Developmental and molecular characterization of novel staminodes in Aquilegia - PubMed (original) (raw)
Developmental and molecular characterization of novel staminodes in Aquilegia
Clara Meaders et al. Ann Bot. 2020.
Abstract
Background and aims: The ranunculid model system Aquilegia is notable for the presence of a fifth type of floral organ, the staminode, which appears to be the result of sterilization and modification of the two innermost whorls of stamens. Previous studies have found that the genetic basis for the identity of this new organ is the result of sub- and neofunctionalization of floral organ identity gene paralogues; however, we do not know the extent of developmental and molecular divergence between stamens and staminodes.
Methods: We used histological techniques to describe the development of the Aquilegia coerulea 'Origami' staminode relative to the stamen filament. These results have been compared with four other Aquilegia species and the closely related genera Urophysa and Semiaquilegia. As a complement, RNA sequencing has been conducted at two developmental stages to investigate the molecular divergence of the stamen filaments and staminodes in A. coerulea 'Origami'.
Key results: Our developmental study has revealed novel features of staminode development, most notably a physical interaction along the lateral margin of adjacent organs that appears to mediate their adhesion. In addition, patterns of abaxial/adaxial differentiation are observed in staminodes but not stamen filaments, including asymmetric lignification of the adaxial epidermis in the staminodes. The comparative transcriptomics are consistent with the observed lignification of staminodes and indicate that stamen filaments are radialized due to overexpression of adaxial identity, while the staminodes are expanded due to the balanced presence of abaxial identity.
Conclusions: These findings suggest a model in which the novel staminode identity programme interacts with the abaxial/adaxial identity pathways to produce two whorls of laterally expanded organs that are highly differentiated along their abaxial/adaxial axis. While the ecological function of Aquilegia staminodes remains to be determined, these data are consistent with a role in protecting the early carpels from herbivory and/or pathogens.
Keywords: Aquilegia; floral organ identity; novelty; staminode.
© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Figures
Fig. 1.
Aquilegia coerulea ‘Origami’ flowers and staminodes. (A) Aquilegia floral diagram. (B) Staminode sheath comprised of six adhered organs. (C) Flower showing two staminode/stamen chimeras (arrowheads). (D) Stage 11a flower. Top, undissected bud; centre, bud with three sepals, two petals and three orthostichies of stamens removed to reveal staminode whorl; bottom, magnified view of the staminode whorl. (E) Stage 11c flower. Top, undissected flower; centre, flower with three sepals, two petals and three orthostichies of stamens removed to reveal the staminode whorl; bottom, magnified view of the staminode whorl surrounding the carpels. (F) Stage 13b flower. Top, undissected flower; centre, flower with two sepals, three petals and three orthostichies of stamens removed to reveal the staminode whorl; bottom, magnified view of the staminode whorl surrounding the carpels. (G) Early stage 15 flower with split staminode sheath. sep = sepal; pet = petal; sta = stamen; std = staminode; car = carpel. Scale bars: (B, C) = 5 mm, (D) = 1 mm; (E–G) = 1 cm.
Fig. 2.
Aquilegia coerulea ‘Origami’ toluidine blue-stained histological sections from three stages of development. (A, D, G) Transverse sections of stage 11a bud. (B, E, H) Transverse sections of stage 11b bud. (C, F, I) Transverse sections of stage 13b flower. (A–C) Continuous staminodial whorls surround the carpels with stamen filaments visible outside the sheath. (D–F) The same images as (A–C) with original colour removed and the staminodes false coloured to highlight both their identity and their alternate marginal curling behaviour. Antesepalous staminodes are coloured pink, while antepetalous staminodes are coloured blue. (G–I) Magnified views of individual staminodes showing marginal junctions between adjacent staminodes and morphologically distinct stamen filaments from the same orthostichy (arrowheads). Scale bars: (A–F) = 500 µm; (G–I) = 200 µm.
Fig. 3.
Cell counting and AqHIS4 in situ hybridization in A. coerulea ‘Origami’. (A) Transverse section of a stage 13b flower stained by calcofluor white showing an adjacent stamen filament (sta) and staminode (std). (B) The same image from (A) processed without colour. Coloured dots represent cells counted using Image J. Light blue dots are abaxial stamen filament epidermal cells; dark blue, adaxial stamen filament epidermal cells; pink, abaxial staminode epidermal cells; and magenta, adaxial staminode epidermal cells. (C) Cell counts from 20 staminodes and adjacent stamens from six flowers. The four classes of cell counts were found to be statistically differentiated by one-way ANOVA, and Tukey HSD was used to determine how the classes differed from one another. This test identified three distinct classes: abaxial staminode cell counts (a), adaxial staminode cell counts (b) and the two stamen cell counts (c). The two staminode classes are significantly different at P < 0.05 while the staminode vs. the stamen classes were significantly different at P < 0.01. (D) Transverse section of stage 8 bud hybridized with the AqHIS4 probe. Cell divisions, as indicated by AqHIS4 expression, are more common in staminode primordia than in stamen primordia. Carpel primordia are outlined in dashed green lines; stamens in blue dashed lines; and selected staminodes in pink dashed lines. Note that the highlighted staminode primordia represent examples of atypical marginal curling, as discussed in the text. (E) Close-up of stamen primordium (blue outline) and staminode primordium (pink outline). (F) Counts of _AqHIS4_-positive cells in five transverse sections of staminode and stamen primordia from stage 8 flower buds. Staminode cell counts are higher than stamen counts as indicated by Student’s _t_-test at P < 0.001. Scale bars: (A, D) = 200 µm; (E) = 100 µm.
