity of RQ-RT-PCR. The broader expression pattern observed in microarrays might result from the non-specificity of the Nuphar AG probe, which was designed from sequences lying between the MADS-box and I regions. Also, BLAST analyses of the probes against the National Center for Biotechnology Information (NCBI) nr database only found other members of the AG gene lineage, supporting the interpretation that they only bind other AG homologs rather than more distantly related MADS-box genes.
The differentially expressed genes identified in the Nuphar floral transcriptome are similar to those of Arabidopsis, i.e. MADS-box genes and other transcription factors. Previous studies of the Arabidopsis transcriptome showed that many potential signaling components, such as protein kinases, transcription factors and other putative signaling proteins, were upregulated during reproductive development (Hennig et al., 2004; Schmid et al., 2005). Wellmer et al. (2006) also showed that in Arabidopsis closely related genes are highly correlated in their temporal gene expression patterns, and the majority of those genes are upregulated during certain developmental stages. In this study, we demonstrated that many transcription factors were differentially expressed in floral tissues, and as in Arabidopsis, their expression patterns are correlated. For example, genes encoding bHLH, homeobox and WRKY transcription factors are expressed in leaves, whereas genes encoding bZIP, MADS and MYB-related transcription factors are mainly expressed in floral tissues (Tables S1 and S2). In particular, all the Nuphar WRKY transcription factor homologs examined are downregulated in floral tissues, whereas MADS-box genes, except the AP1 homolog, are upregulated in reproductive organs. These expression patterns are also observed in Arabidopsis, suggesting functional conservation among major regulatory elements between these two species.
Comparative floral transcriptomics among Nuphar, Persea and Arabidopsis
Our analyses suggest distinct transcriptional programs are deployed in each of the three species examined here. Most of the homologs with similar expression patterns are involved in the basic, and perhaps understandably conserved, processes of cellular metabolism, energy production and biogenesis of cellular components. Also, homologous MADS-box genes from Nuphar, Persea and Arabidopsis exhibit similar expression patterns (Figure 4b; Table S3), suggesting that major components of their function may be conserved. However, homologs of other genes regulating flower development, such as NAP and AFO (ABNORMAL FLORAL ORGANS), are expressed in different floral organs in the three species, suggesting less conservation in broader genetic networks and, perhaps, illustrating one principal mechanism behind floral diversification across the angiosperms. The transcriptional patterns in the three species examined here have also revealed a fundamental difference separating flowers of basal angiosperms from those of eudicots. Both basal angiosperms, Nuphar and Persea, have transcriptional programs that are only weakly partitioned among floral whorls compared with Arabidopsis, and this regulatory shift appears to correlate with a shift in floral morphology. Arabidopsis has well-differentiated floral organs, and the number of genes expressed in more than one floral organ is relatively small, indicating that each floral organ has a nearly unique floral transcriptional program. The transcriptional programs in Nuphar and Persea correspond well with the ‘fading borders’ model of floral organ identity, which explains the morphological intergradation of floral organs in basal angiosperms through fading activities of floral organ identity genes towards the borders of their broad activity zones (Buzgo et al., 2004, 2005). Minor differences in pigmentation can distinguish the outer ‘sepaloid’ tepals from the inner ‘petaloid’ tepals of Nuphar (Warner et al., 2008, 2009), whereas the inner perianth organs of Persea bear glandular patches that are absent from the outer tepals (Chanderbali et al., 2006). The strong linearity between expression profiles of inner and outer tepals in both Nuphar (r = 0.96) and Persea (r = 0.98) evidently reflects their largely undifferentiated morphology. This linearity is also observed between perianth members and stamens of Nuphar (r = 0.85) and Persea (r = 0.82), supporting their grouping in hierarchical clustering analyses. In contrast, a sharp distinction in expression profiles (0.42 ≤ r ≤ 0.66) suggests that the floral organs of Arabidopsis develop under more divergent transcriptional programs, relative to those of the two basal angiosperms.
