Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7.

Endo-β-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of β1[RIGHTWARDS ARROW]4 bonds in mannan polymers and have been associated with the process of weakening the tissues surrounding the embryo during seed germination. In germinating Arabidopsis thaliana seeds, the most highly expressed MAN gene is AtMAN7 and its transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in AtMAN7 have a slower germination than the wild type. To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae MAN7 gene promoters has been done, and these conserved motifs have been used as bait to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library from A. thaliana. The basic-leucine zipper TF AtbZIP44, but not the closely related AtbZIP11, has thus been identified and its transcriptional activation upon AtMAN7 has been validated at the molecular level. In the knock-out lines of AtbZIP44, not only is the expression of the AtMAN7 gene drastically reduced, but these mutants have a significantly slower germination than the wild type, being affected in the two phases of the germination process, both in the rupture of the seed coat and in the breakage of the micropylar endosperm cell walls. In the over-expression lines the opposite phenotype is observed.

Cis-, trans-, transcriptional regulation of the Arabidopsis thaliana gene AtTrxo1 and its response upon germination and to salt

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in redox regulation of many enzymes in different cell compartments. Among them, mitochondrial Trxo has been described to have a response in plants grown under salinity but there is scarce information about its functional role in abiotic stress or its gene regulation. In this work, the transcriptional regulation of the mitochondrial AtTrxo1 gene has been studied for the first time, by identifying functionally relevant cis- elements in its promoter: two conserved motives were found as positive and one as negative regulators. Using them as baits for the screening of an arrayed yeast library containing Arabidopsis Transcription Factors (TF) ORFs, two TFs were selected that are now being validated at the molecular level. We have also studied the response of T-DNA insertion mutant plants for AtTrxo1 to salt stress. The K.O. AtTrxo1 mutants presented several phenotypic changes including the time required to reach 50% germination under salinity, without affecting the final germination percentage.

Transcription factors of the bzip factors of the BZIP family affecting arabidopsis thaliana germination sensu stricto

During seed germination, the endosperm cell walls (CWs) suffer an important weakening process mainly driven by hydrolytic enzymes, such are endo-?- mannanases (MAN; EC. 3.2.1.78) that catalyze the cleavage of ?1?4 bonds in the mannan-polymers. In Arabidopsis thaliana seeds, endo-?-mannanase activity increases during seed imbibition, decreasing after radicle emergence1. AtMAN7 is the most highly expressed MAN gene in seeds upon germination and their transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in this gene (K.O. MAN7) have a slower germination rate than the wild type (t50=34 h versus t50=25 h). To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae orthologous MAN7 gene promoters has been done and these conserved motives have been used as baits to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library of circa 1,200 TFs from A. thaliana. The basic leucine zipper AtbZIP44, but not its closely related ortholog AtbZIP11, has been thus identified and its regulatory function upon AtMAN7 during seed germination validated by different molecular and physiological techniques, such are RT-qPCR analyses, mRNA Fluorescence in situ Hybridization (FISH) experiments, and by the establishment of the germination kinetics of both over-expression (oex) lines and TDNA insertion mutants in AtbZIP44. The transcriptional combinatorial network through which AtbZIP44 regulates AtMAN7 gene expression during seed germination has been further explored through protein-protein interactions between AtbZIP44 and other bZIP members. In such a way, AtbZIP9 has been identified by yeast two-hybrid experiments and its physiological implication in the control of AtMAN7 expression similarly established.

Fungal biodeterioration of colour cinematographic films of the cultural heritage of Cuba

Until recently, cinematographic film was largely cellulose-triacetate-based. However, this material is highly susceptible to biodeterioration, thus placing historic film collections, an important part of the cultural heritage of many countries, at risk. In the present study, samples taken from several biodeteriorated color cinematographic films belonging to the collection of the Cuban Institute for Cinematographic Industry and Arts (ICAIC) were investigated. Infrared spectroscopy showed that all films were of the same composition, i.e., a gelatin emulsion coating one side of a cellulose-triacetate-based film support. The films were analyzed by environmental scanning electron microscopy and scanning electron microscopy to determine the degree of biodeterioration and the type of colonizing microorganisms. Significant fungal colonization was found on both sides of the films in all samples, with a higher concentration of fungi on the gelatin emulsion side. Epifluorescence microscopy of fluorochrome-dyed films demonstrated that some of the fungi were still active, indicating that the films under study, and probably others at the ICAIC, are at risk of further deterioration. Fungi were identified by molecular biology techniques. The fungi mainly responsible for the observed biodeterioration were those belonging to the genera Aspergillus and Cladosporium, although other genera, such as Microascus and Penicillium, were identified as well. In accordance with the findings described herein, the existing guidelines for the prevention and control of film biodeterioration are discussed.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae), a narrow and endangered endemic species of SW Spain