Fig. 4.
Lignin visualization in A. coerulea ‘Origami’. (A) Toluidine blue-stained transverse section of a stage 11b flower bud showing lignification of xylem cells in the staminode and stamen filament (arrowheads), but no evidence in the abaxial (ab) or adaxial (ad) epidermal layers. (B) Transverse section of a staminode from a stage 13 flower showing asymmetric lignification of the adaxial (ad) epidermis, but not the abaxial (ab) epidermis. Lignification is also visible in the xylem (arrowhead). (C) Phloroglucinol staining in fresh staminodes from a stage 13 flower. (D) Lack of phloroglucinol staining in a stamen filament from the same flower as in (A). (E–H) Toluidine blue-stained sections. Left column, light micrograph; centre column, fluorescent micrograph; and right column, merged image. (E) Transverse section of a stage 11b flower. (F) Transverse section of a stage 13b flower. (G) Magnified view of a single staminode from a stage 13b flower. (H) Magnified view of the junction of two adjacent staminodes from a stage 13b flower. The asymmetric lignification does not extend through the region of adherence between two adjacent staminodes (arrow). Scale bars: (A, B) = 1 mm; (C, D) = 10 µm, (D) = 100 µm; (E–E''), (G–G'') = 200 µm; (F–F'') = 500 µm; (H–H'') = 50 μm.
Fig. 5.
Histology of Aquilegia, Semiaquilegia and Urophysa staminodes. (A) A. canadensis. (B) A. coerulea ‘Origami’. (C) A. alpina. (D) A. vulgaris. (E) A. flabellata. (F). S. adoxoides. (G) U. rockii. (A–G) Mature flowers. (A'–G') Transverse sections of the androecium, staminodial whorl and gynoecium, positioned roughly midway between the receptacle and staminode apices of mature flowers. The arrow in F' indicates a solitary staminode. (A''–E'') Magnified view of single staminode showing lignification of the adaxial epidermis. (F'' and G'') Staminodes and stamen filaments (asterisks) of S. adoxoides or U. rockii. No lignification is seen in the staminodes. (A'''–E''') Magnified view of staminode junctions showing adhesion in some cases (B''', E''') but lack of adhesion in others (A''', C''', D'''). Scale bars: (A–E), (G) = 1 cm; (F) = 5 mm; (A'–G') = 500 µm; (A''–G'') = 50 µm; (A'''–E ''') = 200 µm.
Fig. 6.
In situ hybridization of AqFIL in early floral developmental stages of A. coerulea ‘Origami’. (A and B) Stage 3 floral meristems in which the sepals are just emerging. AqFIL is strongly expressed on the abaxial surface of the organs. (C and D) Stage 4–6 buds, as the petal and stamen primordia are initiating. AqFIL is expressed in each arising floral organ. (E) Stage 7 buds, when the carpel primordia (asterisks) appear adjacent to the staminode primordia (arrowheads). (F) Stage 8 buds, during which the floral organ primordia are differentiating. AqFIL expression is restricted to the distal region of the stamens (sta) but is seen throughout the length of the staminodes (arrowheads) as well as the carpels (car). (G) Stage 9 buds, with staminodes (arrowheads) that are clearly differentiated from the stamens (asterisks). AqFIL is still detectable along the abaxial epidermis of the staminodes. (H) Stage 10 floral bud with adjacent stamens and staminode (arrowhead). AqFIL is still detectable along the abaxial epidermis of the staminodes. (I) Stage 7 bud hybridized with a sense probe as a negative control. Arrowheads = staminodes; sta = stamens; pet = petals; asterisks = carpels. Scale bars: (A–D) = 100 µm; (E–I) = 200 µm.
References
- Barcelo AR. 1997. Lignification in plant cell walls. International Review of Cytology 176: 87–132. -PubMed
- Bastida JM, Alcantara JM, Rey PJ, Vargas P, Herrera CM. 2010. Extended phylogeny of Aquilegia: the biogeographical and ecological patterns of two simultaneous but contrasting radiations. Plant Systematics and Evolution 284: 171–185.
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