Takaya Moriguchi

Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin

Five genes of anthocyanin biosynthetic enzymes, chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), anthocyanidin synthase (ANS; EC 1.14.11.X), and UDP glucose:flavonoid 3-O-glucosyltransferase (UFGluT; EC 2.4.1.X), were isolated, and their expression was investigated to elucidate the molecular mechanism for red coloration in apple 〚Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.〛 skin. In ‘Orin,’ a yellow apple cultivar, no significant levels of anthocyanin were detectable, whereas in ‘Jonathan’ and ‘Fuji,’ both red apple cultivars, anthocyanin concentrations increased during fruit development. At the ripe stage, the level of anthocyanin concentration was about three times higher in ‘Jonathan’ than in ‘Fuji.’ The accumulation of transcripts for the five genes was induced at the later developmental stages in all three cultivars. The levels for the expression of the five genes basically corresponded to the anthocyanin concentrations; that is, the induction of the genes in ‘Orin’ was less pronounced, and that in ‘Fuji’ and ‘Jonathan’ was notable, with much higher expression levels in ‘Jonathan’ than in ‘Fuji’. These results indicate that the five genes are coordinately expressed during fruit development and that their levels of expression are positively related to the degree of anthocyanin concentration. This is the first report that characterizes the relationship between the expression of anthocyanin biosynthetic genes and apple fruit coloration.

A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds

RNA isolation is a prerequisite to the study of gene expression at the molecular level and has an increasingly important role in physiological and genetic investigations of fruit trees. However, RNA isolation is difficult in fruit trees that contain large amounts of polysaccharides and polyphenol compounds. Until now, no commercial kit has been developed specifically for, the isolation of high-quality RNA from fruit trees. Furthermore, because of the large amounts of polysaccharides and polyphenol compounds, the common protocols for RNA isolation are tedious and usually result in poor yields when applied to kiwifruit, apples, and peaches. Here we describe a simple method for RNA islation from fruit trees. This procedure involves easy grinding by zirconia balls; washing the fruit samples before extraction; extraction of RNA; and removal of proteins, polyphenols, and polysaccharides by insoluble polyvinylpyrrolidone (PVP) and potassium acetate with ethanol. The protocol results in high-quality RNA, as evidenced by performing reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis.

Identification of a new expansin gene closely associated with peach fruit softening

Abstract Expansins are proteins that have been shown to contribute to fruit softening in tomato. However, expansins that have been correlated with loss of fruit firmness have not yet been identified in peach (Prunus persica (L.) Batsch). Along with the previously isolated PpExp1, two new expansin cDNAs, termed PpExp2 and PpExp3, were isolated from ripe peach fruit, and their mRNA expression patterns were characterized during fruit development and in other tissues, including the flower bud, leaf, and stem. All three expansins were detected in the fruit and not in the other tissues, but each showed differential patterns of mRNA accumulation during fruit development. The PpExp2 mRNA was constitutively expressed throughout fruit development but was abundant in Stage III, when the fruit expands exponentially and then matures. The PpExp1 and PpExp3 mRNAs were up-regulated at the onset of ripening, but PpExp1 was induced at an earlier stage. In order to identify the expansins whose expression correlates with the loss of peach fruit firmness, the mRNA expression levels of the three expansins were compared in the ripe fruit of the ‘Akatsuki’ and ‘Manami’ cultivars during postharvest storage. During storage, the ripe fruit of ‘Akatsuki’ rapidly softened as the level of ethylene increased significantly, while ‘Manami’ fruit remained firm and exhibited very low levels of ethylene production. The PpExp1 and PpExp2 mRNAs were constitutively detectable during the 8-day storage of both cultivars, whereas PpExp3 mRNA was detectable in ‘Akatsuki’ but hardly detectable in ‘Manami,’ suggesting that PpExp3 expression may be related to the changes in fruit firmness. To address the detailed role of PpExp3 in the loss of fruit firmness, the fruit of ‘Manami’ was treated by ethylene to artificially induce softening. The PpExp3 mRNA accumulation in the ethylene-treated ‘Manami’ was detectable and similar to that observed in ‘Akatsuki.’ These results show that, while several expansins show a general increase in expression levels during the later stages of fruit development, some isoforms show a greater association with softening than others. In this regard, PpExp3 is more likely to play a role in peach fruit softening than PpExp1 or PpExp2.

Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus

We isolated a cDNA clone encoding limonoid UDP‐glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Spermidine levels are implicated in heavy metal tolerance in a spermidine synthase overexpressing transgenic European pear by exerting antioxidant activities

To verify whether spermidine synthase (SPDS) can confer long-term multi-heavy metal tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple SPDS (MdSPDS1), as well as a wild type (WT) line, were subjected to stress using either CdCl2, PbCl2, ZnCl2, or a combination thereof. Based on either shoot height increment or fresh weight and morphological changes upon heavy metal stress, the performance of the transgenic line #32 was better than that of WT. Although SPDS expression levels and spermidine (Spd) contents in line #32 were higher than those in WT, possibly due to transgene (MdSPDS1) expression, no obvious inductions of SPDS expression and increases in Spd-content were observed by long-term stress treatments in both lines. When the glutathione (GSH) content was compared with or without stress in each line, GSH was significantly depleted in line #32 with stress, but not as much as in WT. The activities of glutathione reductase and superoxide dismutase and the content of malondialdehyde, an indicator for lipid peroxidation, changed upon stress toward a more favorable status for survival in line #32 than in WT. These antioxidant parameters were positively related to Spd-content. The accumulation of heavy metals tended to be less in line #32 than in WT except for Zn stress, and the Ca content showed an opposite trend. These results suggest that Spd-levels are implicated in enhanced heavy metal tolerance, possibly by exerting an antioxidant activity as well as by the properties of Spd per se including metal chelator.

Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits (Citrus unshiu Marc.)

Abstract The role of sucrose-phosphate synthase (SPS) in sucrose metabolism during citrus fruit development was investigated through sucrose content and enzyme activity in relation to the transcription of three SPS cDNA isoforms. The profiles of sucrose accumulation in edible tissue (juice sacs and segment epidermis) of satsuma mandarin (Citrus unshiu Marc.) agreed with those of SPS activity, that is, an increase in sucrose content accompanied high SPS activity. However, sucrose accumulation in edible tissue preceded the increase in SPS activity. The sucrose accumulation and the activity of SPS in peel tissue (albedo and flavedo) were lower than those in edible tissue. The expressions of three SPS genes (previously isolated and designated as CitSPS1, CitSPS2, and CitSPS3) were analyzed during the fruit developmental stage. Transcripts of the CitSPS1 in edible tissue were expressed at all stages analyzed and the transcript level was highest at the mature stage (223 days after flowering (DAF)). No transcripts of the CitSPS2 were found in edible tissue before 187 DAF, but thereafter transcripts were detected and increased until the mature stage. However, transcripts of CitSPS3 were undetected in edible tissue throughout the developmental stage. The expression of CitSPS1 in peel tissue was low in young fruit, then increased notably at 148 DAF, retaining its level until the mature stage. Transcripts of CitSPS2 in peel tissue showed the same pattern as that of edible tissue. The expression of CitSPS3 in peel tissue was detected at 148, 187 and 223 DAF, although its level was weak compared with CitSPS1 and CitSPS2. The activity of SPS in edible tissue paralleled the levels of transcription of CitSPS1 and CitSPS2, suggesting that the expression of these two genes might have an important role in determining the sucrose composition or accumulation in citrus fruit. In peel tissue, the patterns of SPS activity mostly agreed with the transcripts of CitSPS1, CitSPS2 and CitSPS3. These results also indicate that SPS genes are independently regulated, resulting in distinct temporal and spatial expression patterns.

Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.

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 β-Tubulin, Histone H3, Actin, Elongation factor-1α, 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 ΔCt 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. ΔCt 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 ΔCt 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.

Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase.

Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.

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

AbstractMain conclusionOur 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.

Effects of salt and osmotic stresses on free polyamine content and expression of polyamine biosynthetic genes in Vitis vinifera

Grape (Vitis vinifera L.) seedlings grown in vitro were treated with either 200 mM NaCl or 350 mM mannitol for 7 d. Both salinity and osmotic stress caused significant increase in electrolyte leakage. From the three commonly occurring free polyamines (PA), only conspicuous accumulation of putrescine was found in the NaCl-treated seedlings. Four PA biosynthetic genes encoding arginine decarboxylase (pVvADC), S-adenosylmethionine decarboxylase (pVvSAMDC), spermidine synthase (pVvSPDS) and spermine synthase (pVvSPMS) were successfully isolated. While induction of pVvADC was observed from the 1st day of salt treatment, pVvSAMDC and pVvSPMS were induced only at late stage of stress. As for expression levels of genes in the mannitol-treated seedling, either temporary (pVvADC at day 1) or late (pVvSPMS at days 5 and 7) induction was observed.