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Sharma and Kramer EvoDevo (2017) 8:22 DOI 10.1186/s13227-017-0085-7 EvoDevo SHORT REPORT Open Access Aquilegia B gene homologs promote petaloidy of the sepals and maintenance of the C domain boundary Bharti Sharma1* and Elena M. Kramer2* Abstract The model Aquilegia coerulea x “Origami” possesses several interesting foral features, including petaloid sepals that are morphologically distinct from the true petals and a broad domain containing many whorls of stamens. We under- took the current study in an efort to understand the former trait, but additionally uncovered data that inform on the latter. The Aquilegia B gene homolog AqPI is shown to contribute to the production of anthocyanin in the frst whorl sepals, although it has no major role in their morphology. Surprisingly, knockdown of AqPI in Aquilegia coerulea x “Origami” also reveals a role for the B class genes in maintaining the expression of the C gene homolog AqAG1 in the outer whorls of stamens. These fndings suggest that the transference of pollinator function to the frst whorl sepals included a non-homeotic recruitment of the B class genes to promote aspects of petaloidy. They also confrm results in several other Ranunculales that have revealed an unexpected regulatory connection between the B and C class genes. Keywords: Aquilegia, Homeosis, Floral development, MADS box genes, ABC model, Petaloidy Findings [2]. Te discovery of the genetic program controlling Background foral organ identity, the so-called ABC model [3], gave Botanists make very clear distinctions between petals us candidate genes—homologs of the B class petal iden- and petaloidy. Petals, being synonymous with the corolla, tity genes APETALA3 (AP3) and PISTILLATA (PI)—to are defned by the Plant Ontology Consortium as the explore the molecular basis of petaloidy in all its possible inner whorl of non-reproductive organs that surround iterations. the androecium [1]. While these organs are often showy, Te homeotic nature of the ABC model suggests a the primary traits that defne them are their position simple model in which petaloid organs can arise by spa- in the fower (the second whorl) and the fact that they tial shifts of the B gene expression domain [4, 5]. Tis are sterile. In contrast, “petaloidy” refers to an organ’s appears to be the case in many monocots with undif- appearance and indicates non-photosynthetic organs that ferentiated petaloid perianths, such as tulips or lilies, in are modifed for pollinator attraction. Petaloid organs can which B gene expression is commonly observed in all of occur in any whorl of the fower or even be extra-foral the perianth organs (reviewed [6]). Such undiferentiated (e.g., bracts in Poinsettia). Both second whorl petals and perianths are less common in dicots, but a similar pattern the general appearance of petaloidy have evolved many has been described for lobelioid Clermontia [7]. In these diferent times independently across the angiosperms examples, the frst and second whorl organs are very similar at maturity, suggesting that a single organ iden- tity program is being broadly expressed. In many other *Correspondence: [email protected]; [email protected] instances, however, the sepals may be petaloid, but they 1 Department of Biological Sciences, California Polytechnic State difer considerably in morphology relative to the petals. University Pomona, 3801 West Temple Avenue, Pomona, CA 91768, USA 2 Department of Organismic and Evolutionary Biology, Harvard University, Several such examples have now been studied, and most 16 Divinity Ave., Cambridge, MA 02138, USA of these petaloid sepals lack B gene expression, suggesting © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sharma and Kramer EvoDevo (2017) 8:22 Page 2 of 8 that the development of petaloid features in these organs Down‑regulation of AqPI results in contraction of the is due to convergence rather than any degree of homeosis AqAG1 expression domain and the anthocyanin (e.g., [8–10]). production pathway So are there any instances where B genes contribute We used qRT-PCR to quantify silencing of AqPI in all to the petaloidy of the sepals? Tere is one clear exam- afected foral organs (Fig. 2a). Every tissue tested showed ple, in orchids, in which B class genes appear to be criti- 80–95% silencing except the stamen/petal chimeras, cal to the establishment of separate petaloid identity which is not surprising given their retained petal iden- programs in both the sepals and petals via the deploy- tity. We also examined the expression of the three main ment of AP3 paralogs [11, 12]. In Aquilegia, previous AP3 homologs (Fig. 2b). In