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vázquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known. This species was catalogued in the ?Endangered? category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out an exhaustive census of A. gines-lopezii , and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%?60%) was increased to 97%?99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential ( J w ) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identi fi ed mainly by analysis of DNA sequences, belonged to genera Penicillium, Geotrichum, Mucor , Aspergillus , Microascus and Talaromyces . Particularly, the effect of matric potential ( J m ), and ionic (NaCl) and non-ionic (glycerol) solute potentials ( J s ) on growth rate was studied. The response of strains was highly dependent on the type of J w . For J s, clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for J m in all the strains, with the exception of Aspergillus pseudoglaucus, whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae),a narrow and endangered endemic species of SW Spain.

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vazquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known.This species was catalogued in the "Endangered" category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out anexhaustive census of A. gines-lopezii, and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%-60%) was increased to 97%-99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

bZIP transcription factors in the regulatory networks controlling seed maturation and germination of Arabidopsis thaliana

Se describe la expresión por RTqPCR de los genes que codifican los factores transcripcionales bZIP44 y bZIP9. Asimismo se establece la interacción entre ambas proteínas en el sistema de 2 híbridos de levadura y in planta por complementación bimolecular fluorescente.

Combinatorial interaction between AtbZIP44 and other bZIP transcription factors in the regulation of Arabidopsis thaliana seed maturation and germination

El gen AtbZIP44 se expresa en el endospermo micropilar y en la radícula de semillas en germinación. El mRNA del gen AtbZIP9 se localiza también en la radícula. Se realizan experimentos de interacción proteína-proteína entre los correspondientes factores transcripcionales.

Genes encoding beta-mannanases in Brachypodium distachyon seeds have a role not only in cell wall softening upon germination sensu stricto but also during post-germinative reserve mobilization

En la coleorriza en semillas de Brachypodium son abundantes los mananos y estos van desapareciendo conforme progresa la germinación (entre 12-27 h), al mismo tiempo se observa un pico de actividad endo-beta-mananasa. Se ha establecido que de los 6 miembros de la familia MAN en B. distachyon 3 se expresan en el embrión en germinación y BdMAN3 también es abundante en la aleurona.

Estudio de los factores que condicionan, a escala local, la presencia de Epidendrum rhopalostele (Orchidaceae): Implicaciones para su conservación

