Jun Wasaki

Secreted acid phosphatase is expressed in cluster roots of lupin in response to phosphorus deficiency

The roots of white lupin (Lupinus albus L. cv. Kievskij mutant) secrete acid phosphatase, S-APase, when they grow under conditions of low available phosphorus (P). S-APases hydrolyze organic phosphate compounds in the rhizosphere and supply inorganic phosphate to the plants. Low phosphorus availability also induces vigorous growth of cluster roots. In this study, the function of cluster roots was investigated with reference to S-APase secretion. White lupins were grown in hydroponic culture in a greenhouse under P-deficient and P-sufficient conditions. S-APase in the excised roots after treatment was detected by staining with 4-methylumbelliferone phosphate (MUP). Gene expression of S-APase in cluster and normal roots was also investigated. Activity was greatest in the roots of plants grown under conditions of P -deficiency, particularly in cluster roots. S-APase gene expression was induced by a decrease in internal P concentrations, and was especially high in cluster roots formed under conditions of P -deficiency. It was suggested that decrease of internal P concentration stimulated both of the S-APase expression and cluster root formation.

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase.

The consecutive genes BF0771-BF0774 in the genome of Bacteroides fragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-β-d-mannopyranosyl-d-glucose+Pi→mannose-1-phosphate+glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-β-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.

Prebiotic Properties of Epilactose

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructooligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties.

Cloning and characterization of four phosphate transporter cDNAs in tobacco

Abstract Four distinct cDNAs (NtPT1, NtPT2, NtPT3, and NtPT4) encoding phosphate transporters were isolated from tobacco ( Nicotiana tabacum L.). They show high degrees of similarity to known high-affinity phosphate transporters in higher plants. Northern blot analysis using probes specific to NtPT1 and NtPT2 and to NtPT3 and NtPT4 indicated that large quantities of these four transcripts accumulated under phosphate-deficient conditions. The transcripts of NtPT1 / 2 were detected in immature leaves, mature leaves, old leaves, stems, and roots, while those of NtPT3 / 4 were detected only in old leaves and roots. When tobacco was grown under phosphate-deficient conditions, the transcripts of NtPT1 / 2 and NtPT3 / 4 were systemically enhanced after reduction of the total phosphate concentration in each organ. On the other hand, when phosphate was supplied to phosphate-deficient plants, the transcripts of NtPT1 / 2 and NtPT3 / 4 decreased systemically before an increase in total phosphate concentration. These results suggest that high-affinity phosphate transporter genes are controlled at the level of transcription by two regulatory systems: one responds to the internal phosphate status and the other to the external phosphate status.

Overexpression of the LASAP2 gene for secretory acid phosphatase in white lupin improves the phosphorus uptake and growth of tobacco plants

Abstract Secretion of acid phosphatase (APase) from the roots to take up phosphorus (P) is a well-known strategy of plants under P-deficient conditions. White lupin, which shows vigorous growth in low-P soils, is noted for its ability to secrete APase under P-deficient conditions. The APase secreted by white lupin roots is stable in soil solution and shows low substrate specificity, suggesting that genetic modification of plants using the APase gene LASAP2 might improve their ability to use organic P. The objective of the present study was to evaluate the potential of LASAP2 transgenic plants to increase organic P utilization. Dry matter production and P accumulation were higher in LASAP2 transgenic tobacco plants grown in gel media containing soluble phytate as the sole P source than in wild-type tobacco plants. Phosphorus uptake by the transgenic plants also increased in soil culture conditions. LASAP2 was apparently more effective in the liberation of organic P, including phytate, in the soil than the native tobacco APase. Thus, the enzymatic stability of LASAP2 in the soil appears to be an important factor for P acquisition.

Recent Progress in Plant Nutrition Research : Cross-Talk Between Nutrients, Plant Physiology and Soil Microorganisms

Mineral nutrients taken up from the soil become incorporated into a variety of important compounds with structural and physiological roles in plants. We summarize how plant nutrients are linked to many metabolic pathways, plant hormones and other biological processes. We also focus on nutrient uptake, describing plant-microbe interactions, plant exudates, root architecture, transporters and their applications. Plants need to survive in soils with mineral concentrations that vary widely. Describing the relationships between nutrients and biological processes will enable us to understand the molecular basis for signaling, physiological damage and responses to mineral stresses.

Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).

Structure of a cDNA for an acid phosphatase from phosphate-deficient lupin (Lupinus albus L.) Roots

Abstract We have isolated a cDNA clone, named LASAP1, for an acid phosphatase (EC 3.1.3.2; APase) from a cDNA library constructed from mRNA in phosphatedeficient lupin (Lupinus albus L. cv. Kievskij) roots. LASAP1 was 2,187 bp in length including a single open reading frame of 1,914 nucleotides that encodes 638 amino acid residues containing a putative signal sequence of 31 amino acids. The polypeptide encoded by LASAP1 was highly hydrophilic, while the putative signal peptide was highly hydrophobic. It was predicted that the peptide had a high affinity to the plasma membrane. The deduced amino acid sequence shared a high homology with that of purple APases from germinated seeds of Phaseolus vulgaris and those secreted from Arabidopsis thaliana. Seven amino acids composing the active center of the Phaseolus vulgaris purple APase were conserved in all the homologous APases. Northern blot analysis revealed that the corresponding mRNA accumulated in both shoots and roots under phosphate deficient conditions,...

Molecular Cloning and Root Specific Expression of Secretory Acid Phosphatase from Phosphate Deficient Lupin (Lupinus albus L.)

Abstract During the growth of lupin (Lupinus albus L.) plants, a shortage of phosphate induces the secretion of acid phosphatase (APase) from their roots. We have already isolated and characterized the lasap1 cDNA clone for an APase from phosphate-deficient roots. In this paper, we report on an another cDNA clone, designated as lasap2, encoding secretory APase (S-APase) from phosphate-deficient lupin roots. The lasap2 cDNA consisted of 1,541 bp and had an open reading frame composed of 462 amino acid residues with a signal peptide of 34 residues. Several amino acid sequences of purified lupin S-APase were completely contained in the deduced amino acid sequence (LASAP2). The mature LASAP2 was highly hydrophilic, while the signal peptide was highly hydrophobic. The PSORT prediction program indicated that the mature LASAP2 might be secreted from cells. The primary sequence of LASAP2 shared about 70% homology with that of purple APase from germinating seeds of kidney bean as well as LASAPl. RT-PCR and Western blot analyses revealed that the accumulation of mRNAs and the corresponding proteins for S-APase encoded by lasap2 was induced only in roots under phosphate-deficient conditions, while it was assumed that the LASAP1 hydrolyzed phosphate ester compounds which reached the root surfaces or were released from old organs to the apoplastic space.