Hirokazu Matsui

Secretion of acid phosphatase by the roots of crop plants under phosphorus-deficient conditions and some properties of the enzyme secreted by lupin roots

Nine crop species were grown in P-sufficient and P-deficient nutrient solutions. The activity of acid phosphatase secreted by the roots increased under P-deficient conditions in all the species examined. That of lupin increased most remarkably. The properties of the enzyme secreted by the roots of lupin was investigated. Many isozymes existed in the roots and the leaves, but only one of them was secreted into the rhizosphere in a large amount. The molecular weight of the purified enzyme secreted was estimated to be 72 KD by SDS-PAGE and 140 KD by gel filtration; it was assumed to be a homo-dimer. The iso-electric point of the enzyme was 4.7 and the pH for optimum activity 4.3. When the enzyme was mixed with aqueous solution extracted from a P-deficient soil, its activity declined to 55% of its original activity after 14 days and to 9% after 21 days.

Diverse expression profiles of 21 rice peroxidase genes

Secretory class III plant peroxidases (POXs) catalyze the oxidation of various reductants, and are encoded by a large multigene family. In rice, 42 independent expressed sequence tags for POXs have been identified. By RNA gel blot analysis using specific probes, we show here that 21 rice POX genes are unique in their developmental, organ specific and external stimuli‐responsive expression. This would suggest that encoded POX isoenzymes are involved in a broad range of physiological processes in rice plants, individually.

Purification and properties of acid phosphatase secreted from lupin roots under phosphorus-deficiency conditions

Abstract When lupin plants were grown in phosphorus-deficient nutrient solutions, the activity of acid phosphatase secreted from their roots remarkably increased in comparison with that under adequate phosphorus conditions. Based on activity staining of acid phosphatase on IEF gels, many isozymes were present in roots and leaves, but one of these was mainly secreted into the rhizosphere. This main acid phosphatase secreted from roots was purified from the phosphorus-deficient nutrient solution where lupin was grown, using ion exchange chromatography with DEAE-Sepharose CL-6B, gel filtration with Bio-Gel P-200, and preparative-PAGE. The molecular weight of the purified enzyme was estimated to be 72,000 by SDS-PAGE and approximately 140,000 by gel filtration. Thus, this enzyme was considered to be a homo-dimer. The isoelectric point and the optimum pH of the enzyme were 4.7 and 4.3, respectively.

An HR-Induced Tobacco Peroxidase Gene Is Responsive to Spermine, but Not to Salicylate, Methyl Jasmonate, and Ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Arabidopsis CYP94B3 encodes jasmonyl-l-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate

The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants.

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.

Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes.

Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose‐6‐phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose‐6‐phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose‐6‐phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose‐6‐phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6‐phosphate and about 10 times lower Km values for trehalose 6‐phosphate as compared with trehalose‐6‐phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 °C for 4 min. These characteristics of rice trehalose‐6‐phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant‐specific function of trehalose biosynthesis in response to environmental stresses.

Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants.

A peroxidase gene, poxA, was isolated from a rice (Oryza sativa L.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots, Plant Cell Rep. 13 (1994) 361-366]. The temporal and spatial expression properties of the poxA gene promoter as well as that from a second related peroxidase gene, poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive cis-elements were located within 144 bp from the translational start codon of the poxA gene. The poxN promoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath.

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 of a Gene Cluster Responsible for the Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α

The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase α. Terpenoids are an important class of natural products. However, identification of the biosynthetic gene cluster in terpenoids is relatively rare compared with another important class of natural products, polyketides. To explore a reliable identification method for the biosynthetic gene cluster in fungal diterpenoids, cloning of the biosynthetic gene cluster of aphidicolin was employed. The application of a simple PCR method for genome walking based on the sequence of cDNA encoding aphidicolan-16β-ol synthase (ACS) allowed us to analyze a 15.6-kb region of the Phoma betae genomic DNA. Six ORFs, PbGGS, ACS, PbP450-1, PbP450-2, PbTP, and PbTF were found in this region, and respectively expected to encode geranylgeranyl diphosphate synthase, diterpene synthase, two cytochrome P-450s, the transporter and transcription factor. Their amino acid sequences and introns were deduced by a corresponding cDNA analysis. This study shows that simple PCR-based genome walking without constructing a genomic DNA library is useful for identification of a small gene cluster. We propose a general strategy for the cloning the biosynthetic genes of fungal diterpenoids by using fungal GGS.