Background and Aims
Plant defence traits against herbivores incur production costs that are usually difficult to measure. However, estimating these costs is a prerequisite for characterizing the plant defence strategy as a whole. Myrmecophytes are plants that provide symbiotic ants with specialized nesting cavities, called domatia, in exchange for protection against herbivores. In the particular case of stem domatia, production of extra wood seems to be the only associated cost, making this indirect defence trait a particularly suitable model for estimating the cost of defence.
Measurements were made of growth pattern and cumulative production cost of domatia over secondary growth in the myrmecophyte Leonardoxa africana subsp. africana, whose internodes display both a solid basal segment and a hollow distal part (the domatium), thus allowing paired comparison of investment in wood.
Previous studies showed that ‘overconstruction’ of the hollow part of internodes during primary growth is needed for mechanical support. In this study, it is shown that the relationship between the woody cross-sectional area of the solid and hollow parts of internodes is negatively allometric at the beginning of secondary growth and nearly isometric later on. Thus, in hollow stems, the first phase of slow secondary growth compensates for the ‘overconstruction’ of the ring of wood during primary growth. Moreover, the cumulative production cost of a domatium (estimated as the additional volume of wood required for a hollow stem compared with a solid one) is very high at the beginning of secondary growth and then quickly tends to zero.
Making domatia incurs high costs early in ontogeny, costs that are then amortized later in development of stems and of individual plants. Characterizing ontogenetic variation of the net cost of this peculiar defence mechanism will help us build more accurate theoretical models of resource allocation in myrmecophytes.
Ontogenetic trade-off; resource allocation; plant defence; symbiosis; mutualism; ant-plant; Leonardoxa africana; caulinary domatia; allometry
Background and Aims
Abiotic pollination by wind or water is well established in flowering plants. In some species pollination by rain splashes, a condition known as ombrophily, has been proposed as a floral strategy. However, evidence for this type of abiotic pollination has remained controversial and many reported cases have subsequently been shown to be false. This study investigates ombrophily in the deceptive orchid Acampe rigida to determine the mechanism by which this species is able to maintain high fecundity, despite flowering during the rainy season in south-west China when pollinators are scarce.
The floral mechanisms promoting rain pollination in A. rigida were observed and described in detail. Controlled pollination experiments and observations of floral visitors were conducted. A field experiment using rain shelters at 14 sites in Guangxi, south-west China, evaluated the contribution of rain pollination to fruit-set.
During rainfall, raindrops physically flicked away the anther cap exposing the pollinarium. Raindrops then caused pollinia to be ejected upwards with the strap-like stipe pulling them back and causing them to fall into the stigmatic cavity, resulting in self-pollination. Neither flower nor pollen function were damaged by water. Although A. rigida is self-compatible, it is incapable of autonomous self-pollination without the assistance of rain splashes. The results of the rain-sheltering experiment indicated that rain pollination contributed substantially to increasing fruit-set, although there was variation among sites in the intensity of this effect.
A. rigida flowers during the rainy season, when pollinators are scarce, and ombrophily functions to provide reproductive assurance without compromising opportunities for outcrossing.
Abiotic pollination; Acampe rigida; floral adaptation; ombrophily; rain-mediated self-pollination; deceptive orchid
Background and Aims
Studies on the effects of sub- and/or supraoptimal temperatures on growth and phosphorus (P) nutrition of perennial herbaceous species at growth-limiting P availability are few, and the impacts of temperature on rhizosphere carboxylate dynamics are not known for any species.
The effect of three day/night temperature regimes (low, 20/13 °C; medium, 27/20 °C; and high, 32/25 °C) on growth and P nutrition of Cullen cinereum, Kennedia nigricans and Lotus australis was determined.
