A Lignan from Alnus japonica Activates Myogenesis and Alleviates Dexamethasone-induced Myotube Atrophy.

To find inhibitors against skeletal muscle loss, we isolated a lignan compound ((-)-(2R,3R-1,4-O-diferuloylsecoisolarciresinol, DFS) from the stem of Alnus japonica. C2C12 myoblasts were treated with DFS during differentiation. To induce an in vitro atrophic condition, differentiated myotubes were treated with dexamethasone (a synthetic glucocorticoid). DFS (10 nM) increased expression levels of myogenic factors and the number of multi-nucleated myotubes expressing myosin heavy chain (MHC). The myogenic potential of DFS could be attributed to p38 MAPK activation. DFS also protected against dexamethasone-induced damage, showing increased expression of MHC and mammalian target of rapamycin (mTOR), a major anabolic factor. Under atrophic condition, the anti-myopathy effect of DFS was associated with inactivation of NF-κB signaling pathway and the subsequent suppression of muscle degradative E3 ligases and myostatin. DFS treatment also restored fast muscle fiber (type II a, II b, and II x), known to be susceptible to dexamethasone. These results indicate that DFS isolated from A. japonica can stimulate myogenesis via p38 MAPK activation and alleviate muscle atrophy by modulating the expression of genes associated with muscle protein anabolism/catabolism. Thus, we propose that DFS can be used as a pharmacological and nutraceutical agent for increasing muscle strength or protecting muscle loss.

Nitrogen-fixing red alder trees tap rock-derived nutrients.

Symbiotic nitrogen (N)-fixing trees supply significant N inputs to forest ecosystems, leading to increased soil fertility, forest growth, and carbon storage. Rapid growth and stoichiometric constraints of N fixers also create high demands for rock-derived nutrients such as phosphorus (P), while excess fixed N can generate acidity and accelerate leaching of rock-derived nutrients such as calcium (Ca). This ability of N-fixing trees to accelerate cycles of Ca, P, and other rock-derived nutrients has fostered speculation of a direct link between N fixation and mineral weathering in terrestrial ecosystems. However, field evidence that N-fixing trees have enhanced access to rock-derived nutrients is lacking. Here we use strontium (Sr) isotopes as a tracer of nutrient sources in a mixed-species temperate rainforest to show that N-fixing trees access more rock-derived nutrients than nonfixing trees. The N-fixing tree red alder (Alnus rubra), on average, took up 8 to 18% more rock-derived Sr than five co-occurring nonfixing tree species, including two with high requirements for rock-derived nutrients. The increased access to rock-derived nutrients occurred despite spatial variation in community-wide Sr sources across the forest, and only N fixers had foliar Sr isotopes that differed significantly from soil exchangeable pools. We calculate that increased uptake of rock-derived nutrients by N-fixing alder requires a 64% increase in weathering supply of nutrients over nonfixing trees. These findings provide direct evidence that an N-fixing tree species can also accelerate nutrient inputs from rock weathering, thus increasing supplies of multiple nutrients that limit carbon uptake and storage in forest ecosystems.

Anti-Herbivore Activity of Oregonin, a Diarylheptanoid Found in Leaves and Bark of Red Alder (Alnus rubra).

Plants synthesize a wide range of bioactive secondary metabolites to defend against pests and pathogens. Red alder (Alnus rubra) bark, root, and leaf extract have a long history of use in traditional medicine and hygiene. Diarylheptanoids, especially oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(ß-D-xylopyranosyloxy)-heptan-3-one), have been identified as major bioactive constituents. Diarylheptanoids have become a focus of research following reports of their antioxidant, antifungal, and anti-cancer activities. Recent data suggest that high oregonin concentration is associated with resistance of red alder leaves to western tent caterpillar (Malacosoma californicum) defoliation. Here we test effects of this compound directly on leaf-eating insects. Purified oregonin was examined in insect choice and toxicity tests using lepidopteran caterpillars. The compound exhibited significant anti-feedant activity against cabbage looper (Trichoplusia ni), white-marked tussock moth (Orgyia leucostigma), fall webworm (Hyphantria cunea), and M. californicum at concentrations corresponding to oregonin content of the most resistant alder clones in previous experiments. Toxicity tests were carried out with cabbage looper larvae only, but no contact or ingested toxicity was detected. Our results suggest that oregonin at levels found in red alder leaves early in the growing season may contribute to protecting red alder from leaf-eating insects.

