Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence of Aristolochia fimbriata, a species that, similar to Amborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, the A. fimbriata genome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons between A. fimbriata and other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology in A. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids in A. fimbriata.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

Etiology of Balkan Endemic Nephropathy: An Update on Aristolochic Acids Exposure Mechanisms.

Aristolochic acid released from decaying Aristolochia clematitis weed is contaminating soil and food crops in Eastern Europe and is one of the major causes to Balkan endemic nephropathy. Measures should be taken to prevent people from being exposed to these highly potent phytotoxins. Research needs to develop remediation methods.

Balkan endemic nephropathy: an update on its aetiology.

Balkan endemic nephropathy (BEN) is a unique, chronic renal disease frequently associated with upper urothelial cancer (UUC). It only affects residents of specific farming villages located along tributaries of the Danube River in Bosnia-Herzegovina, Croatia, Macedonia, Serbia, Bulgaria, and Romania where it is estimated that ~100,000 individuals are at risk of BEN, while ~25,000 have the disease. This review summarises current findings on the aetiology of BEN. Over the last 50 years, several hypotheses on the cause of BEN have been formulated, including mycotoxins, heavy metals, viruses, and trace-element insufficiencies. However, recent molecular epidemiological studies provide a strong case that chronic dietary exposure to aristolochic acid (AA) a principal component of Aristolochia clematitis which grows as a weed in the wheat fields of the endemic regions is the cause of BEN and associated UUC. One of the still enigmatic features of BEN that need to be resolved is why the prevalence of BEN is only 3-7 %. This suggests that individual genetic susceptibilities to AA exist in humans. In fact dietary ingestion of AA along with individual genetic susceptibility provides a scenario that plausibly can explain all the peculiarities of BEN such as geographical distribution and high risk of urothelial cancer. For the countries harbouring BEN implementing public health measures to avoid AA exposure is of the utmost importance because this seems to be the best way to eradicate this once mysterious disease to which the residents of BEN villages have been completely and utterly at mercy for so long.

Fatty Acids Composition and Antibacterial Activity of Aristolochia longa L. and Bryonia dioïca Jacq. Growing Wild in Tunisia.

The composition of the fatty acids of the roots and aerial parts of Aritolochia longa (Aristolacheae) and Bryonia dioïca (Cucurbutaceae) was analyzed by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The oils extracted from the aerial parts of both species were rich in polyunsaturated fatty acids with the essential linolenic and linoleic acids being the most prominent compounds. Oleic and linoleic acids were the majors fatty acids in the roots of both species. Whatever the plant part analyzed and the species, the saturated fatty acids were predominantly composed of palmitic and stearic acids. The antibacterial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the lipid extracts were determined against a panel of five bacterial strains. The results showed that the sensitivity to the lipid extracts was different for the test bacterial strains, and the susceptibility of gram positive bacteria was found to be greater than gram negative bacteria. The antibacterial activity of the root lipid extracts was particularly important against Enterococcus feacium (CMI value of 125 µg/mL; CMB values > 250 µg/mL) and Streptococcus agalactiae (CMI value of 125 µg/mL; CMB values 250 µg/mL for A. longa roots). These results indicate that A. longa and B. dioïca could be considered as good sources of essential fatty acids which can act as natural antibacterial agents.

Time-scale dependency of host plant biomass- and trait-mediated indirect effects of deer herbivory on a swallowtail butterfly.

Despite recent attempts to quantify the relative strength of density- and trait-mediated indirect effects, rarely has the issue been properly addressed at the population level. Most research is based on short-term small-scale experiments in which behavioural and/or physiological responses prevail. Here, we estimated the time-scales during which density- and trait-mediated effects manifest, as well as the strength of these effects, using an interaction chain with three organisms (deer-plant-butterfly). A hierarchical Bayesian model was performed by using a long-term data set of deer density in the Boso Peninsula, central Japan (where local densities differ spatially and temporally) as well as densities of the swallowtail butterfly Byasa alcinous and its host plant Aristolochia kaempferi. The time-scale effect of deer on plant quantity and quality was estimated according to the degree of carry-over effects. The negative influence on leaf density showed a temporal saturation pattern over the long term, while the positive influence on leaf quality due to resprouting of leaves after deer browsing showed no clear temporal trend. The net indirect effect changed from positive to negative with time, with the negative density-mediated effect becoming prominent in the long term. Our novel approach is widely applicable in assessing the dynamic impacts of wildlife if the spatio-temporal variability of expansion and/or invasion history is known.

Diurnal pattern of stomatal conductance in the large-leaved temperate liana Aristolochia macrophylla depends on spatial position within the leaf lamina.

BACKGROUND AND AIMS: The large distance between peripheral leaf regions and the petiole in large leaves is expected to cause stronger negative water potentials at the leaf apex and marginal zones compared with more central or basal leaf regions. Leaf zone-specific differences in water supply and/or gas exchange may therefore be anticipated. In this study, an investigation was made to see whether zonal differences in gas exchange regulation can be detected in large leaves. METHODS: The diurnal course of stomatal conductance, gs, was monitored at defined lamina zones during two consecutive vegetation periods in the liana Aristolochia macrophylla that has large leaves. Local climate and stem water potential were also monitored to include parameters involved in stomatal response. Additionally, leaf zonal vein densities were measured to assess possible trends in local hydraulic supply. KEY RESULTS: It was found that the diurnal pattern of gs depends on the position within a leaf in A. macrophylla. The highest values during the early morning were shown by the apical region, with subsequent decline later in the morning and a further gradual decline towards the evening. The diurnal pattern of gs at the marginal regions was similar to that of the leaf tip but showed a time lag of about 1 h. At the leaf base, the diurnal pattern of gs was similar to that of the margins but with lower maximum gs. At the the leaf centre regions, gs tended to show quite constant moderate values during most of the day. Densities of minor veins were lower at the margin and tip compared with the centre and base. CONCLUSIONS: Gas exchange regulation appears to be zone specific in A. macrophylla leaves. It is suggested that the spatial-diurnal pattern of gs expressed by A. macrophylla leaves represents a strategy to prevent leaf zonal water stress and subsequent vein embolism.

Visualization of embolism formation in the xylem of liana stems using neutron radiography.

BACKGROUND AND AIMS: Cold neutron radiography was applied to directly observe embolism in conduits of liana stems with the aim to evaluate the suitability of this method for studying embolism formation and repair. Potential advantages of this method are a principally non-invasive imaging approach with low energy dose compared with synchrotron X-ray radiation, a good spatial and temporal resolution, and the possibility to observe the entire volume of stem portions with a length of several centimetres at one time. METHODS: Complete and cut stems of Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata were radiographed at the neutron imaging facility CONRAD at the Helmholtz-Zentrum Berlin für Materialien und Energie, with each measurement cycle lasting several hours. Low attenuation gas spaces were separated from the high attenuation (water-containing) plant tissue using image processing. KEY RESULTS: Severe cuts into the stem were necessary to induce embolism. The formation and temporal course of an embolism event could then be successfully observed in individual conduits. It was found that complete emptying of a vessel with a diameter of 100 µm required a time interval of 4 min. Furthermore, dehydration of the whole stem section could be monitored via decreasing attenuation of the neutrons. CONCLUSIONS: The results suggest that cold neutron radiography represents a useful tool for studying water relations in plant stems that has the potential to complement other non-invasive methods.

Escaping the lianoid habit: evolution of shrub-like growth forms in Aristolochia subgenus Isotrema (Aristolochiaceae).

PREMISE OF THE STUDY: A large range of growth forms is a notable aspect of angiosperm diversity and arguably a key element of their success. However, few studies within a phylogenetic context have explored how anatomical, developmental, and biomechanical traits are linked with growth form evolution. Aristolochia (∼500 species) consists predominantly of climbers, but a handful of shrub-like species are known from Aristolochia subgenus Isotrema (hereafter, shortened to Isotrema). We test hypotheses proposing that the establishment of functional traits linked to lianescence might limit the ability to evolve structurally diverse growth forms, particularly self-supporting forms. • METHODS: We focus on the origin of the shrub habit in Isotrema, from which we sampled representatives from climbing to self-supporting forms. Morphological, anatomical, and biomechanical characters are optimized on a chloroplast- and nuclear-derived phylogeny. • KEY RESULTS: Character-state reconstructions revealed that the climbing habit is plesiomorphic in Isotrema and shrub-like forms are derived from climbers. However, shrubs do not constitute a monophyletic group. Both shrubs and climbers show large multiseriate rays, but differ in terms of vessel size and proportion of fibers and soft tissues. • CONCLUSION: We suggest that while shrub-like species might have partly escaped from the constraints of life as lianas; their height size and stability are not typical of self-supporting shrubs and trees. Shrubs retained lianoid stem characters that are known to promote flexibility such as ray parenchyma. The transitions to a shrub-like form likely involved relatively simple, developmental changes that may be attributed to heterochronic processes.

Persistently low fruiting success in the Mediterranean pipevine Aristolochia baetica (Aristolochiaceae): a multi-year study.

Low fruit set is common in many plant species and may be caused by a variety of factors, such as predation, resource limitation or deficient pollination, or it may be an evolutionary strategy. In this paper, we investigate factors that affect fruit set in Aristolochia baetica (Aristolochiaceae), a Mediterranean pipevine found in southwest Spain. Fruit production was monitored in two populations over 4 years (2002-2005), and the causes of flower or fruit loss were determined. Experimental hand-pollinations were performed, and germinated pollen grains on the stigmas of open-pollinated flowers were quantified. Fruit set was always very low (4-14%). Floral abscission initially reduced reproductive output by more than 50%; then herbivory (6-12%) and fruit abortion (8-26%) caused further reductions. Given that the number of efficiently pollinated flowers was always higher than that of ripe fruits, and that xenogamous hand-pollination did not increase fruit set in relation to open-pollination, the final fruit production of A. baetica seems not to be pollen-limited. Fruit abortion of effectively pollinated flowers supports the idea that resource availability limits fruit set. In A. baetica, fruit abortion could lead to mate selection of the best quality fruits. Moreover, the initiated fruits that finally abort could also satiate predators, contributing to increase progeny fitness.

Morphological aspects of self-repair of lesions caused by internal growth stresses in stems of Aristolochia macrophylla and Aristolochia ringens.

This study reveals in detail the mechanism of self-repair during secondary growth in the vines Aristolochia macrophylla and Aristolochia ringens based on morphological data. For a comprehensive understanding of the underlying mechanisms during the self-repair of lesions in the sclerenchymatous cylinder of the stem, which are caused by internal growth stresses, a classification of morphological changes in the cells involved in the repair process is required. In an early stage of self-repair, we observed morphological changes as a mere extension of the turgescent cortex cells surrounding the lesion, whereby the cell wall extends locally through visco-elastic/plastic deformation without observable cell wall synthesis. Later stages involve typical cell growth and cell division. Several successive phases of self-repair were investigated by light microscopy of stained samples and confocal laser-scanning microscopy in fluorescence mode. The results indicate that A. macrophylla and A. ringens respond to lesions caused by internal growth stresses with a sophisticated self-repair mechanism comprising several phases of different repair modes.

Quantitative and qualitative changes in primary and secondary stem organization of Aristolochia macrophylla during ontogeny: functional growth analysis and experiments.

The anatomy of young and old stems of Aristolochia macrophylla has been investigated for a better understanding of how secondary growth processes cause changes in the stem anatomy of a lianescent plant. In A. macrophylla, following an increase in volume of secondary vascular tissues, the cortical tissues are deformed and the outer sclerenchymatous cylinder ruptures. Morphometric measurements prove that the inner zone of the cortical parenchymatous tissue is compressed prior to the rupture of the outer sclerenchymatous cylinder. After the rupture has occurred, the radial width of the inner primary cortex slightly increases again. This could be caused by strain relaxation, suggesting that the inner primary cortex mechanically behaves similarly to cellular technical foam rubbers. Two different experiments were undertaken to test the outer cortical cylinders mechanically. The outer cortical cylinders comprise the outer sclerenchymatous cortical tissue and a collenchymatous sheath underneath the epidermis and the epidermis. In a first experiment, transverse compression loads were applied to the outside of the cortical cylinders causing ovalization of the cylinder until failure. This experiment allowed the Young's Modulus of the outer cortical cylinders to be determined. In a second set of experiments, radial hydraulic pressure was applied to the inside of the cortical cylinders, mimicking the mechanical effects of internal growth processes. The increase of the internal pressure finally led to rupture of the cortical cylinders. The circumferential stresses acting on the inner surface of the cortical cylinders were calculated. These data allow quantitative estimates of the radial and circumferential pressures effected by vascular secondary growth processes during ontogeny in A. macrophylla stems. The experimental results further indicate that the outer sclerenchymatous cylinder is the main contributor to mechanical stability of young A. macrophylla stems.