Search

Key Message: The present study showed that the heat stress (40 °C) caused changes in morphophysiological, biochemical, and ultrastructural parameters to the seeds Melanoxylon brauna, ultimately leading to loss of germination capacity. Temperature is an abiotic factor that influences seed germination. In the present study, we investigated morphophysiological, biochemical, and ultrastructural changes during the germination of Melanoxylon brauna seeds under heat stress. Seed germination was evaluated at constant temperatures of 25 and 40 °C. The samples consisted of seeds soaked in distilled and ionized water for 48 and 96 h at both temperatures. For the evaluation of internal morphology, the seeds were radiographed. Ultrastructural parameters were assessed using transmission electron microscopy (TEM). The production of reactive oxygen species (ROS), content of malondialdehyde (MDA) and glucose, carbonylated proteins, and activity of the enzymes (superoxide dismutase-SOD, ascorbate peroxidase-APX, catalase-CAT, peroxidase-POX, glucose-6-phosphate dehydrogenase-G6PDH, lipase, α- and β-amylase, and protease) were measured by spectrophotometric analysis. An 82% reduction in the germination of M. brauna seeds was observed at 25 °C, and 0% at 40 °C. TEM showed that seeds submitted to heat stress (40 °C) had poorly developed mitochondria and significantly reduced respiration rates. The content of ROS and protein carbonylation in seeds subjected to 40 °C increased compared to that at 25 °C. The activity of antioxidant enzymes, namely SOD, APX, CAT, and POX, was significantly reduced in seeds subjected to heat stress. Glucose content, G6PDH, and lipase activity also decreased when the seeds were exposed to heat stress. Conversely, α- and β-amylase enzymes and the protease increased due to the increase in temperature. Our data showed that the increase in temperature caused an accumulation of ROS, increasing the oxidative damage to the seeds, which led to mitochondrial dysfunction, ultimately leading to loss of germination., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The oily resin produced by Copaifera langsdorffii, commonly called oil of copaiba, is widely exploited by the drug, cosmetic, and biodiesel industries. The distribution of oily secretory cavities and canals (secretory spaces) over the vegetative body characterizes this species. Oil is stored inside the lumen of the secretory spaces and only reaches the organ surface after injuries. Nonetheless, translucent oily deposits occur on the adaxial surface of intact young leaves. In this study, we searched for further sources of oil production in C. langsdorffii leaves in addition to the well-known secretory cavities and investigated the mechanisms of secretion. Leaves in different developmental stages were collected from adult plants and processed for studies on light and transmission electron microscopies. The primary finding of this study was the involvement of the chlorenchyma cells in lipid biosynthesis, in addition to the secretory cavities. The secretory activity of cavities and chlorenchyma cells overlapped in young leaves. Ultrastructurally, secretory cavity cells exhibited abundant smooth endoplasmic reticulum profiles and oleoplasts, whereas the chlorenchyma cells had large chloroplasts with oil inclusions. Our data suggest that the oily material on the leaf surface arose from the chlorenchyma and was transported via the apoplast. These findings open new avenues for understanding oil biosynthesis regulation in mesophyll cells and planning of future strategies for the biotechnological application of C. langsdorffii leaves.

A polysaccharide named Senegrain Water-Soluble Polysaccharide (SWSP) was extracted and purified from Senegrain seeds. The physicochemical properties of SWSP were evaluated by Thin-layer chromatography (TLC), High Performance Liquid Chromatography (HPLC), Scanning Electron Microscopy (SEM), and Differential Scanning Colorimeter (DSC). TLC results showed that SWSP is a gluconic acid polymer. It had a network with a large number of cavities. This polysaccharide with a semi-crystalline structure has an average molecular weight of 47.42 kDa. On other trend, obtained data exhibited potent antidiabetic and antibacterial activities of SWSP. Additionally, the antioxidant activities of SWSP were studied in vitro, and demonstrated that this polymer displayed an interesting ABTS radical scavenging activity as well as a protective effect on the hydroxyl radical-induced DNA damage. The effects of SWSP on oxidative processes in minced raw beef during refrigerated (4 °C) storage were investigated. The results proved that SWSP decreased the TBARS value and inhibited the MetMb accumulation during the period of refrigeration storage. Overall, the finding demonstrated the potential applications of SWSP in food industries., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pollen morphology of 10 species and foliar epidermal anatomy of eight species of Papilionaceae from Skardu valley, northern Pakistan has been estimated for the first time. The present study was commenced with an aim to provide a detailed account of the pollen morphology by scanning electron microscopy and foliar epidermal anatomy by light microscopy. The pollen aperture was tricolporate with reticulate exine in the selected species. Stomata types are actinocytic, paracytic, and anomocytic. Irregular or polygonal with undulate or straight walls, epidermal cells were reported. A unique diversity was observed in the foliar trichomes that show the taxonomic significance of the discrimination of taxa. Non-glandular trichomes were observed in the selected species which are unicellular with thin, long and pointed apical cells. Pollen and foliar micro morphological characters proved to be helpful for the identification of taxa at a specific level., (© 2019 Wiley Periodicals, Inc.)