Application of amendments for the phytoremediation of a former mine technosol by endemic pioneer species: alder and birch seedlings.

Metal(loid) pollution of soils has important negative effects on the environment and human health. For the rehabilitation of these soils, some eco-innovative strategies, such as phytoremediation, could be chosen. This practice could establish a plant cover to reduce the toxicity of the pollutants and stabilize the soil, preventing soil erosion and water leaching; this technique is called phytoremediation. For this, plants need to be tolerant to the pollutants present; thus, phytoremediation can have better outcomes if endemic species of the polluted area are used. Finally, to further improve phytoremediation success, amendments can be applied to ameliorate soil conditions. Different amendments can be used, such as biochar, a good metal(loid) immobilizer, compost, a nutrient-rich product and iron sulfate, an efficient arsenic immobilizer. These amendments can either be applied alone or combined for further positive effects. In this context, a mesocosm experiment was performed to study the effects of three amendments, biochar, compost and iron sulfate, applied alone or combined to a former mine technosol, on the soil properties and the phytoremediation potential of two endemic species, Alnus sp. and Betula sp. Results showed that the different amendments reduced soil acidity and decreased metal(loid) mobility, thus improving plant growth. Both species were able to grow on the amended technosols, but alder seedlings had a much higher growth compared to birch seedlings. Finally, the combination of compost with biochar and/or iron sulfate and the establishment of endemic alder plants could be a solution to rehabilitate a former mine technosol.

Host plant frequency and secondary metabolites are concurrently associated with insect herbivory in a dominant riparian tree.

Herbivory is strongly influenced by different sources of plant variation, from traits such as secondary metabolites to features associated with population- and community-level variation. However, most studies have assessed the influence of these drivers in isolation. We conducted a large-scale study to evaluate the associations between multiple types of plant-based variation and insect leaf herbivory in alder ( Alnus glutinosa) trees sampled in riparian forests throughout northwestern Spain. We assessed the associations between insect leaf herbivory and alder mean production of leaf secondary metabolites (phenolic compounds), variation among neighbouring alder trees in leaf phenolics and community-related features including alder relative size and frequency and tree species phylogenetic diversity. Structural equation modelling indicated that increasing concentrations of alder leaf flavonoids (but not other types of phenolic compounds) and increasing variation in phenolics among neighbouring alders were both significantly negatively associated with herbivory. In addition, increasing relative frequency of alder was positively associated with leaf damage, whereas the size of alders relative to other trees and phylogenetic diversity were not significantly associated with herbivory. These results demonstrate the concurrent and independent influences of different sources of plant-based variation on insect herbivory and argue for further future work simultaneously addressing multiple plant-based bottom-up controls.

Physiological effects of major up-regulated Alnus glutinosa peptides on Frankia sp. ACN14a.

Alnus glutinosa has been shown previously to synthesize, in response to nodulation by Frankia sp. ACN14a, an array of peptides called Alnus symbiotic up-regulated peptides (ASUPs). In a previous study one peptide (Ag5) was shown to bind to Frankia nitrogen-fixing vesicles and to modify their porosity. Here we analyse four other ASUPs, alongside Ag5, to determine whether they have different physiological effects on in vitro grown Frankia sp. ACN14a. The five studied peptides were shown to have different effects on nitrogen fixation, respiration, growth, the release of ions and amino acids, as well as on cell clumping and cell lysis. The mRNA abundance for all five peptides was quantified in symbiotic nodules and one (Ag11) was found to be more abundant in the meristem part of the nodule. These findings point to some peptides having complementary effects on Frankia cells.

Effect of Uranium and Thorium Radionuclides on Biochemical Characteristics of Duschekia fruticosa in "Soil-Plant" System.

The biochemical characteristics of Duschekiafruticosa, grown for a long time under a variety of exposure doses of natural background radiation (up to 150 µR/h) was studied. Uranium was found to make the dominant contribution to the y-background exposure doses. The pH-values and the content of organic matter in soils within the surveyed territory remained unchanged. Accumulation of radionuclides of uranium and thorium in the "soil-plant" system was studied. It is shown for the D. fruticosa that U and Th uptake decreased with y-background increasing. Study of anti-free radical and anti-peroxide cells' protection system indicated a balanced activity of prooxidant-antioxidant systems in the cells of the D. fruticosa leaves. The combined effect of incorporated uranium and thorium is accompanied by a significant increase in chlorophyll content in D. fruticosa.

Ecological consequences of the expansion of N2-fixing plants in cold biomes.

Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N2)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N2O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N2-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N2 fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N2-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N2-fixing plants and a rapid subsequent saturation in the ecosystem's capacity to retain N. Therefore, analogous to increases in N deposition, N2-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.

Leaf and tree age-related changes in leaf ecophysiological traits, nutrient, and adaptive strategies of Alnus nepalensis in the central Himalaya.

Leaf ecophysiological traits are known to change with leaf and tree age. In the present study, we measured the effect of leaf and tree age on leaf ecophysiological and morphological traits of nitrogen-fixing Alnus nepalensis (D. Don) which is a pioneer tree species in degraded lands. Three naturally occurring A. nepalensis forest stands, namely young (5-8 years old), mature (40-55 years old), and old (130-145 years old), were considered in this study. We also investigated the seasonal variations in leaf ecophysiological and morphological traits during leaf flushing, fully expanded, and leaf senescence phenological stages. The ecophysiological and morphological traits were compared between leaf and tree ages using a linear mixed-effect model (LMM) and Tukey's HSD test. Fully expanded leaves and young trees demonstrate ecophysiological traits consistent with acquisitive resource-use strategies. Our results revealed that net photosynthetic capacity (Aarea and Amass), leaf stomatal conductance (gswarea and gswmass), transpiration rate (Earea and Emass), specific leaf area (SLA), predawn and midday water potential (Ψ), leaf total chlorophyll concentration, photosynthetic N- and P-use efficiency (PNUE and PPUE) were higher in younger trees than mature and old trees. We found lower wateruse efficiency (WUE) and intrinsic water-use efficiency (WUEi) in young trees than in mature and old ones. Mass-based net photosynthetic capacity (Amass) was positively correlated with PNUE, PPUE, transpiration rate, stomatal conductance, SLA and chlorophyll concentrations but negatively correlated with WUE and WUEi. However, mass-based leaf nitrogen (N) and phosphorus (P) concentrations were the highest in fully expanded leaves and did not vary with tree age despite N concentration being negatively correlated with SLA. Overall, this study provides valuable insights into the age-related changes in leaf ecophysiological traits of A. nepalensis. The findings underscore the importance of considering tree age when studying plant ecophysiology and highlight the acquisitive resource-use strategies employed by young trees for rapid growth and establishment.

From tolerance to acute metabolic deregulation: contribution of proteomics to dig into the molecular response of alder species under a polymetallic exposure.

Alnus spp. are actinorhizal trees commonly found in wet habitats and able to grow effectively in soil slightly contaminated with metal trace- elements. Two clones belonging to two Alnus species, namely, A. incana and A. glutinosa, were grown in hydroponics and exposed for 9 weeks to a Cd + Ni + Zn polymetallic constraint. Although responding by a similar decrease in total biomass production, the proteomic analysis associated with the study of various biochemical parameters including carbohydrate and mineral analyses revealed that the two clones have a distinct stress-responsive behavior. All parameters indicated that the roots, the organ in direct contact with the media, are more affected than the leaves. In fact, in A. glutinosa the response was almost completely confined to the roots, whereas many proteins change significantly in the roots and in the leaves of the treated A. incana. In both clones, the changes affected a broad range of metabolic processes such as redox regulation and energy metabolism and induced the production of pathogenesis-related proteins. In particular, changes in the accumulation of bacterial proteins that were not identified as coming from the known symbionts of Alnus were reported. Further investigation should be performed to identify their origin and exact role in the plant response to the polymetallic exposure tested here.

Effects of riparian plant diversity loss on aquatic microbial decomposers become more pronounced with increasing time.

