Diazotrophic nitrogen fixation through aerial roots occurs in Avicennia marina: implications for adaptation of mangrove plant growth to low-nitrogen tidal flats.

Nitrogen limitation of primary production is common in coastal ecosystems. Mangrove trees maintain high levels of nitrogen fixation around their roots. The interior aerial space of mangrove roots, in which atmospheric gas is supplied through lenticels, could be efficient sites for nitrogen fixation. We measured tidal variations of partial pressure of N2 in root aerenchyma and conducted field experiments using 15 N2 as a tracer to track N2 movement through aerial roots of Avicennia marina. We used the acetylene reduction assay to identify the root parts harboring diazotrophs. The nitrogenase activity and estimated nitrogen fixation through aerenchyma were higher in pneumatophores and absorbing roots than in cable roots. Positive correlations between root nitrogen contents and turnover rates of root nitrogen derived from N2 through aerenchyma suggested that the internal supply of N2 to diazotrophs could be the main source for nitrogen assimilation by A. marina roots. Our results confirmed that N2 is supplied to diazotrophs through aerial roots and that nitrogen fixation occurs in A. marina roots. The aerial root structures, which occur across families of mangrove plants, could be an adaptation to survival in not only low-oxygen environments but also tidal flats with little plant-available nitrogen.

Temperature dependence of O2 respiration in mangrove leaves and roots: implications for seedling dispersal phenology.

Seasonal differences in diaspore dispersal of three mangrove species, Kandelia obovata, Bruguiera gymnorrhiza and Rhizophora stylosa, suggest that respiratory energy production and demand may differ as a result of interspecific differences in temperature dependence of growth and maintenance processes during seedling establishment. We analyzed growth, temperature dependencies of respiratory O2 consumption and amounts of respiratory chain enzymes in seedlings of these species grown at various temperatures. Respiration rates measured at the low reference temperature, RREF , were highest in leaves of 15°C-grown K. obovata, whose dispersal occurs in the cold season, while root RREF of 15°C-grown R. stylosa was 60% those of the other species, possibly because of warm conditions during its establishment phase. In leaves and roots of K. obovata and leaves of R. stylosa, the overall activation energy, Eo , changed with growth temperature associated with changes in the ratios of the amount of protein in the two respiratory pathways. However, Eo of seedlings of B. gymnorrhiza, which has a long dispersal phase, were constant and independent of growth temperature. The different temperature responses of seedling respiration and growth among these three species may reflect the seasonal temperature range of seedling dispersal and establishment in each species.

Mangrove-diazotroph relationships at the root, tree and forest scales: diazotrophic communities create high soil nitrogenase activities in Rhizophora stylosa rhizospheres.

BACKGROUND AND AIMS: The tidal flats on which mangrove plants grow tend to have low soil nitrogen contents because nitrogen-containing litter is repeatedly washed offshore by ebb tides. Under such circumstances, it is unclear how mangrove plants acquire the nitrogen required to support their vigorous growth. In the present work, chemical and biological characteristics of diazotrophy around mangrove plant roots were surveyed under natural conditions to elucidate mangrove-diazotroph relationships. METHODS: Soil nitrogenase activity of a representative mangrove plant, Rhizophora stylosa, which has a broad geographical distribution, was measured using the acetylene reduction assay at forest, tree and prop root scales. In addition, diazotrophic community composition was compared between rhizosphere and bulk soil based on sequencing of nifH genes. KEY RESULTS: Soil nitrogenase activity was high near prop roots, and this pattern was enhanced as soil live root content increased. At the forest scale, we observed high soil nitrogenase activity (acetylene-reducing activity) inside the forest (the highest value was 90.9 µmol C2H2 min-1 cm-3, average 46.8 ± 18.2 µmol C2H2 min-1 cm-3). Rates decreased sharply from the forest to the tidal flat (range 1.2-22.2 µmol C2H2 min-1 cm-3, average 7.9 ± 4.5 µmol C2H2 min-1 cm-3). The nifH operational taxonomic unit composition differed significantly among forest and tree rhizospheres and the bulk soil (P < 0.0001). CONCLUSIONS: Our results suggest that the accumulation of diazotrophs around R. stylosa mangrove trees enhances the supply of biologically fixed nitrogen to the mangrove roots. This supply is especially important when the soil naturally contains little nitrogen. This nitrogen acquisition system may be a key process that explains the high productivity of mangrove ecosystems.