Isolated bean cells were used to understand the contribution of cell wall and cytoplasmic matrix on starch digestibility. Cotyledon cells were treated enzymatically and mechanically to reduce the level of cell intactness. SEM and chemical characterization revealed that enzymatic treatment modified cell wall thickness and porosity without altering the cytoplasmic matrix, whereas mechanical treatment completely disrupted cell structure. Decreasing cell intactness increased the rate but not the extent of starch digestion in-vitro. It was concluded that cell wall serves as a permeable barrier limiting the access of digestive enzymes. Cytoplasmic matrix, on the other hand, reduced further the accessibility of amylase to starch affecting its hydrolysis rate. In addition, it was proven that cell structural changes, if any, occurring during digestion had no effect on starch hydrolysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Foreign bodies and tissue reactions to foreign materials are commonly encountered in the oral cavity. Exogenous materials causing foreign body reactions may be metallic (amalgam) or nonmetallic (suture materials, vegetable matter). Implantation of food particles in the oral tissues has been known to cause reactive lesions such as oral pulse granuloma. Implantation could be through extraction sockets, deep periodontal pockets, associated with tumor growth, interdental areas of teeth, unfilled root canals, and grossly decayed teeth. These get rapidly digested and altered by host responses. Cellulose persists as hyaline material and invokes chronic granulomatous response. This change may mimic other pathologies., Materials and Methods: Representative specimens from commonly consumed food groups were selected, fixed in 10% neutral buffered formalin, processed, sectioned, stained with hematoxylin and eosin stain and observed under light microscope., Results: Each specimen revealed unique, distinct histology of each food type. The plant materials had a characteristic appearance of rigid double cell wall while rigid regular partitions containing nutrient material were revealed in seeds and beans. Starch-contained lentils exhibited clear spaces. Following is a brief description of some of the significant histological findings of each of the specimens processed and stained., Conclusion: Thus, the study of histological structure of vegetables and legumes will enable their easy recognition in oral biopsy samples and help in distinguishing them from other pathologies and artifact., Competing Interests: There are no conflicts of interest.

During germination, orthodox seeds become gradually intolerant to desiccation, and for this reason, they are a good model for recalcitrance studies. In the present work, physiological, biochemical, and ultrastructural aspects of the desiccation tolerance were characterized during the germination process of Anadenanthera colubrina seeds. The seeds were imbibed during zero (control), 2, 8, 12 (no germinated seeds), and 18 hours (germinated seeds with 1 mm protruded radicle); then they were dried for 72 hours, rehydrated and evaluated for survivorship. Along the imbibition, cytometric and ultrastructural analysis were performed, besides the extraction of the heat-stable proteins. Posteriorly to imbibition and drying, the evaluation of ultrastructural damages was performed. Desiccation tolerance was fully lost after root protrusion. There was no increase in 4C DNA content after the loss of desiccation tolerance. Ultrastructural characteristics of cells from 1mm roots resembled those found in the recalcitrant seeds, in both hydrated and dehydrated states. The loss of desiccation tolerance coincided with the reduction of heat-stable proteins.

Vacuoles have been shown to undergo deep modifications in relation to plant developmental stages and in the maintaining the cellular homeostasis. In this context, we studied the variations of the vacuolar membrane size and α-TIP aquaporin distribution at early and advanced seed stages of maturation, germination and embryo growth in Vicia faba cotyledon storage cells., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background and Aims: The study of floral morphology and ontogeny and the re-investigation of existing data help to uncover potential synapomorphic characters and foster our understanding of phylogenetic relationships that rely primarily on molecular analyses. Goniorrhachis marginata is a monotypic caesalpinioid legume (Leguminosae) that shows some interesting floral features, such as a long hypanthium and regular Rosaceae-like flowers. We studied the ontogeny and morphology of the flowers in detail and present our results in a broad phylogenetic context., Methods: Flower buds were collected in the field, fixed in 70 % ethanol and investigated using scanning electron microscopy. Older buds in spirit were carefully opened to investigate the direction of style bending. Characters of the style from 131 taxa from the main legume lineages were analysed and mapped on a Bayesian molecular phylogeny., Key Results: The tetramerous calyx is the result of complete loss of one sepal. The formation of the radially symmetrical corolla starts in a typical caesalpinioid pattern with the adaxial petal innermost (ascending aestivation). The young style bends in the abaxial direction, which is a character found exclusively in all studied detarioid legumes and therefore a newly described synapomorphy for the clade., Conclusions: We show that investigation of unstudied taxa and reinvestigation of published data can uncover new, previously overlooked and important characters. Curvature of the style can be detected in young buds with a hand lens and therefore is an important character for field botanists. Our study reveals the importance of including poorly studied and/or phylogenetically enigmatic taxa in molecular phylogenies and in detailed morphological and ontogenetic analyses., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The possible involvement of chloroplast lipids in the mechanisms of NaCl tolerance was studied in leaves of two varieties of Fabaceae: Sulla carnosa and Sulla coronaria, which were subjected to 200mM NaCl over 20days. Changes in membrane lipid peroxidation, chloroplast lipids content, fatty acids (FA) composition and the ultrastructure of chloroplasts under salt stress were investigated. Chloroplast lipids were separated and quantified by high performance liquid chromatography coupled to evaporative light scattering detection (HPLC/ELSD). The results showed that salinity induced a significant decrease in digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG) and sulfoquinovosylglycerol (SQDG) content in both S. carnosa and S. coronaria leaves, whereas monogalactosyldiacylglycerol (MGDG) content did not change significantly in S. carnosa leaves. The MGDG/DGDG ratio remained stable in S. coronaria leaves but increased in those of S. carnosa. In addition, the unsaturated-to-saturated fatty acids ratio (UFAs:SFAs) did not change under salt stress in S. coronaria leaves, while it decreased significantly in S. carnosa leaves. Moreover, salinity did not induce significant changes in MGDG and DGDG unsaturation level in S. carnosa leaves, in contrast to S. coronaria, in which salinity seems to enhance the unsaturation level in MGDG, DGDG and PG. Furthermore, the level of membrane lipid peroxidation, as expressed by malondialdehyde (MDA) levels, increased at 200mM in S. carnosa leaves, while it did not change significantly in those of S. coronaria. With respect to the ultrastructure of chloroplasts at 200mM NaCl, investigated by transmission electron microscopy (TEM), salt-stress caused the swelling of thylakoids in S. carnosa mesophyll. These ultrastructural changes were observed especially in the spongy tissue in S. coronaria. Taken together, these findings suggest that the stability of MGDG/DGDG ratio, the unchanged unsaturation level, and increasing unsaturation level in MGDG, DGDG and PG may be effective to some degree in suppressing the ultrastructural damage caused by salinity effects and may contribute to protect the chloroplast membrane integrity against salt stress., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The preparation of tropical wood surface sections for time-of-flight secondary ion mass spectrometry imaging is described, and the use of delayed extraction of secondary ions and its interest for the analysis of vegetal surface are shown. The method has been applied to the study by time-of-flight secondary ion mass spectrometry imaging with a resolution of less than one micron of a tropical wood species, Dicorynia guianensis, which is one of the most exploited wood in French Guiana for its durable heartwood. The heartwood of this species exhibits an economical importance, but its production is not controlled in forestry. Results show an increase of tryptamine from the transition zone and a concomitant decrease of inorganic ions and starch fragment ions. These experiments lead to a better understanding of the heartwood formation and the origin of the natural durability of D. guianensis. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Development and innovation of new technologies are necessary especially in food quality; due that most instrumental technique for measuring quality properties involves a considerable amount of manual work. Image analysis is a technique that allows to provide objective evaluations from digitalized images that can estimate quality parameters for consumer's acceptance. The aim of the present research was to study the effect of freeze drying on the microstructure and texture of legume and vegetables using scanning electron microscopy at different magnifications' combined with image analysis. Cooked and cooked freeze dried rehydrated legumes and vegetables were analyzed individually by scanning electron microscopy at different magnifications' (250, 500, and 1000×).Texture properties were analyzed by texture analyzer and image analysis. Significant differences (P < 0.05) were obtained for image and instrumental texture parameters. A linear trend with a linear correlation was applied for instrumental and image features. Results showed that image features calculated from Grey level co-occurrence matrix at 1,000× had high correlations with instrumental features. In rice, homogeneity and contrast can be applied to evaluate texture parameters gumminess and adhesiviness; Lentils: contrast, correlation, energy, homogeneity, and entropy for hardness, adhesiviness, gumminess, and chewiness; Potato and carrots: contrast, energy, homogeneity and entropy for adhesiviness, chewiness, hardness, cohesiviness, and resilence. Results revealed that combing scanning electron microscopy with image analysis can be a useful tool to analyze quality parameters in legumes and vegetables., (© 2016 Wiley Periodicals, Inc.)