The highest temperature was optimal for growth of C. cinereum, while the lowest temperature was optimal for K. nigricans and L. australis. At optimum temperatures, the relative growth rate (RGR), root length, root length per leaf area, total P content, P productivity and water-use efficiency were higher for all species, and rhizosphere carboxylate content was higher for K. nigricans and L. australis. Cullen cinereum, with a slower RGR, had long (higher root length per leaf area) and thin roots to enhance P uptake by exploring a greater volume of soil at its optimum temperature, while K. nigricans and L. australis, with faster RGRs, had only long roots (higher root length per leaf area) as a morphological adaptation, but had a higher content of carboxylates in their rhizospheres at the optimum temperature. Irrespective of the species, the amount of P taken up by a plant was mainly determined by root length, rather than by P uptake rate per unit root surface area. Phosphorus productivity was correlated with RGR and plant biomass.
All three species exhibited adaptive shoot and root traits to enhance growth at their optimum temperatures at growth-limiting P supply. The species with a slower RGR (i.e. C. cinereum) showed only morphological root adaptations, while K. nigricans and L. australis, with faster RGRs, had both morphological and physiological (i.e. root carboxylate dynamics) root adaptations.
Australian native legumes; carboxylates; climate change; growth; perennial pastures; phosphorus; photosynthesis; root morphology; temperature; water-use efficiency
Background and Aims
Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.
The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.
Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.
The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.
Pollen; flow cytometry; unreduced gametes; genome size; nuclei extraction; 2n pollen; cell cycle
Background and Aims
Delayed selfing is the predominant mode of autonomous self-pollination in flowering plants. However, few delayed selfing mechanisms have been documented. This research aims to explore a new delayed selfing mechanism induced by stigmatic fluid in Roscoea debilis, a small perennial ginger.
Floral biology and flower visitors were surveyed. The capacity of autonomous selfing was evaluated by pollinator exclusion. The timing of autonomous selﬁng was estimated by emasculation at different flowering stages. The number of seeds produced from insect-pollination was assessed by emasculation and exposure to pollinators in the natural population. The breeding system was also tested by pollination manipulations.
Autonomous self-pollination occurred after flowers wilted. The stigmatic fluid formed a globule on the stigma on the third day of flowering. The enlarged globule seeped into the nearby pollen grains on the fourth flowering day, thus inducing pollen germination. Pollen tubes then elongated and penetrated the stigma. Hand-selfed flowers produced as many seeds as hand-crossed flowers. There was no significant difference in seed production between pollinator-excluded flowers and hand-selfed flowers. When emasculated flowers were exposed to pollinators, they produced significantly fewer seeds than intact flowers. Visits by effective pollinators were rare.
This study describes a new form of delayed autonomous self-pollination. As the predominant mechanism of sexual reproduction in R. debilis, delayed self-pollination ensures reproduction when pollinators are scarce.
stigmatic fluid; autonomous selfing; reproductive assurance; Roecoea debilis; pollinator failure; ginger
Background and Aims
Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes.
Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato.
Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe.
Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.
Coexistence; contact zone; cytogeography; flow cytometry; fragrant orchid; Gymnadenia; Orchidaceae; hybridization; mixed-ploidy population; polyploidy; sympatry; unreduced gametes
Background and Aims
Photosynthetic thermotolerance (PT) is important for plant survival in tropical and sub-tropical savannas. However, little is known about thermotolerance of tropical and sub-tropical wild plants and its association with leaf phenology and persistence. Longer-lived leaves of savanna plants may experience a higher risk of heat stress. Foliar Ca is related to cell integrity of leaves under stresses. In this study it is hypothesized that (1) species with leaf flushing in the hot-dry season have greater PT than those with leaf flushing in the rainy season; and (2) PT correlates positively with leaf life span, leaf mass per unit area (LMA) and foliar Ca concentration ([Ca]) across woody savanna species.
The temperature-dependent increase in minimum fluorescence was measured to assess PT, together with leaf dynamics, LMA and [Ca] for a total of 24 woody species differing in leaf flushing time in a valley-type savanna in south-west China.
The PT of the woody savanna species with leaf flushing in the hot-dry season was greater than that of those with leaf flushing in the rainy season. Thermotolerance was positively associated with leaf life span and [Ca] for all species irrespective of the time of flushing. The associations of PT with leaf life span and [Ca] were evolutionarily correlated. Thermotolerance was, however, independent of LMA.
