Anther development in tribe Epidendreae: orchids with contrasting pollination syndromes.

BACKGROUND: Epidendreae is one of the most diverse tribes among the orchids with remarkable variation in life form, floral morphology and pollination syndromes. Its circumscription was recently revised and subtribes Agrostophyllinae and Calypsoinae were transferred into this tribe. One of the principal floral characters utilized in classification of orchids is the incumbency or bending of the column. This study records and compares late stages of anther, column and lip development, and discusses anther characters in fifteen representative taxa of five of the six subtribes in Epidendreae with respect to classification and pollination biology. METHODS: A series of late floral stages were sampled and fixed for examination under scanning electron microscope. RESULTS: Anther incumbency or bending in this group varies from 90° to almost 180°. Incumbency in the late stages of development is reached in Bletiinae, Ponerinae, Pleurothallidinae and Laeliinae whereas incumbency is reached early in its development in Corallorhiza and Govenia of Calypsoinae. DISCUSSION: Our observations indicate that the position of Chysis in subtribe Bletiinae needs revision based on differences in a number floral, and in particular of anther characters; and that Coelia only shares the early anther incumbency with Calypsoinae members, but not the rest of floral and anther characters. Anatomical characters such as crystals around the actinocytic stomata on the anther cap and sugar crystals in Laeliinae; lack of rostellum in Bletiinae; coalescent anther with the column, lack of trichomes and papillae on lip keels, and underdeveloped rostellum in Chysis; a mechanism by which the anther cap comes off (it is joined with the grooved lip by a claw) in Isochilus are all related to pollination syndromes and reproductive biology.

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana.

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.

Experimental evidence of pollination in marine flowers by invertebrate fauna.

Pollen transport by water-flow (hydrophily) is a typical, and almost exclusive, adaptation of plants to life in the marine environment. It is thought that, unlike terrestrial environments, animals are not involved in pollination in the sea. The male flowers of the tropical marine angiosperm Thalassia testudinum open-up and release pollen in mucilage at night when invertebrate fauna is active. Here we present experimental evidence that, in the absence of water-flow, these invertebrates visit the flowers, carry and transfer mucilage mass with embedded pollen from the male flowers to the stigmas of the female flowers. Pollen tubes are formed on the stigmas, indicating that pollination is successful. Thus, T. testudinum has mixed abiotic-biotic pollination. We propose a zoobenthophilous pollination syndrome (pollen transfer in the benthic zone by invertebrate animals) which shares many characteristics with hydrophily, but flowers are expected to open-up during the night.

A novel motif in the NaTrxh N-terminus promotes its secretion, whereas the C-terminus participates in its interaction with S-RNase in vitro.

BACKGROUND: NaTrxh, a thioredoxin type h, shows differential expression between self-incompatible and self-compatible Nicotiana species. NaTrxh interacts in vitro with S-RNase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. NaTrxh contains N- and C-terminal extensions, a feature shared by thioredoxin h proteins of subgroup 2. To ascertain the function of these extensions in NaTrxh secretion and protein-protein interaction, we performed a deletion analysis on NaTrxh and fused the resulting variants to GFP. RESULTS: We found an internal domain in the N-terminal extension, called Nß, that is essential for NaTrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. The lack of hydrophobicity as well as the location of the secretion signal within the NaTrxh primary structure, suggest an unorthodox secretion route for NaTrxh. Notably, we found that the fusion protein NaTrxh-GFP(KDEL) is retained in the endoplasmic reticulum and that treatment of NaTrxh-GFP-expressing cells with Brefeldin A leads to its retention in the Golgi, which indicates that NaTrxh uses, to some extent, the endoplasmic reticulum and Golgi apparatus for secretion. Furthermore, we found that Nß contributes to NaTrxh tertiary structure stabilization and that the C-terminus functions in the protein-protein interaction with S-RNase. CONCLUSIONS: The extensions contained in NaTrxh sequence have specific functions on the protein. While the C-terminus directly participates in protein-protein interaction, particularly on its interaction with S-RNase in vitro; the N-terminal extension contains two structurally different motifs: Nα and Nß. Nß, the inner domain (Ala-17 to Pro-27), is essential and enough to target NaTrxh towards the apoplast. Interestingly, when it was fused to GFP, this protein was also found in the cell wall of the onion cells. Although the biochemical features of the N-terminus suggested a non-classical secretion pathway, our results provided evidence that NaTrxh at least uses the endoplasmic reticulum, Golgi apparatus and also vesicles for secretion. Therefore, the Nß domain sequence is suggested to be a novel signal peptide.

How common is self-incompatibility across species of the herkogamous genus Ariocarpus?

PREMISE OF THE STUDY: Self-incompatibility (SI), the most effective mechanism to prevent selfing, may limit the number of compatible mates in populations. The seven species of Ariocarpus are endangered and predominantly outcrossers but fruit set may reach 1-20% after selfing. We aimed to determine whether SI is the underlying mechanism influencing mating in Ariocarpus species. METHODS: We characterized the presence/absence of SI using pollination treatments (self-pollination, cross-pollination, natural pollination) in one population per species. We assessed SI using epifluorescence and generalized linear models (GLMs) to compare the presence of pollen tubes in the stigma, stylar transmitting tissue, and ovary among self- and cross-pollinated pistils 48 h after pollination. Following the same treatments, production of fruit set was noted and related to pollen tube growth. KEY RESULTS: Pollen tubes were found more frequently in the ovaries of natural and cross-pollinated flowers than in ovaries of self-pollinated. Stylar rejection of self-pollen indicated gametophytic SI, although pollen tubes reached the ovaries in six species (4-33% of pistils). Fruit set was lower after hand-pollinations than expected from pollen tube observations. CONCLUSIONS: The low percentages of self-compatibility in all species in pollen tube growth and pollination experiments indicated that no species had complete self-sterility, suggesting the presence of partial SI. Reduced fruit set relative to pollen tube production could result from a threshold of insufficient pollination, early-acting inbreeding depression, or resource limitation. The origin of partial SI in Ariocarpus could respond to pressures such as pollen limitation and population size.

Understanding ontogenetic trajectories of indirect defence: ecological and anatomical constraints in the production of extrafloral nectaries.

BACKGROUND AND AIMS: Early ontogenetic stages of myrmecophytic plants are infrequently associated with ants, probably due to constraints on the production of rewards. This study reports for the first time the anatomical and histological limitations constraining the production of extrafloral nectar in young plants, and the implications that the absence of protective ants imposes for plants early during their ontogeny are discussed. METHODS: Juvenile, pre-reproductive and reproductive plants of Turnera velutina were selected in a natural population and their extrafloral nectaries (EFNs) per leaf were quantified. The anatomical and morphological changes in EFNs during plant ontogeny were studied using scanning electron and light microscopy. Extrafloral nectar volume and sugar concentration were determined as well as the number of patrolling ants. KEY RESULTS: Juvenile plants were unable to secrete or contain nectar. Pre-reproductive plants secreted and contained nectar drops, but the highest production was achieved at the reproductive stage when the gland is fully cup-shaped and the secretory epidermis duplicates. No ants were observed in juvenile plants, and reproductive individuals received greater ant patrolling than pre-reproductive individuals. The issue of the mechanism of extrafloral nectar release in T. velutina was solved given that we found an anatomical, transcuticular pore that forms a channel-like structure and allows nectar to flow outward from the gland. CONCLUSIONS: Juvenile stages had no ant protection against herbivores probably due to resource limitation but also due to anatomical constraints. The results are consistent with the growth-differentiation balance hypothesis. As plants age, they increase in size and have larger nutrient-acquiring, photosynthetic and storage capacity, so they are able to invest in defence via specialized organs, such as EFNs. Hence, the more vulnerable juvenile stage should rely on other defensive strategies to reduce the negative impacts of herbivory.

The fruit of Bursera: structure, maturation and parthenocarpy.

BACKGROUND AND AIMS: The deterioration of seasonally tropical dry forests will stop with the implementation of management plans for this ecosystem. To develop these plans, we require information regarding aspects such as germination and the presence of 'empty seeds' of representative species-like, for example, Bursera, a genus with a high number of endemic species of the Mesoamerican Hotspot-that would enable us to propagate its species. The main purpose of this study is to describe the phenological and structural characteristics of fruits of 12 Bursera species and provide useful data for future studies on germination and seed dispersal, and to acquire new and useful information to understand the phylogenetic relationships of the Burseraceae family. METHODOLOGY: We described the phenology of fruit ripening in 12 species of Bursera. Fruits were collected from the study sites in three different stages of development. The histochemical and anatomical characteristics of fruits of all species were described with the use of inclusion techniques and scanning microscopy. PRINCIPAL RESULTS: There is a time gap between the development of the ovary and the development of the ovule in the 12 studied species. The exposed pseudoaril during the dispersion stage is an indicator of the seed's maturity and the fruit's viability. The Bursera fruit shows the same structural pattern as that of Commiphora, as well as many similarities with species of the Anacardiaceae family. All species develop parthenocarpic fruits that retain the structural characteristics of the immature fruits: soft tissues rich in nitrogen compounds and few chemical and physical defences. Insects were found mainly inside the parthenocarpic fruits in eight species of Bursera. CONCLUSIONS: The dispersion unit in Bursera consists of a seed, a lignified endocarp that protects the seed, and a pseudoaril that helps attract seed dispersers. The production of parthenocarpic fruits is energy saving; however, it is necessary to evaluate the potential benefits of this phenomenon.

Conostegia xalapensis (Melastomataceae): an aluminum accumulator plant.

In acidic soils, an excess of Al³âº is toxic to most plants. The Melastomataceae family includes Al-accumulator genera that tolerate high Al³âº by accumulating it in their tissues. Conostegia xalapensis is a common shrub in Mexico and Central America colonizing mainly disturbed areas. Here, we determined whether C. xalapensis is an Al accumulator, and whether it has internal tolerance mechanisms to Al. Soil samples collected from two pastures in the state of Veracruz, Mexico, had low pH and high Al³âº concentrations along with low Ca²âº levels. Leaves of C. xalapensis from pastures showed up to 19,000 mg Al kg⁻¹ DW (dry weight). In laboratory experiments, 8-month-old seedlings treated with 0.5 and 1.0 mM AlCl3 for 24 days showed higher number of lateral roots and biomass. Pyrocatechol violet and hematoxylin staining evidenced that Al localized in epidermis and mesophyll cells in leaves and in epidermis and vascular pith in roots. Scanning electron microscope-energy dispersive X-ray microanalysis of Al-treated leaves corroborated that Al is in abaxial and adaxial epidermis and in mesophyll cells (31.2%) in 1.0 mM Al-treatment. Roots of Al-treated plants had glutathione reductase (EC 1.6.4.2) and superoxide dismutase (EC 1.15.1.1) activity higher, and low levels of O2*⁻ and H2O2. C. xalapensis is an Al-accumulator plant that can grow in acidic soils with higher Al³âº concentrations, and can be considered as an indicator species for soils with potential Al toxicity.

Functional bisporangiate cones in Pinus johannis (Pinaceae): Implications for the evolution of bisexuality in seed plants.

PREMISE OF STUDY: Bisexuality (male and female function in one structure) has been reported as a key innovation of angiosperms. Although there are several reports of "teratological" bisporangiate (bisexual) cones in gymnosperms, there have been none on the viability of their ovules and pollen. Analyses of the development and arrangement of female and male structures on bisporangiate cones of Pinus johannis enables us to gain insight on the origin of bisexuality in seed plants, for both angiosperms and gymnosperms. METHODS: Viability of bisporangiate cones was assayed by performing manual crosses and using anatomical and histological methods. KEY RESULTS: We determined that bisporangiate cones of P. johannis produce functional pollen and ovules. Male and female organs occupy basal and apical positions, respectively, the same positions found in almost all bisporangiate strobili in gymnosperms and bisexual flowers in angiosperms. CONCLUSIONS: The viability and spatial distribution of female and male organs of bisporangiate cones and their frequent occurrence in gymnosperms suggest a common mechanism in all seed plants for the production of bisporangiate structures. This idea is further supported by the presence of homologous genes for sexual organ identity in gymnosperms and angiosperms as reported by other authors. The lack of bisporangiate structure in gymnosperms may be primarily due to selection to avoid inbreeding rather than to genetic constraint.

Seed reserve composition in 19 tree species of a tropical deciduous forest in Mexico and its relationship to seed germination and seedling growth.

BACKGROUND AND AIMS: The size and composition of seed reserves may reflect the ecological strategy and evolutionary history of a species and also temporal variation in resource availability. The seed mass and composition of seed reserves of 19 co-existing tree species were studied, and we examined how they varied among species in relation to germination and seedling growth rates, as well as between two years with contrasting precipitation (652 and 384 mm). METHODS: Seeds were collected from a tropical deciduous forest in the northwest of Mexico (Chamela Biological Station). The seed dry mass, with and without the seed coat, and the concentrations of lipids, nitrogen and non-structural carbohydrates for the seed minus seed coat were determined. The anatomical localization of these reserves was examined using histochemical analysis. The germination capacity, rate and lag time were determined. The correlations among these variables, and their relationship to previously reported seedling relative growth rates, were evaluated with and without phylogenetic consideration. KEY RESULTS: There were interannual differences in seed mass and reserve composition. Seed was significantly heavier after the drier year in five species. Nitrogen concentration was positively correlated with seed coat fraction, and was significantly higher after the drier year in 12 species. The rate and lag time of germination were negatively correlated with each other. These trait correlations were also supported for phylogenetic independent contrasts. Principal component analysis supported these correlations, and indicated a negative association of seedling relative growth rate with seed size, and a positive association of germination rate with nitrogen and lipid concentrations. CONCLUSIONS: Nitrogen concentration tended to be higher after the drier year and, while interannual variations in seed size and reserve composition were not sufficient to affect interspecific correlations among seed and seedling traits, some of the reserves were related to germination variables and seedling relative growth rate.

Parthenocarpy and seed predation by insects in Bursera morelensis.

BACKGROUND AND AIMS: While parthenocarpy (meaning the production of fruits without seeds) may limit fecundity in many plants, its function is not clear; it has been proposed, however, that it might be associated with a strategy to avoid seed predation. Bursera morelensis is a dioecious endemic plant that produces fruits with and without seeds, and its fruits are parasitized by insects. Its reproductive system is not well described and no published evidence of parthenocarpy exists for the species. The purpose of this work was to describe the breeding system of B. morelensis and its relationship to seed predation by insects. METHODS: The breeding system was described using pollination experiments, verifying the presence of parthenocarpic fruits and apomictic seeds. Reproductive structures from flower buds to mature fruits were quantified. For fruits, an anatomical and histological characterization was made. The number of fruits in which seeds had been predated by insects was correlated with parthenocarpic fruit production. KEY RESULTS: The major abortion of reproductive structures occurred during fruit set. The results discard the formation of apomictic seeds. Flowers that were not pollinated formed parthenocarpic fruits and these could be distinguished during early developmental stages. In parthenocarpic fruits in the first stages of development, an unusual spread of internal walls of the ovary occurred invading the locule and preventing ovule development. Unlike fruits with seeds, parthenocarpic fruits do not have calcium oxalate crystals in the ovary wall. Both fruit types can be separated in the field at fruit maturity by the presence of dehiscence, complete in seeded and partial in parthenocarpic fruits. Trees with more parthenocarpic fruits had more parasitized fruits. CONCLUSIONS: This is the first time the anatomy of parthenocarpic fruits in Burseraceae has been described. Parthenocarpic fruits in B. morelensis might function as a deceit strategy for insect seed predators as they are unprotected both chemically and mechanically by the absence of calcium oxalate crystals.

Developmental gametophyte morphology of seven species of Thelypteris subg. Cyclosorus (Thelypteridaceae).

The morphogenesis of the sexual phase of seven species of Thelypteris subg. Cyclosorus (Thelypteridaceae) is described and compared. Spores of all species are monolete, ellipsoid and have positive photoblastism. They have Vittaria-type germination, germinal filaments are short and uniseriate, and Aspidium-type development. Adult gametophytes are spatulate-cordiform, and have wide wings with numerous glandular, marginal and superficial hairs. Antheridia have a narrow or widened basal cell, an undivided annular cell, and an opercular cell; antherozoids are liberated through three mechanisms. Archegonia have short necks and four triangular cells on the neck's mouth. The sporophyte's first leaf is lobed, with open dichotomous veins, glandular hairs similar to those of the gametophyte, and anomocytic stomata. Differences observed between the studied species are: spore size; ornamentation of the perispore; germination and emergence periods; size of hairs; developmental period of gametangia; and antherozoid liberation mechanisms through total detachment of the operculum, partial detachment of the operculum, and development of an apical pore through tearing of the operculum. These taxonomically valuable characters, combined with the sporophyte characteristics, can be useful tools in the identification of the species studied to the subg. Cyclosorus.

A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro.

Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollen-pistil interaction processes or particularly in S-RNase-based self-incompatibility.

Compatibilidad sexual entre dos tipos de Hylocerus (Cactaceae) / Sexual compatibility between two types of Hylocerus (Cactaceae)

There are two types of pitahaya that are cultivated in Yucatan Peninsula of Mexico. They differ mainly in the skin color of the fruit, one of them has a red skin (Uqroo1), while the other has a light yellow skin (Uqroo2) both belong to Hylocereus undatus (Haworth) Britt. & Rose. The yellow skin pitahaya is the sweetest. The studies were conducted using the methods of self pollination and cross pollination, with direct crosses and reciprocal pollination in order to evaluate the effect related to the quantity of fruits that accomplish their growing process. Some characteristic parameters of fruits, thus obtained, were used to detect the possible xenic effect. The control samples were collected from naturally pollinated flowers. The experimental design used was completely random and the experiment was repeated fifteen times. The results revealed that the Uqroo1 turned out to be self-compatible, and their fruits reached one hundred percent of development. On the other hand, Uqroo2 was self-incompatible, that is, it did not accept self pollen. The control samples of both types reached the same weight, and the fruits of the Uqroo2 produced by cross pollination had the same or more weight than the control samples. Several parameters were positively correlated. The coefficient between the number of seeds and the fruit weight was r = 0.84. The highest correlation that was found (r = 0.97) in the fruit weight and the pulp weight within both types. The fruits obtained by cross-pollination maintained the characteristic of the female progenitor. It is suggest intercalate the yellow pitahaya plants with another types or species of pitahaya, other than the Uqroo1.

[Sexual compatibility between two types of Hylocerus (Cactaceae)]. / Compatibilidad sexual entre dos tipos de Hylocerus (Cactaceae).

There are two types of pitahaya that are cultivated in Yucatan Peninsula of Mexico. They differ mainly in the skin color of the fruit, one of them has a red skin (Uqroo1), while the other has a light yellow skin (Uqroo2) both belong to Hylocereus undatus (Haworth) Britt. & Rose. The yellow skin pitahaya is the sweetest. The studies were conducted using the methods of self pollination and cross pollination, with direct crosses and reciprocal pollination in order to evaluate the effect related to the quantity of fruits that accomplish their growing process. Some characteristic parameters of fruits, thus obtained, were used to detect the possible xenic effect. The control samples were collected from naturally pollinated flowers. The experimental design used was completely random and the experiment was repeated fifteen times. The results revealed that the Uqroo1 turned out to be self-compatible, and their fruits reached one hundred percent of development. On the other hand, Uqroo2 was self-incompatible, that is, it did not accept self pollen. The control samples of both types reached the same weight, and the fruits of the Uqroo2 produced by cross pollination had the same or more weight than the control samples. Several parameters were positively correlated. The coefficient between the number of seeds and the fruit weight was r = 0.84. The highest correlation that was found (r = 0.97) in the fruit weight and the pulp weight within both types. The fruits obtained by cross-pollination maintained the characteristic of the female progenitor. It is suggest intercalate the yellow pitahaya plants with another types or species of pitahaya, other than the Uqroo1.