Toxic effects of copper (Cu) were analyzed in young plants of Inga subnuda subs. luschnathiana, a species that is highly tolerant to flooding and found in Brazil in wetlands contaminated with Cu. Plants were cultivated in fully nutritive solution, containing different concentrations of Cu (from 0.08 μmol to 0.47 mmol L(-1)). Symptoms of Cu toxicity were observed in both leaves and roots of plants cultivated from 0.16 mmol Cu L(-1). In the leaves, Cu clearly induced alterations in the thickness of the epidermis, mesophyll, palisade parenchyma, and intercellular space of the lacunose parenchyma. Also, this metal induced disorganization in thylakoid membranes, internal and external membrane rupture in chloroplasts, mitochondrial alterations, and electrodense material deposition in vacuoles of the parenchyma and cell walls. The starch grains disappeared; however, an increase of plastoglobule numbers was observed according to Cu toxicity. In the roots, destruction of the epidermis, reduction of the intercellular space, and modifications in the format of initial cells of the external cortex were evident. Cell walls and endoderm had been broken, invaginations of tonoplast and vacuole retractions were found, and, again, electrodense material was observed in these sites. Mineral nutrient analysis revealed higher Cu accumulation in the roots and greater macro- and micronutrients accumulation into shoots. Thus, root morphological and ultrastructural changes induced differential nutrients uptake and their translocations from root toward shoots, and this was related to membrane and endoderm ruptures caused by Cu toxicity.

Heavy metals are major environmental pollutant when they present in high concentration in soil and have toxic effects on growth, nodulation and nitrogen fixation of legumes and development of plants. Mercury stress triggers disturbances in cellular structure, and metabolismn is poorly understood. The response of seedling growth and nodulation of Vigna radiata (L) Wilczek to different concentrations (0.1, 0.5, 1.0 and 1.5 ppm) of mercury (Hg) salt solutions were studied. Morphological parameters like root and shoot length, dry weight, nodule number, total leaf area and biochemical constituents (chlorophyll, malondialdehyde and leghaemoglobin) of bean plants were recorded at an interval of 30 days. The successive growth deformaties in seedlings and nodules were recorded at lower concentration (0.1 ppm), but marginal (0.5 ppm) and higher (1.0 ppm) level of Hg salt solution showed significant suppression. The maximum level of Hg concentration (1.5 ppm) shows high level of tolerance index without any nodule. The control treatment shows maximum level of leghaemoglobin (0.219 mM) and all other morpho-physiological and bio-chemical properties of roots and shoots excepting tolerance index (0.00) and chlorophyll 'a' (7.52 mg g(-1) FW). Mercury accumulation pattern follows the sequences: leaf > nodule > root ≈ shoot at lower level of Hg (0.1 and 0.5 ppm). However, higher level of Hg (1.0 and 1.5 ppm) showed shoot > root > leaf > nodule. The scanning electron microscopy (SEM) study of root also highlights the deleterious effect of Hg salt at higher concentration.

A comparative anatomical study was carried out on the secretory structures of leaflets from taxa belonging to the genus Rhynchosia - taxa difficult to delimit because of uncertain interspecific relations - in order to evaluate the potential diagnostic value of these anatomical traits for taxonomic assignment. A further objective was to establish consensual denomination for these secretory structures. The new anatomical features found in these taxa were sufficiently consistent to separate the species evaluated. The presence and localization of glandular-punctate structures bulbous-based trichomes, the number of layers in the palisade parenchyma and the arrangement of vascular units distinguish the taxa investigated and these characteristics can be extended to other species of Papilionoideae. The trichomes analyzed were described and classified into five types. Depicted in diagrams, photomicrographs, and by scanning electron microscopy, and listed for the first time at the genus and species levels. The information obtained served to effectively distinguish the taxa investigated among species of Papilonoideae.