We examined the potential long-term impacts of riparian plant diversity loss on diversity and activity of aquatic microbial decomposers. Microbial assemblages were obtained in a mixed-forest stream by immersion of mesh bags containing three leaf species (alder, oak and eucalyptus), commonly found in riparian corridors of Iberian streams. Simulation of species loss was done in microcosms by including a set of all leaf species, retrieved from the stream, and non-colonized leaves of three, two or one leaf species. Leaves were renewed every month throughout six months, and microbial inoculum was ensured by a set of colonized leaves from the previous month. Microbial diversity, leaf mass loss and fungal biomass were assessed at the second and sixth months after plant species loss. Molecular diversity of fungi and bacteria, as the total number of operational taxonomic units per leaf diversity treatment, decreased with leaf diversity loss. Fungal biomass tended to decrease linearly with leaf species loss on oak and eucalyptus, suggesting more pronounced effects of leaf diversity on lower quality leaves. Decomposition of alder and eucalyptus leaves was affected by leaf species identity, mainly after longer times following diversity loss. Leaf decomposition of alder decreased when mixed with eucalyptus, while decomposition of eucalyptus decreased in mixtures with oak. Results suggest that the effects of leaf diversity on microbial decomposers depended on leaf species number and also on which species were lost from the system, especially after longer times. This may have implications for the management of riparian forests to maintain stream ecosystem functioning.

Annotated genome sequence of a fast-growing diploid clone of red alder (Alnus rubra Bong.).

Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, that of Alnus glutinosa. Our work initiated a detailed comparative analysis of members of the order Fagales and established some similarities with previous reports in this clade, suggesting a biased retention of certain gene functions in the vestiges of an ancient genome duplication when compared with more recent tandem duplications.

Ecosystem carbon stocks and sequestration rates in white oak forests in the central Himalaya: Role of nitrogen-fixing Nepalese alder.

Nitrogen-fixing Nepalese alder (Alnus nepalensis D. Don.), a pioneer species and nurse tree species, forms pure stands, and sometimes occurs in mixed stands in areas affected by landslides. The objective of this study was to understand the influence of A. nepalensis on carbon stock in white oak (Quercus leucotrichophora A. Camus) forests. We investigated the differences in vegetation biomass carbon (tree, sapling, seedling, shrub and herbs, and forest floor mass), soil organic carbon stock, and sequestration rates in five naturally occurring oak mixed alder (OMA) forest stands and five naturally occurring oak without alder (OWA) forest stands along the basal area gradient in order to investigate the role of A. nepalensis on ecosystem carbon stock. The total basal area ranged from 61.20 to 89.51 m2 ha-1 in the OMA stands and from 38.02 to 53.54 m2 ha-1 in the OWA stands. The total tree density of the OMA stands (1120 to 1330 trees ha-1) was higher than that of the OWA stands (950 to 1230 trees ha-1). The total ecosystem carbon stock in the OMA stands was significantly (P<0.05) higher than that in the OWA stands, ranging from 485.3 to 635.6 Mg C ha-1 in the former and from 378.8 to 472 Mg C ha-1 in the latter. Soil was the second largest carbon pool in all the studied stands, with the values ranging from 238.1 to 254.1 Mg C ha-1 in the OMA and 185.5 to 215.8 Mg C ha-1 in the OWA stands. The soil organic carbon (SOC) stock was 1.19 to 1.28 times higher in the OMA than in the OWA stands. Of the total ecosystem carbon stock in different OMA stands, A. nepalensis stored 16.2 to 38.8%. Annual carbon sequestration rates (6.6 to 9.5 Mg C ha-1 yr-1) in the OMA stands were significantly (P<0.05) higher than in the OWA (2.5 to 5.4 Mg C ha-1 yr-1) stands. Among all the species and across the stands, the greatest carbon sequestration was exhibited by A. nepalensis (3.4 to 5.3 Mg C ha-1 yr-1). The present results show the role of A. nepalensis in ecosystem carbon stock and sequestration rates. Significantly higher rates of carbon sequestration by oak in OMA stands than OWA stands clearly indicate the facilitative role of co-occurring nitrogen-fixing A. nepalensis. The results imply that Q. leucotrichophora mixed with a A. nepalensis plantation may be a good option for enhancing ecosystem carbon stock, carbon sequestration, and habitat restoration in the central Himalaya.

Net degradation of methyl mercury in alder swamps.

