The gynostemium: More than the sum of its parts with emerging floral complexities

The gynostemium: More than the sum of its parts with emerging floral complexities Natalia Pabón-Mora 1 and Favio González 2 Abstract Partial or complete floral organ fusion, which occurs in most angiosperm lineages, promotes integration of whorls leading to specialization and complexity. One of the most remarkable floral organ fusions occurs in the gynostemium, a highly specialized structure formed by the congenital fusion of the androecium and the upper portion of the gynoecium. Here we review the gynostemia evolution across flowering plants, the morphological requirements for the synorganization of the two fertile floral whorls, and the molecular basis most likely responsible for such intimate fusion process. Addresses 1 Universidad de Antioquia, Instituto de Biología, Medellín, Colombia 2 Universidad Nacional de Colombia, Facultad de Ciencias, Instituto de Ciencias Naturales, Sede Bogotá, Colombia Corresponding author: Pabón-Mora, Natalia ([email protected]. co) Current Opinion in Plant Biology 2024, 81:102609 This review comes from a themed issue on Growth and development 2024 Edited by Madelaine Bartlett and Annis Richardson For complete overview of the section, please refer the article collection - Growth and development 2024 Available online 30 July 2024 https://doi.org/10.1016/j.pbi.2024.102609 1369-5266/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http:// creativecommons.org/licenses/by-nc/4.0/). Where do true gynostemia occur? A true gynostemium (i.e. the congenital fusion between the stamens and the free styles and/or stigmas) has independently evolved only a few times across the angiosperms, once in the magnoliid genus Aristolochia (Aristolochiaceae) (Figure 1;[1]), and three times in the monocots, once in Corsia (Corsiaceae), once in Pauridia (Hypoxidaceae), and once in the lineage encompassing all Orchidaceae (Figure 2;[2]). In Aristolochia, the gynostemium is a crown-like structure formed by 5 or 6 (rarely more) sessile extrorse anthers, which congenitally fuse to 3, 5, 6 or rarely more commissural stigmatic lobes (Figure 1met; [1,3,4]). Whereas the number of anthers and stigmatic lobes is the same in most Aristolochia species (i.e. those of the subgenera Aristolochia and Pararistolochia), the six anthers are paired and fused to three stigmatic lobes in the species of subgenus Siphisia, which indicates that the fusion itself is independent from the number of primordia involved. In Corsia, the short gynostemium is the result of the fusion between the filaments of six fertile stamens (with extrorse anthers) and the style for about half of their length [2]. In Pauridia, the gynostemium occurs through the fusion of the three outer sterile stamens and the style, forming the so-called “stylar appendages” [2,5], whereas the inner fertile stamens attach to the perianth tube [2,5,6]. In all Orchidaceae, the gynostemium is formed by the staminal whorls and the stigmatic tips. However, there is considerable variation across subfamilies [7]: In the early diverging Apostasioideae, it is formed by the median outer stamen, two inner lateral stamens and the stigmas [8]; in Cypripedioideae, it is formed by the fusion of the two inner lateral stamens and the stigmas [7]; in the remaining subfamilies (Epidendroideae, Orchidoideae and Vanilloideae) the gynostemium is the result of the fusion of the single median outer stamen and the stigmas (Figure 2; [7,9e12]). At least in Orchidoideae and Epidendroideae, the two lateral outer whorl stamens often exist as sterile appendages in the gynostemium [13]. At first glance, the fusion of the floral fertile whorls would promote self-fertilization. However, asynchronous organ maturation (i.e. protandry or protogyny) appears to avoid autogamy except probably in Pauridia [14]. The flowers of Aristolochia are protogynous, where stigmas are receptive prior to anther dehiscence (Figure 1o, p; [1e3,15]). In Corsia and other Corsiaceae, flowers are protandrous [16]. Finally, with over 27.000 orchid species, it is hard to identify a single pattern for the timing of sexual organ maturation. Although self-pollination occurs in many orchids with simultaneous maturation of stamens and carpels [17,18], cross-pollination mediated by protogyny or proterandry is by far more frequent [19e21]. The orchid gynostemium often undergoes subtle position shifts over time, curving forward and backward. This could be a physical constraint to avoid immediate self-pollination, as pollen massulae (pollinia) mature spatially compartmentalized, away from the same flower stigmatic surface (Figure 2;[19,22]). Available online at www.sciencedirect.com ScienceDirect Current Opinion in Plant Biology www.sciencedirect.com Current Opinion in Plant Biology 2024, 81:102609 Figure 1 Floral morphology of representative Aristolochiaceae. (a–h) Asarum canadense;(a) frontal view of a flower in anthesis; (b–d) successive female and male floral phases. (e–g) successive stages in young floral buds showing free stamens which are not fused to the