Ancient Gene Duplications, Rather Than Polyploidization, Facilitate Diversification of Petal Pigmentation Patterns in Clarkia gracilis (Onagraceae).

It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. Clarkia gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, that is, before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.

Chemical composition and in vitro ruminal digestibility of Ludwigia grandiflora.

Ludwigia grandiflora is an amphibious plant that has been designated as a specific alien plant in Japan. Due to the risk of regeneration on land, plants are burned after eradication. Since L. grandiflora is fertile and rich in biomass, this study investigated the use of L. grandiflora as a feed for ruminants. We measured general components, detergent fiber components, and in vitro dry matter digestibility (IVDMD) using rumen microbes for three forms (terrestrial form, emergent form, and floating-leaved form) of L. grandiflora. In addition, concentrations of hazardous metallic elements were also measured from the viewpoint of feed safety. Crude protein, neutral detergent fiber, acid detergent fiber, and acid detergent lignin were contained 10.31-14.62, 27.83-42.28, 20.54-36.05, and 6.94-18.90 (%DM), respectively. IVDMD was 50.75%-68.24%. Toxic elements such as Pb, Cd, and Hg were not detected in all forms. These results suggest that L. grandiflora could be partially used as alternative roughage for ruminants.

R2R3-MYB genes control petal pigmentation patterning in Clarkia gracilis ssp. sonomensis (Onagraceae).

Petal pigmentation patterning is widespread in flowering plants. The genetics of these pattern elements has been of great interest for understanding the evolution of phenotypic diversification. Here, we investigate the genetic changes responsible for the evolution of an unpigmented petal element on a colored background. We used transcriptome analysis, gene expression assays, cosegregation in F2 plants and functional tests to identify the gene(s) involved in petal coloration in Clarkia gracilis ssp. sonomensis. We identified an R2R3-MYB transcription factor (CgsMYB12) responsible for anthocyanin pigmentation of the basal region ('cup') in the petal of C. gracilis ssp. sonomensis. A functional mutation in CgsMYB12 creates a white cup on a pink petal background. Additionally, we found that two R2R3-MYB genes (CgsMYB6 and CgsMYB11) are also involved in petal background pigmentation. Each of these three R2R3-MYB genes exhibits a different spatiotemporal expression pattern. The functionality of these R2R3-MYB genes was confirmed through stable transformation of Arabidopsis. Distinct spatial patterns of R2R3-MYB expression have created the possibility that pigmentation in different sections of the petal can evolve independently. This finding suggests that recent gene duplication has been central to the evolution of petal pigmentation patterning in C. gracilis ssp. sonomensis.

Nutrient enhancement of allelopathic effects of exotic invasive on native plant species.

Many ecosystems may suffer from both nutrient enrichment and exotic plant invasions simultaneously. Much has been known that nutrient inputs can promote growth and expansion of exotic invasive plants in wetlands, and that allelopathic effects of the exotic invasive plants can inhibit the growth of coexisting native plants, contributing to their invasion success. Thus, we hypothesized that allelopathic effects of exotics on natives in invaded ecosystems can be enhanced by nutrient enrichment. To test this hypothesis, we conducted two greenhouse hydroponic experiments. One is the monoculture experiment in which a widespread exotic invasive perennial Alternanthera philoxeroides and a native perennial Ludwigia peploides subsp. stipulacea in monoculture were subjected to five levels of nutrient supply. The other is the mixture experiment in which the two species in mixture were subjected to five levels of nutrient supply, each with and without activated carbon addition. Both A. philoxeroides and L. peploides grew better under higher level of nutrient availability in monoculture experiment. In the mixture experiment, A. philoxeroides formed less total and root biomass while L. peploides formed more in response to activated carbon addition and all of the responses had larger degree at higher level of nutrient availability, indicating A. philoxeroides had significant allelopathic effects on L. peploides and the effects was significantly enhanced by nutrient enrichment. Such results support our hypothesis and reveal a novel mechanism for exotic plant invasion in eutrophicated and invaded wetlands, i.e. nutrient enhancement of allelopathic effects of exotics on natives.

Phytoremediation of contaminated soils containing gasoline using Ludwigia octovalvis (Jacq.) in greenhouse pots.

