EcCRC expression is confined to carpels and mature seeds

The RT-PCR experiments I performed on vegetative and reproductive organs of E. californica revealed that EcCRC expression is confined to carpels, but is excluded from all other floral organs, leaves and green seeds (Figure 2A, C). Additionally, EcCRC expression is detected in mature seeds. Moreover, EcCRC is continuously expressed throughout developmental stages as the expression starts in stage 1-5 in floral buds with 0-1 mm in diameter and decreases in floral buds with 3mm in diameter, when female meiosis occurs (stages according to (BECKER

et al. 2005).

To obtain more detailed information on the spatial and temporal expression of EcCRC, I performed in situ hybridization. In stage 5, EcCRC is expressed in the entire gynoecium, which has just initiated (Figure 2B, Publication I). During stage 6, the EcCRC expression changes dynamically. Longitudinal section through floral buds shows that at the beginning of stage 6, the expression of EcCRC is confined to abaxial domains embracing two-thirds of the carpel walls, but is excluded from the most apical and basal carpel regions (Figure 2C, Publication I). Additionally, EcCRC expression domain is present at the centre of the gynoecium base, where the cell division of the floral meristem was terminated just after gynoecium inception in the previous stage. In a cross section, EcCRC expression occurs in two wide strips surrounding the presumptive replum regions of the gynoecium, but without being expressed inside (Figure 2E, Publication I). In addition, EcCRC transcripts are distributed uniformly in the carpel walls. Longitudinal sections through flowers of stage 6 show that the EcCRC expression in the carpel walls loses its abaxial character and expands

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into the entire gynoecium, while remaining further excluded from its apical part (Figure 2D, Publication I). In longitudinal view of the gynoecium at stage 7, EcCRC expression is apparent as abaxial slender streaks along the carpel walls, enclosing the presumptive replum and placenta, but without being expressed in there (Figure 2F, Publication I). Moreover, the domain of expression at the gynoecium base is maintained in a small group of cells. After ovule initiation, the EcCRC hybridization signal is detected in abaxial domains along the carpel walls enveloping the placenta and presumptive replum (Figure 2G, H, I, Publication I).

EcCRC expression was not detected in the carpel margins, placenta, replum and ovules at any of the examined developmental stages.

The early carpel expression seems to be characteristic for CRC orthologs, suggesting that CRC-like genes control the establishment of carpel features since carpel inception (Figure 4).

I detected initial expression of EcCRC in the just initiated gynoecium at stage 5. Similar to E.

californica, in A. thaliana, the gynoecium also develops at around stage 5 (stages according to (BOWMAN and SMYTH 1999b; SMYTH et al. 1990). CRC expression is firstly detected at stage 6, showing that conceivably EcCRC is required earlier in the carpel development than CRC (Figure 4). Expression of CRC orthologs in the centre of the floral meristem before carpel inception has been reported for AfCRC, the CRC ortholog in the Ranunculales species A.

formosa (LEE et al. 2005d). Besides EcCRC, AfCRC is the only other basal eudicot CRC orthologous gene, on which expression data, although incomplete, is available (Figure 4). The expression in the floral meristem seems to be characteristic also for CRC-like genes in monocot grasses. Such expression is reported for DLin O. sativa, the first identified monocot CRC ortholog, and recently also for DL-like genes within three further grass species, Z. mays, Triticum aestivum (T. aestivum, wheat) and Sorghum bicolor (S. bicolor, sorghum) (ISHIKAWA et al. 2009; YAMAGUCHI et al. 2004) (Figure 4).