Many legumes have been selected as model plants to degrade organic contaminants with their special associated rhizosphere microbes in soil. However, the function of root nodules during microbe-assisted phytoremediation is not clear. A pot study was conducted to examine phenanthrene (PHE) utilizing bacteria associated with root nodules and the effects of cowpea root nodules on phytoremediation in two different types of soils (freshly contaminated soil and aged contaminated soil). Cowpea nodules in freshly-contaminated soil showed less damage in comparison to the aged-contaminated soil, both morphologically and ultra-structurally by scanning electron microscopy. The study of polycyclic aromatic hydrocarbon (PAH) attenuation conducted by high performance liquid chromatography revealed that more PAH was eliminated from liquid culture around nodulated roots than nodule-free roots. PAH sublimation and denaturation gradient gel electrophoresis were applied to analyze the capability and diversity of PAH degrading bacteria from the following four parts of rhizo-microzone: bulk soil, root surface, nodule surface and nodule inside. The results indicated that the surface and inside of cowpea root nodules were colonized with bacterial consortia that utilized PHE. Our results demonstrated that root nodules not only fixed nitrogen, but also enriched PAH-utilizing microorganisms both inside and outside of the nodules. Legume nodules may have biotechnological values for PAH degradation.

The genus Desmodium is represented in Santa Catarina State, Brazil, by 13 species, all with lomen- taceous fruits. Shape, size and isthmus margin of loments vary, while the surface is glabrous, or covered by trichomes of different types. Morphological diversity of trichomes becomes particularly relevant to taxonomic description. The trichome types present on the surface of Desmodium fruits provide data for the identification and classification of species in the State. To assess this, three fruits of each species were collected and deposited at two herbaria, HBR and FLOR, in Santa Catarina, Brazil. Some rehydrated samples were examined using light microscopy (LM); and some sections were exposed to the following histochemical reagents: Sudan III for oils and Thionine for mucilage. The structural aspects of trichomes can be classified into uni- or multicellular and may still be simple, i.e., nonglandular or glandular. Using scanning electron microscopy (SEM), five types of trichomes were identified and analyzed among the Desmodium species studied: uncinate, uniseriate, globose multicellular, globose unicellular and subulate. Characteristics, such as loment margin and article form, glabrescent or pillous indument, trichome type, with or without papillous epidermal cells and epicuticular striations, showed relevant diagnostic value. An identification key was developed for Desmodium species from Santa Catarina State, Brazil, based on macro and micromorphological characters of the fruit.

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.

Daminozide (B9) is a growth inhibitor with important regulatory roles in plant growth and development. Locating and quantifying B9-binding proteins in plant tissues will assist in investigating the mechanism behind the signal transduction of B9. In this study, red fluorescent Mn-doped CdTe quantum dots (CdTeMn QDs) were synthesized by a high-temperature hydrothermal process. Since CdTeMn QDs possess a maximum fluorescence emission peak at 610nm, their fluorescence properties are more stable than those of CdTe QDs. A B9-CdTeMn probe was synthesized by coupling B9 with CdTeMn QDs. The fluorescence intensity of the probe is double that of CdTeMn QDs; its fluorescence stability is also superior under different ambient conditions. The probe retains the biological activity of B9 and is unaffected by interference from the green fluorescent protein present in plants. Therefore, we used this probe to label B9-binding proteins selectively in root tissue sections of mung bean seedlings. These proteins were observed predominantly on the surfaces of the cell membranes of the cortex and epidermal parenchyma., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the present study, hydroxyapatite (HAP) nanoparticles of different particle sizes with high crystallinity and similiar structure were prepared by hydrothermal method. The crystal structure and particle size were characterized by X-ray diffraction pattern (XRD), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. Mung bean sprouts were first used as experimental models. Instead of by MTT assay, the cytoxicity of HAP nanoparticles were proved and evaluated by measuring the hypocotyle length of mung bean sprouts in the culture media. The result showed that the inhibition effect to the growth of mung bean sprouts enhanced when HAP nanoparticles existed. Culture media of HAP nanoparticles with different concentrations and particle sizes was prepared to investigate the level of inhibition effect to the growth of mung bean sprouts. The result found that hypocotyl length of mung bean sprouts were the shortest cultured in 5mg/mL culture media in which the HAP nanoparticles were prepared by hydrothermal method for 24h. It was concluded the inhibition effect depended on the amount of intracellular HAP nanoparticles. The nanostructure and Ca(2+) concentration were considered as the main factors to cause cell apoptosis which was the reason of inhibition. The study provided a preliminary perspective about biotoxicity of HAP nanomaterials to the plant growth., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Premise of the Study: Apocarpy (i.e., free carpels) is considered to be the basal condition for ovary development in angiosperms. Yet it only occurs in 10% of angiosperm species, of which another 10% are monocarpellate. Most legume flowers are monocarpellate. Species with polycarpellate gynoecia occur in about 15 genera with most representatives in Mimosoideae. In the present study, we analyze legumes with polycarpellate flowers with the aim of improving our understanding of gynoecium evolution., Methods: Flowers of nine legume species from five genera were analyzed using a scanning electron microscope (SEM)., Key Results: In Leguminosae, carpels usually form as individual primordia or protuberances. Inga congesta differs slightly from this pattern in that the central apex bulges outward before the formation of individual carpel primordia. While legumes usually develop entirely plicate carpels, flowers of Acacia celastrifolia and Inga bella show an intermediate type of carpel morphology with a distal plicate zone and a small proximal ascidiate zone. Carpels in Inga congesta and Archidendron glabrum are sometimes slightly fused at the ovary base. The orientation of carpel clefts seems to reflect the floral symmetry. They are directed to the floral center in mimosoids and caesalpinioids, whereas in Swartzia dipetala carpel clefts are oriented to the adaxial side., Conclusions: Polycarpelly arose at least seven times independently in Leguminosae. The polycarpellate condition appears to be correlated with polyandry, and in most instances, it is accompanied by a profound change in floral organization from a closed to an open system.

