What a difference a CArG-box makes

New insights into the Development and Evolution of Floral Meristem and Organ Identity in Arabidopsis

Günter Theißen1 & Francois Parcy2

1Friedrich Schiller University Jena, Department of Genetics, Philosophenweg 12, D-07743 Jena, Germany; guenter.theissen@uni-jena.de
2 Laboratory of Plant & Cell Physiology, CNRS, CEA, Univ. Grenoble Alpes, INRA, 38000 Grenoble, France

A report by Ye et al. (2016) about the relatively recent acquisition of a cis-regulatory element during the evolution of a MADS-box gene involved in flower development may help to answer some long-standing questions about flower development and evolution.

The first set of questions concerns the origin and conservation of the floral homeotic A function during evolution. In addition to the B and C functions, the A function is part of the iconic ABC model of flower development (Haughn and Somerville 1988; Coen and Meyerowitz 1991). The ABC model postulates three classes of homeotic functions (A, B, and C) that specify floral organ identity in a combinatorial way: the A function alone specifies sepals in the first floral whorl, A + B defines petals in the second whorl, B + C specifies stamens in the third whorl, and C defines carpels in the fourth whorl (Coen and Meyerowitz, 1991). According to the ABC model an antagonism between the A and C function is responsible for the spatial restriction of the A function in the outer two and the C function to the inner two whorls of the flower (Coen and Meyerowitz, 1991). In consequence, if all three homeotic functions work properly in an developing flower of the model plant Arabidopsis thaliana (Arabidopsis, for short), sepals, petals, stamens and carpels develop in whorls 1-4, respectively, as shown in the picture.

Gunther image.jpg

Photo kindly provided by Grégoire Denay

Even though the ABC model was originally largely based on mutant phenotypes of Arabidopsis (Coen and Meyerowitz 1991), mutational and gene expression analyses during the last 25 years revealed that the B and C functions are highly conserved throughout flowering plants and even in gymnosperms (Becker and Theißen 2003; Melzer et al. 2010).

In contrast, the concept of an A function has always been discussed controversially. For example, almost simultaneously to the ABC model an alternative model was proposed that focused on snapdragon (Antirrhinum majus) rather than Arabidopsis. This model postulated two developmental pathways in addition to a floral ground state (Schwarz-Sommer et al., 1990). These two pathways were historically (and confusingly) named A and B (Schwarz-Sommer et al., 1990), but they are equivalent to the B and C functions, respectively, of the ABC model (Theißen et al., 2000). According to the snapdragon-based model sepal development represents the developmental ‘default state’ of floral organ development and thus does not require an extra A function (Schwarz-Sommer et al., 1990). Indeed, except Arabidopsis, in almost all plants that have been investigated so far one does not find recessive mutants in which the identity of both types of perianth organs (sepals and petals) is affected (discussed by Theißen et al., 2000; Litt 2007).

However, compared to the B and C functions, even in Arabidopsis thaliana the A function appears not well defined (reviewed by Theißen et al., 2000; Litt, 2007; Causier et al., 2010). Originally, only the gene APETALA2 (AP2) was thought to provide the A-function (Coen and Meyerowitz, 1991), but later on also the gene APETALA1 (AP1) was established in the literature as an A-function gene (for a review, see Litt, 2007). According to the ABC model, the loss-of-function of these genes should lead to the development of carpels and stamens in the whorl 1 and 2, respectively, due to the ectopic expression of the C function gene. However, depending on environmental conditions and genetic background, the organs in the outer whorls of ap1 or ap2 loss-of-function mutants may also develop into leaf- or bract-like structures, or can even be missing; in case of ap1 mutants secondary flowers may eventually develop in the axils of leaves that replace sepals (reviewed by Litt, 2007; Causier et al., 2010). Thus, the A function that specifies sepal and petal organ identity and that antagonizes the C function is difficult to separate genetically from a more basic function of AP1 and AP2 in specifying floral meristem identity.

This raises the question as to why then does it seem that A function in Arabidopsis is an exception rather than the rule, and why is it less-well defined than the other floral homeotic functions? The recent publication by Ye et al. (2016) may have brought us much closer to reasonable answers to these questions.

In contrast to AP2, AP1 represents a MADS-box gene and thus encodes a MADS-domain transcription factor (Mandel et al., 1992). MADS-domain transcription factors bind to cis-regulatory elements termed CArG-boxes (for ‘C-A-rich-G’). Ye et al. (2016) have studied the AP1 gene in comparison to its closely related paralog CAULIFLOWER (CAL). While mutants in which CAL has lost its function (cal) have no obvious mutant phenotype, double mutants of AP1 and CAL (ap1 cal) have a much stronger mutant phenotype than ap1 mutants alone, in that cauliflower-like structures composed of proliferating meristems develop (Bowman et al. 1993; Kempin et al., 1995). AP1 and CAL may have originated from an ancestral gene by the α whole genome duplication close to the origin of Brassicaceae roughly about 40 – 50 million years ago (MYA) (Cheng et al., 2013; Franzke et al., 2016).

