AqFIE and AqEMF2 VIGS-treated plants displayed a range of lateral organ phenotypes. Silenced leaves often had ruffled or curled lamina, additional lobing, and an increased frequency of higher order branching. The perianth organs were generally narrower than wild type organs. Sepals were also curled and petals were stunted or had bent spurs, while petal limbs also had a particularly intense yellow coloration seemingly due to an accumulation of carotenoid pigments in these cells. Many of the phenotypes we observed are similar to those seen in clf mutants and FIE cosuppressed A. thaliana, including curled leaves and narrow perianth organs [17, 29]. Unlike clf mutants and AG over-expressers in A. thaliana, dramatic transformation towards carpel identity was not observed in the AqFIE- and AqEMF2-treated sepals or petals. However, the level of AqAG1 expression in these organs was much less than what is seen in wild type Aquilegia carpels. Interestingly, the distinct folded morphology of the sepals may suggest slight transformation towards carpel identity as silenced leaves were folded towards the abaxial surface while the sepals were dramatically folded towards the adaxial surface, which is similar to the folding pattern of the Aquilegia carpel .
It is interesting to note that in AqFIE silenced leaves, AqEMF2 is also down-regulated. The reverse is not true in AqEMF2 silenced leaves, and AqEMF2 expression is not affected in AqFIE silenced floral organs. This result suggests that PRC2 may be directly or indirectly regulating AqEMF2 expression in A. x coerulea leaves, which could account for the generally more severe phenotypes observed in AqFIE silenced leaves compared to AqEMF2 silenced leaves. AqEMF2 is the only member of the complex that appears to be PRC2-regulated as the expression of AqCLF and AqSWN is not affected in PRC2 down-regulated leaves. In general, the potential for this type of cross-regulation is relatively unexplored in A. thaliana and, therefore, bears further study.
In our analysis of candidate target genes, we found that AqAG1 is often ectopically expressed in PRC2 down-regulated tissue. AqAP3-3 and AqSEP3 are also up-regulated in some organs, but expression of the class I KNOX genes and several candidate genes involved in carotenoid production or degradation seem largely unaffected. Mutations in AG and SEP3 are known to suppress the curled leaf phenotype in clf mutant plants while over-expression of these MADS box genes, which themselves function together in a complex , is thought to be the cause of the curled leaf phenotype . It is, therefore, possible that over-expression of AqAG1 and AqSEP3 is similarly responsible for many of the observed phenotypes in AqFIE and AqEMF2 silenced leaves. These findings lead us to conclude that PRC2-based regulation of AG and SEP3 homologs is deeply conserved in eudicots. It has recently been shown that several chromatin remodeling factors associate with MADS complexes and one model is that an important function of MADS domain complexes may be to recruit chromatin remodeling complexes to target loci in order to alter transcription of these genes and direct organ development [50, 51]. For example, RELATIVE OF EARLY FLOWERING 6 (REF6) was enriched in protein complexes that were isolated via immunoprecipitation using tagged ABCE class MADS box proteins . REF6 has been shown to specifically demethylate H3K27me3, the histone modification deposited by PRC2 . Activation of SEP3 by APETALA1 (AP1) in A. thaliana results in the reduction of H3K27me3 at the SEP3 promoter, suggesting that AP1 may recruit REF6 to the SEP3 promoter in order to help induce SEP3 gene function . Our data suggests that this key dependency on epigenetic regulation for the switch from vegetative to floral development may be important outside of A. thaliana. There are some complications, however. Of the two A. x coerulea AG homologs, only one, AqAG1, is strongly regulated by PRC2. Perhaps consistent with this observation, sequencing of the Aquilegia genome (http://www.phytozome.net/search.php?method=Org_Acoerulea) reveals that AqAG1 does contain the large regulatory second intron that is common to AG homologs [53, 54] while AqAG2’s second intron is much smaller. These results suggest that PRC2 regulation can be directed in a paralog-specific fashion and may even play some role in the distinct expression patterns observed among these gene copies .
The class I KNOX genes are directly or indirectly regulated by PRC2 in both A. thaliana and Physcomitrella, however, we detected little or no increase in KNOX gene expression in our AqFIE and AqEMF2 silenced leaves. This is somewhat surprising because of the higher order branching that we observed in silenced leaves, including several of the tested RNA samples. The class I KNOX genes are thought to play a role in compound leaf development in a number of species. In many, but not all, compound leafed taxa where KNOX gene expression has been studied, including Aquilegia, it has been shown that the genes are expressed in the shoot apical meristem and down-regulated in incipient leaf primordia (P0), but subsequently turned back on in early leaf primordia . Down-regulation of class I KNOX genes in the leaves of models such as tomato or Cardamine causes reduced branching while over-expression leads to increased branching [55, 56], suggesting that KNOX genes act to maintain indeterminacy in compound leaves and promote leaflet initiation.
There are several possible explanations for why we did not observe significant ectopic KNOX gene expression in our VIGS-treated leaves. First, it is possible the KNOX genes were ectopically expressed early in leaf development when the higher order branching actually developed, but were later down-regulated by redundant mechanisms, such as ASYMMETRIC LEAVES 1 (AS1)-mediated repression [57, 58]. In A. thaliana AS1 mediated silencing of some of the KNOX genes has been shown to require the PRC2 complex and it is thought that AS1 and AS2 directly recruit the PRC2 complex to KNOX loci . However, it is important to remember that in other taxa with compound leaves, the KNOX and AS1 homologs have lost their mutually exclusive regulatory interactions and are expressed together at later stages . This may suggest that the AS1-dependent epigenetic silencing of KNOX genes that has been described in several simple-leafed models [57, 58] does not hold for plants with compound leaves. Along these lines, it is also possible that the increased branching phenotypes are due to other factors, such as accelerated phase change or novel genetic mechanisms regulating leaflet branching in Aquilegia. For instance, a recent functional study of the gene AqFL1 in A. x coerulea revealed that it promotes proper leaf margin development, a unique finding for homologs of this gene lineage . This raises the possibility that factors other than the KNOX genes contribute to compound leaf branching in Aquilegia.
In addition to the conserved role in regulating AG, AP3, and SEP3, A. x coerulea PRC2 may target novel pathways, including those regulating carotenoid production or degradation. In A. thaliana patches of yellow anther-like tissue are observed on clf mutant petals . However, the yellow pigmentation we observed is due to the accumulation of carotenoids in the plastids rather than to a partial homeotic transformation. While genes in the carotenoid pathway are not known to be suppressed by PRC2, some loci are positively epigenetically regulated in A. thaliana. Previous studies have shown that a major enzyme in the carotenoid biosynthesis pathway, CRTISO, requires the chromatin modifying enzyme SET DOMAIN GROUP 8 (SDG8) to maintain its expression . NCED3, an enzyme that cleaves some types of carotenoids as a part of abscisic acid (ABA) synthesis, is similarly epigenetically regulated by the A. thaliana trithorax homolog ATX1 . While none of the genes we tested were consistently up- or down-regulated in AqFIE and AqEMF2 silenced petals, carotenoid production is very genetically complex and we were unable to test all of the candidate loci . Thus, it seems likely that PRC2 regulates an as yet unidentified enzyme in this pathway in A. x coerulea.