Search

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

Ectomycorrhizal (ECM) fungi improve the host plant's tolerance to abiotic and biotic stresses. Cenococcum geophilum (Cg) is among the most common ECM fungi worldwide and often grows in saline environments. However, the physiological and molecular mechanisms of salt tolerance in this fungus are largely unknown. In the present study, 12 isolates collected from different ecogeographic regions were used to investigate the mechanism of salt tolerance of Cg. The isolates were classified into four groups (salt-sensitive, moderately salt-tolerant, salt-tolerant, and halophilic) based on their in vitro mycelial growth under 0, 50, 125, 250, and 500 mM NaCl concentrations. Hence, the Na, Ca, P, and K concentrations of mycelia and the pH of the culture solution were determined. Compared with salt-tolerant isolates, treatment with 250 mM NaCl significantly increased the sodium concentration and decreased the potassium concentration of salt-sensitive isolates. RNA-sequencing and qRT-PCR analysis were conducted to identify differentially expressed genes (DEGs) involved in transmembrane transport and oxidoreductase activity pathways. The hydrogen peroxide concentration and activities of peroxidase and superoxide dismutase in mycelia were determined, and the accumulation and scavenging of reactive oxygen species in the salt-sensitive isolates were more active than those in the salt-tolerant isolates. The results supply functional validations to RNA-seq and qRT-PCR analysis. This study provides novel insights into the salt-stress response of Cg isolates and provides a foundation for elucidation of the salt-tolerance mechanism of ECM fungi.

Arbuscular mycorrhizal fungi (AMF) and earthworms have potential uses in the bioremediation of contaminated soils. In recent years, heavy metal-contaminated sites have been remediated by adding plants and AMF or earthworms to the soil. However, there are few studies on remediation using combinations of plants, animals, and microbes, especially for the remediation of Cu-contaminated soil. The present study investigated the separate and combined effects of AMF and earthworms on Cu-contaminated soil in which Tagetes patula L. was grown. The results show that the combined application of AMF and earthworms markedly increased the biomass of plant shoots and roots by more than 100%. It also increased Cu extraction by T. patula by 270%. The combined treatment was effective in increasing the CEC, contents of OM, and available Cu, P and K, but reduced the soil pH. Furthermore, the combined treatment significantly increased the abundance and diversity of the soil microbial community. In particular, the abundances of the bacteria Bacteroides, Proteobacteria, and Actinobacteria were increased, with the genera Flavobacterium, Pedobacter, Algoriphagus, Gaetbulibacter, Pseudomonas, Luteimonas, and Arthrobacter dominating. Meanwhile, the abundance of the fungus Zygomycota was increased, with Mortierella dominating. Moreover, inoculation with earthworms greatly improved the structure of the soil microbial community.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H2O2). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆, respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H2O2. Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.

Sedum alfredii and Sedum plumbizincicola typically have high heavy metal (such as Zn and Cd) accumulation capacities with fast growth rates and relatively high Pb tolerance in contaminated soils. We compared the accumulation characteristics of heavy metals in Sedum species through meta-analysis. Furthermore, we analyzed the effects of soil organic matter (SOM) and soil pH on Cd, Pb and Zn accumulation by S. alfredii and S. plumbizincicola and the correlation between various metals. Results showed that the accumulations of Cd and Zn in shoots were higher than that of roots, but Pb accumulated in roots more than shoots. Moreover, there is a significant positive correlation between the accumulation of Zn and Cd in shoots. We found that the heavy metal accumulation rate in shoots was higher with lower soil pH. Sedum species had the highest Cd adsorption capacity in 20–30 g/kg SOM and the highest Zn adsorption capacity in SOM less than 20 g/kg. The accumulation rate of Cd in shoots of S. plumbizincicola was increased with exposure time, while the accumulation rate of Zn was slightly decreased. S. alfredii and S. plumbizincicola are two common Cd and Zn hyperaccumulators. We systematically compared the accumulation characteristics of heavy metals in Sedum plants and the effects of soil organic matter and pH on the accumulation of Cd, Pb and Zn in S. alfredii and S. plumbizincicola through meta-analysis. This provides certain theoretical knowledge for the application of sedum plants to the phytoremediation of heavy metal contaminated soils.

Large-scale genetic population study of the two dominant tropical species of seagrass is conducted in Mindanao, southern Philippines. The goal of the study was to understand population genetic status of the dominant tropical seagrass species, Cymodocea rotundata and Enhalus acoroides for appropriate management. Population genetics structure for the 15 sites was performed by using polymorphic microsatellite markers. The results showed that the clonal richness was high in E. acoroides than C. rotundata. The largest genet found in the study was at Rizal, northern Mindanao wherein throughout the sampling area (i.e. 300 x 40 m) only one genet was identified for C. rotundata. The mean FIS (coefficient of local inbreeding) values was positive (heterozygous deficit) with some sites deviated from Hardy-Weinberg Equilibrium. Isolation by distance (IBD) was detected in C. rotundata (P E. acoroides (P = 0.253), with the Mindanao ocean currents influenced genetic connectivity and structure. Genetic differentiation did not show any relationship between the geographical location and distance exhibiting high FST values for E. acoroides (0.183) and C. rotundata (0.205). The floating, buoyant fruits of E. acoroides may play a role in their long-distance dispersal; however, such dispersal is not frequent. Almost all of the seeds and fruits of C. rotundata are derived from self-recruitment in the natal meadow. This study suggests that C. rotundata and E. acoroides populations possess a weak genetic connectivity, and that the persistence of the meadow is threatened due to the low genetic diversity and high degree of population isolation.

Polyploids recurrently emerge in angiosperms, but most polyploids are likely to go extinct before establishment due to minority cytotype exclusion, which may be specifically a constraint for dioecious plants to evolve polyploid populations. Investigations into the frequency and distribution of polyploids in natural populations is thus necessary for understanding polyploid evolution in plants. This study determined the ploidy levels of 28 populations and 351 individuals of Salix polyclona, and identified the type of polyploidy (auto- vs. allo-) using whole genome re-sequencing data. We further investigated the phylogeny, population genetic diversity and species range shifts to explore the origin and spatiotemporal evolution of the polyploid complex. Our analyses revealed a high frequency (52%) of autopolyploids in it with a clear geographic distribution confined to the western part of its range where complex mountain systems create higher levels of environmental heterogeneity. Comparisons of diploid male and female genomes suggested a female heterogametic sex-determining factor on chromosome 15, which likely also acts in the dioecious polyploids. Fossil-calibrated phylogeny showed a more recent diversification of the polyploids (ca. 2.3 Ma) than the diploid (ca. 6.2 Ma), and population demographic histories largely corroborated the geological and climatic history of the region. Our results suggest that climatic oscillations and uplift of eastern Qinghai-Tibetan Plateau and the connecting mountains may have facilitated the preservation and establishment of polyploid populations. This study provides an example of the evolution of a diploid-polyploid complex in a willow species and illustrates a role of polyploidization in mountain biodiversity.

Many marine species have a pelagic larval phase that undergo dispersal among habitats. Studies on marine larval dispersal have revealed a large variation in the spatial scale of dispersal, and accumulated evidence has shown that seascape patchiness is the major determinant for variation in self-recruitment. However, few studies have investigated the influence of geographic settings on marine larval dispersal. Bays or lagoons generally enhance the retention of larvae, while larvae are more likely to be flushed by strong currents in open coasts. To examine associations between larval dispersal, geographic setting, and hydrodynamics, we compared fin-scale dispersal patterns, self-recruitment, and local retention of two anemonefishes (Amphiprion frenatus and A. perideraion) between a semi-enclosed bay and an open coast in the Philippines combining genetic parentage analysis and biophysical dispersal modelling. Contrary to our expectations, parentage analysis revealed lower estimates of self-recruitment in the semi-closed bay (0–2%) than in the open coast (14–15%). The result was consistent with dispersal simulations predicting lower local retention and self-recruitment in the former (0.4% and 19%) than in the latter (2.9% and 38%). Dispersal modelling also showed that cross-shore currents toward offshore were much stronger around the semi-closed bay and were negatively correlated with local retention and self-recruitment. These results suggest that stronger cross-shore currents around the semi-closed bay transport anemonefish larvae to the offshore and mainly contributed to the lower self-recruitment. Our results highlight difficulty in predicting self-recruitment from geographic setting alone and importance of hydrodynamics on it.

Arbuscular mycorrhizal fungi (AMF) and earthworms have potential uses in the bioremediation of contaminated soils. In recent years, heavy metal-contaminated sites have been remediated by adding plants and AMF or earthworms to the soil. However, there are few studies on remediation using combinations of plants, animals, and microbes, especially for the remediation of Cu-contaminated soil. The present study investigated the separate and combined effects of AMF and earthworms on Cu-contaminated soil in whichCompared with phytoremediation or microbial remediation alone, combined remediation was considered to be a more efficient remediation strategy. This study describes the effects of the combined remediation of

Forest trees are colonised by different species of ectomycorrhizal (ECM) fungi that interact competitively or mutualistically with one another. Most ECM fungi can produce sporocarps. To date, the effects of co-colonising fungal species on sporocarp formation in ECM fungi remain unknown. In this study, we examined host plant growth, mycorrhizal colonisation, and sporocarp formation when roots of Pinus densiflora are colonised by Laccaria japonica and three other ECM fungal species (Cenococcum geophilum, Pisolithus sp., and Suillus luteus). Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45days, 62days, and 1year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may negatively impact mycorrhizal colonisation and sporocarp formation in L. japonica. Sporocarp formation in L. japonica was positively correlated with conspecific mycorrhizal colonisation but negatively correlated with the biomass of seedlings of P. densiflora. The co-occurring ECM fungi largely competed with L. japonica, resulting in various effects on mycorrhizal colonisation and sporocarp formation in L. japonica. A variety of mechanisms may be involved in the competitive interactions among the different ECM fungal species, including abilities to more rapidly colonise root tips, acquire soil nutrients, or produce antibiotics. These mechanisms need to be confirmed in further studies.

Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B-box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue-specific expression patterns and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting the accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9-generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin- and anthocyanin-specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved.

Pine wilt disease (PWD), Bursaphelenchus xylophilus, is an extremely threatening invasion forest disease throughout the world, especially in Asia. B. xylophilus is spread in Asia by vector beetles of Monochamus alternatus, which has long no effective control method. Understanding of landscape effects on the dispersal and outbreaks of forest pests is crucial to establishing effective ecological control strategies. Here, we analyzed the samples of M. alternatus collected at landscapes in order to estimate the effects of landscape types on the genetic structure and dispersal of M. alternatus. The landscapes included the geographical scales, forest types and land uses. The individuals of M. alternatus were genotyped by using whole-genome resequencing. Population genetic structures were clearly differentiated at the intermediate scale, suggesting the intermediate scale is an effective barrier against natural dispersal of M. alternatus. We used the least-coat distances, least-cost transect analysis, and distance-based redundancy analysis to estimate the effects of forest types and land uses within the fine scales. The results showed that the gene flow and genetic diversity were positively correlated with host and mixed forests, whereas negatively with non-host forests. Among land-use landscapes, the roads had the positive effect on gene flow and genetic diversity but farmland and urban uses had negative effects. This highlights that human-mediated transport via roads was likely to be the main factor leading to the long-distance invasion of M. alternatus, whereas non-host landscapes could suppress the spread of this species. These findings may be useful to control the PWD dispersed by M. alternatus.

Currently, the research and application of biochar in the remediation of heavy metal contaminated soil has become a hotspot, especially regarding the remediation of agricultural land. Biochar has been proved to be effective in reducing the content of available heavy metals in the soil as well as the heavy metals in plants. However, the long-term effectiveness of biochar immobilization has not been widely studied. In this review, retrospective search was carried out on the published literature results concerning remediation effects of biochar on different areas of heavy metal contaminated soil in the recent years, its application in field remediation (several years), and some potential abiotic and biotic factors that may weaken the immobilization effects of biochar. This results indicate that: (1) biochar is widely used in the remediation of heavy metal contaminated soil in different areas and has excellent immobilization effect. (2) Most of the research demonstrate that the immobilization effect of biochar is effective for 2-3 years or according to few results even for 5 years. However, there have been various reports claiming that the immobilization effect of biochar decreases with time. (3) Abiotic factors such as acid rain, flooded environment, changes in soil condition (pH, redox and dissolved organic matter) and changes in biochar (Cl- and alkali leaching) can significantly weaken the immobilization effect of biochar. (4) Biotic factors such as plant roots, earthworms and soil microorganisms can also significantly reduce the immobilization effect of biochar. Therefore, field experiments having longer time span with biochar need to be further carried out, and the developmental research of modified biochar with a more stable immobilization effect also needs further attention.

