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2.  Genotype Delimitation in the Nod-Independent Model Legume Aeschynomene evenia 
PLoS ONE  2013;8(5):e63836.
Research on the nitrogen-fixing symbiosis has been so far focused on two model legumes, Medicago truncatula and Lotus japonicus, which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some semi-aquatic Aeschynomene species present the distinctive feature to form nitrogen-fixing nodules on both roots and stems following elicitation by photosynthetic bradyrhizobia that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the molecular mechanisms of this unique Nod-independent nitrogen-fixing symbiosis, we previously identified A. evenia C. Wright as an appropriate model legume, because it displays all the requisites for molecular and genetic approaches. To advance the use of this new model legume species, here we characterized the intraspecific diversity found in A. evenia. For this, the accessions available in germplasm banks were collected and subjected to morphological investigations, genotyping with RAPD and SSR markers, molecular phylogenies using ITS and single nuclear gene sequences, and cross-compatibility tests. These combined analyses revealed an important intraspecific differentiation that led us to propose a new taxonomic classification for A. evenia comprising two subspecies and four varieties. The A. evenia ssp. evenia contains var. evenia and var. pauciciliata whereas A. evenia ssp. serrulata comprises var. serrulata and var. major. This study provides information to exploit efficiently the diversity encountered in A. evenia and proposes subsp. evenia as the most appropriate subspecies for future projects aimed at identifying plant determinants of the Nod-independent symbiotic process.
PMCID: PMC3662760  PMID: 23717496
3.  Optical detection of downy mildew in grapevine leaves: daily kinetics of autofluorescence upon infection 
Journal of Experimental Botany  2012;64(1):333-341.
A 15-day survey of autofluorescence has been conducted upon infection by downy mildew [Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni] of leaves of a susceptible grapevine genotype. Different autofluorescence signals were followed from the cellular to the whole-leaf level by using four types of devices for fluorosensing: a macroscope, a spectrofluorimeter, a portable field optical sensor (the Multiplex 3), and a field fluorescence sensor prototype with 335nm excitation. It was shown for the first time, by the three different techniques and at three different scales, that the stilbene-dependent violet–blue autofluorescence (VBF) had a transitory behaviour, increasing to a maximum 6 days post-inoculation (DPI) and then decreasing to a constant lower level, nevertheless significantly higher than in the control leaf. This behaviour could be sensed from both sides of the leaf. On the abaxial side, VBF could discriminate the presence of infection from 1 DPI, and on the adaxial side from 3 DPI. There was a constant increase in blue-excited green fluorescence starting from 8 DPI, concomitant with a decrease in leaf chlorophyll content sensed by one reflectance and two fluorescence indices available on the Multiplex 3 sensor. These results show that a pre-symptomatic and symptomatic sensing of downy mildew is possible by autofluorescence-based sensors, and this is potentially applicable in the field.
PMCID: PMC3528039  PMID: 23213137
Disease diagnostics; optical proximal sensors; phenolic compounds; phytoalexin fluorescence; Plasmopara viticola; Vitis vinifera.
4.  Cabomba as a model for studies of early angiosperm evolution 
Annals of Botany  2011;108(4):589-598.
The angiosperms, or flowering plants, diversified in the Cretaceous to dominate almost all terrestrial environments. Molecular phylogenetic studies indicate that the orders Amborellales, Nymphaeales and Austrobaileyales, collectively termed the ANA grade, diverged as separate lineages from a remaining angiosperm clade at a very early stage in flowering plant evolution. By comparing these early diverging lineages, it is possible to infer the possible morphology and ecology of the last common ancestor of the extant angiosperms, and this analysis can now be extended to try to deduce the developmental mechanisms that were present in early flowering plants. However, not all species in the ANA grade form convenient molecular-genetic models.
The present study reviews the genus Cabomba (Nymphaeales), which shows a range of features that make it potentially useful as a genetic model. We focus on characters that have probably been conserved since the last common ancestor of the extant flowering plants. To facilitate the use of Cabomba as a molecular model, we describe methods for its cultivation to flowering in the laboratory, a novel Cabomba flower expressed sequence tag database, a well-adapted in situ hybridization protocol and a measurement of the nuclear genome size of C. caroliniana. We discuss the features required for species to become tractable models, and discuss the relative merits of Cabomba and other ANA-grade angiosperms in molecular-genetic studies aimed at understanding the origin of the flowering plants.
PMCID: PMC3170152  PMID: 21486926
Cabomba; Cabombaceae; Trithuria; Hydatellaceae; Nymphaeales; ANA grade; angiosperm; flowering plant evolution; expressed sequence tags; EST; c-value; in situ hybridization; evo-devo
5.  Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus 
Annals of Botany  2011;107(8):1287-1312.
Background and Aims
Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined.
Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species.
Key Results and Conclusions
Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.
PMCID: PMC3101146  PMID: 21527419
Oil-bee pollination; glandular trichomes; elaiophores; lipids; phylogeography; Sisyrinchieae; Olsynium; Solenomelus
6.  An athymic rat model of cutaneous radiation injury designed to study human tissue-based wound therapy 
To describe a pilot study for a novel preclinical model used to test human tissue-based therapies in the setting of cutaneous radiation injury.
