Hideki Murayama

Fruitlet abscission: A cDNA-AFLP approach to study genes differentially expressed during shedding of immature fruits reveals the involvement of a putative auxin hydrogen symporter in apple (Malus domestica L. Borkh)

Apple Malus X domestica fruitlet abscission is preceded by a stimulation of ethylene biosynthesis and a gain in sensitivity to the hormone. This phase was studied by a differential screening carried out by cDNA-AFLP in abscising (AF) and non-abscising (NAF) fruitlet populations. Fifty-three primer combinations allowed for the isolation of 131, 66 and 30 differentially expressed bands from cortex, peduncle and seed, respectively. All sequences were then classified as up- or down-regulated by comparing the profile in AFs and NAFs. Almost all of these sequences showed significant homology to genes encoding proteins with known or putative function. The gene ontology analysis of the TDFs isolated indicated a deep change in metabolism, plastid and hormonal status, especially auxin. Furthermore, some common elements between abscission and senescence were identified. The isolation of the full length of one of these TDFs allowed for the identification of a gene encoding an auxin hydrogen symporter (MdAHS). Bioinformatic analysis indicated that the deduced protein shares some features with other auxin efflux carriers, which include PINs. Nevertheless the 3D structure pointed out substantial differences and a conformation largely dissimilar from canonical ion transporters. The expression analysis demonstrated that this gene is regulated by light and development but not affected by ethylene or auxin.

Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis.

Summary Based on the cDNA sequences encoding sweet cherry self-incompatibility associated ribonucleases (S-RNases), a PCR-based S-allele typing system for sweet cherry cultivars has been recently developed. Using this technique, we determined S-genotypes of the three newly released Japanese cvs Kouka-Nishiki, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was classified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of ‘Rainier’ (S1S4)‘ and ‘Sato-Nishiki (S3S6)’,which leads to the development of a more reliable S-allele identification method of PCR-RFLP for sweet cherry cultivars. Total DNA isolated from leaves of the four cultivars along with those from ten cultivars with known S-genotypes were PCR amplified with two sets of primers that were designed from DNA sequences encoding the signal peptide (Pru-T2) and two conserved domains (Pru-C2 and Pru-C4R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the 5-genotypes of ‘Kouka-Nishiki’, ‘Beni-Sayaka’, ‘Beni-Shuho’ and ‘Merton Glory’ were suggested to be S1S3, S1S6, S4S6, and S4S6, respectively. Two of these three S-genotypes (S1S6 and S4S6) were found for the first time. DNA sequencing of PCR products from S-alleles of ‘Rainier’ and ‘Sato-Nishiki’ revealed that Ban II, Nru I, Apa LI and Ava I sites, respectively, were unique in the S1-, S3-, S4- and S6- sequences flanked by Pru-T2 and Pru-C4R primers. RFLP analysis of the PCR products using these enzymes confirmed that S1-, S3-, S4- and S6-alleles of the four cultivars contained the respective restriction enzyme recognition sites.

Metabolic profiling of developing pear fruits reveals dynamic variation in primary and secondary metabolites, including plant hormones

Metabolites in the fruits of edible plants include sweet sugars, visually appealing pigments, various products with human nutritional value, and biologically active plant hormones. Although quantities of these metabolites vary during fruit development and ripening because of cell division and enlargement, there are few reports describing the actual dynamics of these changes. Therefore, we applied multiple metabolomic techniques to identify the changes in metabolite levels during the development and ripening of pear fruits (Pyrus communis L. ‘La France’). We quantified and classified over 250 metabolites into six groups depending on their specific patterns of variation during development and ripening. Approximately half the total number of metabolites, including histidine and malate, accumulated transiently around the blooming period, during which cells are actively dividing, and then decreased either rapidly or slowly. Furthermore, the amounts of sulfur-containing amino acids also increased in pear fruits around 3–4 months after the blooming period, when fruit cells are enlarging, but virtually disappeared from ripened fruits. Some metabolites, including the plant hormone abscisic acid, accumulated particularly in the receptacle prior to blooming and/or fruit ripening. Our results show several patterns of variation in metabolite levels in developing and ripening pear fruits, and provide fundamental metabolomic data that is useful for understanding pear fruit physiology and enhancing the nutritional traits of new cultivars.

Effects of arbuscular mycorrhizal fungi on the abundance of foliar-feeding insects and their natural enemy

We investigated the effects of symbiotic association between a plant and an arbuscular mycorrhizal fungus (AMF) on the abundance of aboveground foliar-feeding insects that differed in feeding mode and their predator. We examined effects on insect abundance as the result of AMF-related changes in the quality and quantity of plants in the field. The numbers of three insects with different foliar-feeding mode (phloem feeder, chewer, and cell-content feeder) and their generalist predator Orius sauteri Poppius on soybean Glycine max (L.) Merrill with and without the AMF Gigaspora margarita Becker & Hall were compared over time. Symbiotic association between the AMF and the soybean increased shoot biomass, the concentration of phosphorus in the soybean, and the abundance of the phloem feeder Aulacorthum solani Kaltenbach, but did not affect the abundance of generalist chewers. In addition, the effects of the symbiotic association on the abundance of cell-content feeding Thrips spp. and the generalist predator O. sauteri differed between sample dates. These results indicated that the effects of the symbiotic association on the number of foliar-feeding insects depended on feeding mode and the number of predators.

Leaf herbivory by Spodoptera litura increases arbuscular mycorrhizal colonization in roots of soybean

The improvement of host plant growth resulting from mycorrhizal colonization often affects the viability of above-ground herbivorous insects. Conversely, the effects of herbivorous insects on plant growth may indirectly affect below-ground microorganisms. This study investigated the effects of herbivorous insects on arbuscular mycorrhizal (AM) colonization of soybean (Glycine max Merr.). Seeds of soybean were sown in soil inoculated with the AM fungus Gigaspora margarita Becker & Hall and grown in growth chambers. At 39 days after inoculation (DAI), a larva of the common cutworm (Spodoptera litura Fabricius) was placed on a leaf of each soybean plant for 5 days. AM colonization of soybean on 39 DAI was 21%. Although not evident by 62 DAI, by 77 DAI there was a statistically significant increase in AM colonization in plants subjected to herbivory by S. litura relative to control plants. Leaf removal at 46 DAI was also associated with increased AM colonization at 78 DAI. These results suggest that herbivory by chewing insect affects AM colonization and that the effect is likely caused by a decrease in leaf area.