We present the leaf anatomy of seven amphibious species of Aeschynomene L. (Papilionoideae, Leguminosae), interpreting their structures and ecological functions, and also, providing information on which their taxonomy can be based, especially of morphologically similar species. We evaluated Aeschynomene americana, A. ciliata, A. evenia, A. denticulata, A. fluminensis, A. rudis and A. sensitiva. The anatomy corroborates the separation of the series Americanae, Fluminenses, Indicae and Sensitivae, with the shape of the petiole, types of trichomes and quantity of vascular units in the petiole as main characteristics to delimit the species. The petiole shape varies from cylindric in A. americana, A. sensitiva and A. fluminensis, to triangular in A. evenia and quadrangular in A. rudis, A. denticulata and A. ciliata. We observed four types of trichomes: hydathode trichome, long conic trichome, short conic trichome and bulb-based trichome. The hydathode trichome was the most common, except for A. americana and A. fluminensis. Species with higher affinity with water share similar adaptive characteristics, including hydathode trichomes described for the first time for the genus. This article adds unseen descriptions for the genus and on the adaptation factors of the amphibious species.

Background and Aims: The inflorescence structure determines the spatiotemporal arrangement of the flowers during anthesis and is therefore vital for reproductive success. The Leguminosae are among the largest angiosperm plant families and they include some important crop plants. In papilionoid legumes, the raceme is the most common type of inflorescence. However, a range of other inflorescence types have evolved via various developmental processes. A (re-)investigation of inflorescences in Swainsona formosa, Cicer arietinum, Abrus precatorius, Hardenbergia violacea and Kennedia nigricans leads to new insights into reduction mechanisms and to a new hypothesis on the evolution of the papilionoid pseudoraceme., Methods: Inflorescence morphology and ontogeny were studied using scanning electron microscopy (SEM)., Key Results: The inflorescence in S. formosa is an umbel with a rare type of pendulum symmetry which may be triggered by the subtending leaf. Inflorescences in C. arietinum are reduced to a single flower. An early formed adaxial bulge is the sterile apex of the inflorescence (i.e. the inflorescence is open and not terminated by a flower). In partial inflorescences of A. precatorius, the axis is reduced and its meristem is relocated towards the main inflorescence. Flower initiation follows a peculiar pendulum pattern. Partial inflorescences in H. violacea and in K. nigricans show reduction tendencies. In both taxa, initiated but early reduced bracteoles are present., Conclusions: Pendulum symmetry in S. formosa is probably associated with distichous phyllotaxis. In C. arietinum, strong reduction tendencies are revealed. Based on studies of A. precatorius, the papilionoid pseudoraceme is reinterpreted as a compound raceme with condensed lateral axes. From an Abrus-like inflorescence, other types can be derived via reduction of flower number and synchronization of flower development. A plea is made for uniform usage of inflorescence terminology.

Seed biology is a relevant aspect of tropical forests because it is central to the understanding of processes of plant establishment, succession and natural regeneration. Anadenanthera colubrina var. cebil is a timber tree from South America that produces large seeds with thin weak teguments, which is uncommon among legumes. This study describes the morphology and anatomy of the seed coat, the viability, imbibition, and germination in this species. Seeds used during the essays came from 10 trees that grow naturally in Horco Molle, province of Tucumán, Argentina. Seed morphology was described from a sample of 20 units. The seed coat surface was examined with a scanning electron microscope. Transverse sections of hydrated and non-hydrated seeds were employed to describe the histological structure of the seed coat. Hydration, viability and germination experiments were performed under laboratory controlled conditions; and the experimental design consisted of 10 replicas of 10 seeds each. Viability and germination tests were conducted using freshly fallen seeds and seeds stored for five months. Morphologically the seeds of A. colubrina var. cebil are circular to subcircular, laterally compressed, smooth, bright brown and have a horseshoe fissure line (= pleurogram) on both sides. The seed coat comprises five tissue layers and a double (external and internal) cuticle. The outer cuticle (on the epidermis) is smooth and interrupted by microcracks and pores of variable depth. The epidermis consists of macroesclereids with non-lignified secondary walls. This layer is separated from the underlying ones during seed hydration. The other layers of internal tissues are comprised of osteosclereids, parenchyma, osteosclereids, and macrosclereids. The percentage of viable seeds was 93%, decreasing to 75% in seeds with five months old. Seed mass increased 76% after the first eight hours of hydration. Germination percentage was 75% after 76 hours. Germination of seeds stored for five months decreased to 12%. The results showed that seeds of A. colubrina var. cebil are highly permeable and germinate directly without a dormant period.

