Feeding response of the Aldabra giant tortoise (Geochelone gigantea) to island plants showing heterophylly

Aim Heterophylly is present in many plant species on oceanic islands. Almost all of these plants are island endemics, and heterophylly may have evolved as a response to feeding from large insular browsers such as giant tortoises and flightless birds. We tested this anti‐browser hypothesis by feeding Aldabra giant tortoises (Geochelone gigantea) with leaves of native Mauritian plants to see if they distinguished between juvenile and adult leaves and between heteophyllous and homophyllous species. Location Mauritius. Methods In a choice experiment we recorded feeding response of four captive Aldabra giant tortoises to 10 species of Mauritian plants, of which seven were heterophyllous and three homophyllous. Results In general, juvenile leaves of heterophyllous species showed convergence in shape and midrib coloration. Homophyllous foliage was preferred to heterophyllous, and among heterophyllous species adult foliage was preferred to juvenile. Main conclusions Several Mascarene heterophyllous plants show convergence in morphology of juvenile leaves and these are avoided by giant tortoises. This indicates a strong selection history from large browsers such as the giant tortoises. The Mascarene example is in accordance with several other comparable cases of plant‐large browser interactions from other archipelagos.     

Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton

Background

Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton.

Principal Findings

Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection.

Conclusions/Significance

Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions.     

Structural and functional aspects of the Nuphar lutea (L.) Smith heterophylly: Ultrastructure and photosynthesis

The ultrastructure and functional characteristics of the photosynthetic apparatus of floating and submersed leaves of the heterophyllous plant Nuphar lutea (L.) Smith have been examined. Differences have been revealed in mesophyll cell chloroplasts, content of pigments, and chlorophyll fluorescence parameters between floating and submersed leaves and submersed leaves at different depths. A sharp decline in the PSII (photosystem II) efficiency of submersed leaves when exposed to an actinic light intensity of more than 60 ??mol m^?2 s^?1 has been noted. The described differences may be considered as an adaptation mechanism of submersed leaves to life in an aquatic environment with a reduced light intensity and changed light spectral composition.     

Isolation and Characterization of Photosynthetically Active Cells from Submersed and Floating Leaves of the Aquatic Macrophyte Potamogeton nodosus Poir

Photosynthetically active cells were isolated by enzymic digestionof floating and submersed leaves of the heterophyllous aquaticmacrophyte Potamogeton nodosus Poir. The yields of cells isolatedfrom floating leaves represented approximately 25% of the leafprotein or chlorophyll, while cell yields from submersed leaveswere only 3%. Photosynthetic activity was maximal in cells isolatedfrom submersed leaves 10 to 14 days after germination of thewinterbuds. Floating leaves were induced by treatment of theplants with abscisic acid. Cells from induced floating leavesshowed maximum photo synthetic rates between 9 and 21 days posttreatment.Phosphoglycerate, fructose 1,6-bisphosphate, sulfate and phosphatewere without significant effect on photosynthesis in eithercell type indicating that the cells were substantially intact.Half-saturation of photosynthesis for bicarbonate was at 0.6mM (pH 7.6) for cells from both leaf types, and the maximumrate was greater for cells from floating leaves. The light intensityfor half-saturation of photosynthesis was approximately 95 µEm^–2s^–1 for cells from both leaf types, and the maximumrate was greater for cells from floating leaves. (Received September 19, 1984; Accepted December 6, 1984)     

Distribution patterns of Batrachium communities in Mediterranean hard waters

The greatest number of Batrachium plant-communities is reached in base-poor waters of SW Europe where most Batrachium species develop laminar leaves. In contrast, base-rich waters of the Western Mediterranean are characterized by few Batrachium communities and by the only one Batrachium species present in the area with only dissected leaves. This work focused initially on studying the water ecology of Iberian Batrachium communities' developing in hard waters in order to seek to what extend the water physical–chemical gradient determines the occurrence of heterophyllous or homophyllous-dissected species. Floristic data and water physical–chemical data were analyzed using multivariate and comparative methods. We found two main types of Batrachium communities: community of Ranunculus trichophyllus – homophyllous and dissected species–, and community of Ranunculus penicillatus – heterophyllous species–. Alkalinity degree is the main factor separating both communities. Our results of a tentative survey on Batrachium composition in hard waters in the Eastern Mediterranean showed a wider range of physical–chemical water features as well as a greater number of Batrachium species with only dissected leaves in comparison to Western Mediterranean. We conclude that high alkalinity is related to the occurrence of Batrachium communities characterized by species with only dissected leaves in both the western and eastern parts of Mediterranean Europe.     