adjacent stigmas; . (h) transverse section of a floral bud; note incipient stamen/carpel fusion; (i–l) Thottea siliquosa;(i) frontal view of a flower at anthesis. (j–k) successive female (j) and male (k) floral phases; note incipient fusion of the base of stamens with stigmatic lobes; (l) transverse section of a flower; (m–t) Aristolochia fimbriata;(m) frontal view of a flower at anthesis; (n) sagittal section of flower; note the position of the gynostemium at the base of the utricle; (o–p) successive female (o) and male (p) phases of gynostemium (q–s) successive stages of a forming gynostemium showing six stamens fused to six commissural stigmatic lobes; (t) transverse section of a floral bud. Arrowheads point to stamens; asterisks point to stigmatic (commissural) lobes. Arrows indicate the medial carpellary vascular bundle. Abbreviation: O, inferior ovary. 2Growth and development 2024 Current Opinion in Plant Biology 2024, 81:102609 www.sciencedirect.com Outside magnoliids and Monocots, adnation of stamens and stigmas has been previously reported and referred toasa“column”orevena“gynostemium”inmembers of the Stylidoideae (Stylidiaceae: Asterales). However, no true congenital fusion between stamens and stigmasoccursinthisfamily[23], which is reasonable as the proterandrous flowers undergo secondary pollen presentation that requires displacement of the style/stigma free from the androecialderived tube [24]. Figure 2 Telipogon nervosus (Orchidaceae: Epidendroideae) as an example of the most frequent type of gynostemium formed by a single fertile outer stamen and three stigmas. (a) frontal and (b) lateral view of a flower at anthesis, (c) detail of the gynostemium opposite the labellum, (d–f) successive stages of young floral buds showing stamens and stigmas congenitally fused, (g) Transverse section of the fertile stamen with four pollinia and the medial stigmatic lobe (to the right). (h) mature gynostemium; (i) transverse section of a mature gynostemium below the level of the stigmatic surface; (j–m) mature pollinarium; (j) lateral view of a mature pollinarium prior to anther cap dehiscence; (k) transverse section of the medial stigmatic tip (rostellum) and the stipe forming tissues and separation layer; (l) mature pollinarium ready for dispersal (bristles removed); (m) transverse section of pollen massulae from a single theca as dispersed. Arrowheads point to stamens; asterisks point to stigmatic tips. Abbreviations: A1, medial outer stamen; a1, a2, lateral inner stamens;l, labellum; ip, inner pollinia; o, inferior ovary; op, outer pollinia; r, rostellum; s, stipe; sl, separation layer; v, viscidium. The gynostemium Pabón-Mora and González 3 www.sciencedirect.com Current Opinion in Plant Biology 2024, 81:102609 What are the requirements to form a gynostemium? The gynostemium is a homoplasious trait, although no reversals seem to occur once it is evolutionarily fixed. Nevertheless, in all cases several structural conditions are required for the formation of a gynostemium: 1) high floral synorganization; 2) perianth organs partially or completely fused, or tightly appressed to stamens and stigmas; 3) sessile anthers; and 4) inferior ovary. We will examine in detail the gynostemium in the two taxa in which it has been evolutionarily fixed in all the species, namely, the genus Aristolochia (with ca. 650 spp.) and the family Orchidaceae (with over 27.000 spp.) As currently circumscribed, the perianth-bearing Piperales include the families Asaraceae, Aristolochiaceae, Hydnoraceae and Lactoridaceae [25]. Only species in the genus Aristolochia exhibit a gynostemium, contrasting with the free stamens and stigmas found in Asarum,Lactoris,Hydnora,Prosopanche, and Saruma (Figure 1aeh). A transitional stage occurs in the genus Thottea, which exhibits an incipient proximal fusion between stamens and stigmas (Figure 1iel). All perianth bearing Piperales possess an inferior or semi-inferior ovary and highly synorganized flowers with a uniseriate perianth composed of sepals (Figure 1;[1,3,4,26]), with the noticeable exception of the monospecific Saruma. However, only the Aristolochia perianth shows complete fusion and bilateral symmetry versus partial fusion and radial symmetry in the remaining genera (Figure 1m). Finally, pollination in Aristolochia is by far the most specialized as insects (mostly flies) are attracted to the kettle-like shaped trap flowers. Here, they get temporarily sequestered during the female phase of the gynostemium, allowing pollen deposition carried by the insect from another flower. Once pollination has been secured, the gynostemium enters the male phase, when anther dehiscence occurs allowing new pollen to be deposited on the insect (Figure 1mep; [15,27]). Conversely, a gynostemium is thought to have been in place in the ancestor of Orchidaceae [2,8]. Orchids are sister to all other Asparagales [28], yet similar congenital fusions or even transitional stages are not found elsewhere in the order, perhaps with the exception of Pauridia [2]. The orchid gynostemium is a unique and extraordinarily complex pollination