Inga vera, native to South America, is an important leguminous species used for ecological restoration of riparian forests and its seeds are among the most recalcitrant ones described up to date. In this work, we analysed the metabolic profile, cell ultrastructure as well as cell wall polysaccharides of I. vera seeds in order to better understand its maturation, which allows embryo germination without a quiescent phase. Increased amounts of citric, glutamic, pyroglutamic, and aspartic acids from stages I to II (120 and 129 days after flowering (DAF)) corroborate the hypothesis of high metabolism, shifting from fermentative to aerobic respiration at seed maturity. This phase was characterized by an extensive vacuolization of embryonic cells, which also indicate high metabolic activity. The proportion of arabinose in the cell walls of embryonic axis (approx. 20%) was lower than those found in some orthodox seeds (nearly 40%), suggesting that arabinose-containing polysaccharides, which are thought to provide more flexibility to the cell wall during natural drying, are less abundant in I. vera seeds. Taken together, our results provide evidence that the major changes occurred during early stages of seed maturation of I. vera, indicating that the rapid temporary metabolic shift observed between stages I and II may be related to the lack of desiccation phase, moving directly to germination., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The presented paper considered the pollen morphology of thirteen species belonging to seven genera of the Fabaceae family occurring in the Pau-de-Fruta Special Protection Area (SPA), Diamantina, state of Minas Gerais, Brazil. The pollen grains of six species of Chamaecrista [C. cathartica (Mart.) H.S. Irwin & Barneby, C. debilis Vogel, C. flexuosa (L.) Greene, C. hedysaroides (Vogel) H.S. Irwin & Barneby, C. glandulosa (L.) Greene, and C. papillata H.S. Irwin & Barneby] have a similar morphology, characterized by three long colporated apertures with a central constriction. The species share specific morphological features regarding pollen size, endoaperture type (circular, lalongate or lolongate) and SEM ornamentation patterns of the exine (rugulate with perforations or perforate). Andira fraxinifolia Benth., Dalbergia miscolobium Benth, Galactia martii DC, Periandra mediterranea (Vell.) Taub., Senna rugosa (G.Don) H.S. Irwin & Barneby and Zornia diphylla (L.) Pers showed different pollen types in small to large size; oblate spheroidal to prolate form; colpus or colporus apertures; circular, lalongate or lolongate endoapertures and distinctive SEM ornamentation patterns of the exine (perforate, microreticulate, reticulate or rugulate with perforations). Only Stryphnodendron adstringens (Mart.) Coville presents polyads. The pollen morphology variation of these species allowed the Fabaceae family to be characterized as eurypalynous in the SPA Pau-de-Fruta.

Premise of the Study: In angiosperm seeds, the developing embryo acquires nutrients via a suspensor that typically undergoes programmed cell death (PCD) at the early cotyledon stage. However, in Leguminosae (the third largest angiosperm family), the suspensors can disappear at the heart-shaped stage (i.e., prior to the cotyledon stage) or still persist at the cotyledon stage. Here, in a comprehensive survey of legume suspensors and embryos, the variation and the evolutionary direction of timing of suspensor PCD in Leguminosae were characterized, and systematic implications were evaluated., Methods: Suspensor development and morphology for 66 leguminous species from 49 genera, 21 tribes, and 3 subfamilies were comparatively studied using standard paraffin sectioning and light microscopy., Key Results: Three patterns of suspensor PCD were observed at the early cotyledon stage. (A) The suspensor persisted. (B) The suspensor separated from the wall of the embryo sac and persisted as a vestige at the radicle apex. (C) The suspensor disappeared completely, and the absorption of nutrients by embryo was carried out via a "contact zone" between the embryo and the endosperm. Pattern C of early suspensor PCD was found only in the tribe Fabeae. An ancestral character reconstruction revealed that the long-lived suspensors of pattern A represented a plesiomorphic condition in Leguminosae and that the suspensors of pattern C evolved only once in the common ancestor of Fabeae., Conclusions: In Leguminosae, short-lived suspensors have thus evolved multiple times from long-lived suspensors. It remains largely unknown, however, how the embryo acquires nutrients after the early suspensor PCD.

The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to ∼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pterodon pubescens cavities are constituted by lumen and uniseriated epithelium surrounded by multiseriate parenchyma sheath. We studied the development of secretory cavities, including the role of parenchyma sheath, using light and transmission electron microscopy. A Tunel assay was performed to verify whether programmed cell death (PCD) occurs during the process. The lumen is formed by schizogeny and lysigeny occur in later developmental stages of the secretory cavities. Ultrastructurally, epithelial cells in later developmental stages become dark and with sinuous walls; the protoplast becomes retracted and the cytoplasm shows low organelle definition. Degenerated cells are released toward the lumen. Our results showed that PCD occurs during later developmental stages of cavities and plays a critical role in functioning of these glands. New cells originated from the parenchyma sheath differentiate into secretory cells and replace those degenerated ones. This fact associated to PCD guarantees epithelium renovation during the secretory cycle and the maintenance of secretory activity of cavities., (Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Background and Aims: Clianthus maximus is a leguminous perennial with an unusual order of floral organ insertion, and inflorescences produced year round that nearly all abort except during a limited time in autumn. This study aimed to determine at what point in floral organ differentiation abortion occurred and whether the expression of the floral identity genes underlies this cessation in flower development., Methods: Inflorescences were harvested across an annual cycle and flower development was examined by light and scanning electron microscopy. Expression of the C. maximus-equivalents of LEAFY (LFY), APETALA1 (AP1), PISTILLATA (PI) and AGAMOUS (AG) was monitored simultaneously by quantitative, reverse transcriptase PCR., Key Results: Only those inflorescences formed in autumn proceeded to anthesis. Organogenesis had not begun in inflorescences that aborted. The C. maximus-equivalents of AP1, PI and AG were expressed in sepals, petals, carpels and stamens, as expected from the ABC model of floral organ identity specification; furthermore, the order of expression of the three genes reflected the unusual pattern of organ differentiation. Low expression of LFY and AP1 was observed during inflorescence abortion., Conclusions: Predictions of gene expression based on the ABC model were upheld despite the unusual mass abortion of inflorescences and the non-standard pattern of organ formation. The lack of expression of LFY and AP1 in inflorescences may have been the cause of inflorescence abortion.