Wetlands are generally considered to be sources of methyl mercury (MeHg) in northern temperate landscapes. However, a recent input-output mass balance study during 2007-2010 revealed a black alder (Alnus glutinosa) swamp in southern Sweden to be a consistent and significant MeHg sink, with a 30-60% loss of MeHg. The soil pool of MeHg varied substantially between years, but it always decreased with distance from the stream inlet to the swamp. The soil MeHg pool was significantly lower in the downstream as compared to the upstream half of the swamp (0.66 and 1.34 ng MeHg g⁻¹ SOC⁻¹ annual average⁻¹, respectively, one-way ANOVA, p = 0.0006). In 2008 a significant decrease of %MeHg in soil was paralleled by a significant increase in potential demethylation rate constant (k(d), p < 0.02 and p < 0.004, respectively). In contrast, the potential methylation rate constant (k(m)) was unrelated to distance (p = 0.3). Our results suggest that MeHg was net degraded in the Alnus swamp, and that it had a rapid and dynamic internal turnover of MeHg. Snapshot stream input-output measurements at eight additional Alnus glutinosa swamps in southern Sweden indicate that Alnus swamps in general are sinks for MeHg. Our findings have implications for forestry practices and landscape planning, and suggest that restored or preserved Alnus swamps may be used to mitigate MeHg produced in northern temperate landscapes.

Future increase in temperature more than decrease in litter quality can affect microbial litter decomposition in streams.

The predicted increase in atmospheric CO(2) concentration for this century is expected to lead to increases in temperature and changes in litter quality that can affect small woodland streams, where water temperature is usually low and allochthonous organic matter constitutes the basis of the food web. We have assessed the individual and interactive effect of water temperature (5 and 10°C) and alder litter quality produced under ambient CO(2) levels (ambient litter) or under CO(2) concentrations predicted for 2050 (elevated litter) on litter decomposition and on fungal activity and assemblage structure. Litter decomposition rates and fungal respiration rates were significantly faster at 10 than at 5°C, but they were not affected by litter quality. Litter quality affected mycelial biomass accrual at 5 but not at 10°C, while increases in temperature stimulated biomass accrual on ambient but not on elevated litter. A similar pattern was observed for conidial production. All variables were stimulated on elevated litter at 10°C (future scenario) compared with ambient litter at 5°C (present scenario), but interactions between temperature and litter quality were additive. Temperature was the factor that most strongly affected the structure of aquatic hyphomycete assemblages. Our results indicate that if future increases in atmospheric CO(2) lead to only slight modifications in litter quality, the litter decomposition and fungal activities and community structure will be strongly controlled by increased water temperature. This may have serious consequences for aquatic systems as faster litter decomposition may lead to food depletion for higher trophic levels.

Potential macro-detritivore range expansion into the subarctic stimulates litter decomposition: a new positive feedback mechanism to climate change?

As a result of low decomposition rates, high-latitude ecosystems store large amounts of carbon. Litter decomposition in these ecosystems is constrained by harsh abiotic conditions, but also by the absence of macro-detritivores. We have studied the potential effects of their climate change-driven northward range expansion on the decomposition of two contrasting subarctic litter types. Litter of Alnus incana and Betula pubescens was incubated in microcosms together with monocultures and all possible combinations of three functionally different macro-detritivores (the earthworm Lumbricus rubellus, isopod Oniscus asellus, and millipede Julus scandinavius). Our results show that these macro-detritivores stimulated decomposition, especially of the high-quality A. incana litter and that the macro-detritivores tested differed in their decomposition-stimulating effects, with earthworms having the largest influence. Decomposition processes increased with increasing number of macro-detritivore species, and positive net diveristy effects occurred in several macro-detritivore treatments. However, after correction for macro-detritivore biomass, all interspecific differences in macro-detritivore effects, as well as the positive effects of species number on subarctic litter decomposition disappeared. The net diversity effects also appeared to be driven by variation in biomass, with a possible exception of net diversity effects in mass loss. Based on these results, we conclude that the expected climate change-induced range expansion of macro-detritivores into subarctic regions is likely to result in accelerated decomposition rates. Our results also indicate that the magnitude of macro-detritivore effects on subarctic decomposition will mainly depend on macro-detritivore biomass, rather than on macro-detritivore species number or identity.

Volatile emissions from Alnus glutionosa induced by herbivory are quantitatively related to the extent of damage.

