'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity

Abstract

Background: The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated.

Results: Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics.

Conclusions: Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes.

Figure 1

Flower types of C. vulgaris. A: wild-type (Niederohe from Lueneburger Heide, Germany). B: 'bud-flowering' ('Amethyst'); C: bottom of wild-type (Niederohe from Lueneburger Heide, Germany) flower; Labels are: car (carpels), sta (stamens), ugl (uppermost whorls of green leaves). The bipartites perianth is separated in whorl I and whorl II organs.

Figure 2

Comparative SEM observations of abaxial and adaxial epidermal surface structures of C. vulgaris perianth organs. wild-type whorl I, abaxial (A), adaxial (B); wild-type whorl II, abaxial (C), adaxial (D); 'bud-flowering' whorl I, abaxial (E), adaxial (F); 'bud-flowering' whorl II, abaxial (G), adaxial (H);

Figure 3

Comparative SEM observations of abaxial and adaxial epidermal surface structures of C. vulgaris tissues. wild-type leaf tissue (A), 'bud-flowering' leaf tissue (B); wild-type bracts, abaxial side (C), adaxial side (E); 'bud-flowering' bracts, abaxial side (D), adaxial side (F);

Figure 4

Expression analysis of CvAP3 (A) and CvSEP1 (B) in C. vulgaris flower tissues. Normalized (vs. 18S rRNA) expression is presented for both the wild-type and the 'bud-flowering' type as fold change (ΔΔCt) of arbitrary units vs. the reference tissue (leaf tissue).

Figure 5

Sagittal slices of mature flower buds. A: wild-type phenotype (Niederohe from Lueneburger Heide, Germany): Ca⁴Co⁽⁴⁾A⁸G⁽⁴⁾; B: 'bud-flowering' phenotype ('Anneliese'): Ca⁴⁺⁴Co⁰A⁰G⁽⁴⁾; The label indicates stamens (sta) in the wild-type flower.

Figure 6

Comparative investigation of C. vulgaris flower development. Histological slices of 5 μm intervals were fixed in AFE and stained by FCA. Organs and tissues are labelled by veg (vegetative tissue), br (bracts), sep I or sep II (sepals, whorl no.), pet (petals, if available), sta (stamens, if available), ne (nectaroids), car (carpels), ov (ovules) and me (flower meristem), respectively. A-C: different stages of a wild-type inflorescence; D-E: different stages of a 'bud-flowering' inflorescence;

Figure 7

Mature flowers of C. vulgaris. Histological slices of 8 μm intervals were fixed in Bouin-Allen's compound and stained by SGL. Organs and tissues are labelled by veg (vegetative tissue), br (bracts), sep I or sep II (sepals, whorl no.), pet (petals, if available), sta (stamens, if available), ne (nectaroids), car (carpels), ov (ovules) and me (flower meristem), respectively. A: wild-type; B: 'bud-flowering';