齋藤 隆德

We have previously reported that the application of- used as isoprothiolane (IPT), a fungicide against rice blast- 20 to 30 days before harvest improved peel color in satsuma mandarin fruit (Citrus unshiu Marcow). However, the mechanism by which IPT improves peel coloration is not yet fully understood. This study examined the effects of IPT on plant hormones such as gibberellic acid (GA) and abscisic acid (ABA), and carotenoid accumulation. Whole trees were treated with IPT at 25 days before harvest. Concentrations of gibberellic acid-1 (GA1) and GA4 in the flavedo of IPT-treated trees were significantly decreased at 5 days after treatment (DAT) compared to the untreated control. The expressions of CitGA20ox1 in IPT-treated flavedo were lower than those in the untreated controls at 5 and 25 DAT. The CitGA3ox expressions in IPT-treated flavedo were lower than those in the untreated control at 5 DAT. ABA concentrations in IPT-treated flavedo were significantly higher than those in the untreated control at 25 DAT. The concentrations of β-cryptoxanthin in IPT-treated flavedo were higher than those in the untreated control at 25 DAT. The chlorophyll concentrations of IPT-treated flavedo were lower than those in the untreated control at 5 and 20 DAT. These results suggest that IPT advances β-cryptoxanthin accumulation through the regulation of endogenous GA1 and GA4 based on the inhibition of CitGA20ox1 and CitGA3ox expressions. It is possible that IPT can be utilized to improve coloration in other citrus fruit. *These two authors contributed equally to this work.

To examine the dwarfing mechanism in apples, one-year-old Marubakaido (Malus prunifolia Borkh.) (invigorating) apple rootstock stools were foliar-sprayed with 860 mg L-1 of paclobutrazol (PBZ) as a single application or without. M.9 apple rootstock (dwarf) was used as a positive control. The phytohormones were estimated in the shoot bark and sub-apical shoot and gene expression in the apices of terminal shoots. Evident responses to PBZ were observed a fortnight after treatment, as the shoot and internode lengths were suppressed significantly. Endogenous indole-3-acetic acid increased in the PBZ treatment, and the polar auxin transporter genes MdPIN1 and MdLAX1 and the biosynthesis gene MdYUCCA10a were upregulated along with the MdARF2 gene. Additionally, PBZ increased the abscisic acid (ABA) concentration and the biosynthesis-related gene MdNCED1 but repressed the degradation gene MdCYP707A1. The ABA transporter gene MdAITb-like was upregulated by PBZ. The concentrations of the gibberellins (GAs) GA1 and GA4 decreased in the PBZ-treated rootstocks. The GA transporter gene MdNFP3.1-like and the signaling gene MdGID1b-like were strongly downregulated by PBZ, whereas the catabolic gene MdGA2OX2 was upregulated. PBZ treatment significantly reduced trans-zeatin (tZ) levels and downregulated the cytokinin biosynthesis gene MdIPT6 but upregulated the MdCKX7 degradation gene. Additionally, PBZ upregulated the cytokinin-related transporter genes MdPUP7-like and MdPUP9-like. Collectively, our results show that the physiological and molecular effect of PBZ was observed within two weeks, and this was indicated by the modulation of phytohormonal levels as well as transporter and other gene expression in Marubakaido apple rootstocks.

The effects of an inhibitor (Abz-E3M) of abscisic acid (ABA) 8 '-hydroxylase, which is a primary enzyme of ABA catabolism, on dehydration tolerance and root formation in grape cuttings under drought conditions were investigated. Cuttings of 'Kyoho' grape (Vitis labruscaL. x Vitis viniferaL.) were sprayed with 100 mu M of Abz-E3M and subjected to water deficit conditions at the stage when their first leaves fully expanded. The physiological and morphological changes in the leaves and basal portions of the cuttings were determined. In Abz-E3M-treated leaves, lower ABA metabolite and higher ABA and indole-3-acetic acid (IAA) concentrations were observed. Compared to the untreated control leaves, higher water potential was significantly maintained in Abz-E3M-treated leaves. Abz-E3M applications resulted in lower proline accumulation and 2,2-diphenyl-2-picrylhydrazyl radical scavenging activity in the leaves and led to enhanced dehydration tolerance. In addition, the percentage of rooted cuttings was significantly increased by Abz-E3M application. In the basal portion of Abz-E3M-treated cuttings, endogenous IAA concentrations and the gene expressions ofVvARF6andVvARF8, which are positive regulators of adventitious root formation, were significantly increased. Moreover, the expression levels of the negative regulator,VvARF17,were significantly lower. These results suggested that the inhibition of ABA 8 '-hydroxylase enhanced dehydration tolerance and adventitious rooting and may be an effective strategy for achieving drought stress tolerance in grape cuttings.

© 2018 International Society for Horticultural Science. All rights reserved. Salinity imposes stress on plant tissues that can lead to water deficits or ionspecific stresses resulting from altered ion ratios. The effect of exogenous ABA on 'Fuji' apple seedlings exposed to sodium chloride (NaCl) was analyzed by measuring parameters such as water potential, stomata aperture, sodium (Na+) concentration, endogenous abscisic acid (ABA), and total polyamines as well as genes related to their metabolism. The results show that damage to the leaf surface such as sunburn, yellowing and hardening of the leaves became more prominent compared to untreated controls (no ABA or NaCl) as treatment progressed. Regarding endogenous ABA levels, there was a significant difference at 5 days between the ABA+NaCl and NaCl groups. At the end of the treatment, the NaCl group showed higher levels of chlorophyll when compared to the untreated control and ABA+NaCl group. The stomata of the groups under stress showed recovery at the end of the treatment; however, the ABA+NaCl group response more closely resembled the status of the untreated, non-stressed control. Water potential levels dropped and stomata closure was apparent at 5 days for groups under stress; however, while the ABA+NaCl group displayed recovery as treatment continued, the NaCl group remained low even when the plants had recovered from the initial shock. Total polyamine levels were higher for the ABA+NaCl group after ABA application and then decreased with time, whereas that for the NaCl group was higher after stress began, peaking at 5 and 10 days. Taken together, ABA, PA and gene activations suggest that the early response to stress induced by ABA might have helped the seedlings overcome it more efficiently.

© 2018 International Society for Horticultural Science. All rights reserved. There is evidence that abscisic acid (ABA) and ethylene both play important roles in the physiological processes of grape maturation. However, the interaction between ABA and ethylene metabolism in grape maturation is unclear. We applied 50 μM (±) abscinazole-E3 (Abz, ABA 8'-hydroxylase inhibitor) or 500 μM ethephon to grape berries at 44 DAFB (days after full bloom). Grape berries were sampled at 44, 48, 54, 64, 94 DAFB. The ABA, ethylene concentrations and related gene expressions were analyzed. The results showed that Abz or ethephon treatment decreased the firmness and titratable acid concentration. Abz treatment inhibited VvCYP707A1 gene expression levels at 48 DAFB and increased endogenous ABA accumulation at 54 DAFB. Ethephon treatment significantly up-regulated VvNCED1 gene expression levels at 48 DAFB and VvCYP707A1 at 54 DAFB, but had no effect on ABA concentration. Ethylene and gene expression levels of VvACO1 and VvERF2 in Abz- and ethephon-treated berries at 48 DAFB were up-regulated. Abz or ethephon treatment also accelerated chlorophyll breakdown in berry skin with the up-regulation of VvPPH and VvRCCR gene expression levels. The total sugar concentrations slightly increased in both Abz- and ethephon-treated berries. These results suggest that Abz treatment before veraison can stimulate grape maturation by increasing endogenous ABA and the ethephon treatment can promote grape maturation similarly to Abz, through VvACO1 and VvERF2.

The production of aromatic volatiles such as esters during the ripening process in climacteric fruits is known to be controlled by ethylene. However, we here show that abscisic acid (ABA) application accelerated the onset of short-chain ester production (hexyl propionate, ethyl-2-methyl butyrate) and the expression of biosynthesis genes (MdAAT2 and MdBCAT1) during ripening of 'Orin' apple. ABA application also promoted the production of ethylene, and caused ethylene peak shifts correlated with the expression of ethylene synthesis genes (MdACS1/3 and MdACO1), suggesting that ABA may act jointly with ethylene as a positive regulator at the ripening stage of 'Orin' apple. Additionally, endogenous levels and expression of biosynthesis (MdNCED1) and signal transduction genes (MdABF2-like) of ABA increased towards ripening. Finally, the localization of the putative MdABF2-like protein binding element, AREB/ABF, was observed in the 5'-upstream region of MdACS1/3 and MdACO1.

In deciduous trees, including apples (Malus x domestica Borkh.), lipid accumulation in the meristem region towards endodormancy induction has been thought to be an important process for the acquisition of cold tolerance. In this study, we conducted histological staining of crude lipids in the meristem region of 'Fuji' apples and found that lipid accumulation coincided with endodormancy induction. Since a major component of lipid bodies (triacylglycerol) is esterified fatty acids, we analysed fatty acid-derived volatile compounds and genes encoding fatty acid-modifying enzymes (MdLOX1A and MdHPL2A); the reduction of lipid breakdown also coincided with endodormancy induction. We then characterised the expression patterns of lipid body-regulatory genes MdOLE1 and MdLIP2A during endodormancy induction and found that the expression of MdLIP2A correlated well with lipid accumulation towards endodormancy induction. Based on these results, we conducted chromatin remodelling studies and localized the cis-element in the 5'-upstream region of MdLIP2A to clarify its regulatory mechanism. Finally, we revealed that chromatin was concentrated - 764 to - 862 bp of the 5'-upstream region of MdLIP2A, which harbours the GARE [gibberellin responsive MYB transcription factor binding site] and CArG [MADS-box transcription factor binding site] motifs-meristem development-related protein-binding sites.

The effects of an ABA 8'-hydroxylase inhibitor, (+/-)abscinazole-E2B (Abz-E2B), were examined in "Fuji" apple seedlings exposed to sodium chloride (NaCl). Water potential, stomata aperture, and endogenous ABA, proline, and sodium (Na+) levels were analyzed, along with expression of the 9-cis-epoxycarotenoid dioxigenase (MdNCED) and ABA 8'-hydroxylase (MdCYP707A) genes. Water potential and stomatal aperture in the leaves treated with Abz-E2B and NaCl (Abz-E2B(+)NaCl(+) group) were lower than those in the untreated controls, but remained higher than those in the leaves treated with NaCl alone (Abz-E2B(-)NaCl(+) group). Endogenous ABA levels were higher in the Abz-E2B(+)NaCl(+) leaves at 4 days after treatment, whereas those in the Abz-E2B(-)NaCl(+) leaves increased with time and peaked at 12 days after treatment. Expressions of the MdNCED1 and MdNCED2 genes remained lower in Abz-E2B(+)NaCl(+) compared with Abz-E2B(-)NaCl(+) leaves, especially at 12 days after treatment. Proline levels increased sharply at 100-mM NaCl, and Na+ levels increased with time. The Na+ concentrations in the Abz-E2B(-)NaCl(+) leaves increased fourfold compared to those in the Abz-E2B(+)NaCl(+) leaves. The expressions of MdCYP707A1 and MdCYP707A2 were higher in Abz-E2B(+)NaCl(+) at 4 days, but declined at 12 days. The present data suggest that the increased endogenous ABA during the early stages of Fuji apple cultivation under salinity-stress conditions might be a good approach to prevent long-lasting damage and allow plants to recover.

In the pear `Kosui' (Pyrus pyrifolia Nakai), the dormancyassociated MADS-box (PpDAM1 = PpMADS13-1) gene has been reported to play an essential role in bud endodormancy. Here, we found that PpDAM1 up-regulated expression of 9-cis-epoxycarotenoid dioxygenase (PpNCED3), which is a rate-limiting gene for ABA biosynthesis. Transient assays with a dual luciferase reporter system (LUC assay) and electrophoretic mobility shift assay (EMSA) showed that PpDAM1 activated PpNCED3 expression by binding to the CArG motif in the PpNCED3 promoter. PpNCED3 expression was increased toward endodormancy release in lateral flower buds of `Kosui', which is consistent with the induced levels of ABA, its catabolism (ABA 80-hydroxylase) and signaling genes (type 2C protein phosphatase genes and SNF1related protein kinase 2 genes). In addition, we found that an ABA response element (ABRE)-binding transcription factor, PpAREB1, exhibiting high expression concomitant with endodormancy release, bound to three ABRE motifs in the promoter region of PpDAM1 and negatively regulated its activity. Taken together, our results suggested a feedback regulation between PpDAM1 and the ABA metabolism and signaling pathway during endodormancy of pear. This first evidence of an interaction between a DAM and ABA biosynthesis in vitro will provide further insights into bud endodormancy regulatory mechanisms of deciduous trees including pear.

Floral induction is an important event in the annual growth cycle of perennial fruit trees. For pear, this event directly affects fruit production in the following year. The flower buds in many species are induced by FLOWERING LOCUS T (FT), whose effect is repressed by the meristem-expressed gene TERMINAL FLOWER1 (TFL1). In this study, we investigated the functions of pear FT and TFL1 genes during floral development. Expression of pear FTs (PpFT1a and PpFT2a) in reproductive meristems was not obviously induced prior to floral initiation, while expression of TFL1s (PpTFL1-1a and PpTFL1-2a) rapidly decreased. The induction of the productive meristem identity MADS-box gene AP1 after repression of PpTFL1s suggested a primary role for PpTFL1 in floral induction. RNA-seq analysis suggested that plant hormone-related genes and several transcription factors that were coexpressed with PpTFL1 were potentially involved in the PpTFL1-mediated floral induction. Our data indicate the essential function of TFL1 in pear floral induction and add another species in the family Rosaceae in addition to strawberry and rose that shows a role for TFL1 in floral induction.

The effects of ethephon, abscisic acid (ABA), and nordihydroguaiaretic acid (NDGA) application on the ripening of pre-harvest 'Kohi' kiwifruit (Actinidia chinensis) were studied. The fruits were treated on-vine at 155 days after full bloom (DAFB) (mature stage) with 250 mu L/L ethephon, 100 mu mol ABA, or 100 mu mol NDGA. The fruits were sampled at 0, 3, 6, 9, and 12 days after treatment (DAT), and the following were analyzed at each time point: ethylene production, 1-aminocyclopropane-1-carboxylate (ACC) and ABA concentrations, ACC synthase (ACS) and ACC oxidase (ACO) activities, volatile compounds (n-hexanal and (E)-2-hexexnal), and the expressions of AcACS1, AcACO1, and 9-cis-epoxycarotenoid dioxygenase 1 (AcNCED1) genes. ABA concentrations and AcNCED1 gene expression increased in ABA-treated fruit. Malic acid concentrations and fruit firmness decreased in ethephon-treated fruit, but soluble solids concentrations (SSC), ethylene biosynthesis, and both AcACS1 and AcACO1 gene expressions increased. The accumulated fruit drop rate in ethephon-treated fruit was 4% at the edible stage at 9 DAT. Moreover, the production of n-hexanal and (E)-2-hexexnal decreased in ethephon-treated fruit. These results suggest that 'Kohi' kiwifruit may be ripened by on-vine ethephon application at 9 DAT, thus obviating ripening treatment after harvest. (C) 2016 Elsevier B.V. All rights reserved.

Main conclusion Paper-bagging treatment can transform non-transcribed MdMYB1-2 and MdMYB1-3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar 'Mutsu.' Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. 'Mutsu,' a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in 'Mutsu,' paper bagging-induced red pigmentation was due to the activation of nontranscribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5' upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5' upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

The effects of red- and blue-light irradiation at night on abscisic acid (ABA) synthesis and anthocyanin and sugar concentrations were examined in grape vines, which were grown in two different seasons. In grapes cultivated with early heating, the ABA concentrations were highest in blue-light-emitting diode (LED)-treated skin; however, those in grapes cultivated in the ordinary growing season were highest in red-LED-treated skin. The expressions of 9-cis-epoxycarotenoid dioxygenase (VvNCED1) and ABA 8'-hydroxylase (VvCYP707A1) were high in each treatment at veraison regardless of the growing season. In both seasons, anthocyanin concentrations were highest under the blue-LED treatment, followed by the red-LED treatment. The expressions of VlMYBA1-2, VlMYBA2, and VvUFGT coincided with anthocyanin concentrations. Sugar concentrations were increased by the blue-or red-LED treatment dependent on the growing season. The results suggest that blue- or red-LED irradiation at night may influence the ABA and anthocyanin metabolism including VvNCED1, VlMYBA1-2, and VlMYBA2 and sugar synthesis in grape berries, although the degree of the effects differs with the growing season.

In the Japanese pear (Pyrus pyrifolia Nakai) ` Kosui', three developmental stages of lateral flower buds have been proposed to occur during ecodormancy to the flowering phase, i. e. rapid enlargement, sprouting and flowering. Here, we report an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor gene, named pear EARLY BUD-BREAK (PpEBB), which was highly expressed during the rapid enlargement stage occurring prior to the onset of bud break in flower buds. Gene expression analysis revealed that PpEBB expression was dramatically increased during the rapid enlargement stage in three successive growing seasons. PpEBB transcript levels peaked 1 week prior to onset of bud break in ` Kosui' potted plants treated with hydrogen cyanamide or water under forcing conditions. Chromatin immunoprecipitation- quantitative PCR showed that higher levels of active histone modifications (trimethylation of the histone H3 tail at Lys4) in the 50-upstream and start codon regions of the PpEBB gene were associated with the induced expression level of PpEBB during the rapid enlargement stage. In addition, we provide evidence that PpEBB may interact with and regulate pear four D-type cyclin (PpCYCD3) genes during bud break in ` Kosui' lateral flower buds. PpEBB significantly increased the promoter activities of four PpCYCD3 genes in a dual-luciferase assay using tobacco leaves. Taken together, our findings uncovered aspects of the bud break regulatory mechanism in the Japanese pear and provided further evidence that the EBB family plays an important role in bud break in perennial plants.

Background: In woody perennial plants, including deciduous fruit trees, such as pear, endodormancy is a strategy for surviving the cold winter. A better understanding of the mechanism underlying the endodormancy phase transition is necessary for developing countermeasures against the effects of global warming. In this study, we analyzed the sRNAome of Japanese pear flower buds in endodormant and ecodormant stages over two seasons by implementing of RNA-seq and degradome-sequencing. Results: We identified 137 conserved or less conserved miRNAs and 50 pear-specific miRNAs. However, none of the conserved microRNAs or pear-specific miRNAs was differentially expressed between endodormancy and ecodormancy stages. On the contrast, 1540 of 218,050 loci that produced sRNAs were differentially expressed between endodormancy and ecodormancy, suggesting their potential roles on the phase transition from endodormancy to ecodomancy. We also characterized a multifunctional miRNA precursor MIR168, which produces two functional miR168 transcripts, namely miR168.1 and miR168.2; cleavage events were predominantly mediated by the non-conserved variant miR168.2 rather than the conserved variant miR168.1. Finally, we showed that a TAS3 trans-acting siRNA triggered phased siRNA within the ORF of one of its target genes, AUXIN RESPONSE FACTOR 4, via the analysis of phased siRNA loci, indicating that siRNAs are able to trigger phased siRNAs in pear. Conclusion: We analyzed the sRNAome of pear flower bud during dormant phase transition. Our work described the sRNA profiles of pear winter buds during dormant phase transition, showing that dormancy release is a highly coordinated physiological process involving the regulation of sRNAs.

The regulatory mechanisms underlying bud breaking (scale leaf elongation) and flowering in the lateral flower buds of Japanese pear (Pyrus pyrifolia Nakai 'Kosui') are unknown. To more fully characterize these processes, we treated pear trees with different amounts of chilling initiated at different times. Chilling for 900aEuro...h at 6aEuro...A degrees C always induced bud breaking (scale elongation in a parts per thousand yen70% lateral flower bud) when provided between October and February, whereas chilling provided earlier (between October and December) was less effective on flowering (floret growth and development) than later chilling and the flowering rate increased with longer chilling durations. During chilling, the expression of pear DAMs (PpMADS13-1, 13-2 and 13-3) in lateral flower buds decreased as chilling accumulated irrespective of the timing of chilling. In addition, pear TFL1 (PpTFL1-1a) in the lateral flower buds was expressed at higher levels when the time interval for chilling was earlier. On the other hand, during forcing at 15aEuro...A degrees C after chilling, the expression pattern of all three PpMADS13 genes was similar among the treatments, and the expression levels seemed lower in the treatment where scale leaves of the lateral flower bud elongated faster, whereas pear FT (PpFT2a) was expressed at higher levels in the buds whose flower clusters elongated more vigorously during forcing. From these results, we infer that flowering time may be mediated via the balance of flowering-related genes FT and TFL1, whereas bud breaking may be regulated via the DAM genes in Japanese pear.

The effects of the application of the jasmonic acid (JA) derivative n-propyl dihydrojasmonate (PDJ) on lesion diameter, endogenous abscisic acid (ABA), phaseic acid (PA), JA, aroma volatiles, and antioxidant activity were investigated in grapes infected by the pathogen Glomerella cingulata. The berries were immersed in 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 degrees C for 12 days. Lesion diameters from the pathogen decreased upon PDJ application. Endogenous ABA and JA concentrations increased in inoculated 'Kyoho' grape berries (Vitis Iabrusca x Vitisvinifera). PDJ application before inoculation increased ABA and PA concentrations, perhaps through the activation of Vitis vinifera and VvCYP707A1 genes. The results suggest that the synergistic effect of JA and ABA may play a role in the defense mechanism against pathogen infection in grape berries. In addition, PDJ application generally increased the production of aldehydes, esters, and terpenes. These volatiles may also be associated with resistance to pathogen infection. (C) 2015 Elsevier B.V. All rights reserved.

We periodically investigated the lateral flower bud morphology of 1-year shoots of 'Kosui' pears (Pyrus pyrifolia Nakai) in terms of dormancy progression, using magnetic resonance imaging. The size of flower buds did not change significantly during endodormancy, but rapid enlargement took place at the end of the ecodormancy stage. To gain insight into the physiological status during this period, we analyzed gene expression related to cell cycle-, cell expansion-and water channel-related genes, namely cyclin (CYC), expansin (EXPA), tonoplast intrinsic proteins (TIP) and plasma membrane intrinsic proteins (PIP). Constant but low expression of pear cyclin genes (PpCYCD3s) was observed in the transition phase from endodormancy to ecodormancy. The expression levels of PpCYCD3s were consistent with few changes in flower bud size, but up-regulated before the sprouting stage. In contrast, the expression of pear expansin and water channel-related genes (PpEXPA2, PpPIP2A, PpPIP2B, PpI delta TIP1A and PpI delta TIP1B) were low until onset of the rapid enlargement stage of flower buds. However, expression of these genes rapidly increased during sprouting along with a gradual increase of free water content in the floral primordia of buds. Taken together, these results suggest that flower bud size tends to stay constant until the endodormancy phase transition. Rapid enlargement of flower buds observed in March is partly due to the enhancement of the cell cycle. Then, sprouting takes place concomitant with the increase in cell expansion and free water movement.

Main conclusion Our studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

To investigate the effects of light quality (wavelength) on shoot elongation and flower-bud formation in Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai), we treated 1-year-old trees with the following: (i) 8 h sunlight + 16 h dark (SD); (ii) 8 h sunlight + 16 h red light (LD(SD + R)); or (iii) 8 h sunlight + 16 h far-red (FR) light (LD(SD + FR)) daily for 4 months from early April (before the spring flush) until early August in 2009 and 2010. In both years, shoot elongation stopped earlier in the LD(SD + FR) treatment than in the SD and LD(SD + R) treatments. After 4 months of treatments, 21% (2009) or 40% (2010) of LD(SD + FR)-treated trees formed flower buds in the shoot apices, whereas all the shoot apices from SD or LD(SD + R)-treated plants remained vegetative. With an additional experiment conducted in 2012, we confirmed that FR light at 730 nm was the most efficacious wavelength to induce flower-bud formation. Reverse transcription-quantitative polymerase chain reaction revealed that the expression of two floral meristem identity gene orthologues, LEAFY (PpLFY2a) and APETALA1 (PpMADS2-1a), were up-regulated in the shoot apex of LD(SD + FR). In contrast, the expression of a flowering repressor gene, TERMINAL FLOWER 1 (PpTFL1-1a, PpTFL1-2a), was down-regulated. In addition, expression of an orthologue of the flower-promoting gene FLOWERING LOCUS T (PpFT1a) was positively correlated with flower-bud formation, although the expression of another orthologue, PpFT2a, was negatively correlated with shoot growth. Biologically active cytokinin and gibberellic acid concentrations in shoot apices were reduced with LD(SD + FR) treatment. Taken together, our results indicate that pear plants are able to regulate flowering in response to the R : FR ratio. Furthermore, LD(SD + FR) treatment terminated shoot elongation and subsequent flower-bud formation in the shoot apex at an earlier time, possibly by influencing the expression of flowering-related genes and modifying plant hormone concentrations.

We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as beta-Tubulin, Histone H3, Actin, Elongation factor-1 alpha, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by Delta Ct method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. Delta Ct method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by Delta Ct method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out reliable reference genes.

We cloned 10 Japanese pear ( Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA ( AP). 1-like PpMADS3-1 was FRUITFULL ( FUL)/. SQUAMOSA ( SQUA)-like PpMADS4-1 was AGAMOUS-like ( AGL). 6 PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS ( SOC)-like PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA ( SEP)-like while PpMADS15-1 was AGL/. SHATTERPROOF ( SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class ( PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class ( PpMADS15-1), E-class ( PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class ( PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening. © 2013 Elsevier B.V.

Suppression subtractive hybridization (SSH) was employed to identify candidate genes involved in red coloration in apple peel with the ultraviolet (UV)-B-treated 'Mutsu'. After reverse Northern blotting verification, nearly 80 clones were successfully sequenced. Large portions of the expressed sequence tags (ESTs) are well characterized anthocyanin biosynthesis-related genes, such as chalcone synthase (11A5), flavonol synthase (12F3), anthocyanidin synthase (11H5) and UDP-glycosyl transferase (14A12) whose presence proved the success of SSH. Eight ESTs were selected for quantitative real-time polymerase chain reaction analysis and their expressions were all elevated in 'Induction', further confirming the reliability of the SSH library. One EST, 11F4 (CONSTITUTIVE PHOTOMORPHOGENIC 1: COP1) with putative function in light signal relay was further analyzed in 'Mutsu' and 'Tsugaru', along with MdHY5 (ELONGATED HYPOCOTYL 5: the downstream target of COP1), MdMYB22 (a possible flavonol-specific activator under the regulation of HY5, belonging to the SG7/PRODUCTION OF FLAVONOL GLYCOSIDES family) and MdMYBA. Results showed that MdCOP1, MdHY5, MdMYB22 and MdMYBA were all UV-B inducible genes and anthocyanin accumulation occurred after their increased expressions. Moreover, their expressions and anthocyanin content were enhanced under UV-B plus 17°C treatment. The presence of G box, a known consensus binding site of HY5, in the MdMYBA promoter region implicated that it could be regulated by MdHY5, which was verified by the result of the yeast one-hybrid analysis. Our data suggested that UV-B irradiation would induce the utmost upstream light signaling factor, MdCOP1, which activates MdHY5 signaling by binding to the promoter regions of MdMYBs, and finally leads to the red coloration of apple peels. © Physiologia Plantarum 2012.

The transcriptomes of endodormant and ecodormant Japanese pear (Pyrus pyrifolia Nakai 'Kosui') flower buds were analyzed using RNA-seq technology and compared. Among de novo assembly of 114,191 unigenes, 76,995 unigenes were successfully annotated by BLAST searches against various databases. Gene Ontology (GO) enrichment analysis revealed that oxidoreductases were enriched in the molecular function category, a result consistent with previous observations of notable changes in hydrogen peroxide concentration during endodormancy release. In the GO categories related to biological process, the abundance of DNA methylation-related gene transcripts also significantly changed during endodormancy release, indicating the involvement of epigenetic regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed the changes in transcript abundance of genes involved in the metabolism of various phytohormones. Genes for both ABA and gibberellin biosynthesis were down-regulated, whereas the genes encoding their degradation enzymes were up-regulated during endodormancy release. In the ethylene pathway, 1-aminocyclopropane-1-carboxylate synthase (ACS), a gene encoding the rate-limiting enzyme for ethylene biosynthesis, was induced towards endodormancy release. All of these results indicated the involvement of phytohormones in endodormancy release. Furthermore, the expression of dormancy-associated MADS-box (DAM) genes was down-regulated concomitant with endodormancy release, although changes in the abundance of these gene transcripts were not as significant as those identified by transcriptome analysis. Consequently, characterization of the Japanese pear transcriptome during the transition from endormancy to ecodormancy will provide researchers with useful information for data mining and will facilitate further experiments on endodormancy especially in rosaceae fruit trees. © 2013 The Author.

We isolated three dormancy-associated MADS-box (DAM) genes (MADS13-1, MADS13-2 and MADS13-3) and showed regulated expression concomitant with endodormancy establishment and release in the leaf buds of Japanese pear 'Kosui'. Comparative analysis between 'Kosui' and Taiwanese pear TP-85-119 ('Hengshanli'), a less dormant pear cultivar, showed reduction of MADS13-1 expression level in 'Hengshanli' earlier than in 'Kosui' towards endodormancy release, suggesting the possible relationship between chilling requirement and MADS13-1 expression. Application of hydrogen cyanamide accelerated endodormancy release with a reduction in MADS13 expression, whereas heat treatment in autumn inhibited endodormancy establishment without induction of MADS13 expression, indicating a close relationship between the MADS13 expression pattern and endodormancy phase transitions. Moreover, both the cis-acting regulatory elements and the methylation status in the 5' upstream region of the MADS13-1 gene were not largely different between 'Kosui' and 'Hengshanli'. Genomic structures of MADS13-1 from 'Kosui' and 'Hengshanli' revealed a 3218 bp insertion in the first intron of 'Hengshanli' that might be ascribed to the lower expression of MADS13-1tw; however, this insertion was also found in pear genotypes with a high chilling requirement. These results indicated that the low expression of MADS13-1 in 'Hengshanli' towards endodormancy release could not be explained by the identified cis-acting regulatory elements, the methylation status of the putative promoter or by intron insertion.

We analyzed the transcriptome of Japanese pear (Pyrus pyrifolia Nakai) 'Kousui' leaf buds during the dormancy transitional phases using a 10K cDNA microarray. Leaf buds were collected in October (deep endodormancy) and February (ecodormancy). Over 1000 genes were differentially expressed (P<0.05, >= 2-fold change). Most of these genes are functionally related to chloroplast or plastids, electron transport or energy pathways, response to stimuli or stress, signal transduction or transcription. Of these genes detected in this study, 76 and 22 were highly induced (>= 10-fold change) during endodormancy and ecodormancy phases, respectively. Comparison of selected genes between 'Kousui' and the less-dormant Taiwanese pear 'Hengshanli' (TP-85-119) identified two novel transcription factors (NAC and PRR) whose expression varied concomitantly with the dormancy phase changes. Further studies involving the genes uncovered from this microarray analysis is expected to provide new insights into the molecular events underlying their physiological role in plants including dormancy breaking for stable fruit set. (C) 2012 Elsevier B.V. All rights reserved.