Plant volatile organic compounds (VOCs) elicited in response to herbivory serve as cues for parasitic and predatory insects. Knowledge about quantitative relationships between the extent of herbivore-induced damage and the quantities of VOCs released is scarce. We studied the kinetics of VOC-emissions from foliage of the deciduous tree Alnus glutinosa induced by feeding activity of larvae of the geometrid moth Cabera pusaria. Quantitative relationships between the intensity of stress and strength of plant response were determined. Intensity of biotic stress was characterized by herbivore numbers (0-8 larvae) and by the amount of leaf area eaten. The strength of plant response was characterized by monitoring (i) changes in photosynthesis, (ii) leaf ultrastructure, and (iii) plant volatiles. Net assimilation rate displayed compensatory responses in herbivore-damaged leaves compared with control leaves. This compensatory response was associated with an overall increase in chloroplast size. Feeding-induced emissions of products of the lipoxygenase pathway (LOX products; (E)-2-hexenal, (Z)-3-hexenol, 1-hexanol, and (Z)-3-hexenyl acetate) peaked at day 1 after larval feeding started, followed by an increase of emissions of ubiquitous monoterpenes peaking on days 2 and 3. The emission of the monoterpene (E)-ß-ocimene and of the nerolidol-derived homoterpene 4,8-dimethyl-nona-1,3,7-triene (DMNT) peaked on day 3. Furthermore, the emission kinetics of the sesquiterpene (E,E)-α-farnesene tended to be biphasic with peaks on days 2 and 4 after start of larval feeding. Emission rates of the induced LOX products, of (E)-ß-ocimene and (E,E)-α-farnesene were positively correlated with the number of larvae feeding. In contrast, the emission of DMNT was independent of the number of feeders. These data show quantitative relationships between the strength of herbivory and the emissions of LOX products and most of the terpenoids elicited in response to feeding. Thus, herbivory-elicited LOX products and terpenoid emissions may convey both quantitative and qualitative signals to antagonists of the herbivores. In contrast, our data suggest that the feeding-induced homoterpene DMNT conveys the information "presence of herbivores" rather than information about the quantities of herbivores to predators and parasitoids.

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus.

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1- and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Flooding induced emissions of volatile signalling compounds in three tree species with differing waterlogging tolerance.

To gain insight into variations in waterlogging responsiveness, net assimilation rate, stomatal conductance, emissions of isoprene and marker compounds of anoxic metabolism ethanol and acetaldehyde, and stress marker compounds nitric oxide (NO), volatile products of lipoxygenase (LOX) pathway and methanol were studied in seedlings of temperate deciduous tree species Alnus glutinosa, Populus tremula and Quercus rubra (from highest to lowest waterlogging tolerance) throughout sustained root zone waterlogging of up to three weeks. In all species, waterlogging initially resulted in reductions in net assimilation and stomatal conductance and enhanced emissions of ethanol, acetaldehyde, NO, LOX products and methanol, followed by full or partial recovery depending on process and species. Strong negative correlations between g(s) and internal NO concentration and NO flux, valid within and across species, were observed throughout the experiment. Isoprene emission capacity was not related to waterlogging tolerance. Less waterlogging tolerant species had greater reduction and smaller acclimation capacity in foliage physiological potentials, and larger emission bursts of volatile stress marker compounds. These data collectively provide encouraging evidence that emissions of volatile organics and NO can be used as quantitative measures of stress tolerance and acclimation kinetics in temperate trees.

Geometrical similarity analysis of photosynthetic light response curves, light saturation and light use efficiency.

Light absorption and use efficiency (LAUE mol mol(-1), daily gross photosynthesis per daily incident light) of each leaf depends on several factors, including the degree of light saturation. It is often discussed that upper canopy leaves exposed to direct sunlight are fully light-saturated. However, we found that upper leaves of three temperate species, a heliophytic perennial herb Helianthus tuberosus, a pioneer tree Alnus japonica, and a late-successional tree Fagus crenata, were not fully light-saturated even under full sunlight. Geometrical analysis of the photosynthetic light response curves revealed that all the curves of the leaves from different canopy positions, as well as from the different species, can be considered as different parts of a single non-rectangular hyperbola. The analysis consistently explained how those leaves were not fully light-saturated. Light use optimization models, called big leaf models, predicted that the degree of light saturation and LAUE are both independent of light environment. From these, we hypothesized that the upper leaves should not be fully light-saturated even under direct sunlight, but instead should share the light limitation with the shaded lower-canopy leaves, so as to utilize strong sunlight efficiently. Supporting this prediction, within a canopy of H. tuberosus, both the degree of light saturation and LAUE were independent of light environment within a canopy, resulting in proportionality between the daily photosynthesis and the daily incident light among the leaves.