apparatus that incorporates fertile stamens, lateral staminodia, and a strongly zygomorphic upper region of the gynoecium. The median stigma lobe grows faster and usually protrudes above and further out, while the two lateral stigmas remain below (Figure 2;[29e32]). Among the three stigmas, a broad receptive disc is formed for the landing of pollinia (Figure 2). The most exquisite elaboration occurs in monandrous orchids, in which the single fertile stamen is fused to the medial stigmatic lobe, and together, they protrude forming the rostellum, a structure responsible for the formation of the pollinarium. The latter is a compound pollen dispersal unit that corresponds (from proximal to distal) to the four (or 2, or 8) pollinia per flower, their common stalk (stipe or caudicle), and their sticky pad (viscidium), which directly attaches to the pollinator (Figure 2;[30e32]). What do we know about the identity of the gynostemium parts? As the gynostemium is the result of congenital fusion between stamens and stigmas that crown an inferior ovary, two sets of genes can help to better comprehend how such fusion occurs without compromising organ identity. On one hand, given that fusion of primordia from adjacent floral whorls distorts boundaries among organs, boundary gene regulation might be critical to assess gynostemia development. On the other hand, floral organ identity and gynoecium patterning genes could help to identify the androecial versus the gynoecial identity in this compound structure. We will analyze both sets of genes below. Stamen and carpel patterning genes The ABCE model of floral organ identity genes, identified primarily in the model Arabidopsis thaliana, establishes that a specific expression and interaction of MADS-box transcription factors controls the identity of each of the four floral whorls. Specifically, the combination of B, C, and E class genes results in stamen identity, while the C and E class gene combination promotes carpel identity [33e37]. The expression of all floral organ identity MADS-box Aristolochia homologs has been carefully assessed in Aristolochia fimbriata. In this species, the expression of the B-class homologs PISTILLATA (PI) and APETALA3 (AP3) is, in fact, restricted to the staminal region of the gynostemium; however, AfimPI is transiently expressed also early on in the young stigmatic tips (Figure 3;[3]). The expression of AGAMOUS (AG) is only restricted to the ovary, and does not reach the stigmatic region of the gynostemium (Figure 3;[4]). This already points to an alternative genetic control of the stigmatic lobe formation, suggesting that they could correspond either to massive filaments, or to de novo structures that do not share genetic identity with either true stamens or carpels. An additional scenario emerges from the possibility that in situ RNA hybridization is less efficient in highly vacuolated lipid-rich or sugar-rich tissues, and perhaps, alternative experiments should be used to establish gene transcription throughout the gynostemium. In Arabidopsis, downstream of AGAMOUS (AG), other genes are turned on to shape the specific carpel regions and tissues. SPATULA (SPT), a bHLH transcription factor, and CRABS CLAW (CRC), a YABBY homolog, are key factors to establish basal-apical patterning in the gynoecium [38e40]. CRC is a direct target of AG and 4Growth and development 2024 Current Opinion in Plant Biology 2024, 81:102609 www.sciencedirect.com controls carpel growth, style development and apical closure [38e40]. SPT is critical for style and stigma formation and to the development of the medial tissue of the gynoecium [41]. At later stages in Arabidopsis gynoecium patterning, HECATE (HEC) is essential for style development and the subsequent formation of the transmitting tissue [42]. Finally, NGATHA (NGA) genes, belonging to the RAV clade of the B3-domain transcription factor family, also contribute to style and stigma development and proper carpel fusion [43,44]. Expression of carpel patterning homologs in A. fimbriata points to broader roles of AfimCRC,AfimHEC,AfimNGA and AfimSPT in stamen development, ovary wall identity, and style patterning ([45]; Figure 3). However, only AfimHEC,AfimNGA and AfimSPT, are transiently expressed in forming stigmatic lobes, but their expression is lost in fully formed gynostemia [45]. The data suggest that stigmas in gynostemia do not retain the expression of canonical gynoecium patterning genes ([45]; Figure 3). Boundary genes Several genes are critical for the separation of organ primordia, and most of them maintain the boundaries between organs (reviewed in the study by Aida et al. [46]). We will revise only those whose mutation results Figure 3 Summary of gene expression of B and C class MADS box organ identity genes and gynoecium patterning genes in Aristolochia fimbriata.(a) SEM micrograph of young gynostemium and portion of the inferior ovary as reference for figs. b–h.