Foraging dispersion of Ryukyu flying-foxes and relationships with fig abundance in East-Asian subtropical island forests.

BACKGROUND: Figs are widely distributed key resources to many tropical-subtropical animals, and flying-foxes are major consumers and seed dispersers of figs. Bat-fig interrelationships, however, may vary among species differing in fruiting traits, i.e., bat- versus bird-dispersed figs. We examined Ryukyu flying-fox foraging dispersion and the relationships with tree species composition and fig abundance in forests of Iriomote Island. RESULTS: Bat foraging dispersion showed no spatial patterns with respect to different areas of the island, and was not explained by heterogeneity, density, or basal area (BA) of total trees, nor by relative density or BA of fruiting trees or total fruiting figs among sites. Instead, bat densities were positively dependent on the relative density of total figs, and particularly the relative BA of bat-dispersed figs Ficus septica and F. variegata. Both species were dominant figs in forests, fruiting asynchronously with long crop seasons, and were used as predominant foods. Bats foraged mostly solitarily and the mean density was in a hump-shaped relationship with crop sizes of the dominant bat-figs. These two species and Ficus benguetensis are larger-sized bat-figs, all contained more seeds, higher dry-pulp mass and water mass, but not necessarily water content. By approximate estimation, higher proportions of seeds of these bat-figs would have been removed from fruits through the bat consumption, than that of small-sized bird-figs like F. virgata, F. superba, and F. microcarpa. CONCLUSIONS: The foraging dispersion of Ryukyu flying-foxes in forests depends on the availability of the most abundant bat-figs that serve as predominant foods. Intermediate levels of crop sizes of theses figs appear most fit with their solitary foraging. Our results suggest that as density and BA coverage of these dominant bat-figs are below a certain level, their effectiveness to attract bats may dwindle and so would their chance of dispersal by bats.

Genetic differentiation and phylogeography of partially sympatric species complex Rhizophora mucronata Lam. and R. stylosa Griff. using SSR markers.

BACKGROUND: Mangrove forests are ecologically important but globally threatened intertidal plant communities. Effective mangrove conservation requires the determination of species identity, management units, and genetic structure. Here, we investigate the genetic distinctiveness and genetic structure of an iconic but yet taxonomically confusing species complex Rhizophora mucronata and R. stylosa across their distributional range, by employing a suite of 20 informative nuclear SSR markers. RESULTS: Our results demonstrated the general genetic distinctiveness of R. mucronata and R. stylosa, and potential hybridization or introgression between them. We investigated the population genetics of each species without the putative hybrids, and found strong genetic structure between oceanic regions in both R. mucronata and R. stylosa. In R. mucronata, a strong divergence was detected between populations from the Indian Ocean region (Indian Ocean and Andaman Sea) and the Pacific Ocean region (Malacca Strait, South China Sea and Northwest Pacific Ocean). In R. stylosa, the genetic break was located more eastward, between populations from South and East China Sea and populations from the Southwest Pacific Ocean. The location of these genetic breaks coincided with the boundaries of oceanic currents, thus suggesting that oceanic circulation patterns might have acted as a cryptic barrier to gene flow. CONCLUSIONS: Our findings have important implications on the conservation of mangroves, especially relating to replanting efforts and the definition of evolutionary significant units in Rhizophora species. We outlined the genetic structure and identified geographical areas that require further investigations for both R. mucronata and R. stylosa. These results serve as the foundation for the conservation genetics of R. mucronata and R. stylosa and highlighted the need to recognize the genetic distinctiveness of closely-related species, determine their respective genetic structure, and avoid artificially promoting hybridization in mangrove restoration programmes.