Greenhouse experiments were carried out to determine the phytotoxic effects on the plant Ludwigia octovalvis in order to assess its applicability for phytoremediation gasoline-contaminated soils. Using plants to degrade hydrocarbons is a challenging task. In this study, different spiked concentrations of hydrocarbons in soil (1, 2, and 3 g/kg) were tested. The results showed that the mean efficiency of total petroleum hydrocarbon (TPH) removal over a 72-day culture period was rather high. The maximum removal of 79.8 % occurred for the 2 g/kg concentration, while the removal rate by the corresponding unplanted controls was only (48.6 %). The impact of gasoline on plants included visual symptoms of stress, yellowing, growth reduction, and perturbations in the developmental parameters. The dry weight and wet weight of the plant slightly increased upon exposure to gasoline until day 42. Scanning electron microscopy (SEM) indicated change to the root and stem structure in plant tissue due to the direct attachment with gasoline contaminated compared to the control sample. The population of living microorganisms in the contaminated soil was found to be able to adapt to different gasoline concentrations. The results showed that L. octovalvis and rhizobacteria in gasoline-contaminated soil have the potential to degrade organic pollutants.

Examination of secondary metabolites and antioxidant capacity of Anthyllis vulneraria, Fuchsia sp., Galium mollugo and Veronica beccabunga.

Anthyllis vulneraria L., Fuchsia sp., Galium mollugo L., and Veronica beccabunga L. were selected to analyse the phenolic content and the antioxidant activity by ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The highest polyphenol, tannin, and flavonoid contents were measured in Fuchsia species (7.40 ± 0.8, 5.62 ± 0.7 and 0.72 ± 0.1 g/100 g dry weight), while the lowest values were detected in Anthyllis vulneraria (0.68 ± 0.02, 0.17 ± 0.03 and 0.45 ± 0.01 g/100 g dry weight) and Galium mollugo (1.77 ± 0.05, 0.49 ± 0.04 and 0.16 ± 0.06 g/100 g dry weight). The leaf extract of Fuchsia sp. had the highest, while the herb of A. vulneraria had the lowest antioxidant effect measured by both methods, which is probably related to total polyphenol, tannin, and flavonoid contents.

Differences in Copper Absorption and Accumulation between Copper-Exclusion and Copper-Enrichment Plants: A Comparison of Structure and Physiological Responses.

Differences in copper (Cu) absorption and transport, physiological responses and structural characteristics between two types of Cu-resistant plants, Oenothera glazioviana (Cu-exclusion type) and Elsholtzia haichowensis (Cu-enrichment type), were investigated in the present study. The results indicated the following: (1) After 50 µM Cu treatment, the Cu ratio in the xylem vessels of E. haichowensis increased by 60%. A Cu adsorption experiment indicated that O. glazioviana exhibited greater resistance to Cu, and Cu absorption and the shoot/root ratio of Cu were significantly lower in O. glazioviana than in E. haichowensis. (2) An analysis of the endogenous abscisic acid (ABA) variance and exogenous ABA treatment demonstrated that the ABA levels of both plants did not differ; exogenous ABA treatment clearly reduced Cu accumulation in both plants. (3) The leaf stomatal density of O. glazioviana was significantly less than that of E. haichowensis. Guard cells in E. haichowensis plants were covered with a thick cuticle layer, the epidermal hair was more numerous and longer, and the number of xylem conduits in the root was small. (4) The transpiration rate and the stomatal conductance of O. glazioviana were both significantly lower than those of E. haichowensis, regardless of whether the plants were treated with Cu. Taken together, these results indicate that the differences in the structural characteristics between these two plant species, particularly in the characteristics related to plant transpiration, are important factors that govern whether plants acquire or exclude Cu.

Production of the anti-inflammatory compound 6-O-palmitoyl-3-O-ß-D-glucopyranosylcampesterol by Callus cultures of Lopezia racemosa Cav. (Onagraceae).

Lopezia racemosa Cav. is a plant used in Mexican traditional medicine to heal inflammatory diseases. From this plant we isolated the novel compound 6-O-palmitoyl- 3-O-ß-D-glucopyranosylcampesterol (1) and 6-O-palmitoyl-3-O-ß-D-glucopyranosyl-ß-sitosterol (2), previously reported to have cytotoxic activity on several cancer cell lines. We evaluated the anti-inflammatory activity of 1 in vivo by mouse ear edema induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 57.14% inhibition was observed. The aim of our study was to obtain callus cultures derived from this plant species with the ability to produce the compounds of interest. Callus cultures were initiated on MS basal medium amended with variable amounts of naphthaleneacetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with 6-benzylaminopurine (BAP). Ten treatments with these growth regulators were carried out, using in vitro germinated seedlings as source of three different explants: hypocotyl, stem node, and leaf. Highest yield of 1 was observed on callus derived from leaf explants growing in medium containing 1.0 mg/L 2,4-D and 0.5 mg/L BAP. Selected callus lines produced less 1 than wild plants but the in vitro cultured seedlings showed higher production. So we conclude that it could be attractive to further investigate their metabolic potential.

Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae).

Petal spots are widespread in angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two as yet unidentified loci. We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes. We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes showed that the dihydroflavonol-4-reductase 2 (Dfr2) gene has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicated the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development as a result of early expression of Dfr2 and the flavonoid 3' hydroxylase 1 (F3'h1) gene. Pigments in the background appear later, as a result of later expression of Dfr1 and the flavonoid 3'-5' hydroxylase 1 (F3'5'h1) genes. The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network.

Mercury methylation and the microbial consortium in periphyton of tropical macrophytes: effect of different inhibitors.

Macrophyte-associated periphyton is known as a site of Hg accumulation and methylation in tropical environments. Sulfate-reducing bacteria (SRB) is found in periphyton and its role in Hg methylation is acknowledged. However, the contribution of other microorganisms to this process is largely unknown. We tested the effect of inhibitors for different microorganisms on methylmercury (MMHg) formation on distinct macrophyte species from lakes of the Bolivian Amazon basin and in Brazil. We also tested the effect of inhibitors on bacterial secondary activity at two lakes in Brazil. Samples were incubated on-site with (203)Hg and Me(203)Hg was extracted and measured by liquid scintillation. MMHg formation on macrophytes varied among species ranging from 0.2% to 36%. Treatments with specific inhibitors resulted in reduction of MMHg production on most sites and inhibitors. The most successful treatment was the co-inhibition of SRB and methanogens. The inhibitions of algae and fungi activity showed fewer effects on methylation rates at all sites analyzed. Bacterial secondary activity was slightly affected by algae and fungi inhibition, and largely influenced by prokaryotic, SRB and methanogens inhibition. The data suggest that MMHg formation may not be directly performed by all microorganisms in periphyton but depends on complex interactions among them.

Plasticity in response to phosphorus and light availability in four forest herbs.

The differential ability of forest herbs to colonize secondary forests on former agricultural land is generally attributed to different rates of dispersal. After propagule arrival, however, establishing individuals still have to cope with abiotic soil legacies from former agricultural land use. We focused on the plastic responses of forest herbs to increased phosphorus availability, as phosphorus is commonly found to be persistently bioavailable in post-agricultural forest soils. In a pot experiment performed under field conditions, we applied three P levels to four forest herbs with contrasting colonization capacities: Anemone nemorosa, Primula elatior, Circaea lutetiana and Geum urbanum. To test interactions with light availability, half of the replicas were covered with shade cloths. After two growing seasons, we measured aboveground P uptake as well as vegetative and regenerative performance. We hypothesized that fast-colonizing species respond the most opportunistically to increased P availability, and that a low light availability can mask the effects of P on performance. All species showed a significant increase in P uptake in the aboveground biomass. The addition of P had a positive effect on the vegetative performances of two of the species, although this was unrelated to their colonization capacities. The regenerative performance was affected by light availability (not by P addition) and was related to the species' phenology. Forest herbs can obviously benefit from the increased availability of P in post-agricultural forests, but not all species respond in the same way. Such differential patterns of plasticity may be important in community dynamics, as they affect the interactions among species.

Arsenate tolerance mechanism of Oenothera odorata from a mine population involves the induction of phytochelatins in roots.

We investigated the arsenate tolerance mechanisms of Oenothera odorata by comparing two populations [i.e., one population from the mine site (MP) and the other population from an uncontaminated site (UP)] via the exposure of hydroponic solution containing arsenate (i.e., 0-50 microM). The MP plants were significantly more tolerant to arsenate than UP plants. The UP plants accumulated more As in their shoots and roots than did the MP plants. The UP plants translocated up to 21 microg g(-1) of As into shoots, whereas MP plants translocated less As (up to 4.5 microg g(-1)) to shoots over all treatments. The results of lipid peroxidation indicated that MP plants were less damaged by oxidative stress than were UP plants. Phytochelatin (PC) content correlated linearly with root As concentration in the MP (i.e., [PCs](root)=1.69x[As](root), r(2)=0.945) and UP (i.e., [PCs](root)=0.89x[As](root), r(2)=0.979) plants. This relationship means that increased PC to As ratio may be associated with increased tolerance. Our results suggest that PC induction in roots plays a critical role in As tolerance of O. odorata.