(b–h) in situ hybridization expression patterns of PISTILLATA (PI)(b),APETALA3 (AP3)(c),AGAMOUS (AG)(d),CRABS CLAW (CRC)(e),SPATULA (SPT)(f),HECATE 3 (HEC3)(g), and NGATHA (NGA)(h). Arrowheads point to stamens; asterisks point to the stigmatic (commissural) lobes. Abbreviations: a, anthers; o, inferior ovary. The gynostemium Pabón-Mora and González 5 www.sciencedirect.com Current Opinion in Plant Biology 2024, 81:102609 in fusion between the androecial and gynoecial whorls in the model A. thaliana. It is worth noting, however, that none of the mutant phenotypes characterized so far resembles a true gynostemium in A. thaliana, as expected, given that their flowers have a superior ovary. Boundary genes that play critical roles in establishing free stamens and distinct androecial versus gynoecial whorls are the CUP-SHAPED COTYLEDON (CUC)homologs, CUC1 and CUC2 [47]. In cuc1/cuc2 double mutants, some stamens resulted fused up to the anthers or at least halfway through the filaments, and in some cases, stamens were fused to carpels [47]. SUPERMAN, a C2H2-type Zinc finger transcriptional repressor, also reinforces the limit between stamens and carpels. SUP acts to repress B-class genes, and stamen identity in the fourth whorl, in fact, the sup-1 mutants exhibit supernumerary stamens in the position of carpels [48]. Interestingly, the study of a sup allelic series indicates that SUP also functions in floral meristem termination by repressing the activity of WUSCHEL (WUS), and in establishing the male-female boundary specification mechanism [49]. The floral meristem maintenance homeostasis loops have a number of players repressing WUS [50], like the mobile small peptide CLAVATA3 and its receptor CLAVATA1 [50]. This is the most plausible reason to explain why sup/clv double mutants exhibit strong stamen-carpel fusion, with strong expansion of apical stigmatic tissues. These phenotypes closely resemble those occurring during the formation of a gynostemium, suggesting that in addition to fusion, gynostemium formation may require accompanying gynoecial apical expansion [49]. Finally, it is worth noting that C2H2 Zn-finger factors are very diverse, and that other members in the family, such as RABBIT EARS (RBE) and KNUCKES (KNU) have been linked to the formation of the petalestamen boundaries and the carpel-placenta boundaries, respectively [51,52]. However, it is unknown how these genes have evolved during angiosperm evolution and how the genetic complements have changed in model taxa, such as Arabidopsis, as compared to non-core eudicots, such as our case studies Aristolochiaceae and Orchidaceae. Future research avenues Partial and total congenital stamen/carpel fusion is rare in angiosperms; however, once it arises, no reversals occur. Because profound morpho-anatomical specializations occur when a gynostemium is formed, it is unlikely that this compound structure is the result of the action of a single gene; rather, it would have to be caused by the coordinated action of various genes involved in identity, boundaries, ovary position, and the floral meristem termination pathway. Expression patterns of more candidate genes should be tested in selected species of Aristolochiaceae and Orchidaceae to assess their roles in the development and evolution of such remarkable structure. In parallel, comparative transcriptomic analyses may shed light into new candidate genes underlying fusion and elaboration in the gynostemium. Emerging models, such as those in Aristolochiaceae and Orchidaceae, are key to test gene function and evolution of gynostemia in this remarkable floral convergence. Declaration of competing interest Authors declare that they do not have competing financial interests or personal relationships that have influenced the work reported here. Data availability All data is available under the creative commons agreement. Acknowledgments We thank Madelaine Bartlett and Annis Richardson for the invitation to contribute to this special issue. We thank Paula Rudall (Royal Botanic Gardens, Kew) and an anonymous reviewer for valuable comments on the final version of the manuscript. We would like to thank the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 101007738 Evofruland for funding. Glossary Gynostegium complex structure as a result of postgenital fusion between stamens and stigmas Gynostemium complex structure as a result of congenital fusion between stamens and stigmas Protandry maturation and dehiscence of stamens prior to the receptive phase of the stigma in the same flower Protogyny maturation and dehiscence of stamens after the receptive phase of the stigma in the same flower Synorganization coordinate elaboration of floral whorls resulting in an atypical, yet highly efficient and evolutionarily fixed, floral bauplan. It involves symmetry shifts in one or more whorls, congenital or postgenital fusion events, heterotopy and/or secondary displacement of floral parts References Papers of particular interest, published within the period of review, have been highlighted as: * of special interest * * of outstanding interest 1. González F, Stevenson DW: Gynostemium development in Aristolochia (Aristolochiaceae).Bot. Jahrb. 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