Variation in leaves and petals of Ranunculus subgenus Batrachium on the Aegean Islands, analysed by multivariate analysis

Variation in leaves and petals was studied using canonical variate and cluster analyseS. In total of 33 populations from eight islands leaf variation in laminar, floating leaves was studied in Ranunculus peltatus subsp. peltatus, subsp. baudotii and subsp. saniculifolius, and in R. Tripartitus, all of which are heterophyllous in the Aegean area. The petal data set also included the homophyllous R. Trichophyllus. The results of the analyses show that the populations are morphologically well differentiated and that petal shape has a tendency to discriminate between the populations somewhat better than leaf shape does. There was no basis for subdividing the populations into taxa on these characterS. Analyses showed that up to 70% of the variation was distributed between populations, the remainder (5–18%) being within populations.     

Adaptive phenotypic plasticity: the case of heterophylly in aquatic plants

Phenotypic plasticity may play a key role in the adaptation of organisms to changing environmental conditions. A special case of plasticity is represented by heterophylly, the ability of semi-aquatic plants to produce different types of leaves below and above water. Submerged leaves are thin and lack both a cuticle and stomata, whereas aerial leaves are thicker, cutinized and bear stomata. The striking variability in the submerged, floating and aerial leaves of heterophyllous aquatics has historically been considered a paradigmatic example of adaptive phenotypic plasticity. An extensive body of developmental and physiological research reveals that heterophylly is quite often mediated by similar environmental cues across diverse taxa, which may imply a common underlying mechanism. Patterns of plasticity in response to environmental cues in the laboratory are consistent with the hypothesis of individual adaptation to heterogeneous environments, and the distribution of this trait among phylogenetically related aquatic angiosperms suggests either convergent or parallel evolution in their descent from terrestrial ancestors. Yet, critical evaluations of the ecological and evolutionary significance of this trait are scarce. In this essay, we discuss the patterns of plasticity revealed by experimental manipulative studies of heterophylly in the context of the general problem of adaptive phenotypic plasticity, and suggest avenues for future research that are needed in assessing the ecological and evolutionary significance of this trait.     

Variation in flavonoid pattern in leaves and flowers of Primula veris of different origin and impact of UV-B

Flavonoids can serve as chemotaxonomic markers and play an important role in protection against ultraviolet (UV) radiation. Primula veris originating from two natural field sites in Albania and one cultivar from Austria were used to investigate whether flavonoid pattern may differ between populations and to determine their response to UV. Plants were grown in a common environment and shortly before flowering transferred in two greenhouses with 80% and 4% UV-B transmission, respectively. After two weeks, young leaves and open flowers were harvested and flavonoids analyzed by high performance liquid chromatography. The flavonoid profiles of leaves and flowers were highly distinct for each population, with certain flavonoids occurring only in plants of particular field sites. These flavonoids may be useful biomarkers to identify the origin of plant material. The differences in UV-treatment at that stage had no effect on the total flavonoid contents of both leaves and flowers. However, individual flavonoids of both leaves and flowers responded sensitively to UV, suggesting that they may be involved in protection against UV.     

Identification of flavonols in leaves of Maytenus ilicifolia and M. aquifolium (Celastraceae) by LC/UV/MS analysis

A comparative analysis of the flavonoid components of the leaves of two medicinal plants known in Brazil as "espinheira santa", namely, Maytenus ilicifolia Mart. ex Reiss. and M. aquifolium Mart. (Celastraceae), and a hybrid plant, M. aquifoliumxM. ilicifolia, has been carried out using high-performance liquid chromatography coupled with photodiode array UV detection and mass spectrometry. One methoxyflavonoid glycoside and 18 flavonol-3-O-glycosides were identified in the extracts on the basis of their on-line UV spectra (measured in the absence and presence of shift reagents) and multiple stage mass spectral data. Fingerprint analysis of the flavonoid extracts revealed significant differences in the profiles of the two Maytenus species, while the hybrid plant contained flavonoids found in both parent species.     

Comparative feeding selectivity of herbivorous insects on water lilies: aquatic vs. semi‐terrestrial insects and submersed vs. floating leaves

