Bud endodormancy in deciduous fruit trees: advances and prospects.

Bud endodormancy is a complex physiological process that is indispensable for the survival, growth, and development of deciduous perennial plants. The timely release of endodormancy is essential for flowering and fruit production of deciduous fruit trees. A better understanding of the mechanism of endodormancy will be of great help in the artificial regulation of endodormancy to cope with climate change and in creating new cultivars with different chilling requirements. Studies in poplar have clarified the mechanism of vegetative bud endodormancy, but the endodormancy of floral buds in fruit trees needs further study. In this review, we focus on the molecular regulation of endodormancy induction, maintenance and release in floral buds of deciduous fruit trees. We also describe recent advances in quantitative trait loci analysis of chilling requirements in fruit trees. We discuss phytohormones, epigenetic regulation, and the detailed molecular network controlling endodormancy, centered on SHORT VEGETATIVE PHASE (SVP) and Dormancy-associated MADS-box (DAM) genes during endodormancy maintenance and release. Combining previous studies and our observations, we propose a regulatory model for bud endodormancy and offer some perspectives for the future.

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.

Embryogenic callus induction and Agrobacterium-mediated genetic transformation of 'Shine Muscat' grape.

We established a method for embryogenic callus induction and highly efficient Agrobacterium-mediated genetic transformation of a table grape cultivar 'Shine Muscat' (Vitis labruscana). Embryogenic calli were induced using flower bud filaments from a dormant cane. Agrobacterium strain LBA4404 harboring the binary plasmid pBin19-sgfp, which contains the sgfp and nptII genes, was used to infect embryogenic calli. Infected calli were selected on 1/2 MS medium containing 5% maltose and 2% agar supplemented with 15 mg l-1 kanamycin. Efficiency of transformation of regenerated plants reached nearly 100% as determined by PCR and Southern blot analyses. The developed method will open a new avenue for genome editing of 'Shine Muscat' and contribute to the advancement of grape breeding.

Insertion of a transposon-like sequence in the 5'-flanking region of the YUCCA gene causes the stony hard phenotype.

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

Repression of TERMINAL FLOWER1 primarily mediates floral induction in pear (Pyrus pyrifolia Nakai) concomitant with change in gene expression of plant hormone-related genes and transcription factors.

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.

Dormancy-Associated MADS-Box (DAM) and the Abscisic Acid Pathway Regulate Pear Endodormancy Through a Feedback Mechanism.

In the pear 'Kosui' (Pyrus pyrifolia Nakai), the dormancy-associated 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 8'-hydroxylase) and signaling genes (type 2C protein phosphatase genes and SNF1-related 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.

CRISPR/Cas9-mediated targeted mutagenesis in grape.

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Epigenetic regulation of MdMYB1 is associated with paper bagging-induced red pigmentation of apples.

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 non-transcribed 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.

Involvement of EARLY BUD-BREAK, an AP2/ERF Transcription Factor Gene, in Bud Break in Japanese Pear (Pyrus pyrifolia Nakai) Lateral Flower Buds: Expression, Histone Modifications and Possible Target Genes.

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 5'-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.

Small RNA and PARE sequencing in flower bud reveal the involvement of sRNAs in endodormancy release of Japanese pear (Pyrus pyrifolia 'Kosui').

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.

Physiological differences between bud breaking and flowering after dormancy completion revealed by DAM and FT/TFL1 expression in Japanese pear (Pyrus pyrifolia).

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 ∼900 h at 6 °C always induced bud breaking (scale elongation in ≥70% 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 15 °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 crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype.