Chinese savanna woody species are adapted to hot-dry habitats. However, the current maximum leaf temperature during extreme heat stress (44·3 °C) is close to the critical temperature of photosystem II (45·2 °C); future global warming may increase the risk of heat damage to the photosynthetic apparatus of Chinese savanna species.
Photosynthetic thermotolerance; foliar calcium concentration; chlorophyll fluorescence; critical temperature; leaf life span; leaf mass per area; thermostability; woody savanna species; global warming
Background and Aims
Ovary swelling, and resultant fruit malformation, in bell pepper flowers is favoured by low night temperature or a high source–sink ratio. However, the interaction between night temperature and source–sink ratio on ovary swelling and the contribution of cell size and cell number to ovary swelling are unknown. The present research examined the interactive effects of night temperature and source–sink ratio on ovary size, cell number and cell size at anthesis in bell pepper flowers.
Bell pepper plants were grown in growth chambers at night temperatures of either 20 °C (HNT) or 12 °C (LNT). Within each temperature treatment, plants bore either 0 (non-fruiting) or two developing fruits per plant. Ovary fresh weight, cell size and cell number were measured.
Ovary fresh weights in non-fruiting plants grown at LNT were the largest, while fresh weights were smallest in plants grown at HNT with fruits. In general, mesocarp cell size in ovaries was largest in non-fruiting plants grown at either LNT or HNT and smallest in fruiting plants at HNT. Mesocarp cell number was greater in non-fruiting plants under LNT than in the rest of the night temperature/fruiting treatments. These responses were more marked in ovaries sampled after 18 d of treatment compared with those sampled after 40 d of treatment.
Ovary fresh weight of flowers at anthesis increased 65 % in non-fruiting plants grown under LNT compared with fruiting plants grown under HNT. This increase was due primarily to increases in mesocarp cell number and size. These results indicate that the combined effects of LNT and high source–sink ratio on ovary swelling are additive. Furthermore, the combined effects of LNT and low source–sink ratio or HNT and high source–sink ratio can partially overcome the detrimental effects of LNT and high source–sink ratio.
Capsicum annuum; fruit quality; pepper ovary swelling; source–sink effects
Background and Aims
A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.
The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.
Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.
In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.
Acetylcholinesterases; acetylesterases; carboxylesterases; DIFP; ebelactones; germination; lipases; neostigmine; olive; Olea europaea; pollen; pollen tube; sulfydryl reagents
Land plants (embryophytes) are monophyletic and encompass four major clades: liverworts, mosses, hornworts and polysporangiophytes. The liverworts are resolved as the earliest divergent lineage and the mosses as sister to a crown clade formed by the hornworts and polysporangiophytes (lycophytes, monilophytes and seed plants). Alternative topologies resolving the hornworts as sister to mosses plus polysporangiophytes are less well supported. Sporophyte development in liverworts depends only on embryonic formative cell divisions. A transient basal meristem contributes part of the sporophyte in mosses. The sporophyte body in hornworts and polysporangiophytes develops predominantly by post-embryonic meristematic activity.
This paper explores the origin of the sporophyte shoot in terms of changes in embryo organization. Pressure towards amplification of the sporangium-associated photosynthetic apparatus was a major driver of sporophyte evolution. Starting from a putative ancestral condition in which a transient basal meristem produced a sporangium-supporting seta, we postulate that in the hornwort–polysporangiophyte lineage the basal meristem acquired indeterminate meristematic activity and ectopically expressed the sporangium morphogenetic programme. The resulting sporophyte body plan remained substantially unaltered in hornworts, whereas in polysporangiophytes the persistent meristem shifted from a mid-embryo to a superficial position and was converted into an ancestral shoot apical meristem with the evolution of sequential vegetative and reproductive growth.