1. The rate of grazing damage experienced by submersed and floating leaves of water lilies (Nuphar variegata and Nymphaea odorata) was monitored in lakes in the Upper Peninsula of Michigan, U.S.A. Herbivores damaged 0.2–1.7% of the leaf surface of water lilies per day. These grazing rates differed between plant species, between submersed and floating leaves, and between lakes. Some leaves had more than 60% of their surface damaged and an overall mean of 16% damage occurred during the 2–3 week monitoring period of this study. 2. Snapshot measurements of grazing damage on randomly collected submersed and floating leaves of Nuphar showed that submersed leaves were more damaged (11.0 ± 1.6%, n = 84) than floating leaves (3.8 ± 0.6%, n = 92). Overall, these 176 Nuphar leaves had 7.2% of their area damaged. 3. Five species of herbivorous insects were commonly found on water lilies (Nymphaeacea). One primarily aquatic insect (sensu 1 ), a caddisfly larva (Trichoptera: Limniphilidae), had a generalized diet of water lilies, other macrophytes, algae, and detritus. Four of the five insects were from primarily terrestrial insect groups (Coleoptera and Diptera;‘secondary invaders’, sensu 1 ) and consumed only water lilies in food preference experiments. 4. The feeding preferences of the generalist trichopteran were altered when the macrophytes were freeze-dried, ground into a powder, and reconstituted in an alginate gel. This suggests that plant structure may be an important feeding determinant for this insect. In contrast, a specialist weevil preferred its host plant in choice assays, regardless of whether fresh tissue or reconstituted macrophytes were used, suggesting this insect cued on a unique, non-structural property of its host plant. 5. These results suggest that herbivory on freshwater macrophytes is of a similar magnitude to that on terrestrial plants. The findings of this study are consistent with the hypothesis that herbivorous insects of primarily terrestrial groups have a narrower diet breadth than insects of primarily aquatic groups.     

Photosynthetic use of inorganic carbon among primary and secondary water plants in streams

1. We analysed photosynthetic rates and inorganic carbon use of thirty-five vascular macrophyte species collected submerged in eight nutrient- and CO₂-rich Danish lowland streams. The species were classified in four groups as mainly terrestrial, homophyllous and heterophyllous amphibious and truly submerged. These groups represent plant species differently adapted to life in water. 2. Photosynthetic rates measured in water increased in the gradual transition from mainly terrestrial, through amphibious to truly submerged species. Species normally in contact with air adapted to submergence by increasing the photosynthetic rate at limiting CO₂. Photosynthetic rates of submerged parts of heterophyllous amphibious species were close to those of submerged species. Submerged species with thin or finely dissected leaves had the highest photosynthetic rates, probably because of low diffusional resistance to uptake of nutrients and gases. 3. In contrast to submerged species, terrestrial and amphibious species were unable to use HCO₃^?. Extensive oversaturation with CO₂ in the streams allows, however, many amphibious species to photosynthesize well under water, based on CO₂-use alone. Amphibious CO₂-users, with very few structural adaptations to life under water, can therefore be as dominant in the submerged vegetation of lowland streams as HCO₃^?-using water plants. Moreover, the streams provide open space for colonization from the dense vegetation ashore. 4. Among the 1265 Danish herbaceous species no less than seventy-five terrestrial species occasionally grow submerged, forty-five species are amphibious, and fifty-one are true water plants. These numbers suggest that adaptation to permanent or temporary submergence is an ongoing process involving many species and that the land-water interface does not represent as difficult a barrier as often believed.     

Epigenetic correlates of plant phenotypic plasticity: DNA methylation differs between prickly and nonprickly leaves in heterophyllous Ilex aquifolium (Aquifoliaceae) trees

Phenotypic plasticity is central to the persistence of populations and a key element in the evolution of species and ecological interactions, but its mechanistic basis is poorly understood. This article examines the hypothesis that epigenetic variation caused by changes in DNA methylation are related to phenotypic plasticity in a heterophyllous tree producing two contrasting leaf types. The relationship between mammalian browsing and the production of prickly leaves was studied in a population of Ilex aquifolium (Aquifoliaceae). DNA methylation profiles of contiguous prickly and nonprickly leaves on heterophyllous branchlets were compared using a methylation‐sensitive amplified polymorphism (MSAP) method. Browsing and the production of prickly leaves were correlated across trees. Within heterophyllous branchlets, pairs of contiguous prickly and nonprickly leaves differed in genome‐wide DNA methylation. The mean per‐marker probability of methylation declined significantly from nonprickly to prickly leaves. Methylation differences between leaf types did not occur randomly across the genome, but affected predominantly certain specific markers. The results of this study, although correlative in nature, support the emerging three‐way link between herbivory, phenotypic plasticity and epigenetic changes in plants, and also contribute to the crystallization of the consensus that epigenetic variation can complement genetic variation as a source of phenotypic variation in natural plant populations. © 2012 The Linnean Society of London     

Origins of non-linear and dissimilar relationships between epidermal UV absorbance and UV absorbance of extracted phenolics in leaves of grapevine and barley