A protocol was designed to irradiate the skin of athymic rats while sparing the body and internal organs by utilizing a non-occlusive skin clamp along with an x-ray image guided stereotactic irradiator. Each rat was irradiated both on the right and the left flank with a circular field at a 20 cm source-to-surface distance (SSD). Single fractions of 30.4 Gy, 41.5 Gy, 52.6 Gy, 65.5 Gy, and 76.5 Gy were applied in a dose-finding trial. Eight additional wounds were created using the 41.5 Gy dose level. Each wound was photographed and the percentage of the irradiated area ulcerated at given time points was analyzed using ImageJ software.
No systemic or lethal sequelae occurred in any animals, and all irradiated skin areas in the multi-dose trial underwent ulceration. Greater than 60% of skin within each irradiated zone underwent ulceration within ten days, with peak ulceration ranging from 62.1% to 79.8%. Peak ulceration showed a weak correlation with radiation dose (r = 0.664). Mean ulceration rate over the study period is more closely correlated to dose (r = 0.753). With the highest dose excluded due to contraction-related distortions, correlation between dose and average ulceration showed a stronger relationship (r = 0.895). Eight additional wounds created using 41.5 Gy all reached peak ulceration above 50%, with all healing significantly but incompletely by the 65-day endpoint.
We developed a functional preclinical model which is currently used to evaluate human tissue-based therapies in the setting of cutaneous radiation injury. Similar models may be widely applicable and useful the development of novel therapies which may improve radiotherapy management over a broad clinical spectrum.
PMCID: PMC3403853  PMID: 22568958
Acute cutaneous radiation injury; Normal tissue toxicity; Kilovoltage x-ray irradiation; Immunodeficient athymic rat model; Adipose-derived stem cell
7.  In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves 
Journal of Experimental Botany  2012;63(10):3697-3707.
Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.
PMCID: PMC3388817  PMID: 22412183
3D fluorescence microscopy imaging; autofluorescence; defence response; downy mildew; phytoalexins; resistance to pathogen; resveratrol; spectrofluorimetry; Vitaceae (Vitis vinifera L.)
8.  Biomechanical Properties of The Vaginal Wall: Effect of Pregnancy, Elastic Fiber Deficiency, and Pelvic Organ Prolapse 
To identify pregnancy-induced changes in biomechanical properties of the vaginal wall and compare these with Fibulin-5 knockout mice (Fbln5-/-) with and without prolapse.
Study Design
Mid-vaginal segments of nonpregnant and late-pregnant wild type (WT), Fbln5-/- with prolapse, and Fbln5-/- mice without prolapse were studied. Tissue length at failure, maximal strain, maximal stress, and tissue stiffness were determined.
Compared with nonpregnant mice, vaginas of pregnant and Fbln5-/- (with prolapse) mice exhibited decreased maximal stress, increased distensibility and strain, and decreased stiffness. Tissues from Fbln5-/- mice without prolapse were similar to nonpregnant WT animals.
Pregnancy confers remarkable changes in the vaginal wall including increased distensibility and decreased stiffness and maximal stress. Elastinopathy alone is insufficient to cause significant changes in these properties, but prolapse confers additional alterations in distensibility and stiffness similar to those observed in pregnancy. These changes may contribute to the poor durability of many restorative surgical procedures for prolapse.
PMCID: PMC2760217  PMID: 18455541
stress-strain; vaginal muscularis; pelvic organ support; fibulin-5; elastic fibers
9.  Nuclear Localization of Cytoplasmic Poly(A)-Binding Protein upon Rotavirus Infection Involves the Interaction of NSP3 with eIF4G and RoXaN▿  
Journal of Virology  2008;82(22):11283-11293.
Rotavirus nonstructural protein NSP3 interacts specifically with the 3′ end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.
PMCID: PMC2573281  PMID: 18799579
10.  Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining 
Applied and Environmental Microbiology  2001;67(10):4662-4670.
The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
PMCID: PMC93217  PMID: 11571170
11.  First Cytogenetic Investigation in Populations of Acacia heterophylla, Endemic from La Réunion Island, with Reference to A. melanoxylon 
Annals of Botany  1995;75(1):95-100.
Five populations of tetraploid Acacia heterophylla, endemic from La Réunion island, were compared together and with their Australian diploid relative A. melanoxylon for cytogenetic and DNA characteristics. A. melanoxylon (2n = 26) had 1·59 pg nuclear DNA; A. heterophylla (2n = 4x = 52) had double this value (3·19 pg), and there was no difference between populations within species. Both species had 39 % GC. Interchromosome connections were evident at metaphase and mitotic irregularities at anaphase were twice as frequent in A. heterophylla as in A. melanoxylon, again with no difference between populations within species. These results argue for a recent autotetraploid origin of A. heterophylla from A. melanoxylon. Yet, fluorochrome banding showed that in some A. heterophylla populations, GC-rich bands had slightly changed from the supposed ancestral pattern, probably by means of translocations involving parts of nuclear organizer areas. No clear relation was found between banding patterns and ecological factors.
PMCID: PMC3023669  PMID: 21247917
Acacia heterophylla Willd.; Acacia melanoxylon R. Br. (Blackwood); interpopulation variation; autotetraploidy; insular evolution; flow cytometry; DNA content; base ratio; cytogenetics; fluorochrome banding

Results 1-11 (11)