Effects of Pieris japonica (Ericaceae) dominance on cool temperate forest altered-understory environments and soil microbiomes in Southern Japan.

The number of plants unpalatable to deer increases with increasing deer numbers. In the Kyushu Mountain area of Southern Japan, Pieris japonica (Ericaceae), an unpalatable shrub, has become the monodominant vegetation under evergreen conifer and deciduous broad-leaved tree stands. The monodominance of unpalatable plants in the understory has potential advantages and drawbacks; however, the effects of Pieris dominance are not well understood. To assess the effects of P. japonica dominances on forest environments and ecosystems, we investigated understory environments and soil microbiomes in Pieris-dominant sites. Under the deciduous broad-leaved trees, Pieris dominance leads to considerable Pieris leaf litter and humus weights and low soil bulk density and canopy openness. In the soil fungal community and fungal functional groups, the relative abundance of symbiotrophic fungi, particularly ectomycorrhizal fungi in Pieris-dominant sites were lower than in other-vegetation understory sites and saprotrophic fungi vice versa. Because few seedlings and saplings were found under Pieris shrubs, Pieris dominance in the understory might exclude other plant species. The results of this study will contribute to the Pieris population and forest management following deer overgrazing.

Upregulation of MAP kinase HOG1 gene of white-rot fungus Phlebia sp. MG-60 inhibits the ethanol fermentation and mycelial growth.

Wood biomass conversion for fossil resource replacement could result in the sustainable production of chemicals, although lignin represents an obstacle to efficient polysaccharide use. White-rot fungus Phlebia sp. MG-60 reportedly selectively and aerobically degrades lignin in hardwood, then it begins cellulose saccharification from the delignified wood to produce ethanol. Environmental conditions might change white-rot fungi-driven biomass conversion. However, how the environmental response sensor affects ethanol fermentation in white-rot fungi remains elusive. In this study, we focused on MGHOG1, the yeast Hog1 homolog in Phlebia sp. MG-60, a presumably important player in osmoresponse. We generated MGHOG1 overexpressing (OE) transformants in Phlebia sp. MG-60, exhibiting slower mycelial growth compared with the wild-type under salinity stress. MGHOG1 overexpressing liquid cultures displayed suppressed mycelial growth and ethanol fermentation. Therefore, MGHOG1 potentially influences ethanol fermentation and mycelial growth in Phlebia sp. MG-60. This study provides novel insights into the regulation of white-rot fungi-mediated biomass conversion.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume-rhizobia symbiosis.

Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants.

Gene coexpression network for trans-1,4-polyisoprene biosynthesis involving mevalonate and methylerythritol phosphate pathways in Eucommia ulmoides Oliver.

Eucommia ulmoides, a deciduous dioecious plant species, accumulates trans-1,4-polyisoprene (TPI) in its tissues such as pericarp and leaf. Probable TPI synthase (trans-isoprenyl diphosphate synthase (TIDS)) genes were identified by expressed sequence tags of this species; however, the metabolic pathway of TPI biosynthesis, including the role of TIDSs, is unknown. To understand the mechanism of TPI biosynthesis at the transcriptional level, comprehensive gene expression data from various organs were generated and TPI biosynthesis related genes were extracted by principal component analysis (PCA). The metabolic pathway was assessed by comparing the coexpression network of TPI genes with the isoprenoid gene coexpression network of model plants. By PCA, we dissected 27 genes assumed to be involved in polyisoprene biosynthesis, including TIDS genes, genes encoding enzymes of the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and genes related to rubber synthesis. The coexpression network revealed that 22 of the 27 TPI biosynthesis genes are coordinately expressed. The network was clustered into two modules, and this was also observed in model plants. The first module was mainly comprised of MEP pathway genes and TIDS1 gene, and the second module, of MVA pathway genes and TIDS5 gene. These results indicate that TPI is likely biosynthesized by both the MEP and MVA pathways and that TIDS gene expression is differentially controlled by these pathways.