The sporophyte shoot is interpreted as a sterilized sporangial axis interpolated between the embryo and the fertile sporangium. With reference to the putatively ancestral condition found in mosses, the sporophyte body plans in hornworts and polysporangiophytes are viewed as the product of opposite heterochronic events, i.e. an anticipation and a delay, respectively, in the development of the sporangium. In either case the result was a pedomorphic sporophyte permanently retaining juvenile characters.
bryophytes; embryo; development; meristems; plant evolution; sporophyte shoot; stomata
Background and Aims
The present study aims to assess the diversity and distribution of cytotypes of Aster amellus in central and eastern Europe, contributing with data to improve understanding of the evolutionary dynamics of the contact zone between diploids and hexaploids of this polyploid complex.
Large-scale cytotype screening of 4720 individuals collected in 229 populations was performed using 4′,6-diamidino-2-phenylindole (DAPI) flow cytometry. Fine-scale cytotype screening was performed in the mixed-ploidy population. Reproductive variables, such as number of florets per flower head, seed set and seedling emergence, as well as ploidy level of seeds and seedlings were recorded in this population.
The diploid–hexaploid contact zone is large and complex, reaching the Czech Republic in the west, Austria in the south, Poland in the north-east and Romania in the extreme east of the surveyed areas. Most populations presented only one cytotype, either diploid or hexaploid. In several areas of the contact zone both cytotypes were found to grow in parapatry. One mixed-ploidy population of diploids and hexaploids was detected for the first time, but no signs of hybridization were detected. In this population, diploids had a significantly lower reproductive success, and significantly higher production of intercytotype offspring, being in reproductive disadvantage in comparison with hexaploids.
The contact zone of diploid and hexaploid A. amellus in central and eastern Europe seems to be highly dynamic and diffuse, with both primary and secondary contacts being possible. The obtained results suggest the origin of hexaploids through diploids, overall supporting previous hypotheses that this species is autopolyploid. Data from the only mixed-ploidy population detected so far suggest that the minority cytotype exclusion is an important evolutionary mechanisms driving the prevalence of single-cytotype populations, and thus contributing to the current distributional patterns of the cytotypes of A. amellus.
Aster amellus; contact zone; cytotypes; diploids; DNA ploidy level; flow cytometry; hexaploids; hybridization; polyploid aggregate; spatial distribution; tetraploids
Background and Aims
European beech epicormics have received far less attention than epicormics of other species, especially sessile oak. However, previous work on beech has demonstrated that there is a negative effect of radial growth on trunk sprouting, while more recent investigations on sessile oak proved a strong positive influence of the presence of epicormics. The aims of this study were, first, to make a general quantification of the epicormics present along beech stems and, secondly, to test the effects of both radial growth and epicormic frequency on sprouting.
In order to test the effect of radial growth, ten forked individuals were sampled, with a dominant and a dominated fork of almost equal length for every individual. To test the effects of primary growth and epicormic frequency, on the last 17 annual shoots of each fork arm, the number of axillary buds, shoot length, ring width profiles, epicormic shoots and other epicormics were carefully recorded.
The distribution of annual shoot length, radial growth profiles and parallel frequencies of all epicormics are presented. The latter frequencies were parallel to the annual shoot lengths, nearly equivalent for both arms of each tree, and radial growth profiles included very narrow rings in the lowest annual shoots and even missing rings in the dominated arms alone. The location of the latent buds and the epicormics was mainly at branch base, while epicormic shoots, bud clusters and spheroblasts were present mainly in the lowest annual shoots investigated. Using a zero-inflated mixed model, sprouting was shown to depend positively on epicormic frequency and negatively on radial growth.
Support for a trade-off between cambial activity and sprouting is put forward. Sprouting mainly depends on the frequency of epicormics. Between- and within-tree variability of the epicormic composition in a given species may thus have fundamental and applied implications.
European beech; Fagus sylvatica; epicormics; ontogeny; mixed ZIP models; sprouting; radial growth
Background and Aims
Leaf and wood plasticity are key elements in the survival of widely distributed plant species. Little is known, however, about variation in stomatal distribution in the leaf epidermis and its correlation with the dimensions of conducting cells in wood. This study aimed at testing the hypothesis that Podocarpus lambertii, a conifer tree, possesses a well-defined pattern of stomatal distribution, and that this pattern can vary together with the dimensions of stem tracheids as a possible strategy to survive in climatically different sites.