Salt-dependent increase in triterpenoids is reversible upon transfer to fresh water in mangrove plants Kandelia candel and Bruguiera gymnorrhiza.

This study examined the salinity dependence of triterpenoid content and triterpenoid synthase gene expression in mangrove plants, Kandelia candel and Bruguiera gymnorrhiza (Rhizophoraceae) after long-term exposure to salinity and subsequent re-adaptation. Seedlings of the two mangrove species grown in varying salt concentrations for 4 months were divided into two treatment groups and grown for another 4 months, one group continued under the respective saline condition and the other in fresh water for re-adaptation. The total content of triterpenoids increased with increasing salinity in roots and leaves of K. candel, but only in roots in B. gymnorrhiza. This increase was reversed to a variable extent, depending on the species and organ, after transfer to fresh water. In contrast, the total content of phytosterols showed no correlation with salinity throughout the experiment. The increase in total triterpenoids was accompanied by an up-regulation of several triterpenoid synthase genes: KcMS, a multifunctional triterpenoid synthase, in roots and leaves of K. candel and BgLUS, a lupeol synthase, and BgbAS, a ß-amyrin synthase, in roots of B. gymnorrhiza. The expression of root KcCAS, a cycloartenol synthase, which is involved in phytosterol biosynthesis, was not modulated by the salinity conditions but decreased with increasing salinity in leaves, followed by the restoration to the initial level after transfer to fresh water. The concentrations of individual triterpenoids, but not of phytosterols, in the roots positively correlated with the salinity. These results reinforced the importance of triterpenoids in the adaptation of mangroves to withstand salt and/or water stress.

Expression of terpenoid synthase mRNA and terpenoid content in salt stressed mangrove.

The halophytic Kandelia candel and Bruguiera gymnorrhiza are ideal model for studying the molecular mechanisms of salinity tolerance in mangrove plants. The correlation between mRNA expression of four oxidosqualene cyclase (OSC) genes namely, KcMS multifunctional terpenoid synthase and KcCAS cyloartenol synthase (K. candel), BgbAS beta-amyrin synthase and BgLUS lupeol synthase (B. gymnorrhiza) and salt concentration was examined. mRNA level of KcMS was increased with salt concentration in both roots and leaves of K. candel. Similarly, salt stress increased the mRNA levels of BgLUS and BgbAS in the root of B. gymnorrhiza. This result suggests that the function of terpenoids in root is associated with the salt stress. In contrast to these observations, the mRNA level of KcCAS was not modulated by salt stress in the roots, and decreased in the leaves. These results therefore suggest that the terpenoids but not phytosterols play an important role to cope with the salt stress in mangrove root. The content and proportion of beta-amyrin and lupeol increased with salinity in the root of K. candel and B. gymnorrhiza, providing additional evidence for the protective role of terpenoids. However, beta-amyrin and lupeol in B. gymnorrhiza leaves decreased with salt concentration, suggesting that the physiological significance for the terpenoids in the leaf may differ from that for the root.

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae.

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.

Cloning and functional expression of cycloartenol synthases from mangrove species Rhizophora stylosa Griff. and Kandelia candel (L.) Druce.

To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC-MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce.

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.

Isoprene emission from tropical trees in Okinawa Island, Japan.