A recent review of climate patterns in Southern Germany has suggested significant increases in ultraviolet (UV) radiation due to decreases in cloud coverage and in cloud frequency which compound the effects of stratospheric ozone depletion. Whether such UV radiation increases result in UV damage of higher plant leaves depends partly on the capacity of UV-absorbing hydroxycinnamic acids and flavonoids located in the plant epidermis to screen out UV radiation. Epidermal UV screening is most often assessed from UV absorbance of whole-leaf extracts but in the present work, this method is critically examined. In grapevine (Vitis vinifera L.), hydroxycinnamic acid as well as mono-hydroxylated and ortho-dihydroxylated flavonoid concentrations increased in parallel with fluorometrically detected adaxial epidermal UV absorbance but only the latter class of flavonoids was associated with epidermal UV absorbance in barley (Hordeum vulgare L). For both species, curvilinear relationships between epidermal and total phenolic UV absorbance were established: initial slopes of the curves differed markedly between species. Modelling suggested that curvilinearity arises from UV-transparent epidermal areas located between vacuoles which are particularly UV-absorbing due to high levels of phenolics. The species-dependent differences were related to allocation of high amounts of phenolics in the mesophyll and abaxial epidermis in barley but not in grapevine. Both factors, optical heterogeneity and variable distribution of phenolics, severely restrict the use of phenolic absorbance to estimate true epidermal screening.     

The responsiveness of the <Emphasis Type="Italic">IAA2</Emphasis> promoter to IAA and IBA is differentially affected in <Emphasis Type="Italic">Arabidopsis</Emphasis> roots and shoots by flavonoids

The structural features of flavonoids which are involved in the modulation of auxin distribution in Arabidopsis thaliana were evaluated. An auxin-inducible promoter IAA2 fused to a reporter gene (GUS) was used to monitor the tissue responsiveness to auxins. The following aspects were investigated: 1) the influence of flavonoids (quercetin, naringenin, kaempferol, myricetin and isorhamnetin) on the distribution of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in roots and leaves, 2) differences in flavonoid uptake into roots and shoots depending on flavonoid concentration in the medium, and 3) influence of structurally different flavonoids on the gravitropic response and growth of roots. The same flavonoids differently affected IAA and IBA distribution in leaves and roots. There were several structural requirements for the flavonoids which resulted in the changes of auxin response/distribution. Great differences between the ability of shoots and roots to take up quercetin were showed. Also, flavonoids influenced gravitropism and root growth of Arabidopsis seedlings in a structure-dependent manner.     

Studies on the Polyphenolic Composition and the Antioxidant Properties of the Leaves of Poplar (Populus spp.) Various Species and Hybrids

The chemical composition in terms of flavonoid and salicylic compounds of leaves from 6 species and 3 hybrids of poplars (Populus) was identified with the use of TLC and HPLC-DAD/ESI-MS methods. Chromatographic analyses were carried out with 21 standard compounds including salicylic compounds (2), phenolic acids (3) and flavonoids (16). Moreover, on the basis of the obtained chromatographic data from the HPLC-DAD/ESI-MS and TLC separations, the presence of salicortin, tremulacin and chlorogenic acid was confirmed, depending on the analyzed poplar species or hybrid. The content of salicylic compounds was determined by HPLC-UV method and expressed on salicin as free and total fraction. Total flavonoid content was determined by spectroscopic method as quercetin equivalent. Significant qualitative and quantitative differences in the chemical composition of the analyzed leaves were demonstrated. The highest concentration of flavonoids (8.02 mg/g) was found in the leaves of Populus nigra, while the highest content of salicylic compounds (47.14 mg/g) was found in the leaves of P.×berolinensis. The antioxidant and xanthine oxidase inhibition properties of extracts from poplar leaves were investigated by TLC bioautography. It has been shown that the richest set of compounds with antioxidant properties are present in the leaves of P. alba, P.×candicans and P. nigra.     

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae,Lamiaceae, Rosaceae

Background

The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to family Rosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties.

Results

Among observed plant species, the highest amount of total flavonoids has been found in leaves of Helianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU).

Conclusions

This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.     

Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics

Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.     

Localization and antioxidant capacity of flavonoids from intertidal and subtidal Halophila johnsonii and Halophila decipiens

In this study, flavonoid localization, content and total antioxidant capacity in leaves of subtidal Halophila decipiens were compared to intertidal and subtidal Halophila johnsonii. H. johnsonii leaves had significantly higher flavonoid content (3.5 and 3.8 nmol quercetin equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) and antioxidant capacity (101.7 and 224.2 nmol Trolox equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) than H. decipiens leaves (1.4 nmol quercetin equivalent mm⁻² leaf and 21.0 nmol Trolox equivalent mm⁻² leaf). Flavonoid content did not significantly differ between intertidal and subtidal H. johnsonii, however, antioxidant capacity was significantly higher in subtidal plants. Confocal laser scanning microscopy of fresh leaf cross sections indicated that both species contained flavonoids in the cuticle, but only H. johnsonii contained intracellular flavonoids. Intracellular flavonoids are better situated to perform antioxidant functions in planta. These results suggest that flavonoid compounds in H. johnsonii are capable of sunscreen and antioxidant functions while an antioxidant role for flavonoids within H. decipiens is not supported.