Leaves and wood were sampled from trees growing in a cold, wet site in south-eastern Brazil and in a warm, dry site in north-eastern Brazil. Stomata were thoroughly mapped in leaves from each study site to determine a spatial sampling strategy. Stomatal density, stomatal index and guard cell length were then sampled in three regions of the leaf: near the midrib, near the leaf margin and in between the two. This sampling strategy was used to test for a pattern and its possible variation between study sites. Wood and stomata data were analysed together via principal component analysis.
The following distribution pattern was found in the south-eastern leaves: the stomatal index was up to 25 % higher in the central leaf region, between the midrib and the leaf margin, than in the adjacent regions. The inverse pattern was found in the north-eastern leaves, in which the stomatal index was 10 % higher near the midrib and the leaf margin. This change in pattern was accompanied by smaller tracheid lumen diameter and length.
Podocarpus lambertii individuals in sites with higher temperature and lower water availability jointly regulate stomatal distribution in leaves and tracheid dimensions in wood. The observed stomatal distribution pattern and variation appear to be closely related to the placement of conducting tissue in the mesophyll.
Podocarpus lambertii; stomatal index; stomatal density; stomatal distribution pattern; stem tracheids; transfusion tissue; anatomical plasticity
Background and Aims
The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.
Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed.
The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production.
A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.
Cell survival; confocal microscopy; dehydration rate; desiccation; diaminobenzidine; dichlorodihydrofluorescein diacetate; Fontinalis antipyretica; oxygen consumption; reactive oxygen species; ROS
Background and Aims
Stamen movements directly determine pollen fates and mating patterns by altering positions of female and male organs. However, the implications of such movements in terms of pollination are not well understood. Recently, complex patterns of stamen movements have been identified in Loasaceae, Parnassiaceae, Rutaceae and Tropaeolaceae. In this study the stamen movements in Ruta graveolens (Rutaceae) and their impact on pollination are determined.
Pollination effects of stamen movements were studied in Ruta graveolens, in which one-by-one uplifting and falling back is followed by simultaneous movement of all stamens in some flowers. Using 30 flowers, one stamen was manipulated either to be immobilized or to be allowed to move freely towards the centre of the flower but be prevented from falling back. Pollen loads on stigmas and ovule fertilization in flowers with or without simultaneous stamen movement were determined.
Pollen removal decreased dramatically (P < 0·001) when the stamen was stopped from uplifting because its anther was seldom contacted by pollinators. When a stamen stayed at the flower's centre, pollen removal of the next freely moved anther decreased significantly (P < 0·005) because of fewer touches by pollinators and quick leaving of pollinators that were discouraged by the empty anther. Simultaneous stamen movement occurred only in flowers with low pollen load on the stigma and the remaining pollen in anthers dropped onto stigma surfaces after stamens moved to the flower's centre.
In R. graveolens pollen removal is promoted through one-by-one movement of the stamen, which presents pollen in doses to pollinators by successive uplifting of the stamen and avoids interference of two consecutively dehisced anthers by falling back of the former stamen before the next one moves into the flower's centre. Simultaneous stamen movement at the end of anthesis probably reflects an adaptation for late-acting self-pollination.
Dichogamy; experimental manipulation; pollen presentation; pollen removal; pollination; reproductive assurance; Ruta graveolens; stamen movement
Background and Aims
Mutualistic ant–plant associations are common in a variety of plant families. Some myrmecophytic plants, such as the epiphytic orchid Caularthron bilamellatum, actively form hollow structures that provide nesting space for ants (myrmecodomatia), despite a substantial loss of water-storage tissue. This study aimed at assessing the ability of the orchid to take up nitrogen from ant-inhabited domatia as possible trade-off for the sacrifice of potential water storage capacity.