The higher proportion of genes commonly expressed in perianth members and stamens compared with the number of genes expressed in stamens alone suggests that the staminal developmental program is not as unique in Nuphar and Persea as it is in Arabidopsis. Moreover, genes deployed primarily in Nuphar and Persea stamens tend to have broader expression domains than those expressed primarily in carpels (Table 2). These features of the floral transcriptomes of Nuphar and Persea may support the ‘out-of-male’ or ‘mostly-male’ hypotheses for the origin of the flower. According to these hypotheses, female structures and perianth organs evolved from male structures through the contraction of the male developmental program in the upper region of male cones, and sterilization of the outer stamens, respectively (Frohlich and Parker, 2000; Baum and Hileman, 2006; Theissen and Melzer, 2007). Therefore, perhaps these expression features are a vestige of an ancient event: the evolution of female organs and perianth organs from male organs.
In organ relationships based on expression data, Nuphar and Persea perianth organs clustered together with stamens, as do Arabidopsis petals and stamens (Figure 4). According to the traditional view, petals (or in some cases inner tepals) in basal angiosperms are thought to have derived from bracts (bracteopetals), whereas petals of eudicots are considered to be derived from stamens (andro-petals) (Hiepko, 1965; Takhtajan, 1991; Endress, 2001). However, the hierarchy of organ relationship indicates that Nuphar and Persea perianth organs share a genetic program with their respective stamens, and, therefore, may also have a staminal origin (i.e. their tepals would therefore be andropetals). Other data also support our hypothesis that the tepals in Nuphar are derived from stamens, and are hence andropetals (Yoo et al., 2010), as has also been proposed for Persea (Chanderbali et al., 2006).
Although spatial expression patterns and differentially expressed gene proportions are similar in Nuphar and Persea (Figure 4; Table S4), the carpels and stamens of Persea appear to have a slightly more distinct transcriptome than those of Nuphar, as many more genes are twofold upregulated in these organs in Persea than in Nuphar (Table 2). Perhaps, therefore, Nuphar has a less-defined transcriptional program than does Persea, and this broader expression profile might represent the more ancient (or ancestral) transcriptional pattern, considering the basal phylogenetic position of Nuphar relative to Persea (Figure 1), and the floral morphologies of these two genera, especially their different stamen features. The stamens of Persea possess well-differentiated anthers and filaments. Hence, more genes might be involved in the morphogenesis of stamens of Persea compared with stamens of Nuphar, where stamens are petal-like without well-differentiated anthers and filaments.
The stronger similarities in the transcriptional profiles of the floral organs of Nuphar and Persea compared with those of Arabidopsis indicate an evolutionary transformation across angiosperm history towards the organ-specific refinement of ancestrally overlapping transcriptional programs. However, Nuphar and Persea also show differences in some spatial gene expression patterns, although they are similar in some aspects of floral morphology (e.g. both have largely undifferentiated perianths). In Nuphar, most of the floral genes are involved in the development of perianth members and stamens, and the floral transcriptional programs of these organs overlap substantially. In contrast, in Persea, inner and outer tepals share similar gene expression patterns, with proportionally more genes expressed in either stamens or carpels. Thus, each floral organ category (tepal, stamen and carpel) of Persea is controlled by a slightly more specific transcriptional program compared with that of Nuphar. Phylogenetic data, floral morphology and expression profiling, taken together, indicate that the broad patterns of expression observed across floral organs of the basal angiosperms Nuphar and Persea are likely to represent the ancestral condition in angiosperms, whereas the organ-specific transcriptional profiles observed in Arabidopsis are derived. Homologs of the B- and C-function genes, namely PI, AP3 and AG, have similar expression patterns in Nuphar and Persea, but are more broadly expressed than in Arabidopsis. As a consequence, the transcriptional cascades they regulate in Nuphar and Persea are also more broadly deployed. Perhaps the regulatory changes that maintain the strict spatial domains of ABCE functions have led to more spatially discrete downstream transcriptional programs during the course of floral evolution and diversification.