Akihiro Itai

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

Abstract The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear.

Silicon‐mediated changes in polyamines participate in silicon‐induced salt tolerance in Sorghum bicolor L.

Silicon (Si) is generally considered a beneficial element for the growth of higher plants, especially under stress conditions, but the mechanisms remain unclear. Here, we tested the hypothesis that Si improves salt tolerance through mediating important metabolism processes rather than acting as a mere mechanical barrier. Seedlings of sorghum (Sorghum bicolor L.) growing in hydroponic culture were treated with NaCl (100 mm) combined with or without Si (0.83 mm). The result showed that supplemental Si enhanced sorghum salt tolerance by decreasing Na(+) accumulation. Simultaneously, polyamine (PA) levels were increased and ethylene precursor (1-aminocyclopropane-1-carboxylic acid: ACC) concentrations were decreased. Several key PA synthesis genes were up-regulated by Si under salt stress. To further confirm the role of PA in Si-mediated salt tolerance, seedlings were exposed to spermidine (Spd) or a PA synthesis inhibitor (dicyclohexylammonium sulphate, DCHA) combined with salt and Si. Exogenous Spd showed similar effects as Si under salt stress whereas exogenous DCHA eliminated Si-enhanced salt tolerance and the beneficial effect of Si in decreasing Na(+) accumulation. These results indicate that PAs and ACC are involved in Si-induced salt tolerance in sorghum and provide evidence that Si plays an active role in mediating salt tolerance.

Genetic relationships of pear cultivars in Xinjiang, China, as measured by RAPD markers

Summary Thirteen native pear species have been identified in China, of which P. armeniacaefolia Yu and P. sinkiangensis Yu are specific to Xinjiang. P. armeniacaefolia grows wild and a few cultivars have been assigned to this species. Cultivars of P. sinkiangensis have been suspected to be of hybrid origin involving P. communis L. and P. bretschneideri Rehd. In this study, traditional pear cultivars in Xinjiang were evaluated using RAPD markers and compared with representatives of Occidental pear species, cultivars of P. communis and East Asian pear accessions. The combination of 72 pear accessions and 20 selected primers produced 231 scorable polymorphic RAPD bands, of which some were specific to certain species. Five main groups of pear accessions could be distinguished from UPGMA analysis: 1) P. xerophila Yu, its relatives and one cultivar of P. ussuriensis Max., 2) cultivars of P. sinkiangensis, 3) cultivars of P. pyrifolia Nakai and P. bretschneideri, 4) wild Occidental species, cultivars of P. communis and P. armeniacaefolia, and 5) hybrids between P. communis and Chinese or Japanese pear cultivars. The result of PCA generally agrees with that based on UPGMA. Based on RAPD data, some cultivars traditionally classified as P. bretschneideri should be assigned to P. sinkiangensis. Some heirloom cultivars assigned to P. communis were found to be of hybrid origin involving the Chinese white pear (P. bretschneideri) or sand pear (P. pyrifolia). Our results confirmed that P. sinkiangensis is of hybrid origin and at least P. communis, P. armeniacaefolia and Chinese white pears or sand pears have been involved. A further study is needed to understand how pear species and cultivars in Xinjiang are related to those originated from countries in Central Asia.

Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers

Abstract In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F2 population, which revealed that the recombination frequency between the two markers was 20.8 ± 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.

Synthetic cytokinins control persimmon fruit shape, size and quality

SummaryThe effect of three synthetic cytokinins on fruit development of persimmon (Diospyros kaki L.) ‘Hiratanenashi’ was studied by spraying flowers or young fruitlets with 10 or 100 ppm 4PU-30 and TDZ, and 100 or 1000 ppm BA. The most effective for promoting fruit growth was 4PU-30 at 100 ppm, followed by TDZ at 100 ppm and 4PU-30 at 10 ppm. BA at 100 or 1000 ppm and TDZ at 10 ppm had no effect on the fruit enlargement. At 100 ppm, 4PU-30 produced flatter fruit, by promoting transverse growth. Both 4PU-30 and TDZ retarded ripening, as evidenced by delayed chlorophyll degradation and low sugar accumulation. Comparison between prebloom and postbloom applications on cvs Tonewase and Saijo revealed that the former treatment produced flatter, heavier fruit than did the latter. The treated fruit also contained less sugars, especially the reducing sugars, than did the control fruits, indicating that the ripening process was also retarded in these cultivars.

