Liao

Unraveling the mechanism underlying the glycosylation and methylation of anthocyanins in peach

Diversification of anthocyanins in peach is attributed to glycosylation and methylation. Modification of anthocyanin plays an important role in increasing its stability in plants. Here, six anthocyanins were identified in peach (Prunus persica), and their structural diversity is attributed to glycosylation and methylation. Interestingly, peach is quite similar to the wild species Prunus ferganensis but differs from both Prunus davidiana and Prunus kansueasis in terms of anthocyanin composition in flowers. This indicates that peach is probably domesticated from P. ferganensis. Subsequently, genes responsible for both methylation and glycosylation of anthocyanins were identified, and their spatiotemporal expression results in different patterns of anthocyanin accumulation in flowers, leaves, and fruits. Two tandem-duplicated genes encoding flavonoid 3-O-glycosyltransferase (F3GT) in peach, PpUGT78A1 and PpUGT78A2, showed different activity toward anthocyanin, providing an example of divergent evolution of F3GT genes in plants. Two genes encoding anthocyanin O-methyltransferase (AOMT), PpAOMT1 and PpAOMT2, are expressed in leaves and flowers, but only PpAOMT2 is responsible for the O-methylation of anthocyanins at the 3′ position in peach. In addition, our study reveals a novel branch of UGT78 genes in plants that lack the highly conserved intron 2 of the UGT gene family, with a great variation of the amino acid residue at position 22 of the plant secondary product glycosyltransferase box. Our results not only provide insights into the mechanisms underlying anthocyanin glycosylation and methylation in peach but will also aid in future attempts to manipulate flavonoid biosynthesis in peach as well as in other plants.

Inactivation of a Gene Encoding Carotenoid Cleavage Dioxygenase (CCD4) Leads to Carotenoid-Based Yellow Coloration of Fruit Flesh and Leaf Midvein in Peach

Yellow fruit flesh color, resulting from the accumulation of carotenoids, is one of the most important commercial traits of peach. Yellow flesh is controlled by a single locus (Y), with white flesh dominant over yellow flesh. In this study, the Y locus was narrowed to a 2.6-cM interval flanked by two markers, SSRy and W2691. SSRy, which is located on the first exon of a gene encoding carotenoid cleavage dioxygenase (CCD4), was cosegregated with the Y locus in two peach F1 populations. RNA-Seq and qRT-PCR analysis revealed transcript level of CCD4 was consistent with carotenoid degradation in peach fruits. All these results suggest that CCD4 is responsible for white and yellow coloration of peach fruit flesh. In fruits of white-fleshed peach, carotenoids are synthesized but subsequently degraded into colorless compounds, leading to the formation of white color. CCD4 is likely to utilize β-carotene as the substrate in peach. Interestingly, CCD4 also controls white and yellow coloration of leaf midveins of peach. Moreover, LCYE was highly expressed in peach leaves, whereas its transcript was not detectable in fruits. This suggests the difference of carotenoid biosynthesis between peach fruits and leaves. Our study not only shows for the first time the pleiotropic effects of CCD4 gene in peach but also provides a morphological marker for easy selection of new peach cultivars with desirable white or yellow flesh colors.

Multiple R2R3-MYB Transcription Factors Involved in the Regulation of Anthocyanin Accumulation in Peach Flower

Anthocyanin accumulation is responsible for flower coloration in peach. Here, we report the identification and functional characterization of eight flavonoid-related R2R3-MYB transcription factors, designated PpMYB10.2, PpMYB9, PpMYBPA1, Peace, PpMYB17, PpMYB18, PpMYB19, and PpMYB20, respectively, in peach flower transcriptome. PpMYB10.2 and PpMYB9 are able to activate transcription of anthocyanin biosynthetic genes, whilst PpMYBPA1 and Peace have a strong activation on the promoters of proanthocyanin (PA) biosynthetic genes. PpMYB17-20 show a strong repressive effect on transcription of flavonoid pathway genes such as dihydroflavonol 4-reductase. These results indicate that anthocyanin accumulation in peach flower is coordinately regulated by a set of R2R3-MYB genes. In addition, PpMYB9 and PpMYB10.2 are closely related but separated into two groups, designated MYB9 and MYB10, respectively. PpMYB9 shows a strong activation on the PpUGT78A2 promoter, but with no effect on the promoter of PpUGT78B (commonly called PpUFGT in previous studies). In contrast, PpMYB10.2 is able to activate the PpUFGT promoter, but not for the PpUGT78A2 promoter. Unlike the MYB10 gene that is universally present in plants, the MYB9 gene is lost in most dicot species. Therefore, the PpMYB9 gene represents a novel group of anthocyanin-related MYB activators, which may have diverged in function from the MYB10 genes. Our study will aid in understanding the complex mechanism regulating floral pigmentation in peach and functional divergence of the R2R3-MYB gene family in plants.

Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

A gene encoding aluminum‐activated malate transporter (ALMT) was previously reported as a candidate for the Ma locus controlling acidity in apple (Malus × domestica Borkh.). In this study, we found that apple ALMT genes can be divided into three families and the Ma1 gene belongs to the ALMTII family. Duplication of ALMTII genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the Ma1 gene and its homologs in the ALMTII family and only the Ma1 gene is significantly associated with malic acid content. The Ma locus consists of two alleles, Ma1 and ma1. Ma1 resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, ma1 encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the Ma1Ma1 genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the Ma1 gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the Ma locus. This suggests that the Ma gene is not the only genetic determinant of fruit acidity in apple.

Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

A small indel mutation in an anthocyanin transporter causes variegated colouration of peach flowers

Highlight An anthocyanin transporter gene that contains a high frequency of non-transposable element-related mutations is responsible for variegated colouration of flowers in peach.

Constitutive Activation of an Anthocyanin Regulatory Gene PcMYB10.6 Is Related to Red Coloration in Purple-Foliage Plum

Cherry plum is a popular ornamental tree worldwide and most cultivars are selected for purple foliage. Here, we report the investigation of molecular mechanism underlying red pigmentation in purple-leaf plum ‘Ziyeli’ (Prunus cerasifera Ehrhar f. atropurpurea (Jacq.) Rehd.), which shows red color pigmentation in fruit (flesh and skin) and foliage. Six anthocyanin-activating MYB genes, designated PcMYB10.1 to PcMYB10.6, were isolated based on RNA-Seq data from leaves of cv. Ziyeli. Of these PcMYB10 genes, five (PcMYB10.1 through PcMYB10.5) show distinct spatial and temporal expression patterns, while the PcMYB10.6 gene is highly expressed in all the purple-coloured organs of cv. Ziyeli. Constitutive activation of PcMYB10.6 is closely related to red pigmentation in the leaf, fruit (flesh and skin), and sepal. However, the PcMYB10.6 activation cannot induce red pigmentation in the petal of cv. Ziyeli during late stages of flower development due to due to a lack of expression of PcUFGT. The inhibition of red pigmentation in the petal of cherry plum could be attributed to the high-level expression of PcANR that directs anthocyanidin flux to proanthocyanidin biosynthesis. In addition, PcMYB10.2 is highly expressed in fruit and sepal, but its expression cannot induce red pigmentation. This suggests the PcMYB10 gene family in cherry plum may have diverged in function and PcMYB10.2 plays little role in the regulation of red pigmentation. Our study provides for the first time an example of constitutive activation of an anthocyanin-activating MYB gene in Prunus although its underlying mechanism remains unclear.

Assessment of Sugar Components and Genes Involved in the Regulation of Sucrose Accumulation in Peach Fruit

Soluble sugar contents in mature fruits of 45 peach accessions were quantified using gas chromatography analysis. Sucrose is the predominant sugar in mature fruit, followed by glucose and fructose, which have similar concentrations. Overall, sucrose metabolism and accumulation are crucial determinants of sugar content in peach fruit, and there is a wide range of sucrose concentrations among peach genotypes. To understand the mechanisms regulating sucrose accumulation in peach fruit, expression profiles of genes involved in sucrose metabolism and transport were compared among four genotypes. Two sucrose-cleaving enzyme genes (SUS4 and NINV8), one gene involved in sucrose resynthesis (SPS3), and three sugar transporter genes (SUT2, SUT4, and TMT2) were prevalently expressed in peach fruit, and their expression levels are significantly correlated with sucrose accumulation. In contrast, the VAINV genes responsible for sucrose cleavage in the vacuole were weakly expressed in mature fruit, suggesting that the sucrose-cleaving reaction is not active in the vacuole of sink cells of mature peach fruit. This study suggests that sucrose accumulation in peach fruit involves the coordinated interaction of genes related to sucrose cleavage, resynthesis, and transport, which could be helpful for future peach breeding.

Variation of ascorbic acid concentration in fruits of cultivated and wild apples

Ascorbic acid (AsA) content in mature fruits of 457 apple accessions were measured, and a great variation in AsA concentration was detected. Wild fruits showed significantly higher level of AsA than cultivated fruits. Fruit AsA content was positively correlated with malic acid content, but negatively correlated with fruit weight and soluble solid content. Thus, the difference in AsA content between the wild and cultivated fruits could be attributed to an indirect consequence of human selection for larger fruit size, less acidity, and increased sweetness during apple domestication. Additionally, AsA concentration was extremely high in fruit at the juvenile stage, but dramatically decreased at the expanding and mature stages. The expression levels of three genes controlling AsA accumulation, MdGGP1, MdDHAR3-3, and MdNAT7-2, were significantly negatively correlated with AsA contents in fruits, suggesting a feedback regulation mechanism in AsA-related gene expression. Our results could be helpful for future apple breeding.