the frst whorl sepals, we only work has shown that the B class genes do not con- examined AqAP3-1 and AqAP3-2 because AqAP3-3 is tribute to the identity of the sepals, either in terms of expressed at extremely low levels in these organs [13]. their gross morphology or their cell types [13]. How- AqAP3-1 expression was slightly lower in the sepals and ever, these functional tests were always done using the transformed stamens, but increased in the transformed ANTHOCYANIDIN SYNTHASE (AqANS) gene as a second whorl petals. We have previously observed marker (e.g., Fig. 1b), so it cannot be ruled out that the increased AqAP3-1 expression in AqPI-VIGS tissue B gene homologs contribute to anthocyanin produc- [13], so this is not particularly surprising. AqAP3-2 and tion. We, therefore, decided to repeat this experiment AqAP3-3 expression was generally decreased in all the using a virus-induced gene silencing (VIGS) construct organs, except for an increase of AqAP3-3 in the stamen that only contained AqPI in order to determine whether to petal transformed organs, which is consistent with the color production in the sepals was afected. We have petal-specifc expression of this paralog [13]. found that expression levels of multiple members of the In Aquilegia, there are two homologs of the C class anthocyanin pathway are reduced in these fowers, but, gene AGAMOUS, AqAG1 and AqAG2. In the mature in addition, we recovered a novel phenotype, suggesting organs used to assess expression in VIGS experiments, that the B class genes are required for the maintenance AqAG1 is expressed in both stamens and carpels, but of AGAMOUS (AG) homolog expression in the outer AqAG2 is only detectable in carpels [13], making it whorls of stamens. unsuitable for analysis of stamen transformations. Exam- ination of AqAG1 revealed that its expression was, in fact, Results dramatically reduced in the sterilized organs (Fig. 2c); AqPI‑VIGS plants exhibit a range of foral phenotypes however, when stamens were transformed into carpels, After treating 90 plants with TRV2-AqPI, we recov- AqAG1 expression remained strong. ered 50 fowers with homeotic phenotypes, which fell Finally, we tested the expression of three members of into two broad classes. In the frst class, there were the anthocyanin synthesis pathway: ANTHOCYANIDIN 25 fowers that displayed canonical B gene mutant SYNTHASE (AqANS), FLAVONOID 3-HYDROXYLASE phenotypes with petal to sepal and stamen to carpel (AqF3H), and DIHYDROFLAVONOL 4-REDUCTASE transformations (Fig. 1c). Aquilegia has a ffth class of (AqDFR) (Fig. 2d). Note that AqF3H and AqDFR would foral organs, the sterile staminodia, which are simi- be expected to be upstream in the anthocyanin synthesis larly transformed into carpels in these fowers. Tese pathway relative to AqANS [14]. When AqANS is silenced fowers had no more than ten sepals in total, represent- alone, only this member of the synthesis pathway ing the frst whorl sepals and the transformed second decreases; however, when AqPI is silenced alone, all three whorl organs (Table 1). Surprisingly, we also recov- synthesis genes show decreased expression compared to ered 25 fowers that presented novel phenotypes due untreated sepals, although the decrease in AqF3H expres- to sterilization of the outer reproductive whorls. In sion was too variable to be signifcant. these fowers, there were commonly extra whorls of sepals that appear to be in place of the outer whorls Conclusions of fertile organs (Fig. 2d, g, i, m; Table 1). Consistent Te primary goal of this experiment was to test whether with the variable nature of VIGS, this transforma- the B class genes in Aquilegia contribute to color pro- tion was incomplete in some cases, yielding sepal/car- duction in the petaloid sepals, which required conduct- pel (Fig. 1e–h), petal/carpel (Fig. 1j–l) or sepal/petal ing AqPI silencing without the presence of the marker (Fig. 1m) chimeras. In all of these fowers, the sepals AqANS. Our results reveal that the B gene homologs do, show various degrees of color loss, regardless of their in fact, promote activation of the anthocyanin synthesis position in the fower (Fig. 1c–e, g, i, m). Tese sepals pathway in sepals, but we also recovered unexpected evi- are not white as in AqANS-VIGS fowers, but rather dence for a role of the B class genes in maintaining the pale green (compare Fig. 1b–d, g, or i). outer extent of the C class domain. Sharma and Kramer EvoDevo (2017) 8:22 Page 3 of 8 Fig. 1 Phenotypes recovered in Aquilegia x coerulea AqPI-VIGS fowers. a Wild-type fower with labeled frst whorl sepals (1) and second whorl petals (2).

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