Identification of QTLs controlling harvest time and fruit skin color in Japanese pear (Pyrus pyrifolia Nakai)

Using an F1 population from a cross between Japanese pear (Pyrus pyrifolia Nakai) cultivars ‘Akiakari’ and ‘Taihaku’, we performed quantitative trait locus (QTL) analysis of seven fruit traits (harvest time, fruit skin color, flesh firmness, fruit weight, acid content, total soluble solids content, and preharvest fruit drop). The constructed simple sequence repeat-based genetic linkage map of ‘Akiakari’ consisted of 208 loci and spanned 799 cM; that of ‘Taihaku’ consisted of 275 loci and spanned 1039 cM. Out of significant QTLs, two QTLs for harvest time, one for fruit skin color, and one for flesh firmness were stably detected in two successive years. The QTLs for harvest time were located at the bottom of linkage group (LG) Tai3 (nearest marker: BGA35) and at the top of LG Tai15 (nearest markers: PPACS2 and MEST050), in good accordance with results of genome-wide association study. The PPACS2 gene, a member of the ACC synthase gene family, may control harvest time, preharvest fruit drop, and fruit storage potential. One major QTL associated with fruit skin color was identified at the top of LG 8. QTLs identified in this study would be useful for marker-assisted selection in Japanese pear breeding programs.

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.

Characterization of Trichoderma species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan

Native Trichoderma spp. were isolated from agricultural fields in several regions of Ecuador. These isolates were characterized via morphological observation as well as molecular phylogenetic analysis based on DNA sequences of the rDNA internal transcribed spacer region, elongation factor-1α gene and RNA polymerase subunit II gene. Fifteen native Trichoderma spp. were identified as T. harzianum, T. asperellum, T. virens and T. reesei. Some of these strains showed strong antagonistic activities against several important pathogens in Ecuador, such as Fusarium oxysporum f. sp. cubense (Panama disease) and Mycosphaerella fijiensis (black Sigatoka) on banana, as well as Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease) on cacao. The isolates also showed inhibitory effects on in vitro colony growth tests against Japanese isolates of Fusarium oxysporum f. sp. lycopersici, Alternaria alternata and Rosellinia necatrix. The native Trichoderma strains characterized here are potential biocontrol agents against important pathogens of banana and cacao in Ecuador.

Watercore in Fruits

Watercore is a physiological internal disorder affecting apples and pears, in which the intercellular air spaces of the flesh become filled with liquid, resulting in tissues with translucent appearance. Watercore is associated with fruit maturity as well as the presentation of varietal differences in susceptibility in apples and pears. Susceptibility is thus considered a heritable character. Watercore is promoted by low or high air temperatures during the preharvest period, large fruit, poor calcium concentration, high nitrogen and boron nutrition, a high leaf-to-fruit ratio, excessive fruit thinning, high or low light exposure, growth in volcanic ash soil, ethrel (ethephon) and gibberellin treatment, and girdling of the trunk and limbs. Mild watercore symptoms can disappear in storage, but when severe, internal browning and large cavities can develop. The fleshy tissue of apples with watercore has a higher sorbitol and sucrose concentration and lower glucose concentration than tissue without watercore. Watercore is also accompanied by changes in membrane permeability during maturation and ripening. A decrease in the expression of sorbitol transporter, leading to sorbitol accumulation in the intercellular spaces and subsequent flooding of tissues, has also been suggested.