SummaryThe RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, and its ripening inhibitor (rin) mutation yields non-ripening fruit. In this study, the molecular properties of RIN and the rin mutant protein were clarified. The results revealed that the RIN protein accumulates in ripening fruit specifically and is localized in the nucleus of the cell. In vitro studies revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites. Analysis of binding site selection experiments revealed that the consensus binding sites of RIN highly resemble those of the SEPALLATA (SEP) proteins, which are Arabidopsis MADS box proteins that control the identity of floral organs. RIN exhibited a transcriptionactivating function similar to that exhibited by the SEP proteins. These results indicate that RIN exhibits similar molecular functions to SEP proteins although they play distinctly different biological roles. In vivo assays revealed that RIN binds to the cis-element of LeACS2. Our results also revealed that the rin mutant protein accumulates in the mutant fruit and exhibits a DNA-binding activity similar to that exhibited by the wild-type protein, but has lost its transcription-activating function, which in turn would inhibit ripening in mutant fruit.

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(L-leucyl-L-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 ‫؍(‬ NRRL2999) was 0.20 mg ml Aflatoxins are highly toxic, carcinogenic, and teratogenic secondary metabolites that are produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (reviewed in reference 20). Recently, several strains of Aspergillus nomius, Aspergillus pseudotamarii, Aspergillus bombycis, and Aspergillus ochraceoroseus have also been reported to produce aflatoxin. These fungi are ubiquitous and grow on a variety of agricultural products under appropriate temperature and moisture conditions. Aflatoxins have been detected in numerous agricultural commodities, such as cereal grains, whole wheat, rye breads, oil seeds, cottonseed, etc. (25). The toxicity and carcinogenicity of aflatoxins have made contaminated commodities a significant health hazard all over the world. In fact, the incidence of liver cancer is high in regions with high endemic aflatoxin concentrations. Furthermore, the annual costs resulting from crop losses due to aflatoxin contamination and the costs involved in monitoring and disposal of contaminated commodities affect the agricultural economy (34).Many studies have focused on developing aflatoxin control strategies, including genetic engineering for crop resistance, biological control with competitive, nonaflatoxigenic strains of the fungus A. flavus (6, 9), and regulation of aflatoxin biosynthesis by fungicides, pesticides, inhibitory substances originating from plants, and microbial substances (15,24,27). However, most of these strategies have been shown to be limited in effectiveness. Many microorganisms have been studied to control aflatoxin production. Sweedy and Dobson (32) have reported that bacteria, yeast, molds, actinomycetes, and algae can be used to lower aflatoxin levels in foods and feeds. Ono et al. have reported that aflastatin A, which has been isolated from mycelial extracts of Streptomyces sp., effectively inhibits aflatoxin production (17, 23). Saprophytic yeasts, such as Pichia anomala, Candida krusei, and others, have...

The ripening inhibitor (rin) mutation of tomato yields non‐ripening fruit, and the gene corresponding to RIN, LeMADS‐RIN, is known to encode a transcriptional factor that controls ripening‐related genes. In this study, to evaluate the heterozygosity effect of rin on fruit ripening, we developed eight F1 hybrid lines of the rin mutant from various crosses between the lines of the rin mutant and wild type. In the fruit of these F1 hybrid lines, the shelf‐life was improved, but both the shelf‐life and colouring varied between the lines. We then chose one line of the F1 hybrids and investigated the physiological and transcriptional properties of the fruit. Compared with the wild‐type parent, this F1 line showed about half the lycopene content, lower fruit softening and lower mRNA accumulation of the genes that encode phytoene synthase (Psy), polygalacturonase (PG), β‐galactosidase (TBG4) and expansin (LeEXP1). The characteristic climacteric rise in ethylene production typically observed in the wild‐type parent during fruit maturation was not observed in the fruit of this F1 line. The genes that encode ethylene biosynthetic enzymes, namely 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase (ACS2 and ACS4) and ACC oxidase (ACO1), were, however, significantly expressed in the F1 hybrid, suggesting that the post‐transcriptional regulator for activating these enzymes is affected by LeMADS‐RIN. These results suggest that the heterologous effect of LeMADS‐RIN in F1 hybrids affects the gene transcription and activation of ripening‐related factors, resulting in changes in fruit properties, including the extension of the shelf‐life.

The pathway oxoaverantin (OAVN) 3 averufin (AVR) 3 hydroxyversicolorone (HVN) 3 versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.Aflatoxins (AF) are a group of polyketide-derived secondary metabolites produced mainly by certain strains of the common molds Aspergillus flavus and Aspergillus parasiticus (16). Some other strains of Aspergillus nomius (13), Aspergillus pseudotamarii (8), Aspergillus bombycis (17), and Aspergillus ochraceoroseus (12) have also been reported to produce aflatoxins. These toxins are highly toxic and carcinogenic in animals and humans, leading to hepatotoxicity, teratogenicity, immunotoxicity, and even death (3). Among the naturally occurring aflatoxins, the four major ones are aflatoxin B 1 (AFB 1 ), AFB 2 , AFG 1 , and AFG 2 , of which AFB 1 is the most toxic and carcinogenic compound. Contamination of food and feed crops, such as wheat, corn, cotton, peanuts, and tree nuts, with AFB 1