Molecular genetic diversity and population structure analysis were used to clarify the controversial botanical classification of Stylosanthes guianensis. The accessions were clustered in nine groups, each of which was mainly composed of only one of the four botanical varieties.
Background and aims
The botanical classification of Stylosanthes guianensis is controversial, and few studies have used molecular markers to analyse this species. We used microsatellite markers to study the genetic diversity and population structure of S. guianensis and compare our results with the current infraspecific botanical classification.
A representative sample from the S. guianensis Brazilian germplasm collection (150 accessions) was analysed using 20 microsatellite loci. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign accessions into clusters. A dendrogram was constructed based on Roger's genetic distances.
The number of alleles per locus varied from 2 to 11, with an average of 4.7. The observed (HO) and expected (HE) heterozygosity values varied from 0 to 0.58 (mean of 0.18) and from 0.04 to 0.83 (mean of 0.55), respectively. Nine groups were assembled in STRUCTURE, and these groups were consistent with clusters inferred from the genetic distances and taxonomic varieties described for S. guianensis. The GST among the nine groups was 0.46.
The low HO and the GST values observed are in agreement with the outcrossing rate (26 %) estimated for this species. The data indicate a high genetic diversity among and within the botanical varieties and suggest that microsatellite-based information can be combined with classical taxonomy to elucidate infraspecific levels.
The genome size and organization of the important medicinal plant Catharanthus roseus is shown to correspond to 1C = 0.76 pg (~738 Mbps) and 2n = 16 chromosomes. The data provide a sound basis for future studies including cytogenetic mapping, genomics and breeding.
Background and aims
Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size.
The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology.
The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp.
The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.
The ating evidence suggests non-symbiotic hemoglobins affect hormone responses by scavenging NO. Auxin, jasmonic acid, salicylic acid, ethylene and abscisic acid have altered responses when hemoglobins are expressed. Non-symbiotic hemoglobin is a factor during plant development, biotic and abiotic stress.
Background and aims
Non-symbiotic haemoglobins have been an active research topic for over 30 years, during which time a considerable portfolio of knowledge has accumulated relative to their chemical and molecular properties, and their presence and mode of induction in plants. While progress has been made towards understanding their physiological role, there remain a number of unanswered questions with respect to their biological function. This review attempts to update recent progress in this area and to introduce a hypothesis as to how non-symbiotic haemoglobins might participate in regulating hormone signal transduction.
Advances have been made towards understanding the structural nuances that explain some of the differences in ligand association characteristics of class 1 and class 2 non-symbiotic haemoglobins. Non-symbiotic haemoglobins have been found to function in seed development and germination, flowering, root development and differentiation, abiotic stress responses, pathogen invasion and symbiotic bacterial associations. Microarray analyses under various stress conditions yield uneven results relative to non-symbiotic haemoglobin expression. Increasing evidence of the role of nitric oxide (NO) in hormone responses and the known involvement of non-symbiotic haemoglobins in scavenging NO provide opportunities for fruitful research, particularly at the cellular level.
Circumstantial evidence suggests that non-symbiotic haemoglobins may have a critical function in the signal transduction pathways of auxin, ethylene, jasmonic acid, salicylic acid, cytokinin and abscisic acid. There is a strong need for research on haemoglobin gene expression at the cellular level relative to hormone signal transduction.
Filter cubes made with machine-vision dichroic filters and illuminated with a royal blue light emitting diode can be used to produce an epifluorescent digital camera attachment that improves whole organism green fluorescent protein (GFP) photography. Mean pixel intensity responds linearly to purified GFP titration.
Background and aims
Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants.
Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically.
Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29).
Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets.
This paper compiles and discusses all currently available nuclear genome size data for red algae in relation to their most recent taxonomic classification.
Background and aims
The red algae are an evolutionarily ancient group of predominantly marine organisms with an estimated 6000 species. Consensus higher-level molecular phylogenies support a basal split between the unicellular Cyanidiophytina and morphologically diverse Rhodophytina, the later subphylum containing most red algal species. The Rhodophytina is divided into six classes, of which five represent early diverging lineages of generally uninucleate species, whose evolutionary relationships are poorly resolved. The remaining species compose the large (27 currently recognized orders), morphologically diverse and typically multinucleate Florideophyceae. Nuclear DNA content estimates have been published for <1 % of the described red algae. The present investigation summarizes the state of our knowledge and expands our coverage of DNA content information from 196 isolates of red algae.
The DNA-localizing fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and RBC (chicken erythrocytes) standards were used to estimate 2C values with static microspectrophotometry.