Abaxial carpel expression

The abaxial expression of EcCRC in the gynoecium wall of E. californica resembles the expression of CRC-like genes across eudicots (Figure 4). Such expression pattern has been reported for CRC orthologs in the core eudicot species A. thaliana and P. hybrida (BOWMAN

and SMYTH 1999b; LEE et al. 2005a). Abaxial carpel expression has been demonstrated also for AfCRC (Figure 4). In mature flowers, AfCRC is expressed abaxially around the central vascular bundle of the carpel (LEE et al. 2005b). Abaxial expression in the gynoecium is also reported for A. trichopoda, considered to be the earliest diverged angiosperm species,

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indicating that very likely, the abaxial expression of CRC-like genes has developed already in the lineage leading to A. trichopoda (FOURQUIN et al. 2005). Furthermore, the abaxial pattern of expression seems to be characteristic for the ancestral CRC gene and suggests an ancestral function of CRC-like genes in elaboration of abaxial cell fate in the gynoecium wall. The characteristic abaxial expression is independently lost only in grasses, where the DL genes are expressed uniformly in the entire carpel (ISHIKAWA et al. 2009; YAMAGUCHI et al. 2004). In contrast, the expression of the CRC ortholog in the non-grass monocot A. asparagoides, AaDL, resembles rather the expression of CRC-like genes in eudicots than the ones in monocot grasses as this persists only in the abaxial gynoecium wall (Figure 4). This indicates that CRC orthologs acquired ubiquitous carpel expression only within grasses after their split from non-grasses (NAKAYAMA et al. 2010). Another possibility is that the abaxial expression has been remained only within the Asparagus lineage, which branched off earlier than Poaceae within monocots, but has been lost in grass monocots. The differential expression of CRC-like genes in grasses shows that they might have acquired an additional function in establishment of the adaxial carpel wall in difference to the eudicot CRC orthologs, which function only in the abaxial tissue differentiation.

Apical carpel expression

In contrast to CRC homologs in core eudicots, monocots and even in A. formosa, which are expressed continuously in the apical region of the gynoecium, EcCRC is not expressed there (BOWMAN and SMYTH 1999b; ISHIKAWA et al. 2009; LEE et al. 2005c; NAKAYAMA et al.

2010; YAMAGUCHI et al. 2004) (Figure 4). Therefore, EcCRC possibly does not control carpel fusion in E. californica pointing out a functional diversification of the E. californica CRC ortholog from the other CRC-like genes in carpel fusion. It is possible that the apical domain of expression has been lost in the members of Papaveraceae or only in the lineage leading to Eschscholzia. Due to the lack of expression data on CRC-like genes outside of E. californica and A. formosa, both scenarios seems to be plausible.

Adaxial carpel expression

EcCRC is expressed uniformly in the carpel walls, comprising also the adaxial regions, in stage 6 (Figure 2E, Publication I). Also CRC is expressed in adaxial domains at stage 7, but these comprise only the outermost adaxial cell layer of the carpels (BOWMAN and SMYTH

1999a) (Figure 4). Adaxial internal domains of AaDL expression within the carpel walls, similar to the ones reported for CRC, persists in later developmental stages of the monocot A.

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asparagoides (NAKAYAMA et al. 2010) (Figure 4). This puts forward that such temporal adaxial expression might be acquired independently in some eudicot and monocot species.

Placenta expression

We did not observed EcCRC expression in the placenta at any of the developmental stages analysed with in situ hybridization. Placental expression is reported for PhCRC in P. hybrida, but not for CRC (BOWMAN and SMYTH 1999b; LEE et al. 2005a) (Figure 4). It was hypothesized that the pattern of placentation determines the timing of meristem termination (COLOMBO et al. 2008). In E. californica and A. thaliana, the placenta develops from the inner ovary wall. In contrast, in P. hybrida, the placenta originates from the central part of the floral meristem, which in difference to E. californica and A. thaliana is not terminated after gynoecium inception (ANGENENT et al. 1995; COLOMBO et al. 2008). This might explain the absence of placenta expression of CRC orthologs in E. californica and A. thaliana in comparison to P. hybrida.

Replum expression

We also did not observe EcCRC expression in the replum (Figure 4). Replum expression has been reported only for CRC and it might have been acquired independently in the lineage leading to A. thaliana (BOWMAN and SMYTH 1999a).

Carpel margin expression

In the gynoecium at stage 6, EcCRC expression occurs in two distinct stripes along the lateral carpel margins (Figure 4). Similar expression has been reported for CRC in flowers at stage 6, suggesting that both EcCRC and CRC function in the establishment of the lateral carpel margins (BOWMAN and SMYTH 1999b).

Ovule expression

EcCRC, similar to other reported CRC-like genes across eudicots and monocots, is not expressed in the ovules (BOWMAN and SMYTH 1999a; LEE et al. 2005b; NAKAYAMA et al.

2010; YAMAGUCHI et al. 2004). In E. californica, A. thaliana and A. asparagoides, the ovules develop from the placenta, whereas in O. sativa, the ovules arise directly from the floral meristem (BOWMAN and SMYTH 1999a; ITOH et al. 2005; NAKAYAMA et al. 2010).

45 Mature seeds’ expression

The EcCRC expression in mature seeds may hint to a function of EcCRC in late embryogenesis or seed maturation, but such expression has not been reported for any other CRC-like gene.

Nectary expression

Expression in the nectary seems to be restricted to core eudicots, because such expression has been reported only for CRC orthologs in core eudicots (BOWMAN and SMYTH 1999a; LEE et al. 2005b) (Figure 4). CRC homologs are not expressed in the nectaries either in the basal eudicot A. formosa or in the monocot A. asparagoides. E. californica does not develop nectaries. This let assuming that the nectary expression arose independently only in the core eudicot lineage after it diverged from basal eudicots and monocots (FOURQUIN et al. 2005;

LEE et al. 2005b).

Leaf expression

Our RT-PCR experiments did not reveal expression of EcCRC in leaves of E. californica.

Such expression is reported only for DL genes in monocots (ISHIKAWA et al. 2009;

NAKAYAMA et al. 2010; YAMAGUCHI et al. 2004) (Figure 4). This indicates that the DL genes might have acquired additional expression in leaves independently of eudicots.

In summary, the EcCRC expression patterns change dynamically throughout developmental stages. EcCRC exhibit the conserved expression of CRC-like genes outside of grasses in the abaxial gynoecium wall, but in addition shows a unique expression domain at the base of the gynoecium, which is not reported for any other CRC ortholog. Furthermore, EcCRC expression is excluded from the apical region of the gynoecium, in contrast to the rest of the eudicot CRC orthologs, suggesting that EcCRC does not function there. The EcCRC expression patterns put forward that EcCRC shares the conserved function of core eudicot CRC orthologs in establishment the abaxial polarity of the gynoecium, and may function in floral meristem. Furthermore, the EcCRC expression patterns illustrate the dynamic nature of CRC-like gene expression across angiosperm lineages, and particularly in E. californica.

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Figure 4 Schematic diagram showing a simplified phylogeny of the major angiosperm lineages (left side) and summarizing the expression patterns of CRC orthologs (right side) as well as the gain and loss in CRC-like gene function (left side) across angiosperms.

Symbols represent a gain and a loss of function of CRC orthologs in different angiosperm lineages. Mapping of CRC-like gene function in the angiosperm phylogeny tree is restricted to those CRC-like genes, for which functional data is available. Flower developmental stages are described only for A. thaliana, E. californica and P. hybrida

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(ANGENENT et al. 1995; BECKER et al. 2005; SMYTH et al. 1990).

Abbreviations: ab, abaxial; ad, adaxial; ca, carpel; cap, carpel primordium; caw, carpel wall;

cvb, central vascular bundle; fm, floral meristem; gp, gynoecia primordium le, leaf; ne, nectary; ov, ovule; r, replum; pe, petal; pl, placenta; st, stamen.

3.1.1.2 EcCRC functions in floral meristem determinacy, gynoecium differentiation and