Ye et al. (2016) wanted to determine the molecular changes that are responsible for the differences of AP1 and CAL in gene expression and function. They identified a number of putative transcription factor binding sites (TFBS) that were gained or lost during evolution, the most striking one being a CArG-box in the promoter region of AP1 but not of CAL. The authors provide several lines of evidence suggesting that this ‘functional’ CArG-box brought AP1 under auto-regulatory and cross-regulatory control by the AP1 protein itself and the CAL protein, respectively. It was probably the gain of this CArG-box and the resulting regulatory control by AP1 and CAL that brought about an up-regulation of AP1 in sepals and petals during relatively late stages of flower development. This change in gene expression was obviously crucial for the functional importance of AP1 in the specification of the identity of these organs, and hence the contribution of AP1 to the A function.

It is then interesting to note that Ye et al. (2016) identified the crucial CArG-box only in two Arabidopsis species, but not in the closely related Capsella rubella or any other Brassicaceae species they investigated. This finding suggests that the CArG-box originated in the lineage that led to extant Arabidopsis, but after the lineage that led to Capsella had already branched-off. This would imply a relatively recent origin of this CArG-box roughly about 10 – 20 MYA. In mutational terms the origin of this cis-regulatory element seems not to have been a big deal; sequence comparisons comprising orthologous sequences from diverse Brassicaceae species suggest that it may have required only very few (possibly even only one or two) point mutations to transform a 10 base pair long precursor sequence into a sequence that is able to bind the MADS-domain proteins AP1 and CAL (Ye et al., 2016).

It is tempting to speculate that the involvement of AP1 in the floral homeotic A function, or even the whole A function as we know it from Arabidopsis thaliana, is thus a relatively recent evolutionary acquisition of the Arabidopsis lineage rather than a conserved feature of angiosperm flower development. This would be very much in line with the difficulties to identify an A function proper outside of Arabidopsis. So if any other species may turn up with AP1-like genes being involved in the specification of perianth organ identity, and hence a floral homeotic A function, they very likely acquired that function independently from the A function in Arabidopsis.

If our hypothesis is right, in Brassicaceae species other than Arabidopsis, an ap1 mutant may have no mutant phenotype, because the AP1 gene should have only a function in specifying floral meristem identity redundant with CAL. However, since in any lineage neo-functionalization cannot be excluded, a less strong prediction would be that the ap1 mutant in these species should at least do not show a class A loss-of-function phenotype. Fortunately, given that genome editing with CRISPR/Cas9 works very well in plants, testing such hypotheses in species such as Capsella rubella is not rocket science anymore.

The recent acquisition of a CArG-box in AP1 may also explain why the loss-of-function phenotype of the floral meristem identity gene LEAFY (LFY) is not as drastic as that of its orthologue in snapdragon, termed FLORICAULA (FLO) (Coen et al., 1990). In Arabidopsis lfy mutant, flowers are converted into leaves plus shoots at lower positions of the inflorescence, but at higher ones, LFY-independent activation of AP1 triggers the development of flower-like structures (albeit without class B gene activity) (Weigel and Meyerowitz, 1993). When both LFY and AP1 are mutated (lfy ap1), these pseudo-flowers disappear, showing that CAL cannot compensate for the loss of AP1 in this case (Huala and Sussex, 1992; Weigel et al., 1992). This strongly suggests that CAL, as opposed to AP1, cannot be induced independently of LFY (Parcy, 2005). The LFY-independent AP1 induction was shown to involve SVP and AGL24 (Grandi et al., 2012), two MADS-domain transcription factors that could also act through the very same CArG-box, although this remains to be directly established (Grandi et al., 2012; Gregis et al., 2013). The presence of this CArG-box could have thus blurred the central role that LFY plays in most species being the prime inductor of AP1. It is also interesting to note that AP1 and CAL, despite born by duplication from an ancestral single LFY target, appear to be induced through different mechanisms. AP1 is induced directly through identified LFY binding sites (Benlloch et al., 2011; Winter et al., 2011; Moyroud et al., 2011) whereas CAL, as opposed to what was originally proposed (William et al., 2004), appear to have no LFY binding site (Minguet et al., 2015; Winter et al., 2011; Moyroud et al., 2011) and is induced by LFY indirectly, through the action of LMI1 (Saddic et al., 2006). The two paralogs thus appear to have evolved by loss and gain of different TFBS.

Acknowledgements

GT thanks Rainer Melzer and Elliot M. Meyerowitz for helpful discussions about the history of the A function.

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