Nuclear DNA contents are reported for 196 isolates of red algae, almost doubling the number of estimates available for these organisms. Present results also confirm the reported DNA content range of 0.1–2.8 pg, with species of Ceramiales, Nemaliales and Palmariales containing apparently polyploid genomes with 2C = 2.8, 2.3 and 2.8 pg, respectively.
Early diverging red algal lineages are characterized by relatively small 2C DNA contents while a wide range of 2C values is found within the derived Florideophyceae. An overall correlation between phylogenetic placement and 2C DNA content is not apparent; however, genome size data are available for only a small portion of red algae. Current data do support polyploidy and aneuploidy as pervasive features of red algal genome evolution.
Little is known about the genome of Anthurium other than chromosome observations, which frequently indicate supernumerary (“B”) chromosomes. New genome size estimates for 34 species and nine cultivars presented here provide insights into genome organization and evolution in this very large genus.
Background and aims
Anthurium is an important horticultural crop from the family Araceae, order Alismatales, a lineage considered to have diverged from other monocots prior to the cereals. Genome size and its distribution in Anthurium were investigated to gain a basic understanding of genome organization in this large genus and to forge a firm foundation for advancement of molecular approaches for the study of Anthurium. Currently, genome size estimates have been reported for only two Anthurium samples.
Bulk nuclear DNA content estimates were obtained by flow cell cytometry using leaf tissue collected from Anthurium species of different subgeneric groups and from commercial cultivars. The most current and well-supported topology of subgeneric, sectional relationships was applied to present genome size estimates in the context of reported chromosome counts, karyotypes, putative phylogenetic relationships, observed phenotypes and pedigree.
Genome size estimates based on bulk nuclear DNA content for 77 accessions representing 34 species and 9 cultivars were obtained, including initial estimates for 33 Anthurium species, and both the smallest (Anthurium obtusum; Tetraspermium) and largest (Anthurium roseospadix; Calomystrium) Anthurium genome sizes reported to date. Genome size did not distinguish any subgeneric section, but ranged 5-fold (4.42–20.83 pg/2 C) despite consistent 2N= 30 chromosome counts. Intraspecies genome size variation >20 % is reported for Anthurium ravenii, A. watermaliense and A. gracile.
Genome size estimates for Anthurium species spanning 13 recognized subgeneric sections indicate that genome size does not generally correlate with chromosome count or phylogenetic relationships. Mechanisms of genome expansion and contraction, including amplification and reduction of repetitive elements, polyploidy, chromosome reorganization/loss, may be involved in genome evolution in Anthurium as in other species. The new information on Anthurium genome sizes provides a platform for molecular studies supporting further research on genome evolution as well as cultivar development.
Ecological traits of the circumboreal plant Viburnum opulus were examined to improve understanding of the variation of populations occurring in the same biome but on different continents. Seedling development/emergence is shown to be highly similar but some degree of variation was present in other traits, among populations.
Background and aims
Temperate forests are disjunct in the Northern Hemisphere, having become fragmented from the earlier widespread (Tertiary) boreotropical forest. We asked ‘What are the contemporary patterns of population variation in ecological traits of a Tertiary relict in a macroecological context?’. This issue underpins our understanding of variation in populations occurring in the same biome but on different continents.
We examined characters associated with root and shoot emergences among populations of Viburnum opulus in temperate forests of Asia, North America and Europe. This species has complex seedling emergence extending over several years and requiring various temperature cues.
Populations varied in germination responses and clustered into groups that were only partly related to varietal status. Whereas roots (at warm temperatures) and shoots (following a cold period) simultaneously emerged from seeds of all populations when simulated dispersal occurred in winter, they were delayed in some populations when dispersal occurred in summer.
Viburnum opulus populations, some separated by 10 300 km, showed high similarity in seedling development and in germination phenology, and we suggest that stabilizing selection has played a key role in maintaining similar dormancy mechanisms. Nevertheless, there was some degree of variation in other germination characters, suggesting local adaptation.
Grapevines growing in Australia suffer from high temperatures which have major effects on photosynthesis and transpiration. To learn more, gas exchange was measured over several seasons and then modelled across temperatures from 20 to 45°C and validated with independent data.
Background and aims
Grapevines growing in Australia are often exposed to very high temperatures and the question of how the gas exchange processes adjust to these conditions is not well understood. The aim was to develop a model of photosynthesis and transpiration in relation to temperature to quantify the impact of the growing conditions on vine performance.