This study surveyed isoprene emission from 42 indigenous and exotic tropical trees in subtropic Okinawa, Japan. Of the 42 trees studied, 4 emitted isoprene at a rate in excess of 20 microg g(-1)h(-1), and 28 showed the rates of 1-10 microg g(-1)h(-1). The remainder emitted less than 1 microg g(-1)h(-1). The majority of trees in this study may therefore fall within the lower emitting species. However, species in Moraceae that is indigenous in Okinawa emitted isoprene at relatively higher rates with an average of 14.2 microg g(-1)h(-1). The highest emission rate of 107.1 microg g(-1)h(-1) for Ficus virgata yielded the area basis rate of 47.4 nmol m(-2)s(-1), which is almost equivalent to the rate of high emitting species. Furthermore, a linear relationship between light intensity and isoprene emission was noted with Ficus virgata up to 1700 micromol m(-2)s(-1). These findings may show the potential importance of subtropical areas as sources of isoprene to the atmosphere.

Effect of salt stress on the metabolism of ethanolamine and choline in leaves of the betaine-producing mangrove species Avicennia marina.

Glycinebetaine synthesis from [methyl-14C]choline and [1,2-14C]ethanolamine in leaf disks of Avicennia marina, was increased by salt stress (250 and 500 mM NaCl). After 18 h incubation with [methyl-14C]choline, phosphocholine and CO(2) were found to be heavily labelled. Phosphocholine contained 39% of the total radioactivity taken up by non-salinised (control) leaf disks and 15% of the total for salinised leaf disks stressed with 500 mM NaCl. Eighteen and 49% of the radioactivity absorbed by control and salinised disks, respectively, were released as CO(2). Metabolic studies of [1,2-14C]ethanolamine revealed that the radioactivity taken up by the leaf disks was recovered as the following compounds after 18 h: phosphorylated compounds (mainly phosphoethanolamine, phosphodimethylethanolamine and phosphocholine) (40-50%); choline (1-2%); glycinebetaine (3-5%); lipids (20-28%); CO(2) (6-10%). Unlike glycinebetaine, incorporation into phosphorylated compounds and lipids were reduced by salt stress. Incorporation of [methyl-14C]S-adenosyl-L-methionine (SAM) into choline, phosphocholine and glycinebetaine in leaf disks was stimulated by salt stress. In vitro activities of adenosine kinase and adenosine nucleosidase, which are implicated in stimulating the SAM regeneration cycle, increased after the leaf disks were incubated with 250 and 500 mM NaCl for 18 h. Changes in metabolism involving choline and glycinebetaine due to salt stress are discussed.

Lipid composition of mangrove and its relevance to salt tolerance.

Lipid compositions of mangrove trees were studied in relation to the salt-tolerance mechanism. Leaves and roots were obtained from seven mature mangrove trees on Iriomote Island, Okinawa: Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia candel, Lumnitzera racemosa, Avicennia marina, Pemphis acidula and Sonneratia alba. Lipids of mangrove leaves mainly consisted of 11 lipid classes: polar lipids, unknown (UK) 1-6, sterols, triacyl glycerols, wax ester and sterol ester (UK 3 and 4 were found to be tri-terpenoid alcohol in this study). Of these lipid classes, sterol ester was the main lipid in all species comprising 17.6-33.7% of total lipids. Analysis of the chemical structure found that the sterol esters mainly consisted of fatty acid esters of tri-terpenoid alcohols. One major tri-terpenoid alcohol was identified to be lupeol by interpretation of infrared resonance, nuclear magnetic resonance and mass spectrometry. Because of the unique anatomy of the mangrove root, lipid analyses were made separately for epidermis, cortex and innermost stele, respectively. The concentration of free tri-terpenoid alcohols showed a higher tendency in the outside part than in the inside portion of the roots, suggesting their protective roles. Relevance of lipid composition to salt tolerance was studied with propagules of K. candel and B. gymnorrhiza planted with varied salt concentrations. The proportions of free tri-terpenoids increased with salinity in both leaves and roots of K. candel, and only in roots of B. gymnorrhiza. No salt-dependent changes were noted in the phospholipid and fatty acid compositions in both species. These findings suggested that salt stress specifically modulated the terpenoid concentrations in mangroves.