El análisis de los factores que determinan el establecimiento y supervivencia de orquídeas epífitas, incluyen: a) las condiciones microambientales de los bosques que las mantienen, b) preferencias por las características de los hospederos donde crecen, c) limitación en la dispersión de semillas, d) interacciones planta-planta, y e) asociaciones micorrízicas para la germinación y resultan esenciales para el desarrollo de estrategias para la conservación y manejo de este grupo de plantas. Este trabajo ha evaluado la importancia de estos factores en Epidendrum rhopalostele, orquídea epífita del bosque de niebla montano, a través de los análisis de los patrones espaciales de los árboles que la portan y de la propia orquídea, a escala de población, estudios de asociación y métodos moleculares. Estos últimos han consistido en el uso de marcadores AFLP para el análisis de la estructura genética de la orquídea y en la secuenciación-clonación de la región ITS para la identificación de los hongos micorrízicos asociados. El objetivo de esta tesis es, por tanto, una mejor comprensión de los factores que condicionan la presencia de orquídeas epífitas en los remanentes de bosque de niebla montano y una evaluación de las implicaciones para la conservación y mantenimiento de sus hábitats y la permanencia de sus poblaciones. El estudio fue realizado en un fragmento de bosque de niebla montano de sucesión secundaria situado al este de la Cordillera Real, en los Andes del sur de Ecuador, a 2250 m.s.n.m y caracterizado por una pendiente marcada, temperatura media anual de 20.8°C y precipitación anual de 2193 mm. En este fragmento se mapearon, identificaron y caracterizaron todos los árboles presentes con DBH > 1 cm y todos los individuos de Epidendrum rhopalostele. Así mismo se tomaron muestras de hoja para obtener ADN de todas las orquídeas registradas y muestras de raíces de individuos con flor de E. rhopalostele, uno por cada forófito, para el análisis filogenético de micorrizas. Análisis espaciales de patrones de puntos basados en la K de Ripley y la distancia al vecino más cercano fueron usados para los árboles, forófitos y la población de E. rhopalostele. Se observó que la distribución espacial de árboles y forófitos de E. rhopalostele no es aleatoria, ya que se ajusta a un proceso agregado de Poisson. De ahí se infiere una limitación en la dispersión de las semillas en el fragmento estudiado y en el establecimiento de la orquídea. El patrón de distribución de la población de E. rhopalostele en el fragmento muestra un agrupamiento a pequeña escala sugiriendo una preferencia por micro-sitios para el establecimiento de la orquídea con un kernel de dispersión de las semillas estimado de 0.4 m. Las características preferentes del micro-sitio como tipos de árboles (Clusia alata y árboles muertos), tolerancia a la sombra, corteza rugosa, distribución en los dos primeros metros sugieren una tendencia a distribuirse en el sotobosque. La existencia de una segregación espacial entre adultos y juveniles sugiere una competencia por recursos limitados condicionada por la preferencia de micro-sitio. La estructura genética de la población de E. rhopalostele analizada a través de Structure y PCoA evidencia la presencia de dos grupos genéticos coexistiendo en el fragmento y en los mismos forófitos, posiblemente por eventos de hibridización entre especies de Epidendrum simpátricas. Los resultados del análisis de autocorrelación espacial efectuados en GenAlex confirman una estructura genético-espacial a pequeña escala que es compatible con un mecanismo de dispersión de semillas a corta distancia ocasionada por gravedad o pequeñas escorrentías, frente a la dispersión a larga distancia promovida por el viento generalmente atribuida a las orquídeas. Para la identificación de los micobiontes se amplificó la región ITS1-5.8S-ITS2, y 47 secuencias fueron usadas para el análisis filogenético basado en neighborjoining, análisis bayesiano y máximum-likelihood que determinó que Epidendrum rhopalostele establece asociaciones micorrízicas con al menos dos especies diferentes de Tulasnella. Se registraron plantas que estaban asociadas con los dos clados de hongos encontrados, sugiriendo ausencia de limitación en la distribución del hongo. Con relación a las implicaciones para la conservación in situ resultado de este trabajo se recomienda la preservación de todo el fragmento de bosque así como de las interacciones existentes (polinizadores, micorrizas) a fin de conservar la diversidad genética de esta orquídea epífita. Si fuere necesaria una reintroducción se deben contemplar distancias entre los individuos en cada forófito dentro de un rango de 0.4 m. Para promover el reclutamiento y regeneración de E. rhopalostele, se recomienda que los forófitos correspondan preferentemente a árboles muertos o caídos y a especies, como Clusia alata, que posean además corteza rugosa, sean tolerantes a la sombra, y en el área del sotobosque con menor luminosidad. Además es conveniente que las orquídeas en su distribución vertical estén ubicadas en los primeros metros. En conclusión, la limitación en la dispersión, las características del micro-sitio, las interacciones intraespecíficas y con especies congenéricas simpátricas y las preferencias micorrízicas condicionan la presencia de esta orquídea epífita en este tipo de bosque. ABSTRACT The analysis of factors that determine the establishment and survival of epiphytic depends on factors such as a) microenvironmental conditions of forest, b) preference for host characteristics where orchids grow, c) seed dispersal limitation, d) plant-plant interaction, e) priority mycorrhizal associations for germination, are essential for the development of strategies for management and conservation. This work evaluated the importance of these factors in Epidendrum rhopalostele, an epiphytic orchid of montane cloud forest through the analysis of spatial patterns of host trees and the orchid, in a more specific scale, with association studies and molecular methods, including AFLPs for orchid population genetic structure and the sequencing of the ITS region for associated mycorrhizal fungi. The aim of this thesis is to understand the factors that condition the presence of epiphytic orchids in the remnants of montane cloud forest and to assess the implications for the conservation and preservation of their habitats and the persistence of the orchid populations. The study was carried out in a fragment of montane cloud forest of secondary succession on the eastern slope of Cordillera Real in the Andes of southern Ecuador, located at 2250 m a.s.l. characterized by a steep slope, mean annual temperature of 20.8°C and annual precipitation of 2193 mm. All trees with DBH > 1 cm were mapped, characterized and identified. All E. rhopalostele individuals present were counted, marked, characterized and mapped. Leaf samples of all orchid individuals were collected for DNA analysis. Root samples of flowering E. rhopalostele individuals were collected for phylogenetic analysis of mycorrhizae, one per phorophyte. Spatial point pattern analysis based on Ripley`s K function and nearest neighbor function was used for trees, phorophytes and orchid population. We observed that spatial distribution of trees and phorophytes is not random, as it adjusts to a Poisson cluster process. This suggests a limitation for seed dispersal in the study fragment that is affecting orchid establishment. Furthermore, the small-scale spatial pattern of E. rhopalostele evidences a clustering that suggests a microsite preference for orchid establishment with a dispersal kernel of 0.4 m. Microsite features such as types of trees (dead trees or Clusia alata), shade tolerance trees, rough bark, distribution in the first meters suggest a tendency to prefer the understory for their establishment. Regarding plant-plant interaction a spatial segregation between adults and juveniles was present suggesting competition for limited resources conditioned for a microsite preference. Analysis of genetic structure of E. rhopalostele population through Structure and PCoA shows two genetic groups coexisting in this fragment and in the same phorophyte, possibly as a result of hybridization between sympatric species of Epidendrum. Our results of spatial autocorrelation analysis develop in GenAlex confirm a small-scale spatial-genetic structure within the genetic groups that is compatible with a short-distance dispersal mechanism caused by gravity or water run-off, instead of the long-distance seed dispersal promoted by wind generally attributed to orchids. For mycobionts identification ITS1-5.8S-ITS2 rDNA region was amplified. Phylogenetic analysis was performed with neighborjoining, Bayesian likelihood and maximum-likelihood for 47 sequences yielded two Tulasnella clades. This orchid establishes mycorrhizal associations with at least two different Tulasnella species. In some cases both fungi clades were present in same root, suggesting no limitation in fungal distribution. Concerning the implications for in situ conservation resulting from this work, the preservation of all forest fragment and their interactions (pollinators, mycorrhiza) is recommended to conserve the genetic diversity of this species. If a reintroduction were necessary, distances between individuals in each phorophyte within a range of 0.4 m, are recommended. To promote recruitment and regeneration of E. rhopalostele it is recommended that phorophytes correspond to dead or fallen trees or species, such as Clusia alata. Trees that have rough bark and are shade tolerant are also recommended. Furthermore, regarding vertical distribution, it is also convenient that orchids are located in the first meter (in understory, area with less light). In conclusion, limitation on seed dispersal, microsite characteristics, plant-plant interactions or interaction with cogeneric sympatric species and mycorrhizal preferences conditioned the presence of this epiphytic orchid in this fragment forest.

Seed germination characteristics of Phillyrea angustifolia L. and P. latifolia L. (Oleaceae), two Mediterranean shrub species having lignified endocarp

The aim of this study was to determine the germination characteristics of Phillyrea angustifolia L. and P. latifolia L. seeds in order to develop an optimized propagation protocol for Phillyrea species. Seeds of P. angustifolia and P. latifolia were collected from wild plants growing in Cáceres province (CW Spain) and Andalucía (S Spain), respectively. Percentage of water uptake for P. latifolia seeds was calculated. Seeds with and without endocarp were germinated at different constant and alternating temperatures. Seeds without endocarp were soaked in distilled water or gibberellic acid, and then set to germinate. Seeds with endocarp of both species were stratified at 5 ºC for 30 or 90 days and then the endocarp was completely removed from the seeds before they were sowed. Chemical scarification with sulfuric acid and mechanical scarification were tested on P. angustifolia seeds with endocarp. Phillyrea endocarp was permeable to water, since Phillyrea seeds with endocarp imbibed water, but water uptake was faster when the endocarp was removed. Moreover, the encodarp could interfere mechanically in the emergence of the radicle, since seed germination of Phillyrea species was promoted by the complete removal of the lignified endocarp surrounding each seed. Optimal germination temperature for both species was 15 ºC, and lower temperatures produced secondary dormancy. Soaking in distilled water or gibberellic acid did not significantly enhance seed germination. Cold stratification and chemical scarification treatments were detrimental for seed germination. Keywords cold stratification, Phillyrea species, treatments before sowing, seed germination, seed scarification, lignified endocarp.

Mannans and endo-beta-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24–30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36–42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.