BACKGROUND: Red coloration of fruit skin is one of the most important traits in peach (Prunus persica), and it is mainly due to the accumulation of anthocyanins. Three MYB10 genes, PpMYB10.1, PpMYB10.2, and PpMYB10.3, have been reported as important regulators of red coloration and anthocyanin biosynthesis in peach fruit. In this study, contribution of PpMYB10.1/2/3 to anthocyanin accumulation in the fruit skin was investigated in the Japanese peach cultivars, white-skinned 'Mochizuki' and red-skinned 'Akatsuki'. We then investigated the relationships between allelic type of PpMYB10.1 and skin color phenotype in 23 Japanese peach cultivars for future establishment of DNA-marker. RESULTS: During the fruit development of 'Mochizuki' and 'Akatsuki', anthocyanin accumulation was observed only in the skin of red 'Akatsuki' fruit in the late ripening stages concomitant with high mRNA levels of the last step gene leading to anthocyanin accumulation, UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). This was also correlated with the expression level of PpMYB10.1. Unlike PpMYB10.1, expression levels of PpMYB10.2/3 were low in the skin of both 'Mochizuki' and 'Akatsuki' throughout fruit development. Moreover, only PpMYB10.1 revealed expression levels associated with total anthocyanin accumulation in the leaves and flowers of 'Mochizuki' and 'Akatsuki'. Introduction of PpMYB10.1 into tobacco increased the expression of tobacco UFGT, resulting in higher anthocyanin accumulation and deeper red transgenic tobacco flowers; however, overexpression of PpMYB10.2/3 did not alter anthocyanin content and color of transgenic tobacco flowers when compared with wild-type flowers. Dual-luciferase assay showed that the co-infiltration of PpMYB10.1 with PpbHLH3 significantly increased the activity of PpUFGT promoter. We also found close relationships of two PpMYB10.1 allelic types, MYB10.1-1/MYB10.1-2, with the intensity of red skin coloration. CONCLUSION: We showed that PpMYB10.1 is a major regulator of anthocyanin accumulation in red-skinned peach and that it activates PpUFGT transcription. PpMYB10.2/3 may be involved in functions other than anthocyanin accumulation in peach. The peach cultivars having two MYB10.1-2 types resulted in the white skin color. By contrast, those with two MYB10.1-1 or MYB10.1-1/MYB10.1-2 types showed respective red or pale red skin color. These findings contribute to clarifying the molecular mechanisms of anthocyanin accumulation and generating gene-based markers linked to skin color phenotypes.

Polyamines function in stress tolerance: from synthesis to regulation.

Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity, and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine, spermidine, and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS) due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested.

Development of flower buds in the Japanese pear (Pyrus pyrifolia) from late autumn to early spring.

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, PpIδTIP1A and PpIδTIP1B) 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.

ICE1 of Poncirus trifoliata functions in cold tolerance by modulating polyamine levels through interacting with arginine decarboxylase.

ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix-loop-helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H2O2 and O2 (-), was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene.

Histone modification and signalling cascade of the dormancy-associated†MADS-box gene, PpMADS13-1, in Japanese pear (Pyrus pyrifolia) during endodormancy.

Dormancy-associated MADS-box (DAM) genes play an important role in endodormancy phase transition. We investigated histone modification in the DAM homolog (PpMADS13-1) from Japanese pear, via chromatin immunoprecipitation-quantitative PCR, to understand the mechanism behind the reduced expression of the PpMADS13-1 gene towards endodormancy release. Our results indicated that the reduction in the active histone mark by trimethylation of the histone H3 tail at lysine 4 contributed to the reduction of PpMADS13-1 expression towards endodormancy release. In contrast, the inactive histone mark by trimethylation of the histone H3 tail at lysine 27 in PpMADS13-1 locus was quite low, and these levels were more similar to a negative control [normal mouse immunoglobulin G (IgG)] than to a positive control (AGAMOUS) in endodormancy phase transition. The loss of histone variant H2A.Z also coincided with the down-regulation of PpMADS13-1. Subsequently, we investigated the PpMADS13-1 signalling cascade and found that PpCBF2, a pear C-repeated binding factor, regulated PpMADS13-1 expression via interaction of PpCBF2 with the 5'-upstream region of PpMADS13-1 by transient reporter assay. Furthermore, transient reporter assay confirmed no interaction between the PpMADS13-1 protein and the pear FLOWERING LOCUS T genes. Taken together, our results enhance understanding of the molecular mechanisms underlying endodormancy phase transition in Japanese pear.

An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis.

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.

Effect of extending the photoperiod with low-intensity red or far-red light on the timing of shoot elongation and flower-bud formation of 1-year-old Japanese pear (Pyrus pyrifolia).

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.