Fumio Tamura

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.

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.

Partitioning patterns of photosynthates from different shoot types in ‘Nijisseiki’ pear (Pyrus pyrifolia Nakai)

Summary Partitioning patterns of photosynthates from different shoot types on 16 year old ‘Nijisseiki’ pears were investigated 1, 2, 4, 7 and 13 weeks after anthesis (WAA). When leaves were at 45% of final expansion at 1 WAA, net leaf photosynthetic rate reached about two-thirds of its maximum value. One WAA, unfolded leaves could export photosynthates, although most photosynthates remained in source leaves, regardless of shoot type. The proportion of fruiting-spur-leaf-derived photosynthates entering fruit increased from 13±14% at 1 WAA to 50–65% at 13 WAA. In contrast, bourse leaves contributed only 1.1–1.8% of photosynthates to fruitlets 1 WAA. By 13 WAA this value increased to more than 21%. Nonfruiting spurs could export photosynthates to fruit on an adjacent spur as early as 1 WAA; this was accelerated by removing the fruiting spur leaves. From 2 WAA, nonfruiting spurs showed an apparent export of photosynthates out of the spur complex. The fruiting-spur-leaf-derived photosynthates were exported out of the spur complex after 4 WAA. No export of bourse-derived photosynthates out of the spur complex was found from 1 to 7 WAA, except some export at 2 WAA. By 13 WAA, both spur and bourse leaves exported photosynthates out of the spur complex. The extension-shoot-derived photosynthates might play a complementary role in the growth of fruit on spurs with few leaf numbers. Competition for current photosynthates between bourse shoots and fruit during the early developmental stage of fruit was not recognized.

Comparative Transcriptome Analysis of the Less-Dormant Taiwanese Pear and the Dormant Japanese Pear during Winter Season

The flower bud transcriptome in the less dormant Taiwanese pear ‘Hengshanli’ and high-chilling requiring Japanese pear strain TH3 subjected to the same chilling exposure time were analyzed during winter using next-generation sequencing. In buds sampled on January 10th and on February 7th in 2014, 6,978 and 7,096 genes, respectively, were significantly differentially expressed in the TH3 and ‘Hengshanli’ libraries. A comparative GO analysis revealed that oxidation-reduction process (biological process) and ATP binding (molecular function), were overrepresented during the ecodormancy period (EP) when compared to the endodormancy deepest period (DP), indicating that ATP synthesis was activated during the transition between these dormancy stages. Among the 11 differently expressed genes (DEGs) annotated as probable dehydrins or LEA protein-related genes, 9 DEGs showed higher transcript levels in the DP than in the EP. In order to focus on transcription factors induced by low temperature or drought, 7 differently expressed genes (DEGs) annotated as probable ICE1 or DREB proteins were analyzed by real-time PCR. Expression levels of 3 genes were higher in TH3 than in ‘Hengshanli’ on all sampling days. Their expression increased during the endodormancy deepest period (DP) and then decreased before endodormancy breaking in TH3 buds. Taken together, these results suggest that these genes annotated as ICE1, DREB and ERF are involved in endodormancy maintenance and in the transition from endodormancy to ecodormancy.

Fate of photosynthates from spur leaves of ‘Nijisseiki’ pear during the period of rapid fruitgrowth

Summary Spurs of ‘Nijisseiki’ pear (Pyrus pyriflora Nakai) were allowed to assimilate 10033;CO2 at 87.d after anthesis (DAA) and at 108 DAA during the period of rapid fruit growth. Then the spurs were sampled periodically until fruit harvest time to trace the time course and amount of movement of assimilates from spur leaves to individual organs in the spur. The amount of 10033;C absorbed by fruit within 3.d after labelling was constant until harvest, regardless of the labelling date. However, the total amount of 10033;C in the spur decreased continually until harvest.Of the total amount of 10033;C recovered in the spur labelled at 87 DAA, by harvest, 43.2% of 13C was found in the fruit flesh (cortex of receptacle), 5% in the core (pith of receptacle 1 pericarp 1 seeds), 5.6% in spur stem, 5.4% in the source leaves, and 40.8% was respired and exported from the spur. Of total amount of initial 10033;C labelled at 108 DAA, at harvest, the proportion of 10033;C translocated to flesh, core, stem, respired, and remaining in leaves was 61%, 6.1%, 3.0%, 24% and 5.4%, respectively. Photosynthates fixed by the spur early in the rapid growth stage of the fruit contribute more to the formation of starch and structural materials and less to solublecarbohydrates in fruit than do those fixed later.

Identification of the expressed protein and the impact of change in ascorbate peroxidase activity related to endodormancy breaking in Pyrus pyrifolia

Endodormancy is an important feature of perennial deciduous fruit trees that survive in the extreme climates brought about by seasonal variation. To acquire a comprehensive knowledge of the biochemical processes occurring just before endodormancy breaking, the buds collected in the pre-breaking period (PP) phase were used as samples to identify the proteins related to the breaking of endodormancy in the Japanese pear (Pyrus pyrifolia Nakai). Using nano-ESI-LC-MS/MS analysis, 96 proteins were overlapped by analyses of three times and identified as expressed proteins at the PP stage. Among these proteins, dehydrin, several classes of heat shock proteins (HSP), auxin-binding protein, and auxin-induced protein were identified in the floral bud in the PP stage. The majority of these proteins were involved primarily in the oxidation-reduction process. We focused on catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) as enzymes regulating the levels of hydrogen peroxide (H2O2) in the bud. From measurements taken during the deepest period (DP), PP, mid-breaking period (MP), and late-breaking period (LP) of endodormancy, CAT activity decreased gradually, while APX activity also decreased from DP to MP, but then increased rapidly during LP. Protein data for PP and the rapid increase in APX activity observed in LP provided knowledge of the biochemical processes that regulate the consecutive transition from endodormancy breaking to ecodormancy induction in the Japanese pear.

Putrescine content of Japanese pear (Pyrus pyrifolia) styles increases in response to pollination

Abstract Although poly amines (Pas) are suggested to be involved in the pollination process, the kinetics of this process in Japanese pear (Pyrus pyrifolia) are not currently understood. In this work, the content of putrescine increased (α ≤ 0.05) 5 h after in vitro pollination in styles of five different cultivars of Japanese pear. The evolution of spermidine and spermine within the cultivars used was not consistent; whereas spermidine contents decreased, increased, or did not have statistically significant changes (α ≤ 0.05), spermine content either decreased or increased. Moreover, the regulation of the ethylene and putrescine responses to pollination seemed to be linked to the cultivars gynoecious genotype, and was influenced by the pollen source. These results agree with the idea that putrescine increases in the styles during the first few hours after pollination in this species.

Draft genome sequence of Japanese pear (Pyrus pyrifolia)

The Japanese pear (Pyrus pyrifolia), a member of the family Rosaceae, is one of the most important fruit trees in Japan. This article documents the public availability of the partial draft genome sequence data of the Japanese pear strain TH3, which is the S1 of ‘Osa-Nijisseiki’ and is homozygous for the S4sm gene. This dataset may be used to prepare molecular markers for breeding of new cultivars having a crisp texture and feel. This data will also help research on physiological disorders affecting Japanese pear fruit. We sequenced paired-end libraries using Illumina HiSeq. 2500 and generated approximately 212M reads. Data on the draft genome obtained in this study has been deposited to the DNA Data Bank of Japan (DDBJ). The read data were submitted to the DDBJ Read Archive (BioProject: PRJDB6878, BioSample: SAMD00117051).