Nitrogen uptake capabilities and uptake kinetics of 15N-labelled compounds (NH4+, urea and l -glutamine) were studied in field-grown Caularthron bilamellatum plants in a tropical moist forest in Panama. Plants were either labelled directly, by injecting substrates into the hollow pseudobulbs or indirectly, by labelling of the associated ants in situ.
Caularthron bilamellatum plants were able to take up all tested inorganic and organic nitrogen forms through the inner surface of the pseudobulbs. Uptake of NH4+ and glutamine followed Michaelis–Menten kinetics, but urea uptake was not saturable up to 2 mm. 15N-labelled compounds were rapidly translocated and incorporated into vegetative and reproductive structures. By labelling ants with 15N in situ, we were able to prove that ants transfer N to the plants under field conditions.
Based on 15N labelling experiments we were able to demonstrate, for the first time, that a myrmecophytic orchid is capable of actively acquiring different forms of nitrogen from its domatia and that nutrient flux from ants to plants does indeed occur under natural conditions. This suggests that beyond anti-herbivore protection host plants benefit from ants by taking up nitrogen derived from ant debris.
Ant–plant interactions; mutualism; nutrient uptake; Michaelis–Menten kinetics; 15N labelling; myrmecophytes; epiphytes; Caularthron bilamellatum; Orchidaceae; BCNM
Background and Aims
Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL.
Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry.
The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds.
As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.
Abscisic acid; blue light; germination; nitric oxide; osmotic stress; Solanum lycopersicum; tomato; 7B-1 mutant
Background and Aims
Wild soybean (Glycine soja), a native species of East Asia, is the closest wild relative of the cultivated soybean (G. max) and supplies valuable genetic resources for cultivar breeding. Analyses of the genetic variation and population structure of wild soybean are fundamental for effective conservation studies and utilization of this valuable genetic resource.
In this study, 40 wild soybean populations from China were genotyped with 20 microsatellites to investigate the natural population structure and genetic diversity. These results were integrated with previous microsatellite analyses for 231 representative individuals from East Asia to investigate the genetic relationships of wild soybeans from China.
Analysis of molecular variance (AMOVA) revealed that 43·92 % of the molecular variance occurred within populations, although relatively low genetic diversity was detected for natural wild soybean populations. Most of the populations exhibited significant effects of a genetic bottleneck. Principal co-ordinate analysis, construction of a Neighbor–Joining tree and Bayesian clustering indicated two main genotypic clusters of wild soybean from China. The wild soybean populations, which are distributed in north-east and south China, separated by the Huang-Huai Valley, displayed similar genotypes, whereas those populations from the Huang-Huai Valley were different.
The previously unknown population structure of the natural populations of wild soybean distributed throughout China was determined. Two evolutionarily significant units were defined and further analysed by combining genetic diversity and structure analyses from Chinese populations with representative samples from Eastern Asia. The study suggests that during the glacial period there may have been an expansion route between south-east and north-east China, via the temperate forests in the East China Sea Land Bridge, which resulted in similar genotypes of wild soybean populations from these regions. Genetic diversity and bottleneck analysis supports that both extensive collection of germplasm resources and habitat management strategies should be undertaken for effective conservation studies of these important wild soybean resources.
Wild soybean; Glycine soja; microsatellites; genetic diversity; population structure
Background and Aims
Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood.
Eight hardwood species were inclined at 30–45° at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate.
Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S1 and S2 layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S1 + S2 + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution.
Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.
Phloem fibre; lignification; reaction phloem; Cercidiphyllum japonicum; Melia azedarach; Acer rufinerve; Mallotus japonicus; Celtis sinensis; Aphananthe aspera; Corylus sieboldiana; Magnolia salicifolia
Background and Aims
Teak forms xylem rings that potentially carry records of carbon sequestration and climate in the tropics. These records are only useful when the structural variations of tree rings and their periodicity of formation are known.
The seasonality of ring formation in mature teak trees was examined via correlative analysis of cambial activity, xylem and phloem formation, and climate throughout 1·5 years. Xylem and phloem differentiation were visualized by light microscopy and scanning electron microscopy.