The data obtained in this study proved that Mycoplasma gallisepticum S6 known as avian pathogen had a phytopathogenic potential. The vegetative forms and the viable but nonculturable (VBNC) forms of this mycoplasma could infect the plants via an assemblage of rootlets, invade different tissues, persist there and cause destructive events characteristic to phytomycoplasmoses. In comparison with the vegetative forms, the VBNC forms induced more prominent destructive changes. This phenomenon might be connected to increasing expression of proteins responsible for virulence in the bacterial cells. The fact that M. gallisepticum S6 could demonstrate virulent features (infectivity, invasiveness, persistence and toxigenicity) in regard to plants seems to require a development of new ways for controlling phytomycoplasmoses taking into account the probable presence of asymptomatic carriers of this bacterium., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Boron (B) deficiency has a strong effect on molecular and cellular plant-bacteria interactions during the development of the legume-rhizobia symbiosis, leading to reduced infection and early necrosis of nodules, resembling a pathogenic-like rather than a symbiotic interaction. Therefore, induction of pathogenesis-related (PRs) proteins was investigated here in legume root nodules. Following two-dimensional electrophoresis and MALDI-TOF spectrometry analysis of proteins extracted from Pisum sativum B-sufficient (+B) or B-deficient (-B) root nodules, two proteins from the family PR10, ABR17 and PR10.1, were identified as highly induced in -B nodules. Analysis of gene expression and the use of anti-ABR17 confirmed that induction occurred in B-deficient young nodules and increased during nodule development. ABR17 was also induced in -B nodules of Phaseolus vulgaris. Boron deficiency did not significantly increase the expression of these PR10 in uninfected plant tissues. Moreover, independent of B, induction was detected in senescent tissues, although at a level weaker than in -B nodules. The immunochemical study of ABR17 antigen distribution showed that it was localized in all tissues of poorly invaded B-deficient nodules and accumulated around bacteria, which showed advanced degradation. These results suggest that, under B deficiency, the rhizobia-legume dialogue fails and the bacterium is recognized as a pathogen by the plant, which reacts to prevent infection by inducing at least these two identified PR10 proteins., ((c) 2010 Elsevier GmbH. All rights reserved.)

Reports concerning colleters in Fabaceae have been scarce, mainly in the Caesalpinioideae subfamily. The present work reports the occurrence, structure, and functional aspects of the colleters of Copaifera langsdorffii. Shoot apices and developing leaves were fixed and processed for examination by light and electron microscopy. Secretion samples were studied to determine their chemical nature and physical properties. The colleters are clavate and occur on the adaxial face of the stipules, petiole and rachis. The secretory stage of the colleters occurs during the leaf expansion, after which these structures turn brown and senesce. The secretion is composed of highly hygroscopic acidic polysaccharides and lipids. The colleters are composed of cells with thin walls, large nuclei, and dense cytoplasm with dictyosomes, mitochondria, plastids and the endoplasmic reticulum. Analyses of the secretion, placement, and functional aspects of the colleters present in C. langsdorffii indicate that these structures help protect young leaves from desiccation.

Background and Aims: The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes., Methods: Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae., Key Results: The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species., Conclusions: Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established.

Suspension-cultured bean cells habituated to growth in a lethal concentration of dichlobenil were cultured for 3-5 years in a medium lacking the inhibitor in order to obtain long-term dehabituated cell lines. The growth parameters, cell morphology and ultrastructure of cells in the absence of dichlobenil reverted to that of non-habituated cells. The cellulose content and Fourier transform infrared (FTIR) spectra of crude cell walls from long-term dehabituated cells were also similar to those of non-habituated cells. However, long-term dehabituated cells showed three times more tolerance to dichlobenil than non-habituated cells. The incorporation of [(14)C]Glc into cellulose was reduced by 40% in dehabituated cells when compared with non-habituated cells. However, the addition of dichlobenil to dehabituated cells increased the incorporation of [(14)C]Glc into cellulose 3.3-fold with respect to that of non-habituated cells. Dehabituated cells showed a constitutively increased peroxidase activity when compared with non-habituated cells. Results reported here indicate that the habituation of bean cultured cells to dichlobenil relied partially on a stable change in the cellulose biosynthesis complex and is associated with high guaiacol peroxidase activity.

Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

Discrepant and incomplete interpretations of fruits of Pterodon have been published, especially on the structural interpretation of the pericarp portion that remain attached to the seed upon dispersal. The present work clarified these doubts and analyzed ultrastructural aspects of the Pterodon emarginatus diaspores using light and transmission electron microscopes. Cell divisions are prevalent among the initial phases of development, and the subadaxial and adaxial meristems form the fibrous inner mesocarp and the endocarp composed of multi-seriate epidermis, respectively. At the median mesocarp, numerous secretory ducts differentiate between the lateral bundles, by lytic process. After lysis of the central cells and the formation of the lumen, the ducts show unistratified secretory epithelium with dense cells; oil droplets are observed on the secretory epithelium and the subadjacent tissues. At maturity, the uniseriate exocarp and the outer mesocarp slough off in an irregular fashion, leaving the diaspore composed of a papery and brittle wing linked to a seed chamber that includes the median mesocarp composed of lignified cells, bordering vascular bundles and many secretory ducts whose epithelial cells develop large vacuoles that accumulate oleoresins. The Pterodon emarginatus fruit is a cryptosamara.

Extrafloral nectaries are nectar-secreting structures that are especially common among the woody flora of the Brazilian cerrado, a savanna-like vegetation. In this study, we provide morphological and anatomical descriptions of extrafloral nectaries (EFNs) occurring on vegetative and reproductive organs of several plant species from the cerrado, and discuss their function and ecological relevance. We describe the morphology and anatomy of EFNs of 40 species belonging to 15 woody families using scanning electron microscopy and light microscopy. We categorise EFNs following a structural-topographical classification, and characterise the vascularised and complex nectaries, amorphous nectaries and secretory trichomes. Fabaceae, Bignoniaceae, Malpighiaceae and Vochysiaceae were the plant families with the majority of species having EFNs. Ten species possess more than one morphotype of gland structure. Observations and experimental field studies in the cerrado support the anti-herbivore role of EFN-gathering ants in this habitat. Additional morphological studies of EFNs-bearing plants, including other growth forms (e.g. herbs and lianas), are being undertaken and will hopefully cast further light on the ecological relevance of these glands in the cerrado, especially with respect to their attractiveness to multiple visitors.