Tricholoma matsutake is known to be the dominant fungal species in matsutake fruitbody neighboring (shiro) soil. To understand the mechanisms behind matsutake dominance, we studied the bacterial communities in matsutake dominant shiro soil and non-shiro soil, isolated the strains of Streptomyces from matsutake mycorrhizal root tips both from shiro soil and from the Pinus densiflora seedlings cultivated in shiro soil. Further, we investigated three Streptomyces spp. for their ability to inhibit fungal growth and Pinus densiflora seedling root elongation as well as two strains for their antifungal and antioxidative properties.Our results showed that Actinobacteria was the most abundant phylum in shiro soil. However, the differences in the Actinobacterial community composition (phylum or order level) between shiro and non-shiro soils were not significant, as indicated by PERMANOVA analyses. A genus belonging to Actinobacteria, Streptomyces, was present on the matsutake mycorrhizas, although in minority. The two antifungal assays revealed that the broths of three Streptomyces spp. had either inhibitory, neutral or promoting effects on the growth of different forest soil fungi as well as on the root elongation of the seedlings. The extracts of two strains, including one isolated from the P. densiflora seedlings, inhibited the growth of either pathogenic or ectomycorrhizal fungi. The effect depended on the medium used to cultivate the strains, but not the solvent used for the extraction. Two Streptomyces spp. showed antioxidant activity in one out of three assays used, in a ferric reducing antioxidant power assay. The observed properties seem to have several functions in matsutake shiro soil and they may contribute to the protection of the shiro area for T. matsutake dominance.

The pine wilt disease is caused by the pinewood nematode Bursaphelenchus xylophilus and it results in serious ecological and economic losses. Therefore, effective prevention and control methods for the pinewood nematode are urgently required. Bacillus thuringiensis (Bt), a widely used microbial insecticide, produces toxins that are toxic to several species of parasitic nematodes, however, its effects on B. xylophilus have not been determined. In this study, Cry5Ba3, App6Aa2, Cry12Aa1, Cry13Aa1, Cry14Aa1, Cry21Aa3, Cry21Fa1, Xpp55Aa1, and Cyt8Aa1 toxins' nematocidal activity against B. xylophilus was evaluated, six toxins with high toxicity were identified: App6Aa2 (LC

Wall-associated kinases (WAKs) are a family of receptor-like kinases (RLKs) with important roles against biotic and abiotic stresses in plants. Here, we reported that a rice WAK gene, OsWAK11, detoxifies excess copper by regulating the methylesterification of the cell wall. RNA interference- (RNAi-) mediated OsWAK11 knockdown in rice resulted in hypersensitivity to copper toxicity. This Cu hypersensitivity in OsWAK11-RNAi transgenic lines might result from more Cu uptake, which accumulated in the cytoplasm rather than the cell wall. Surprisingly, less Cu was accumulated in the cell wall pectin and hemicellulose of OsWAK11-RNAi transgenic lines than in wild-type plants, possibly because of decreased pectin methylesterase (PME) activity. We detected dramatically reduced expression of a key PME encoding gene, OsPME14, in OsWAK11-RNAi plants. Furthermore, disruption of OsPME14 expression could mimic the effects of OsWAK11 downregulation in plants challenged by excess Cu. Given that OsWAK11 expression is induced by excess Cu, our results indicate that plant cells activate OsWAK11 activity to modify the properties of the cell wall, thereby retaining Cu on the wall when subjected to excess Cu.

Seagrasses are marine angiosperms and play an essential ecological role in coastal ecosystems; however, seagrass meadows are threatened locally by anthropogenic disturbances. Understanding the dispersal patterns of seagrasses is essential for appropriate ecosystem management and establishment of marine protected areas (MPAs) in coastal ecosystems. In the Guimaras Strait in the Philippines, Banate (BAN) has been established as an MPA. However, there is a lack of information on the genetic diversity of seagrasses in BAN and the surrounding areas. In the present study, population genetics analysis of Enhalus acoroides was performed by using polymorphic microsatellite markers, for the estimation of genetic diversity, differentiation, and migration patterns of seagrasses within the regional geographical scale (~200 km) around the Guimaras Strait. The results showed that the genetic diversity of BAN is extremely low, although the Guimaras Strait is located in the tropical central habitat. Guimaras Island geographically divides the populations of E. acoroides into south and north. However, the genetic structure did not show any relationship between the geographical location and distance. The floating, buoyant fruits of E. acoroides may play a role in their long-distance dispersal; however, such dispersal is not frequent. Almost all of the seeds and fruits are derived from self-recruitment in the natal meadow. This study suggests that E. acoroides populations possess a weak genetic connectivity, and that the persistence of the meadow is threatened due to the low genetic diversity and high degree of population isolation in BAN. To maintain and enhance the genetic diversity of seagrasses within the MPA, the seagrass meadows in the surrounding areas should also be conserved.

Within the domain of phytoremediation research, the proper disposal of harvestable plant parts, that remove pollutants from contaminated soil, has been attracted extensive attention. Here, the bioenergy generation capability of trace metals (Cu, Pb, Zn, Cd, Mn, and As) polluted plants was assessed. The biogas production potential of accumulators or hyperaccumulator plants, Elsholtzia haichowensis, Sedum alfredii, Solanum nigrum, Phytolacca americana and Pteris vittata were 259.2 ± 1.9, 238.7 ± 4.2, 135.9 ± 0.9, 129.5 ± 2.9 and 106.8 ± 2.1 mL/g, respectively. The presence of Cu (at approximately 1000 mg/kg) increased the cumulative biogas production, the daily methane production and the methane yield of E. haichowensis. For S. alfredii, the presence of Zn (≥ 500 mg/kg) showed a significant negative impact on the methane content in biogas, and the daily methane production, which decreased the biogas and methane yield. The biogas production potential increased when the content of Mn was at 5 000–10,000 mg/kg, subsequently, decreased when the value of Mn at 20,000 mg/kg. However, Cd (1–200 mg/kg), Pb (125–2000 mg/kg) and As (1250–10,000 mg/kg) showed no distinctive change in the cumulative biogas production of S. nigrum, S. alfredii and P. vittata, respectively. The methane yield showed a strong positive correlation (R2 = 0.9704) with cumulative biogas production, and the energy potential of the plant residues were at 415–985 kW h/ton. Thus, the anaerobic digestion has bright potential for the disposal of trace metal contaminated plants, and has promising prospects for the use in energy production.

Tricholoma matsutake is an economically valuable fungus in Japan. Its fruit bodies occur in colonies established in natural pine forests. We traced colony establishment processes of T. matsutake in 2001 and 2010 by using microsatellite markers. T. matsutake genets persisted at least for a decade. Comparing colonies found in 2001 and 2010, five novel colonies were observed in 2010, where the organic layer was removed or pine trees were regenerated less than 50 y ago. The novel colonies were genotyped, and all of them were different from colonies found in 2001, indicating that these colonies were established by dispersed basidiospores. Novel genet and candidate parent genets were located in the same ridge, and the mean geographical distance between a novel genet and a parent genet was 19.59 ± 14.73 m, indicating that basidiospores may colonize more easily on roots of hosts near parental basidiocarps. In conclusion, sufficient basidiospore supply and organic layer removal or pine tree regeneration were essential factors for colony establishment of T. matsutake.

Juniperus sabina L. is an evergreen coniferous shrub, with a widespread distribution in Asia and Europe. It is one of the key species for restoration of the ecosystem in desertification areas. In the present study, we isolated and characterized eight microsatellite loci of this species. The number of alleles per locus ranged from two to 27, with observed heterozygosity values ranging from 0.256 to 0.744 and expected heterozygosity from 0.276 to 0.939. The markers developed in this study could be useful for population genetics studies of J. sabina.

Pinus parviflora Sieb. et Zucc. var. parviflora is a coniferous tree species distributed in mountainous areas of temperate zones from southern Tohoku to the Kyushu area in Japan. Some P. parviflora populations have become small and endangered due to massive death resulting from pine wilt disease, scab canker, and presumably climate change. We developed 11 microsatellite markers for P. parviflora by the dual-suppression technique and next-generation sequencing using 32 individuals of P. parviflora collected from Aokigahara at the foot of Mt. Fuji. The number of alleles for each locus ranged from two to 10. The averages of observed and expected heterozygosities were 0.61 and 0.59, respectively. These markers will become powerful tools for assessing genetic diversity, genetic connectivity, and genetic structure in P. parviflora populations, which will facilitate our understanding and conservation of P. parviflora.

MYB transcription factors have a wide range of functions in plant growth, hormone signaling, salt, and drought tolerances. In this study, two homologous transcription factors, PtrMYB55 and PtrMYB121, were isolated and their functions were elucidated. Tissue expression analysis revealed that PtrMYB55 and PtrMYB121 had a similar expression pattern, which had the highest expression in stems. Their expression continuously increased with the growth of poplar, and the expression of PtrMYB121 was significantly upregulated in the process. The full length of PtrMYB121 was 1395 bp, and encoded protein contained 464 amino acids including conserved R2 and R3 MYB domains. We overexpressed PtrMYB121 in Arabidopsis thaliana, and the transgenic lines had the wider xylem as compared with wild-type Arabidopsis. The contents of cellulose and lignin were obviously higher than those in wild-type materials, but there was no significant change in hemicellulose. Quantitative real-time PCR demonstrated that the key enzyme genes regulating the synthesis of lignin and cellulose were significantly upregulated in the transgenic lines. Furthermore, the effector-reporter experiment confirmed that PtrMYB121 bound directly to the promoters of genes relating to the synthesis of lignin and cellulose. These results suggest that PtrMYB121 may positively regulate the formation of secondary cell wall by promoting the synthesis of lignin and cellulose.

Amorphophallus variabilis Blume, a member of Araceae, is a fleshy perennial tuber crop endemic in Java Island, Indonesia. The plant produces white edible corm; and it was used as food during famine time before 1960s. Rapid ecological changes and land fragmentations in Java in recent times threaten populations of A. variabilis . Here, compound microsatellite markers were developed in order to develop conservation strategies in the populations. Twelve primers pairs produced high polymorphism ranging from 5 to 22 alleles per locus. The observed and expected heterozygosities ranged from 0.191 to 0.851 and 0.380 to 0.943, respectively. This high allelic diversity indicates that these markers are suitable for the study on population genetic structure . Cross-amplification on related and non-related species was performed. Application of the markers on populations from Dramaga Conservation Forest revealed high allelic richness, high diversity within and among populations. Genetic distance among populations increased with an increase of geographic distance. Present study suggested that, it is important to study population of A. variabilis in Java in order to understand the population genetic structure and develop effective in situ conservation programs.

To advance our understanding of the effects of inoculation with ectomycorrhizal fungi (EMF) on seedling colonization in mine wastelands, we conducted a field experiment in a copper tailing. Six-month-old seedlings of Japanese red pine (Pinus densiflora) and oak (Quercus variabilis) separately inoculated with three EMF species (Pisolithus sp., Cenococcum geophilum, Laccaria laccata) were transplanted to the copper tailing. The survival rates of tree seedlings were monitored monthly, and growth (biomass and height), contents of nutrients and heavy metals (K, P, Ca, Mg, Cu, Zn), and mycorrhizal infection rates of seedlings were determined 6 months after planting. Oak seedlings exhibited higher survival rates than pine seedlings after 6 months of growth on the tailing. EMF inoculations of pine seedlings significantly enhanced their survival, growth, and nutrient uptake. In contrast, EMF inoculations of oak seedlings improved growth only in terms of biomass. Additionally, EMF inoculation caused pine seedlings to accumulate more Cu and Zn in roots compared to non-inoculated seedlings, whereas inoculation inhibited the accumulation of heavy metals in shoots. However, similar results were not observed in oak seedlings. Observations of roots indicated that the rates of mycorrhizal infection of both tree species had dramatically declined at harvest time. In conclusion, ectomycorrhizal symbioses can improve the survival and performance of pine seedlings in mine tailings. The present study provided direct evidence of the importance of EMF inoculation of seedlings to the reforestation of mine wastelands.

Recently, seagrasses have been declining along coastlines worldwide. We genetically analyzed 1058 ramets of Syringodium isoetifolium collected from 21 populations in the Philippines and 9 in the Ryukyu Islands using nine microsatellite markers, to provide a baseline of genetic information on seagrasses in the western North Pacific region under the influence of the Kuroshio Current, and aid in their genetic conservation. We detected 421 genotypes and the average clonal richness (R) was relatively low (R = 0.38), suggesting that S. isoetifolium populations were formed mainly through asexual reproduction. The index of genetic differentiation among populations for S. isoetifolium was high ( $$D_{ST}^{\prime }$$ = 0.238 and $$G_{ST}^{\prime }$$ = 0.337), and most pairwise F ST values were significantly greater than zero, suggesting that gene flow among populations is rare. Clear genetic structure in S. isoetifolium populations were detected, indicating four genetically-similar bioregions, that is, the Ryukyu, northwest Philippines, northeast Philippines, and central Philippines populations. This genetic structure indicated that the Kuroshio strongly affects the formation of S. isoetifolium populations and should be factored into conservation efforts for S. isoetifolium.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in numerous genera and species, but their functions in abiotic stress tolerance remain unclear. Here, a MT gene from Oryza sativa, OsMT2c, was isolated and characterized, encoding a type 2 MT, and observed expression in the roots, leaf sheathes, and leaves, but only weak expression in seeds. OsMT2c was upregulated by copper (Cu) and hydrogen peroxide (H2O2) treatments. Excessive Cu elicited a rapid and sustained production and release of H2O2 in rice, and exogenous H2O2 scavengers N,N'-dimethylthiourea (DMTU) and ascorbic acid (Asc) decreased H2O2 production and OsMT2c expression. Furthermore, the expression of OsMT2c increased in the osapx2 mutant in which the H2O2 levels were higher than in wild-type (WT) plants. These results showed that Cu increased MT2c expression through the production and accumulation of Cu-induced H2O2 in O. sativa. In addition, the transgenic OsMT2c-overexpressing Arabidopsis displayed improved tolerance to Cu stress and exhibited increased reactive oxygen species (ROS) scavenging ability compared to WT and empty-vector (Ev) seedlings.

Phytolacca americana, a globally invasive species, is able to flourish in heavy metal-contaminated habitats. To improve understanding of the adaptive evolutionary mechanisms of plants under heavy metal stress, we investigated key factors contributing to variation in leaf manganese (Mn) content in P. americana. Genetic surveys and common garden experiments were conducted simultaneously in an analysis of P. americana populations growing on Mn-contaminated and uncontaminated soil. Our field survey detected a significant relationship between leaf Mn concentrations in P. americana and concentrations in the soils from which plants were collected. Microsatellite analyses identified low levels of genetic diversity within and between populations; 32 of 39 populations (82 %) were genetically monomorphic. No genetic differentiation was detected between populations from contaminated and uncontaminated soils. Our common garden experiments showed that Mn concentrations in P. americana were related only to the growth habitat, regardless of the origin of the seeds. Combining the results of our ecological and genetic analyses, we concluded that genetic variation is not likely to be responsible for the wide ecological distribution of P. americana in China. Rather, phenotypic plasticity is probably the major contributor to its successful colonisation of stressful habitats, such as heavy metal-contaminated soils.

Persistence of populations at their distributional ranges relies on local population dynamics and the fitness of species with low dispersal potential. We analyzed the population genetic diversity and structure of a tropical seagrass species, Cymodocea serrulata, at 34 sites spanning Philippine (diversity hotspot) and Ryukyu Islands (northern limit of distribution) populations using microsatellite (SSR) markers. Seagrass populations in the diversity hotspot are hypothesized to contain higher genetic diversity and clonal richness than those resulting from expansion or geographic range limits. We tested this hypothesis by comparing the genetic diversity, genetic structure and clonal richness of C. serrulata populations in the Philippines and Ryukyu Islands. C. serrulata populations showed decreased genetic diversity and clonal richness at their northern limit. Clonal reproduction predominated at the northern limit, while sexual reproduction prevailed in the diversity hotspot. Decreased genetic diversity and clonal richness at the northern limit may be the consequence of drift resulting from founder effect, reduced habitat, sea surface temperature and low gene flow and/or natural selection across life stages, wherein clonal reproduction confers greater environmental fitness. Analysis of molecular variance (AMOVA) and the fixation index, F ST, showed significant genetic differentiation within and among geographic populations. STRUCTURE analysis revealed that the Ryukyu Islands populations were mosaics of genets from the eastern Philippines, likely carried by the Kuroshio Current.