A 3 month dry season resulted in semi-deciduousness, cambial dormancy and formation of annual xylem growth rings (AXGRs). Intra-annual xylem and phloem growth was characterized by variable intensity. Morphometric features of cambium such as cambium thickness and differentiating xylem layers were positively correlated. Cambium thickness was strongly correlated with monthly rainfall (R2 = 0·7535). In all sampled trees, xylem growth zones (XGZs) were formed within the AXGRs during the seasonal development of new foliage. When trees achieved full leaf, the xylem in the new XGZs appeared completely differentiated and functional for water transport. Two phloem growth rings were formed in one growing season.
The seasonal formation pattern and microstructure of teak xylem suggest that AXGRs and XGZs can be used as proxies for analyses of the tree history and climate at annual and intra-annual resolution.
Growth rings; teak; Tectona grandis; vascular cambium; xylem and phloem formation
Background and Aims
Multi-stemmed trees (tree clusters) in Nothofagus pumilio, a dominant tree species in Patagonia, are very uncommon and are restricted to the edge of second-growth forests following human-provoked fires. No vegetative reproduction has been reported so far. The genetic structure of multi-stemmed trees of this species was investigated and it was hypothesized that genets within a cluster were more closely related than average in the population.
Fifteen clusters (composed of at least three purported stems) and 15 single trees were sampled at the edge of a second-growth forest and genotyped using two amplified fragment length polymorphism (AFLP) primer pairs. We obtained 119 polymorphic markers that allowed clonality to be determined, together with sibship structure and relatedness among samples.
Clonality was detected in seven clusters but all clusters had at least two different genotypes. Full sibs were found exclusively within clusters and in all clusters. Within a cluster, stems that were not identified as full sibs were often half sibs. Relatedness values for the full sibs and half sibs were higher than the theoretical values of 0·5 and 0·25 but the relatedness between clusters was very low.
Tree clusters that are merged at the edge of the second-growth forest of N. pumilio are composed of stems of the same genotype and of other genotypes that are highly related (but not always). It is suggested that this peculiar genetic structure results from a combination of several causes, including selection for merging of related individuals.
AFLP; multi-stemmed trees; Patagonia; post-fire regeneration; sibship structure
Background and Aims
Despite general agreement regarding the adaptive importance of plasticity, evidence for the role of environmental resource availability in plants is scarce. In arid and semi-arid environments, the persistence and dominance of perennial species depends on their capacity to tolerate drought: tolerance could be given on one extreme by fixed traits and, on the other, by plastic traits. To understand drought tolerance of species it is necessary to know the plasticity of their water economy-related traits, i.e. the position in the fixed–plastic continuum.
Three conspicuous co-existing perennial grasses from a Patagonian steppe were grown under controlled conditions with four levels of steady-state water availability. Evaluated traits were divided into two groups. The first was associated with potential plant performance and correlated with fitness, and included above-ground biomass, total biomass, tillering and tiller density at harvest. The second group consisted of traits associated with mechanisms of plant adjustment to environmental changes and included root biomass, shoot/root ratio, tiller biomass, length of total elongated leaf, length of yellow tissue divided by time and ﬁnal length divided by the time taken to reach ﬁnal length.
Key Results and Conclusions
The most plastic species along this drought gradient was the most sensitive to drought, whereas the least plastic and slowest growing was the most tolerant. This negative relationship between tolerance and plasticity was true for fitness-related traits but was trait-dependent for underlying traits. Remarkably, the most tolerant species had the highest positive plasticity (i.e. opposite to the default response to stress) in an underlying trait, directly explaining its drought resistance: it increased absolute root biomass. The niche differentiation axis that allows the coexistence of species in this group of perennial dryland grasses, all limited by soil surface moisture, would be a functional one of fixed versus plastic responses.
Dryland; perennial grasses; mechanistic traits; performance traits; trade-off; Bromus pictus;
Poa ligularis; Pappostipa speciosa