Background and Aims: Molecular phylogenies have suggested a new circumscription for Fabales to include Leguminosae, Quillajaceae, Surianaceae and Polygalaceae. However, recent attempts to reconstruct the interfamilial relationships of the order have resulted in several alternative hypotheses, including a sister relationship between Quillajaceae and Surianaceae, the two species-poor families of Fabales. Here, floral morphology and ontogeny of these two families are investigated to explore evidence of a potential relationship between them. Floral traits are discussed with respect to early radiation in the order., Methods: Floral buds of representatives of Quillajaceae and Surianaceae were dissected and observed using light microscopy and scanning electron microscopy., Key Results: Quillajaceae and Surianaceae possess some common traits, such as inflorescence morphology and perianth initiation, but development and organization of their reproductive whorls differ. In Quillaja, initiation of the diplostemonous androecium is unidirectional, overlapping with the petal primordia. In contrast, Suriana is obdiplostemonous, and floral organ initiation is simultaneous. Independent initiation of five carpels is common to both Quillaja and Suriana, but subsequent development differs; the antesepalous carpels of Quillaja become fused proximally and exhibit two rows of ovules, and in Suriana the gynoecium is apocarpous, gynobasic, with antepetalous biovulate carpels., Conclusions: Differences in the reproductive development and organization of Quillajaceae and Surianaceae cast doubt on their potential sister relationship. Instead, Quillaja resembles Leguminosae in some floral traits, a hypothesis not suggested by molecular-based phylogenies. Despite implicit associations of zygomorphy with species-rich clades and actinomorphy with species-poor families in Fabales, this correlation sometimes fails due to high variation in floral symmetry. Studies considering specific derived clades and reproductive biology could address more precise hypotheses of key innovation and differential diversification in the order.

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67(phox), a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of DeltabcnoxR mutants is identical to that of DeltabcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.

Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf's movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf's movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia diphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species.

Background and Aims: Molecular phylogenies have suggested a new circumscription for Fabales to include Leguminosae, Quillajaceae, Surianaceae and Polygalaceae. However, recent attempts to reconstruct the interfamilial relationships of the order have resulted in several alternative hypotheses, including a sister relationship between Quillajaceae and Surianaceae, the two species-poor families of Fabales. Here, floral morphology and ontogeny of these two families are investigated to explore evidence of a potential relationship between them. Floral traits are discussed with respect to early radiation in the order., Methods: Floral buds of representatives of Quillajaceae and Surianaceae were dissected and observed using light microscopy and scanning electron microscopy., Key Results: Quillajaceae and Surianaceae possess some common traits, such as inflorescence morphology and perianth initiation, but development and organization of their reproductive whorls differ. In Quillaja, initiation of the diplostemonous androecium is unidirectional, overlapping with the petal primordia. In contrast, Suriana is obdiplostemonous, and floral organ initiation is simultaneous. Independent initiation of five carpels is common to both Quillaja and Suriana, but subsequent development differs; the antesepalous carpels of Quillaja become fused proximally and exhibit two rows of ovules, and in Suriana the gynoecium is apocarpous, gynobasic, with antepetalous biovulate carpels., Conclusions: Differences in the reproductive development and organization of Quillajaceae and Surianaceae cast doubt on their potential sister relationship. Instead, Quillaja resembles Leguminosae in some floral traits, a hypothesis not suggested by molecular-based phylogenies. Despite implicit associations of zygomorphy with species-rich clades and actinomorphy with species-poor families in Fabales, this correlation sometimes fails due to high variation in floral symmetry. Studies considering specific derived clades and reproductive biology could address more precise hypotheses of key innovation and differential diversification in the order.

Background and Aims: Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae)., Methods: Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families., Key Results: Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a beta-rhizobial strain can effectively nodulate papilioinoid legumes., Conclusions: Papilionoid legumes from widely different tribes can be nodulated by beta-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.

Chitinases (EC 3.2.1.14) are hydrolytic enzymes found in different organisms. In plants, they have been described in different tissues and organs, including seeds. This study was triggered by the isolation of a 30-kDa thermostable chitinase from Adenanthera pavonina L. seeds. The enzyme was submitted to N-terminal amino acid sequencing, and the analysis revealed a high degree of homology with class III chitinases. Bidimensional electrophoresis of the 30-kDa band showed the presence of three isoforms with pIs of 5.2, 5.5 and 5.8. A chitinase was also found in exudates released from the same seeds, which was seen to be immunorelated to the above 30-kDa protein. It was also submitted to N-terminal amino acid sequencing and seen as highly homologous to class III chitinases. In addition, the expression of chitinases during A. pavonina L. seed germination and seedling development was investigated. Seeds were allowed to germinate in the absence of light for approximately 5 days and were grown, for different times, in the absence or presence of light. After each seedling developmental time, samples of exudates, roots and cotyledonary leaves were collected and submitted to protein extraction. The presence of proteins immunorelated to the 30-kDa chitinase was detected in all analyzed samples. Further analyses showed that light significantly interfered with the chitinase expression in some organs. The tissue and subcellular chitinase location in seedling roots was also investigated, and it was majorly localized in the cell wall and in the intercellular spaces of the root hair zone.

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.

Ultra-structural studies were conducted on root nodules of Samanea saman (Jacq.) Merr. collected from trees growing under natural conditions. Nodules were distributed singly as well as in clusters on the main and lateral roots. Mature nodules were elongated, branched and coralloid. Root hair curling was found but infection threads could not be observed. Rhizobia entered through the epidermis and moved intercellularly through the cortical region. Mature nodules of S. saman could be differentiated into meristem, cortex, vascular tissue and bacteroid tissue. The latter showed both infected and non-infected cells mixed together. Vascular bundles were inversely collateral and distributed around the bacteroid tissue. The bacteroids were enclosed in peribacteroid membrane in groups and showed prominent granules of polyhydroxybutyrate in their cytoplasm. Mycorrhizal hyphae were also observed along with rhizobia in the bacteroid tissue. S. saman with dual rhizobial and mycorrhizal infection is a potential tree for plantation in arid soils of Pakistan.

The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots.