Characterization of the basal angiosperm Aristolochia fimbriata: a potential experimental system for genetic studies

Abstract

Background: Previous studies in basal angiosperms have provided insight into the diversity within the angiosperm lineage and helped to polarize analyses of flowering plant evolution. However, there is still not an experimental system for genetic studies among basal angiosperms to facilitate comparative studies and functional investigation. It would be desirable to identify a basal angiosperm experimental system that possesses many of the features found in existing plant model systems (e.g., Arabidopsis and Oryza).

Results: We have considered all basal angiosperm families for general characteristics important for experimental systems, including availability to the scientific community, growth habit, and membership in a large basal angiosperm group that displays a wide spectrum of phenotypic diversity. Most basal angiosperms are woody or aquatic, thus are not well-suited for large scale cultivation, and were excluded. We further investigated members of Aristolochiaceae for ease of culture, life cycle, genome size, and chromosome number. We demonstrated self-compatibility for Aristolochia elegans and A. fimbriata, and transformation with a GFP reporter construct for Saruma henryi and A. fimbriata. Furthermore, A. fimbriata was easily cultivated with a life cycle of just three months, could be regenerated in a tissue culture system, and had one of the smallest genomes among basal angiosperms. An extensive multi-tissue EST dataset was produced for A. fimbriata that includes over 3.8 million 454 sequence reads.

Conclusions: Aristolochia fimbriata has numerous features that facilitate genetic studies and is suggested as a potential model system for use with a wide variety of technologies. Emerging genetic and genomic tools for A. fimbriata and closely related species can aid the investigation of floral biology, developmental genetics, biochemical pathways important in plant-insect interactions as well as human health, and various other features present in early angiosperms.

Figure 1

Angiosperm phylogeny based on Stevens [9], modified (http://www.mobot.org/MOBOT/research/APweb/welcome.html). Important model systems and the proposed model, Aristolochia fimbriata, are shown next to the corresponding clade. Liriodendron, Persea, Populus, and Carica are tree species. Species that have been used as flower development models are indicated with an asterisk.

Figure 2

Overall approach for selecting a basal angiosperm model system. General criteria are indicated; for full description refer to methods. Taxa eliminated after initial application of each criterion are indicated. Some taxa may have been eliminated for more than one reason. For example, Illicium, in family Illiciaceae, is increasingly available in cultivation, unlike most Austrobaileyales, but was eliminated due to its woody growth habit. Many taxa in Piperales were generally accessible and of amenable growth habit, yet family Lactoridaceae was eliminated due to inaccessibility and woodiness. In genus Aristolochia, subgenus Pararistolochia was eliminated due to large genome size. Basal angiosperm family characteristics and those of Aristolochiaceae species cultivated are described in Tables 1 and 2.

Figure 3

Genome sizes in basal angiosperm families. Bennett and Leitch [38] updated with Cui et al. [39], shown on logarithmic scale. Filled symbols indicate taxa used in The Floral Genome Project (http://www.floralgenome.org) or The Ancestral Angiosperm Genome Project (http://ancangio.uga.edu/), and for which EST resources are available. Compare basal angiosperm genome sizes to Arabidopsis at 125 Mb [40] and Oryza at 389 Mb [41]. The symbol representing Aristolochia is the proposed model Aristolochia fimbriata. Other species of Aristolochia have smaller genome sizes (see Figure 5, Table 4).

Figure 4

Diversity of flower and growth forms in Aristolochiaceae. Herbaceous perennials with radially symmetric 3-merous flowers include A. Asarum chingchengenseB. Saruma henryi C. Thottea siliquosa, a small shrub D. A. arborea (subgenus Isotrema), a tree-like shrub with flowers that mimic fungi E. A. triactina (subgenus Pararistolochia) F. A. trilobata (subgenus Aristolochia) with three lobed, evergreen leaves, grows as a vine with woody branches (liana) from which new growth emerges. G. A. passiflorafolia (subgenus Aristolochia) (photo used with permission from Changbin Chen) and H. A. fimbriata (subgenus Aristolochia).

Figure 5

Phylogenetic relationships among sampled Aristolochiaceae, with Tasmannia lanceolata (Canellales) as outgroup. Maximum parsimony strict consensus tree with genome sizes (Mbp/1C) and chromosome counts indicated. If different genome sizes were obtained from different plants belonging to the same species, the smallest size was plotted on the tree. For range of genome sizes within one species and standard deviation refer to Table 4. Bootstrap values from 1000 replicates are indicated on the branches.

Figure 6

Green fluorescent protein expression in Aristolochiaceae. A, C, E, G, I . Light images. B, D, F, H, J. Fluorescent images. A-D. Leaf explants 10 days after Agrobacterium tumefaciens infection A, B. Saruma henryi C, D. A. fimbriata E, F. Regenerating A. fimbriata stem explant. G, H, I, J. Regenerating A. fimbriata roots (one root is shaded from light source in G). K. Gel image of PCR products; Lane L- 200bp ladder, bright band at 1K bp with corresponding bands at each 200bp; Lane 2- A. fimbriata (WT) DNA; Lane 3- In vitro transformed callus 1; Lane 4- In vitro transformed callus 2; Lane 5- In vitro transformed callus; Lane 6- Negative Control; Lane 7-Plasmid PC (1 ng/ul).

Figure 7

Aristolochia fimbriata. A. Twelve three-year old stock plants maintained in pots (12 cm diameter) occupy 1 m x 1.5 m bench space in the greenhouse B. Plants in use for genetic crosses, seed or tissue collection are trellised C. Close-up of vine showing flowers and floral buds at successive developmental stages on one stem [with arrows].

Figure 8

Aristolochia fimbriata genotype and perianth detail.A, B. VL genotype C, D. NV genotype A, C. Presence, absence of leaf variegation B, D. Perianth varies in shape and color E. Perianth is highly modified for insect pollination. Modifications include limb (li), tube (tu), syrinx (sy), utricle (ut) and gynostemium (gy), which has stamen locules on the outside and interior stigmatic surfaces. Glass model by Leopold and Rudolph Blatschka made near Dresden, Germany illustrated by Fritz Kredel (reproduced with permission).