Temperature-dependent changes of cell shape during heterophyllous leaf formation in Ludwigia arcuata (Onagraceae).

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.

Differential responses of the freshwater wetland species Juncus effusus L. and Caltha palustris L. to iron supply in sulfidic environments.

Sulfur pollution can lead to serious problems in freshwater wetlands, including phosphorus eutrophication and sulfide toxicity. We tested the effects of anaerobic iron-rich groundwater discharge in fens, simulated by iron injection, on two characteristic species (Juncus effusus and Caltha palustris) in a sulfidic environment. Biomass production of C. palustris roots showed an optimum response to the combined addition of iron and sulfide, with highest values at intermediate concentrations of both substances. Iron deficiency apparently occurred at low iron concentrations, while at high iron concentrations, growth was decreased. For J. effusus, in contrast, no toxic effects were found of both iron and sulfide. This could be explained by larger radial oxygen loss (ROL) of J. effusus and could not be explained by differences in phosphorous concentrations. The results of our experiments confirm that iron-rich groundwater discharge has the potential to affect vegetation composition through toxicity modification in sulfidic environments.

Ecological studies on the macrohydrophytes in Egypt II. Ludwigia stolonifera (Guill. and Perr.) P.H. Raven.

The present study contributes information about the environmental factors controlling the distribution, variation in standing crop phytomass and chemical compositional change of Ludwigia stolonifera. Also, treatment with cattle manure, as source of organic waste, was made to evaluate the response of the plant to the environmental pollution. The sandy textured bottom sediments of Ludwigia habitat at Damira irrigating canal had higher percentages of organic carbon and total soluble salts in spring than in the other seasons while the overlying water was characterized by low total soluble salts. The phytomass and assimilating surface area were increased in spring and summer months and appeared to decline in winter. The phytochemical constituents were highly concentrated during August. As common in hydrophyte, the plant organs of L. stolonifera are characterized by abundance of aerenchyma, absence of cork cells and reduction of vascular tissues. Addition of cattle manure to the aquatic habitat of Ludwigia plant resulted in an increase of organic carbon, salinity, chloronity and in a decrease of pH value of hydrosoil and water. Heavy metals accumulation showed considerable increase due to application of cattle manure and this appeared to be a reflection to the increased concentrations of these metals in the environment.

Seasonal and temperature dependence of photosynthesis and respiration for two co-occurring broad-leaved tree species with contrasting leaf phenology.

We measured the seasonal and temperature responses of leaf photosynthesis and respiration of two co-occurring native New Zealand tree species with contrasting leaf phenology: winter-deciduous fuchsia (Fuchsia excorticata J. R. Forst & G. Forst) and annual evergreen wineberry (Aristotelia serrata J. R. Forst & G. Forst). There was no difference in the amount of nitrogen per unit leaf area (Narea, range 40-160 mmol m-2, P = 0.18) or specific leaf area (S, range 8-27 m2 kg-1, P = 0.87) in summer leaves of wineberry or fuchsia. The amount of nitrogen per unit leaf area and S varied significantly with height of leaves in the canopy for both species (r2 range 0.61-0.87). Parameters describing the maximum rates of rubisco carboxylation (Vcmax) and electron transport (Jmax) were related significantly to Narea, and were 60% higher on average in spring and summer leaves than in autumn and winter leaves for both species. The seasonal effect remained significant (P < 0.001) when Narea was included in a regression model, indicating that seasonal changes were not only due to changes in Narea. Values for Vcmax and Jmax were 30% lower in wineberry leaves than in fuchsia leaves on average, although the difference ranged from 15% in summer leaves to 39% in autumn leaves. Activation energies describing the temperature dependence of Vcmax and Jmax in wineberry were 111 and 114% of corresponding values for fuchsia (Ea (Vcmax) = 39.1 kJ mol-1, Ea (Jmax) = 32.9 kJ mol-1). Respiration at night was the same (P = 0.34) at 10 degrees C for both species (R10 = 0.7 micromol m-2 s-1), although activation energies (E0) were higher in wineberry than in fuchsia (47.4 and 32.9 kJ mol-1 K-1, respectively). These results show that rates of photosynthesis are higher in winter-deciduous fuchsia than in annual evergreen wineberry.

Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri.

A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile.