L-Canavanine, a Root Exudate From Hairy Vetch (Vicia villosa) Drastically Affecting the Soil Microbial Community and Metabolite Pathways.

L-Canavanine, a conditionally essential non-proteinogenic amino acid analog to L-arginine, plays important roles in cell division, wound healing, immune function, the release of hormones, and a precursor for the synthesis of nitric oxide (NO). In this report, we found that the L-canavanine is released into the soil from the roots of hairy vetch (Vicia villosa) and declines several weeks after growth, while it was absent in bulk proxy. Hairy vetch root was able to exudate L-canavanine in both pots and in vitro conditions in an agar-based medium. The content of the L-canavanine in pots and agar conditions was higher than the field condition. It was also observed that the addition of L-canavanine significantly altered the microbial community composition and diversity in soil. Firmicutes and Actinobacteria became more abundant in the soil after the application of L-canavanine. In contrast, Proteobacteria and Acidobacteria populations were decreased by higher L-canavanine concentration (500 nmol/g soil). Prediction of the soil metabolic pathways using PICRUSt2 estimated that the L-arginine degradation pathway was enriched 1.3-fold when L-canavanine was added to the soil. Results indicated that carbon metabolism-related pathways were altered and the degradation of nitrogen-rich compounds (i.e., amino acids) enriched. The findings of this research showed that secretion of the allelochemical L-canavanine from the root of hairy vetch may alter the soil microbial community and soil metabolite pathways to increase the survival chance of hairy vetch seedlings. This is the first report that L-canavanine acts as an allelochemical that affects the biodiversity of soil microbial community.

Ultrafine bubble water mitigates plant growth in damaged soil.

Water containing ultrafine/nano bubbles (UFBs) promoted the growth of tomato (Solanum lycopersicum) in soil damaged by cultivation of tomato in the previous year or bacterial wilt-like disease and also promoted the growth of lettuce (Lactuca sativa) when lettuce was grown in the soil damaged by repeated cultivation of lettuce. On the other hand, UFB supply did not affect plant growth in rock wool or healthy soil. Furthermore, the growth of lettuce was not affected by UFB water treatment in the soil damaged by the cultivation of tomato. UFB water partly suppressed the growth of the pathogen of bacteria wilt disease, Ralstonia solanacearum in vitro. These data suggest that UFB water is effective to recover the plant growth from soil damage.

Field multi-omics analysis reveals a close association between bacterial communities and mineral properties in the soybean rhizosphere.

The plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere are different from the outer soil region (bulk soil). They establish characteristic conditions including microbiota, metabolites, and minerals, and they can directly affect plant growth and development. However, comprehensive insights into those characteristic environments, especially the rhizosphere, and molecular mechanisms of their formation are not well understood. In the present study, we investigated the spatiotemporal dynamics of the root-associated environment in actual field conditions by multi-omics analyses (mineral, microbiome, and transcriptome) of soybean plants. Mineral and microbiome analyses demonstrated a characteristic rhizosphere environment in which most of the minerals were highly accumulated and bacterial communities were distinct from those in the bulk soil. Mantel's test and co-abundance network analysis revealed that characteristic community structures and dominant bacterial taxa in the rhizosphere significantly interact with mineral contents in the rhizosphere, but not in the bulk soil. Our field multi-omics analysis suggests a rhizosphere-specific close association between the microbiota and mineral environment.

Structural characterization of highly branched glucan sheath from Ceriporiopsis subvermispora.

Wood rotting basidiomycetes produce extracellular mucilaginous sheaths interfacing fungal hyphae and plant biomass. While the versatility of these fungal sheaths has been addressed, sheaths generated by selective white-rot fungi remain poorly understood. To fill this gap, the sheath produced by the basidiomycete Ceriporiopsis subvermispora, which degrades lignin while inflicting limited cellulose damage, was analyzed in this study. Fluorescence and transmission electron microscopy revealed that the sheath formed three days after inoculation into a beech wood slice on an agar plate and was embedded at the interface between fungal hyphae and wood cell walls. The sheath's chemical structure was evaluated from fungus cultures in a liquid medium containing [U-13C6]-d-glucose and beech wood slices. Compositional analysis, methylation analysis, and 13C NMR demonstrated that the sheath mainly consisted of a comb-like ß-1,6-glucopyranose residue-branched ß-1,3-glucan, which is advantageous to retain water and extracellular secondary metabolites.

Functions of xyloglucan in plant cells.

While an increase in the number of xyloglucan tethers between the cellulose microfibrils in plant cell walls increases the walls' rigidity, the degradation of these tethers causes the walls to loosen. Degradation can occur either through the integration of xyloglucan oligosaccharides due to the action of xyloglucan endotransglucosylase or through direct hydrolysis due to the action of xyloglucanase. This is why the addition of xyloglucan and its fragment oligosaccharides causes plant tissue tension to increase and decrease so dramatically. Experiments involving the overexpression of xyloglucanase and cellulase have revealed the roles of xyloglucans in the walls. The degradation of wall xyloglucan in poplar by the transgenic expression of xyloglucanase, for example, not only accelerated stem elongation in the primary wall, but also blocked upright-stem gravitropism in the secondary wall. Overexpression of cellulase also reduced xyloglucan content in the walls as cellulose microfibrils were trimmed at their amorphous region, resulting in increased cell volume in Arabidopsis leaves and in sengon with disturbed leaf movements. The hemicellulose xyloglucan, in its function as a tether, plays a key role in the loosening and tightening of cellulose microfibrils: it enables the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.

Acceleration of cell growth by xyloglucan oligosaccharides in suspension-cultured tobacco cells.

The incorporation of xyloglucan oligosaccharide (XXXG) into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Xyloglucan for generating tensile stress to bend tree stem.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Activation of beta-glucan synthases by wall-bound purple acid phosphatase in tobacco cells.

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

Purple acid phosphatase in the walls of tobacco cells.

Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220kDa homotetramer composed of 60kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k(cat)/K(m)) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120kDa dimer in the cytoplasm and as a 220kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.

Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon.

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells.

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.