Changes in phytohormone content and associated gene expression throughout the stages of pear (Pyrus pyrifolia Nakai) dormancy.

To elucidate the role of phytohormones during bud dormancy progression in the Japanese pear (Pyrus pyrifolia Nakai), we investigated changes in phytohormone levels of indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA) and trans-zeatin (tZ). Using ultra-performance liquid chromatography/mass spectrometry/mass spectrometry, we monitored phytohormone levels in the buds of field-grown and potted trees that were artificially heated to modify the timing of dormancy and flowering (spring flush) progression. We also analyzed the expression of GA- and ABA-metabolic genes during dormancy. Indole acetic acid and tZ levels were low during dormancy and increased toward the flowering stage. Gibberellic acid levels were maintained at relatively high concentrations during the dormancy induction stage, then decreased before slightly increasing prior to flowering. The low GA concentration in potted trees compared with field-grown trees indicated that GA functions in regulating tree vigor. Abscisic acid levels increased from the dormancy induction stage, peaked near endodormancy release and steadily decreased before increasing again before the flowering stage. The ABA peak levels did not always coincide with endodormancy release, but peak height correlated with flowering uniformity, suggesting that a decline in ABA concentration was not necessary for resumption of growth but the abundance of ABA might be associated with dormancy depth. From monitoring the expression of genes related to GA and ABA metabolism, we inferred that phytohormone metabolism changed significantly during dormancy, even though the levels of bioactive molecules were consistently low. Phytohormones regulate dormancy progression not only upon the reception of internal signals but also upon sensing ambient conditions.

QTL mapping of male sterility and transmission pattern in progeny of Satsuma mandarin.

Seedlessness is one of the important traits in citrus breeding. Male sterility derived from Satsuma mandarin (Citrus unshiu) has been used in Japanese citrus breeding programs to obtain seedless cultivars. The efficiency of seedless cultivar breeding would be improved by developing a selection marker linked to seedlessness. In this study, we performed QTL mapping in 'Okitsu No. 46' × 'Okitsu No. 56' (O46-O56) crosses for the number of pollen grains per anther (NPG) and apparent pollen fertility (APF), two traits used as an index of male sterility, and detected a candidate QTL for NPG (MS-P1) on linkage group 8 with a significant LOD score (7.31) and 47% of variance explained. The QTL for APF (MS-F1) was detected on linkage group 6 with a significant LOD score (5.71) and 63.6% of variance explained. The role of both MS-P1 in reducing NPG and MS-F1 in decreasing APF were confirmed with the 'Okitsu No.46' × 'Kara' (O46-K) cross. Pedigree analysis inferred that both MS-P1 and MS-F1 in 'Okitsu No. 46' were derived from kunenbo (Citrus nobilis) through hassaku (C. hassaku) and 'Sweet Spring'. Cytoplasm analysis revealed that both male-sterile 'Sweet Spring' and 'Okitsu No. 46' have cytoplasm derived from Kishu (C. kinokuni hort. ex Tanaka), but the cytoplasm of male-sterile kunenbo and hassaku were derived from other varieties rather than Kishu. These results suggest that MS-P1 and MS-F1 primarily reduce the NPG and decrease APF, but their expression requires a cytoplasm derived from Kishu. These findings will improve our understanding of the molecular mechanism of male sterility in citrus and help to develop a DNA marker for seedless breeding in citrus.

Segregation and Heritability of Male Sterility in Populations Derived from Progeny of Satsuma Mandarin.

Male sterility derived from Satsuma mandarin (Citrus unshiu) has been used in Japanese citrus breeding programs to obtain seedless cultivars, which is a desirable trait for consumers. Male sterility has often been evaluated by anther development or pollen fertility; however, the inheritance and heritability of male sterility derived from Satsuma is poorly understood. In this study, we investigated the mode of inheritance and broad-sense heritability of male sterility derived from Satsuma. Initially, we evaluated the total number of pollen grains per anther and apparent pollen fertility, as indicated by lactophenol blue staining, in 15 citrus cultivars and selections to understand the male sterility of Satsuma. The results indicated that male sterility was primarily caused by decreased number of pollen grains per anther in progeny of Satsuma. We also evaluated these traits in three F1 populations (hyuganatsu × 'Okitsu No. 56', 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara'), of which the parents are derived from Satsuma. Individuals in these populations showed strong segregation for number of pollen grains per anther. The apparent fertility of pollen also showed segregation but was almost constant at 70%-90%. The estimated broad-sense heritability for the number of pollen grains per anther was as high as 0.898 in the 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara' populations. These results indicated that the number of pollen grains per anther primarily determined male sterility among progeny of Satsuma, and this trait was inherited by the progeny. Development of DNA markers closely linked to male sterility using the F1 populations of 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara' is expected to contribute to the breeding of novel seedless citrus cultivars.

Characterization of genomic sequence showing strong association with polyembryony among diverse Citrus species and cultivars, and its synteny with Vitis and Populus.

Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony.

Effect of jasmonates on ethylene biosynthesis and aroma volatile emission in Japanese apricot infected by a pathogen (Colletotrichum gloeosporioides).

The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.

Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers.

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

Carotenoid accumulation in Japanese apricot (Prunus mume Siebold & Zucc.): molecular analysis of carotenogenic gene expression and ethylene regulation.

To elucidate the regulatory mechanisms of carotenogenesis in Japanese apricot (Prunus mume Siebold & Zucc.), the relationships between carotenoid accumulation and the expression of the carotenogenic genes, phytoene synthase (PmPSY-1), phytoene desaturase (PmPDS), zeta-carotene desaturase (PmZDS), lycopene beta-cyclase (PmLCYb), lycopene epsilon-cyclase (PmLCYe), beta-carotene hydroxylase (PmHYb), and zeaxanthin epoxidase (PmZEP), were analyzed in two cultivars with different ripening traits, 'Orihime' and 'Nanko.' In 'Orihime' fruits, large amounts of carotenoids accumulated on the tree, concomitant with the induction of PmPSY-1 and the downstream carotenogenic genes PmLCYb, PmHYb, and PmZEP. In 'Nanko' fruits, carotenoids accumulated mainly after harvest, correlating with an appreciable induction of PmPSY-1 expression, but the downstream genes were not notably induced, which may explain the lower total carotenoid content in 'Nanko' than in 'Orihime.' In both cultivars, a decrease in PmLCYe expression and increased or constant PmLCYb expression could cause the metabolic shift from beta,epsilon-carotenoid synthesis to beta,beta-carotenoid synthesis that occurs as ripening approaches. Next, the effects of ethylene on the expression of PmPSY-1 and carotenoid accumulation were investigated in 'Nanko' fruits treated with propylene or 1-methylcyclopropene (1-MCP). Propylene treatment induced both ethylene production and carotenoid accumulation. PmPSY-1 was constitutively expressed, but propylene treatment accelerated its induction. 1-MCP treatment caused a slight inhibition of carotenoid accumulation along with the repression, although not complete, of PmPSY-1. Collectively, although PmPSY-1 expression was not exclusively regulated by ethylene, both the notable induction of PmPSY-1 accelerated by ethylene and the subsequent induction of the downstream carotenogenic genes, especially PmLCYb, could be necessary for the massive carotenoid accumulation that occurs during ripening. Furthermore, the switch from PmLCYe expression to PmLCYb expression could cause beta,beta-carotenoid accumulation in both Japanese apricot cultivars.

Acetate and ethanol production from H2 and CO2 by Moorella sp. using a repeated batch culture.

The growth inhibition of Moorella sp. HUC22-1 by undissociated acetic acid was analyzed using a non-competitive inhibition model coupled with a pH inhibition model. In the cells grown on H2 and CO2, the inhibition constant, K(p) of the undissociated acetic acid was 6.2 mM (164 mM as the total acetate at pH 6.2, pKa = 4.795, 55 degrees C), which was 1.5-fold higher than that obtained in cells grown on fructose. When a pH-controlled batch culture was performed using a fermentor at pH 6.2 with H2 and CO2, a maximum of 0.92 g/l of dry cell weight and 339 mM of acetate were produced after 220 h, which were 4.4- and 6.8-fold higher than those produced in the pH-uncontrolled batch culture, respectively. In order to reduce acetate inhibition in the culture medium, a repeated batch culture with cell recycling was performed at a constant pH with H2 and CO2. At a pH of 6.2, the total acetate production reached 840 mmol/l-reactor with 4.7 mmol/l-reactor of total ethanol production after 420 h. When the culture pH was maintained at 5.8, which was the optimum for ethanol production, the total ethanol production reached 15.4 mmol/l-reactor after 430 h, although the total acetate production was decreased to 675 mmol/l-reactor.

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses.

Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5' upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart.

Putative PIP1 genes isolated from apple: expression analyses during fruit development and under osmotic stress.

To gain insight into the function of plasma membrane intrinsic protein (PIP) genes in apple, two genes, MdPIP1a and MdPIP1b, were isolated. MdPIP1 expression was in accordance with the volume increase during fruit development, which is a loading process of water and solutes. In addition, the expression of MdPIP1 was up-regulated in the stems by osmotic stress. These results indicate that MdPIP1 may play important roles not only in fruit expansion, but also in maintaining water homeostasis under stress conditions.

Flavonol synthase gene expression during citrus fruit development.

We isolated a cDNA clone (CitFLS) encoding flavonol synthase (FLS) from the satsuma mandarin (Citrus unshiu Marc.) fruit and investigated the steady state of CitFLS RNA expression during the fruit development. The CitFLS was 1274 bp long, encoded 335 amino acid residues, and belonged to a family of 2-oxoglutarate-dependent dioxygenases. The level of CitFLS transcript was higher in the young leaves than in the old leaves, and it was high at the early developmental stage and low at the mature stage in the juice sacs/segment epidermis (edible part). On the other hand, the CitFLS transcript increased in the peel during fruit maturation. These results indicated that the satsuma mandarin CitFLS was differentially regulated in the developmental stage and in a tissue-specific manner. Additionally, satsuma mandarin peel tissues produced rutin (a flavonol glycoside) from an exogenous dihydroquercetin (taxifolin), indicating the ability of these tissues to produce flavonols.