Flexible mating: cross-pollination affects sex-expression in a marine clonal plant

Many functionally hermaphroditic plants have evolved mechanisms to reduce interference between the sex functions and to optimize reproductive output. In addition to physical mechanisms such as the spatial (herkogamy) and temporal (dichogamy) separation of male and female functions, plasticity in sex expression by means of mate-recognition (flexible mating) could be important in plants with variable access to cross-pollen. This applies particularly to clonal plants because of their modular growth form. We experimentally tested for the effects of pollen source and vegetative neighbourhood on instantaneous sex ratio and seed production in the self-compatible clonal marine angiosperm Zostera marina L. To this end, we exposed the (monoecious) flowering shoots to self and cross-pollen and to neighbourhoods of their own and a mix of foreign vegetative shoots. Flowering shoots that had been exposed to cross-pollen showed (1) a significantly lower female/male ratio at peak flowering, evidence for mate-recognition, and (2) a significantly higher seed set by the end of the season. Both effects were independent of the genetic composition of their vegetative neighbourhood. The results suggest that Z. marina maintains a cryptic self-incompatibility system not previously described for angiosperms with sub-aqueous pollination. In Z. marina, and possibly other self-compatible clonal plant species, mate-recognition could be a means of increasing the out-crossing probability for flowering shoots with central positions within their clone.     

Plants intruding Laretia acaulis (Umbelliferae), a high Andean cushion plant

Several plants invade the cushions of Laretia acaulis in the high Andean communities of central Chile. Humus develops among the rosettes, and 42 different species settle on the cushions. Most of these species are also found outside the cushions. Semi-woody chamaephytes, hemicryptophytes and cryptophytes have a similar cover on both substrates, while the cover of woody chamaephytes and therophytes is smaller on the cushions. The diversity and cover of invading plants, which vary with altitude, result from interactions among the environmental conditions, cushion morphology and the surrounding vegetation.This study is part of the Chilean contribution to the Man and the Biosphere (MAB) Programme, within the framework of the UNEP/UNESCO Andean Project (1105-77-01).Nomenclature is given in the Appendix.We thank M. T. K. Arroyo, C. Marticorena and O. Matthei, and the Herbaria of the Universidad Concepción and the Museo de Historia Natural, for help with identification of species.     

Drift patterns in a high Andean stream

The drifting of invertebrates was sampled for six 24-hour periods from September 1976 to July 1977 in a small stream of the paramo of the Ecuadorian Andes. The composition of the drift is similar to that of the benthos, though percentages may differ markedly. Drift is relatively constant throughout the year, except in March when unusually high rates were noted; at this same period a marked reduction in the benthos was also observed. Diel periodicities in the drift are unclear, although on the whole drift is more important during the daytime; when individual data series are analyzed, weak patterns of day or night drifting can be recognized in some groups. The input of insects through drift from the small streams is thought to be an important source of food for the salmonid fish inhabiting the larger torrents.     

Induced plant volatiles allow sensitive monitoring of plant health status in greenhouses

A novel approach to support the inspection of greenhouse crops is based on the measurement of volatile organic compounds emitted by unhealthy plants.This approach has attracted some serious interest over the last decade. In pursuit of this interest, we performed several experiments at the laboratory-scale to pinpoint marker volatiles that can be used to indicate certain health problems. In addition to these laboratory experiments, pilot and model studies were performed in order to verify the validity of these marker volatiles under real-world conditions. This paper provides an overview of results and gives an outlook on the use of plant volatiles for plant health monitoring.Key words: plant health, volatiles, plant pathogens, plant infection     

European plant science: a field of opportunities

Plants have a pivotal role in eco- and agricultural systems.Genomics is driving a rapid expansion of our understanding ofhow genes, individually and in networks, determine plant function.Technological developments in breeding and genomics are providingstrategies to translate this knowledge into crop improvement.The possibilities range from improvement of existing crops andthe systematic use of natural diversity through to the domesticationof completely new species. As examples of possible goals, itis discussed how profiling of composition will integrate plantbreeding and agronomic practice with emerging knowledge aboutnutrition and health, how improved and novel crops will contributeto the creation of new bio-based economies revolving aroundplant products, and how advances in our knowledge about plant–environmentand plant–pathogen interactions will provide novel strategiesto stabilize agricultural yield in a fluctuating environmentand contribute to integrated approaches in which modern agricultureis carried out in concert with the environment. In addition,knowledge generated by plant science will be needed to monitor,understand, and cope with climate change and its impact on agricultureand ecosystems. Realization of these goals will require closeinteractions with related disciplines including agronomy andecology. Further, it will be important to continue and deepenopen support for research in the developing world. Key words: Agronomic practice, biodiversity, domestication, ecosystems, environment, genomics, novel crops, plant breeding, plant products, yield     

Cold resistance mechanisms in high desert Andean plants

Freezing tolerance and freezing avoidance were studied, during the growing season, in plant species from two different elevations (3200 m and 3700 m) in a desert region of the high Andes (29° 45S, 69° 59W) in order to determine whether there was a relationship between plant height and cold resistance mechanisms. Freezing injury and supercooling capacity were determined in plants of different height, from ground-level (<20 cm tall) to tall shrubs (27–90 cm). All ground-level plants showed freezing tolerance as the main mechanism for resistance to freezing temperatures. Tall shrubs avoided freezing temperatures, mainly through supercooling. Supercooling was only present in plants occupying the lower elevation (i.e., 3200 m). Both avoidance and tolerance mechanisms are present in a single genus (i.e., Adesmia).     

Repeated evolution of vertebrate pollination syndromes in a recently diverged Andean plant clade

Although specialized interactions, including those involving plants and their pollinators, are often invoked to explain high species diversity, they are rarely explored at macroevolutionary scales. We investigate the dynamic evolution of hummingbird and bat pollination syndromes in the centropogonid clade (Lobelioideae: Campanulaceae), an Andean‐centered group of ∼550 angiosperm species. We demonstrate that flowers hypothesized to be adapted to different pollinators based on flower color fall into distinct regions of morphospace, and this is validated by morphology of species with known pollinators. This supports the existence of pollination syndromes in the centropogonids, an idea corroborated by ecological studies. We further demonstrate that hummingbird pollination is ancestral, and that bat pollination has evolved ∼13 times independently, with ∼11 reversals. This convergence is associated with correlated evolution of floral traits within selective regimes corresponding to pollination syndrome. Collectively, our results suggest that floral morphological diversity is extremely labile, likely resulting from selection imposed by pollinators. Finally, even though this clade's rapid diversification is partially attributed to their association with vertebrate pollinators, we detect no difference in diversification rates between hummingbird‐ and bat‐pollinated lineages. Our study demonstrates the utility of pollination syndromes as a proxy for ecological relationships in macroevolutionary studies of certain species‐rich clades.     

Demographic effects of large, introduced herbivores on a long-lived endemic plant

The introduction of alien ungulates is a major threat for the survival of endangered plants, especially in island ecosystems. However, very few studies have investigated the potential damage of large herbivores on plant diversity in Mediterranean protected areas. In this study, we describe the population structure and the long-term dynamics of the main populations of the Sardinian narrow endemic Centaurea horrida Bad. (Asteraceae), by means of permanent plots where individual plants were tagged and monitored through 6?years (2004?C2009). We monitored this endangered plant at three sites: two were protected areas where introduced and feral ungulates are present, and the other one was a non-protected site without introduced ungulates. We found that adults and saplings were more abundant at the non-protected site. Through matrix models, we also highlighted that the non-protected population showed the highest population growth rate. Finally, by means of an exclusion experiment for ungulates at one protected site, we demonstrated that herbivores had a negative effect on the survival of seedlings and adult plants, and reduced the stochastic population growth rate. An LTRE analysis showed that differences in the survival, especially of adult individuals, had the highest responsibility in explaining the higher population growth rate when herbivores are excluded. Our study constitutes a clear example on how the protection of alien large herbivores can have opposite effects on the conservation of an endangered plant. Some management options are proposed, and the urgent need of manipulative experiments on species-specific interactions between protected plants and alien herbivore species is invoked.     

Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.     

Field and genetic studies testing optimal outcrossing in Agave schottii,a long-lived clonal plant

In thisstudy we combine field experiments, designed to test the predictions of optimal outcrossing theory in Agave schottii, with molecular genetic studies, using RAPD (random amplified polymorphic DNA), polymerase chain reaction to assess the underlying genetic hypothesis of optimal outcrossing theory. Initially, 48 females of A. schottii were hand-pollinated with pollen collected from 1 m, 10 m, 100 m, and 2500 m distances. Each female received all four distance treatments. Additionally, a subset of the focal females and their pollen donors were used in an analysis of genetic similarity across the four distances. Results of hand-pollinations showed that crosses of 1 m had significantly lower seed set than 10 m and 100 m crosses. Crosses of 2500 m had intermediate seed set. Combined relative fitness was significantly lower for 1 m crosses compared to 10 m crosses, while 100 m and 2500 m crosses were intermediate. Thus, A. schottii experiences inbreeding depression and a trend toward outbreeding depression. Genetic analyses showed a similar pattern: individuals 1 m apart had on average higher genetic similarity (proportion of bands shared) than individuals separated by greater distances, with a trend toward lower genetic similarity for plants located 2500 m distant. The observed spatial genetic patterns are likely maintained by the combined effects of clonal reproduction, clone longevity, limited seed dispersal and the substantial number of inbred progeny produced, counteracting distant allele transfer which tends to reduce population genetic structure. The correspondence between our ecological and genetic results indicates that RAPD markers are useful tools for assessing ecological phenomena.     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.     

The best for the guest: high Andean nurse cushions of Azorella madreporica enhance arbuscular mycorrhizal status in associated plant species

Positive interactions between cushion plant and associated plants species in the high Andes of central Chile should also include the effects of fungal root symbionts. We hypothesized that higher colonization by arbuscular mycorrhizal (AM) fungi exists in cushion-associated (nursling) plants compared with conspecific individuals growing on bare ground. We assessed the AM status of Andean plants at two sites at different altitudes (3,200 and 3,600 m a.s.l.) in 23 species, particularly in cushions of Azorella madreporica and five associated plants; additionally, AM fungal spores were retrieved from soil outside and beneath cushions. 18 of the 23 examined plant species presented diagnostic structures of arbuscular mycorrhiza; most of them were also colonized by dark-septate endophytes. Mycorrhization of A. madreporica cushions showed differences between both sites (68% and 32%, respectively). In the native species Hordeum comosum, Nastanthus agglomeratus, and Phacelia secunda associated to A. madreporica, mycorrhization was six times higher than in the same species growing dispersed on bare ground at 3,600 m a.s.l., but mycorrhiza development was less cushion dependent in the alien plants Cerastium arvense and Taraxacum officinale at both sites. The ratio of AM fungal spores beneath versus outside cushions was also 6:1. The common and abundant presence of AM in cushion communities at high altitudes emphasizes the importance of the fungal root symbionts in such situations where plant species benefit from the microclimatic conditions generated by the cushion and also from well-developed mycorrhizal networks.     

A new aerobic chemolithoautotrophic arsenic oxidizing microorganism isolated from a high Andean watershed

Biological arsenic oxidation has been suggested as a key biogeochemical process that controls the mobilization and fate of this metalloid in aqueous environments. To the best of our knowledge, only four aerobic chemolithoautotrophic arsenite-oxidizing (CAO) bacteria have been shown to grow via direct arsenic oxidation and to have the essential genes for chemolithoautotrophic arsenite oxidation. In this study, a new CAO bacterium was isolated from a high Andean watershed evidencing natural dissolved arsenic attenuation. The bacterial isolate, designated TS-1, is closely related to the Ancylobacter genus, in the Alphaproteobacteria class. Results showed that TS-1 has genes for arsenite oxidation and carbon fixation. The dependence of bacterial growth from arsenite oxidation was demonstrated. In addition, a mathematical model was suggested and the kinetic parameters were obtained by simultaneously fitting the biomass growth, arsenite depletion curves, and arsenate production. This research increases the knowledge of chemolithoautotrophic arsenic oxidizing microorganisms and its potential role as a driver for natural arsenic attenuation.     

Exploring the impact of neutral evolution on intrapopulation genetic differentiation in functional traits in a long-lived plant

Most plant species, particularly long-lived plants, harbor a large amount of genetic variation within populations. A central issue in evolutionary ecology is to explore levels of genetic variation and understand the mechanisms that influence them. In this study, our goals were to examine the impact of neutral evolutionary processes on the genetic variance and functional diversity within three populations of a long-lived plant (Quercus suber L.). For this purpose, we genotyped the progeny of 45 open-pollinated mother trees from three populations originating from Spain, Portugal, and Morocco using six microsatellite markers. Seedlings were planted in a common garden trial and were phenotypically characterized by seven leaf functional traits. Molecular analyses revealed weak genetic differences between Iberian and Moroccan populations. Nevertheless, high genetic differentiation was observed among maternal families within populations. Differentiation between particular maternal families from the same population reached values of 29.2 %, which far exceeds the values reported between the most genetically distant populations for this species (11.7 %). Maternal families differed also in phenology, leaf size, and shape traits. In the Moroccan population, there were correlations among matrices of distances for molecular markers, leaf shape traits (e.g., leaf circularity index), and phenology, indicating that maternal families with contrasting phenologies were genetically and functionally distinct. This, together with the moderate heritability for phenology in Moroccan population, suggests that besides selective forces, neutral evolutionary processes have promoted intrapopulation genetic divergence and contribute to maintain high levels of genetic variation within this population. Overall, our results reinforce the importance of intrapopulation studies in long-lived plants under an evolutionary context.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Pollen transfer in fragmented plant populations: insight from the pollen loads of pollinators and stigmas in a mass‐flowering species

Pollinator and/or mate scarcity affects pollen transfer, with important ecological and evolutionary consequences for plant reproduction. However, the way in which the pollen loads transported by pollinators and deposited on stigmas are affected by pollination context has been little studied. We investigated the impacts of plant mate and visiting insect availabilities on pollen transport and receipt in a mass‐flowering and facultative autogamous shrub (Rhododendron ferrugineum). First, we recorded insect visits to R. ferrugineum in plant patches of diverse densities and sizes. Second, we analyzed the pollen loads transported by R. ferrugineum pollinators and deposited on stigmas of emasculated and intact flowers, in the same patches. Overall, pollinators (bumblebees) transported much larger pollen loads than the ones found on stigmas, and the pollen deposited on stigmas included a high proportion of conspecific pollen. However, comparing pollen loads of emasculated and intact flowers indicated that pollinators contributed only half the conspecific pollen present on the stigma. At low plant density, we found the highest visitation rate and the lowest proportion of conspecific pollen transported and deposited by pollinators. By contrast, at higher plant density and lower visitation rate, pollinators deposited larger proportion of conspecific pollen, although still far from sufficient to ensure that all the ovules were fertilized. Finally, self‐pollen completely buffered the detrimental effects on pollination of patch fragmentation and pollinator failure. Our results indicate that pollen loads from pollinators and emasculated flowers should be quantified for an accurate understanding of the relative impacts of pollinator and mate limitation on pollen transfer in facultative autogamous species.     

Slurry-producing agents provide buoyancy and allow growth of plant suspension cells in stationary culture

Summary Suspension cultures of carrot, soybean, tobacco, Black Mexican sweet corn, and photoautotrophic soybean have been successfully grown in stationary slurry culture. Cell colonies grew loosely and slowly in clumps which were easily dispersable, increasing in size only 4–5 fold by 48 days, which facilitated subculture.