Leaf gas exchange was measured along the grapevine shoots in accordance with their growth and development over several growing seasons. Using a general linear statistical modelling approach, photosynthesis and transpiration were modelled against leaf temperature separated into bands and the model parameters and coefficients applied to independent datasets to validate the model.
Photosynthesis, transpiration and stomatal conductance varied along the shoot, with early emerging leaves having the highest rates, but these declined as later emerging leaves increased their gas exchange capacities in accordance with development. The general linear modelling approach applied to these data revealed that photosynthesis at each temperature was additively dependent on stomatal conductance, internal CO2 concentration and photon flux density. The temperature-dependent coefficients for these parameters applied to other datasets gave a predicted rate of photosynthesis that was linearly related to the measured rates, with a 1 : 1 slope. Temperature-dependent transpiration was multiplicatively related to stomatal conductance and the leaf to air vapour pressure deficit and applying the coefficients also showed a highly linear relationship, with a 1 : 1 slope between measured and modelled rates, when applied to independent datasets.
The models developed for the grapevines were relatively simple but accounted for much of the seasonal variation in photosynthesis and transpiration. The goodness of fit in each case demonstrated that explicitly selecting leaf temperature as a model parameter, rather than including temperature intrinsically as is usually done in more complex models, was warranted.
Austrobaileya has long served as a model for ancient angiosperm pollen structure. Its pollen germination is relatively rapid and requires < 10 % of the progamic phase. Extensive evidence suggests pollen germination underwent acceleration early in angiosperm history.
Background and aims
The pollination to fertilization process (progamic phase) is thought to have become greatly abbreviated with the origin of flowering plants. In order to understand what developmental mechanisms enabled the speeding of fertilization, comparative data are needed from across the group, especially from early-divergent lineages. I studied the pollen germination process of Austrobaileya scandens, a perennial vine endemic to the Wet Tropics area of northeastern Queensland, Australia, and a member of the ancient angiosperm lineage, Austrobaileyales.
I used in vivo and in vitro hand pollinations and timed collections to study development from late pollen maturation to just after germination. Then I compared the contribution of pollen germination timing to progamic phase duration in 131 angiosperm species (65 families).
Mature pollen of Austrobaileya was bicellular, starchless and moderately dehydrated—water content was 31.5 % by weight and volume increased by 57.9 % upon hydration. A callose layer in the inner intine appeared only after pollination. In vivo pollen germination followed a logarithmic curve, rising from 28 % at 1 hour after pollination (hap) to 97 % at 12 hap (R2 = 0.98). Sufficient pollen germination to fertilize all ovules was predicted to have occurred within 62 min. Across angiosperms, pollen germination ranged from 1 min to >60 h long and required 8.3 ± 9.8 % of the total duration of the progamic phase.
Pollen of Austrobaileya has many plesiomorphic features that are thought to prolong germination. Yet its germination is quite fast for species with desiccation-tolerant pollen (range: <1 to 60 h). Austrobaileya and other early-divergent angiosperms have relatively rapid pollen germination and short progamic phases, comparable to those of many insect-pollinated monocots and eudicots. These results suggest that both the pollen germination and pollen tube growth periods were marked by acceleration of developmental processes early in angiosperm history.
Cells of embryonic axes in recalcitrant horse chestnut seeds are associated with a high water content and functionally preserved vacuoles with active aquaporins and invertase. These vacuoles function to the support rapid cell elongation of the hypocotyl that results in radicle emergence and thus the start of visible germination.
Backgrounds and aims
In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport.
Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H+-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties.
Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H+-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins.
Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due to the short duration. The retained physiological activity of vacuoles allows them to function rapidly as dormancy is lost and when external conditions permit. Cell vacuolation precedes cell elongation in both hypocotyl and radicle, and provides impetus for rapid germination.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
The paper uses modified population viability models and spatial structure via block analysis to assess population demography of Trillium recurvatum a clonal understory plant. The population is expanding, a likely outcome of the relatively high proportion of juvenile and non-flowering adult ramets and fast-replicating non-flowering adults. Further work is needed to elucidate the relative contributions of clonal vs seed recruitment to genetic structure and demography.
Background and aims
Understanding the demography of long-lived clonal herbs, with their extreme modularity, requires knowledge of both their short- and long-term survival and ramet growth patterns. The primary objective of this study was to understand the dynamics of a clonal forest herb, Trillium recurvatum, by examining temporal and small-scale demographic patterns. We hypothesized: (i) there would be more variability in the juvenile age class compared with non-flowering adult and flowering adult classes due to year-to-year fluctuations in recruitment; (ii) rates of population growth (λ) and increase (r) would be highest in non-flowering ramets due to a combination of transitions from the juvenile stage and reversions from flowering adults; and (iii) inter-ramet distances would be most variable between flowering and juvenile ramets due to a combination of clonal growth, seed dispersal by ants and ramet death over time.
Census data were collected on the total number of stems in the population from 1990 to 2007, and placed within one of three life stages (juvenile, three-leaf non-flowering and three-leaf flowering). Modified population viability equations were used to assess temporal population viability, and spatial structure was assessed using block krigging. Correlations were performed using current and prior season weather to current population demography.
The first hypothesis was rejected. The second hypothesis was supported: population increase (r) and growth rate (λ) were highest in non-flowering ramets. Finally, the third hypothesis was rejected: there was no apparent density dependence within this population of Trillium and no apparent spatial structure among life stages.
Overall population density fluctuated over time, possibly due to storms that move soil, and prior year's temperature and precipitation. However, density remained at some dynamic stable level. The juvenile age class had greater variability for the duration of this study and population growth rate was greatest for non-flowering ramets.
This paper show that inter-subspecies hybridization among certain Vigna unguiculata subspecies, occurred during the course of evolution. This has affected several regions of the genome and is interfering with the dependable assessment of sub-species relationships using single (rRNA regions) or multilocus markers.
Background and aims
Intra-species hybridization and incompletely homogenized ribosomal RNA repeat units have earlier been reported in 21 accessions of Vigna unguiculata from six subspecies using internal transcribed spacer (ITS) and 5S intergenic spacer (IGS) analyses. However, the relationships among these accessions were not clear from these analyses. We therefore assessed intra-species hybridization in the same set of accessions.
Arbitrarily primed polymerase chain reaction (AP-PCR) analysis was carried out using 12 primers. The PCR products were resolved on agarose gels and the DNA fragments were scored manually. Genetic relationships were inferred by TREECON software using unweighted paired group method with arithmetic averages (UPGMA) cluster analysis evaluated by bootstrapping and compared with previous analyses based on ITS and 5S IGS.
A total of 202 (86 %) fragments were found to be polymorphic and used for generating a genetic distance matrix. Twenty-one V. unguiculata accessions were grouped into three main clusters. The cultivated subspecies (var. unguiculata) and most of its wild progenitors (var. spontanea) were placed in cluster I along with ssp. pubescens and ssp. stenophylla. Whereas var. spontanea were grouped with ssp. alba and ssp. tenuis accessions in cluster II, ssp. alba and ssp. baoulensis were included in cluster III. Close affinities of ssp. unguiculata, ssp. alba and ssp. tenuis suggested inter-subspecies hybridization.
Multi-locus AP-PCR analysis reveals that intra-species hybridization is prevalent among V. unguiculata subspecies and suggests that grouping of accessions from two different subspecies is not solely due to the similarity in the ITS and 5S IGS regions but also due to other regions of the genome.
The review discusses recent advances in H2O2 plant biology, focusing on its signalling capabilities, the transduction pathways involved and its potential for interfering with other information transfer mechanisms in plant cells.
Hydrogen peroxide (H2O2) was initially recognized as a toxic reactive oxygen species, able to cause damage to a variety of cellular structures. However, it became clear in the last decade that H2O2 can also act as a potent signalling molecule, involved in a plethora of physiological functions.
In the present review, we offer a brief summary of H2O2 signalling events and focus on the mechanisms of its perception and signal transduction, the factors that act downstream, as well as H2O2 interference with other information transfer mechanisms.
The significant scientific effort in the last 10 years to determine the position of H2O2 in signal transduction networks in plants demonstrated that it is essential for both the communication with external biotic and abiotic stimuli and the control of developmentally regulated processes. In addition, H2O2 complements, synergizes or antagonizes many cellular regulatory circuits by active interaction with other signals and plant hormones during growth, development and stress responses. Therefore, further understanding of H2O2 signal transduction is not only of fundamental, but also of practical importance, since this knowledge may contribute to improve agricultural practices and reduce stress-induced damage to crops.
This is a large scale investigation of morphological diversity in Juniperus excelsa excelsa. It offers complementary results to those obtained for the same populations using molecular markers. These two approaches are complementary and should be considered together in order to obtain a comprehensive view of the variability of J. excelsa excelsa.
Background and aims
Juniperus excelsa M.-Bieb. is a major forest element in the mountains of the eastern part of Mediterranean and sub-Mediterranean regions. This study comprises the first morphological investigation covering a large part of the geographical range of J. excelsa and aims to verify the congruency between the morphological results and molecular results of a previous study.
We studied 14 populations sampled from Greece, Cyprus, Ukraine, Turkey and Lebanon, 11 of which have previously been investigated using molecular markers. Three hundred and ninety-four individuals of J. excelsa were examined using nine biometric features characterizing cones, seeds and shoots, and eight derived ratios. Statistical analyses were conducted in order to evaluate the intra- and inter-population morphological variability.
The level of intra-population variability observed did not show any geographical trends. The total variation mostly depended on the ratios of cone diameter/seed width and seed width/seed length. The discrimination analysis, the Ward agglomeration method and barrier analysis results showed a separation of the sampled populations into three main clusters. These results confirmed, in part, the geographical differentiation revealed by molecular markers with a lower level of differentiation and a less clear geographical pattern. The most differentiated populations using both markers corresponded to old, isolated populations in the high altitudes of Lebanon (>2000 m). Moreover, a separation of the northern Turkish population from the southern Turkish populations was observed using both markers.
Morphological variation together with genetic and biogeographic studies make an effective tool for detecting relict plant populations and also populations subjected to more intensive selection.
Species traits, such as breeding system, phylum and growth form and habitat characteristics are shown to influence reproductive performance of liverworts and mosses in the Brazilian Atlantic Rainforest, and drive life-history differentiation among species and populations.
Background and aims
Short life cycles and trade-offs linked to breeding systems make bryophytes good models for the study of plant reproductive strategies. Our aim was to test if differences in sexual reproductive performance of bryophytes in tropical rainforests are driven by the breeding system of the species (monoicous or dioicous) or are mainly affected by the habitat.
The reproductive performance (sexual branches, gametangia (sex organs), fertilization and sporophyte production) of 11 species was repeatedly monitored and analysed from populations at sea-level and montane sites of a Brazilian Atlantic rainforest over 15 months.
Monoicous species had the highest reproductive performance, particularly for sexual branches, fertilized gametangia and sporophyte production. Species at the sea-level site produced more sexual branches and had more female-biased sex ratios of gametangia than species in the montane site. Fertilizations were more frequent at the montane site, but sporophyte frequency was similar between the two sites. Fertilization tended to occur mostly in the periods of heavy rain (October to December).
Breeding system is not the only major influence on the reproductive performance of bryophytes. We show that habitat is also an important factor determining life-history differentiation. Female-biased sex ratios and low rates of fertilization are seen to be compensated for by high production of reproductive structures at the initial phases of the reproductive cycle.
The experiments demonstrate that stress treatments to large Artemisia annua plants have a minor promoting effect on the initiation of glandular trichomes in developing leaves, and a maturing effect on glandular trichomes later in the life time of the individual trichome.
Background and aims
Glandular trichomes (GT) of Artemisia annua produce valuable compounds for pharmaceutical and industrial uses, most notably the anti-malarial artemisinin. Our aim was to find out whether the density, number and size of GT can be manipulated to advantage by environmental stress. A range of external stress treatments, including stress response regulators, was therefore given to fully grown plants under field and greenhouse conditions.
In a field experiment (Ex1), seed-grown plants were subjected to chemical or physical stress and plants analysed after 5 weeks. In a greenhouse experiment (Ex2), three groups of clonally derived plants were stressed at weekly intervals for 5 weeks. Stress treatments included sandblasting, leaf cutting and spraying with jasmonic acid, salicylic acid, chitosan oligosaccharide (COS), H2O2 (HP) and NaCl (SC)at different concentrations. Leaves from an upper and a lower position on the plants were analysed by fluorescence microscopy to determine the density and size of GT.
Densities of GT on upper leaves of full-grown A. annua plants generally showed no response to external stress and only plants from one clone of Ex2 supported the hypothesis that increased density of GT was inducible in upper leaves by stress (significant for SC, HP and COS). The density of GT on lower leaves was not affected by stress in any experiment. Glandular trichomes were significantly smaller on the lower leaves in response to stress in Ex2, and a similar non-significant trend was observed in Ex1.
The results indicate a dynamic system in which stress treatments of large A. annua plants had a minor promoting effect on the initiation of GT in developing leaves, and a maturing effect of GT later in the lifetime of the individual GT. The hypothesis that applying stress can induce larger GT or more numerous GT was rejected.
The present study reveals significant genetically determined differences in a range of growth and ecophysiological traits between different Phragmites australis genotypes, and provides evidence that the differences are neither related to ploidy level per se nor to the phylogeographic relationships of the genotypes.
Background and aims
Phragmites australis is a wetland grass with high genetic variability, augmented by its cosmopolitan distribution, clonal growth form and large variation in chromosome numbers. Different ploidy levels and ecotypes differ in morphology and ecophysiological traits, and may possess different levels of phenotypic variation. The aim of this study was to quantify the natural variation in ecophysiological characteristics of P. australis, and to explore whether differences in ecophysiological traits can be related to ploidy levels or to the geographic origin of the clones.
Fifteen clones of P. australis from Europe and Asia/Australia, representing five ploidy levels (4x, 6x, 8x, 10x and 12x), were grown in a common garden design for 119 days. Plant growth and light-saturated rate of photosynthesis (Pmax), stomatal conductance (gs), water use efficiency (WUE) and concentrations of photosynthetic pigments and mineral ions in the leaves were measured.
The growth of the plants and most ecophysiological parameters differed significantly between clones. The mean maximum shoot height varied from 0.9 to 1.86 m, Pmax from 9.7 to 27 µmol m−2 s−1, gs from 0.22 to 1.41 mol m−2 s−1 and WUE from 13 to 47 µmol mol−1. The concentrations of chlorophylls did not vary significantly between clones, but the chlorophyll a/b ratio and the concentrations of total carotenoids did. The observed differences were not explained either by the ploidy level per se or by the geographic origin or phylogenetic relationships of the clones.
Phylogeographic relationships in P. australis on a global scale do not mirror the environment where the adaptations have evolved, and high phenotypic variation among and within clones complicates comparative studies. Future studies aimed at explaining differences in plant behaviour between P. australis populations should be careful in the selection of target genotypes and/or populations, and should avoid generalizing their findings beyond the genotypes and/or populations studied.
Proton Transfer Reaction-Mass Spectrometry (PTR-MS) analyses revealed that damaged Brassica roots emit sulfur-containing volatiles. B. nigra, B. juncea and B. napus emitted isothiocyanate markers, whereas B. rapa, B. oleracea, and B. carinata emitted methanethiol. These compounds can be used as markers for root damage by insect larvae and other below-ground herbivores.
Background and aims
Plants damaged by herbivores emit a variety of volatile organic compounds (VOCs). Here we used proton-transfer reaction mass spectrometry (PTR-MS) as a sensitive detection method for online analysis of herbivore-induced VOCs. Previously, it was found that Brassica nigra plants emit several sulfur-containing VOCs when attacked by cabbage root fly (Delia radicum) larvae with m/z 60 as a marker for the formation of allylisothiocyanate from the glucosinolate sinigrin. We tested the hypothesis that m/z 60 emission occurs only in plants with sinigrin in their roots. Additionally, we tested the hypothesis that methanethiol, dimethylsulfide and dimethyldisulfide are only emitted after larval infestation.
Proton-transfer reaction mass spectrometry was used to track sulfur-containing VOCs from six different species of Brassica over time. The roots were either artificially damaged or infested with cabbage root fly larvae. Glucosinolate profiles of the roots were analysed using high-pressure liquid chromatography and compared with VOC emissions.
Brassica nigra, B. juncea and B. napus primarily emitted m/z 60 directly after artificial damage or root fly infestation. Sulfide and methanethiol emissions from B. nigra and B. juncea also increased after larval damage but much later (6–12 h after damage). Brassica rapa, B. oleracea and B. carinata principally emitted methanethiol after artificial and after larval damage. Brassica oleracea and B. carinata showed some increase in m/z 60 emission after larval damage. Comparison with root glucosinolate profiles revealed that sinigrin cannot be the only precursor for m/z 60.
The principal compound emitted after root damage is determined by the plant species, and not by damage type or root glucosinolate composition. Once determined, the principal compounds may be used as markers for identifying damaged or infested plants. Further analyses of plant enzymes involved in the breakdown of sulfur compounds is needed to reveal the origin of sulfur-containing VOCs from plants.
Direct seeding is replacing transplanting in rice. Early flooding suppresses weeds but selective action is compromised by the sharing of flood-tolerance traits. Understanding adaptive traits in both species is therefore a prerequisite for developing direct seeding systems that control weeds while leaving rice seedlings relatively unharmed.
Background and aims
Direct seeding of rice is being adopted in rainfed and irrigated lowland ecosystems because it reduces labour costs in addition to other benefits. However, early flooding due to uneven fields or rainfall slows down seed germination and hinders crop establishment. Conversely, early flooding helps suppress weeds and reduces the costs of manual weeding and/or dependence on herbicides; however, numerous weed species are adapted to lowlands and present challenges for the use of flooding to control weeds. Advancing knowledge on the mechanisms of tolerance of flooding during germination and early growth in rice and weeds could facilitate the development of improved rice varieties and effective weed management practices for direct-seeded rice.
Rice genotypes with a greater ability to germinate and establish in flooded soils were identified, providing opportunities to develop varieties suitable for direct seeding in flooded soils. Tolerance of flooding in these genotypes was mostly attributed to traits associated with better ability to mobilize stored carbohydrates and anaerobic metabolism. Limited studies were undertaken in weeds associated with lowland rice systems. Remaining studies compared rice and weeds and related weed species such as Echinochloa crus-galli and E. colona or compared ecotypes of the same species of Cyperus rotundus adapted to either aerobic or flooded soils.
Tolerant weeds and rice genotypes mostly developed similar adaptive traits that allow them to establish in flooded fields, including the ability to germinate and elongate faster under hypoxia, mobilize stored starch reserves and generate energy through fermentation pathways. Remarkably, some weeds developed additional traits such as larger storage tubers that enlarge further in deeper flooded soils (C. rotundus). Unravelling the mechanisms involved in adaptation to flooding will help design management options that will allow tolerant rice genotypes to adequately establish in flooded soils while simultaneously suppressing weeds.
European Phragmites australis is one of four main cp-DNA haplotype clusters present worldwide. The European gene pool extends from North America to Far East Asia and South Africa. Extensive gene flow occurs only within the temperate region of Europe.
Background and aims
Two Phragmites australis taxa are recognized in Europe: P. australis ssp. altissimus, also known as Phragmites isiaca, in the Mediterranean region and P. australis in the temperate region. Another taxonomic group in the Mediterranean is Phragmites frutescens. European genotypes are diverse genetically, cytologically and morphologically, and are related to African, Asiatic and American genotypes. We investigated chloroplast DNA (cpDNA) diversity in Europe and defined the current borders of the European gene pool.
We analysed chloroplast variation with parsimony and genetic distance methods, and compared it with that of nuclear amplified fragment length polymorphism and microsatellites. We also investigated the phenological pattern of 188 genotypes collected worldwide in a common garden in Denmark. We assumed that non-flowering genotypes could indicate climatic, geographic and/or reproductive barriers to dispersal and would have been recorded in the genetic pattern as groups genetically isolated from, or within, the European pool.
The European P. australis gene pool extends from North America to the Far East and South Africa. However, African and North American genotypes are differentiating from the European genotypes. Mediterranean P. australis is genetically different from temperate P. australis and shares several similarities with Phragmites mauritianus in Africa and Phragmites karka in Asia. Phragmites frutescens shares the cpDNA sequences with both these tropical species. Two DNA bands can distinguish Mediterranean P. australis from P. frutescens and P. mauritianus and from temperate P. australis, and reveal possible hybrids among these species in the Mediterranean region. Phenological data confirmed possible gene flow within the temperate region of Europe, whereas the Mediterranean genotypes did not set inflorescences in Denmark, suggesting reproductive barriers between temperate and Mediterranean P. australis.
European P. australis appears as one of four main Phragmites groups known in the world. Further research is needed to understand the implications of long-distance dispersal at the population level.
Hybridization of Phragmites has occurred in the Gulf Coast and likely is occurring elsewhere in North America. However, detection failure may be due to limited genetic tools. Additionally, nomenclature confusion necessitates a revision of the current classification system.
Background and aims
We review evidence for hybridization of Phragmites australis in North America and the implications for the persistence of native P. australis ssp. americanus populations in North America. We also highlight the need for an updated classification system, which takes P. australis intraspecific variation and hybridization into account.
We reviewed available published, in press and in preparation literature to assess the likelihood of hybridization and interbreeding in genotypes of P. australis present in North America.
Experimental results demonstrate that hybridization among introduced and native haplotypes is possible within the genus Phragmites, yet evidence that hybridization has occurred naturally is only starting to emerge. The lag in identifying hybridization in Phragmites in North America may be related to under-sampling in some parts of North America and to a lack of molecular tools that provide the capability to recognize hybrids.
Our understanding of the gene flow within and between species in the genus Phragmites is moving at a fast pace, especially on the east and Gulf coasts of North America. More attention should also be focused on the Great Lakes region, the southwestern and the west coast of the USA, where sympatry has created opportunities for hybridization. Where hybridizations have been detected, there are currently no published data on how hybridization affects plant vigour, morphology, invasiveness or conservation of the genetic integrity of the North American native subspecies. We conclude that the detection of more hybridization is highly likely and that there is a need to develop new markers for the different Phragmites species and lineages to fill current knowledge gaps. Finally, we suggest that the classification system for P. australis should be updated and published to help clarify the nomenclature.
The present investigation was undertaken to mass propagate Cymbidium mastersii, an ornamental orchid of Northeast India by in vitro propagation method. This approach could also help for the conservation as well as commercialization of C. mastersii and other threatened and ornamental orchids.
Background and aims
Cymbidium mastersii is an epiphytic orchid distributed mainly in Northeast India. Owing to its high commercial value in the floricultural industry, natural populations are under threat from over-exploitation. Mass propagation provides an alternative means of satisfying the demand. Unfortunately, conventional propagation is slow and difficult, suggesting in vitro methods for mass multiplication may be more appropriate. The objective of this study was to develop an efficient protocol.
Methodology and principal results
Four nutrient media were evaluated for seed germination and early protocorm development: Murashige and Skoog (MS), half-strength MS, Knudson ‘C’ (KC), and Vacin and Went (VW). In addition, the effects of plant growth regulators 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were studied alone and in combination. The maximum percentage seed germination (93.58 ± 0.56) was obtained in MS basal medium after 8–9 weeks of culture. Secondary protocorms (protocorm-like bodies) were developed from primary protocorms on MS medium fortified with different concentrations and combinations of cytokinins (BAP and KN) and auxins (NAA and IBA). The highest numbers of secondary protocorms (20.55 ± 0.62)/primary protocorms were obtained in MS medium supplemented with 5.0 µM BAP and 2.5 µM NAA. The most effective auxin source promoting root production (7.46 ± 0.09 per shoot) was 10.0 µM IBA. The plants were acclimatized effectively (survival percentage 88 %) in a greenhouse using a rooting medium of crushed sterile brick and charcoal (1 : 1 v/v) and vermicompost (leaf litter + cow dung, 1 : 1 v/v).
An efficient protocol was established for in vitro propagation of C. mastersii using seed as the starting material. The percentage seed germination varied with the composition of the nutrient media and was highest in full-strength MS basal medium. The number of secondary protocorms that developed from primary protocorms was increased by the addition of 5.0 µM BAP and 2.5 µM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.
The paper supports the view that ethylene plays a significant role in maintaining tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels and increased tomato pollen sensitivity to heat stress. On the other hand, increasing ethylene levels before heat-stress improved pollen quality.
Background and aims
Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene involvement in the pollen heat-stress response. Here we elucidated ethylene's involvement in pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling pathway and altering ethylene levels on tomato pollen functioning under heat stress.
Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or heat-stress growing conditions, and pollen quality was determined. Starch and carbohydrates were measured in isolated pollen grains from these plants. The effect of pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor of ethylene biosynthesis on pollen quality was assessed.
Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant reduction in the total number of pollen grains, reduction in the number of viable pollen and elevation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls.
Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels increased tomato pollen sensitivity to heat stress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.
This study reveals a negative relationship between leaf phenolic compounds and photosynthetic Amax among different plant species. This indicates a functional integration among carbon gain and the concentration of leaf phenolic compounds that reflects the trade-off between growth and defence/protection demands.
Background and aims
Phenolic compounds are the most commonly studied of all secondary metabolites because of their significant protective–defensive roles and their significant concentration in plant tissues. However, there has been little study on relationships between gas exchange parameters and the concentration of leaf phenolic compounds (total phenolics (TP) and condensed tannins (CT)) across a range of species. Therefore, we addressed the question: is there any correlation between photosynthetic capacity (Amax) and TP and CT across species from different ecosystems in different continents?
A plethora of functional and structural parameters were measured in 49 plant species following different growth strategies from five sampling sites located in Greece and Australia. The relationships between several leaf traits were analysed by means of regression and principal component analysis.
The results revealed a negative relationship between TP and CT and Amax among the different plant species, growth strategies and sampling sites, irrespective of expression (with respect to mass, area or nitrogen content). Principal component analysis showed that high concentrations of TP and CT are associated with thick, dense leaves with low nitrogen. This leaf type is characterized by low growth, Amax and transpiration rates, and is common in environments with low water and nutrient availability, high temperatures and high light intensities. Therefore, the high TP and CT in such leaves are compatible with the protective and defensive functions ascribed to them.
Our results indicate a functional integration between carbon gain and the concentration of leaf phenolic compounds that reflects the trade-off between growth and defence/protection demands, depending on the growth strategy adopted by each species.