Lilac (Syringa oblata) genome provides insights into its evolution and molecular mechanism of petal color change.

Color change during flower opening is common; however, little is understood on the biochemical and molecular basis related. Lilac (Syringa oblata), a well-known woody ornamental plant with obvious petal color changes, is an ideal model. Here, we presented chromosome-scale genome assembly for lilac, resolved the flavonoids metabolism, and identified key genes and potential regulatory networks related to petal color change. The genome assembly is 1.05 Gb anchored onto 23 chromosomes, with a BUSCO score of 96.6%. Whole-genome duplication (WGD) event shared within Oleaceae was revealed. Metabolome quantification identified delphinidin-3-O-rutinoside (Dp3Ru) and cyanidin-3-O-rutinoside (Cy3Ru) as the major pigments; gene co-expression networks indicated WRKY an essential regulation factor at the early flowering stage, ERF more important in the color transition period (from violet to light nearly white), while the MBW complex participated in the entire process. Our results provide a foundation for functional study and molecular breeding in lilac.

Identification of leucoanthocyanidin reductase and anthocyanidin reductase genes involved in proanthocyanidin biosynthesis in Malus crabapple plants.

Proanthocyanidins (PAs) from plants are a nutritionally valuable component of the human diet and play important roles in defense against pests and diseases. PAs are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. The enzymes leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are involved in PA biosynthesis. The PA biosynthetic pathway has been characterized in several plant species, but the relationship between its expression and PA accumulation in Malus crabapple remains unclear. Here, we cloned the LAR genes MrLAR1, 2, and the ANR genes MrANR1, 2, from the red leaved Malus crabapple cultivar 'Royalty'. The contents of PAs and the expression levels of the LAR and ANR genes were investigated in different organs of the two crabapple cultivars. The transcript levels of two LAR genes and two ANR genes correlated with the contents of the catechin and epicatechin, which are proanthocyanidin precursors. Over-expression of the MrLAR1, 2 and MrANR1, 2 in tobacco (Nicotiana tabacum) promoted the accumulation of PAs, while transient silencing of their expression in crabapple resulted in reduced PA levels. In addition, a negative correlation between quercetin, anthocyanin, and PA biosynthesis was also found during crabapple leaf and fruit peel development. We also found that MrLAR1 and 2 may contribute to epicatechin biosynthesis. In summary, the LAR and ANR genes are critical factors in PA biosynthesis, and there is competition between the quercetin, anthocyanin, and PA biosynthetic pathways during leaf and fruit peel development in Malus crabapple.

McMYB10 Modulates the Expression of a Ubiquitin Ligase, McCOP1 During Leaf Coloration in Crabapple.

In higher plants, anthocyanins are protective secondary metabolites, which contribute to the color of leaves, stems, flowers, and fruits, and have been found to have an antioxidant role in human health. In this study, we determined the expression of McMYB10 and its specific E3 ubiquitin ligase, McCOP1, in crabapple leaves during the course of a day and in five leaf development stages. Interestingly, the results showed that the transcription level of McCOP1 genes was higher in daylight than at night, and the transcripts of McMYB10 presented a positive correlation with the transcription of McCOP1-1 and McCOP1-2 and anthocyanin accumulation in a crabapple cultivar with red-colored leaves. Several MYB transcription factor (TF) binding sites of the MYBCORE type were found in the McCOP1-1 and McCOP1-2 promoters, and we deduced that there may be a relationship between McMYB10 and McCOP1-1 and McCOP1-2 at the transcriptional level. Yeast one hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) demonstrated that the McMYB10 TF binds specifically to the promoter of McCOP1-1 and McCOP1-2. Furthermore, increased levels of McMYB10 promoted anthocyanin biosynthesis and the expression level of McCOP1-1 and McCOP1-2 in crabapple leaves during continuous light treatments, and overexpression or silencing of McMYB10 in crabapple leaves and apple fruits also result in an increase or decrease, respectively, in the expression of McCOP1-1 and McCOP1-2 and in anthocyanin biosynthesis. Our results reveal a new self-regulation mechanism in where McMYB10 modulates its own expression by activating McCOP1-1 and McCOP1-2 expression to promote ubiquitination of the McMYB10 protein by McCOP1.

Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars.

Anthocyanins are secondary metabolites in land plants that contribute to the colors of leaves and flowers, and are nutritionally valuable components of the human diet. The DFR gene plays an important role in the anthocyanin biosynthetic pathway. In this study, we investigated the regulation of DFR expression and in different Malus crabapple cultivars that show distinct patterns of leaf coloration, and how it influences leaf anthocyanin accumulation and coloration. Specifically, we studied the ever-red leaved cultivar 'Royalty', the ever-green leaved cultivar 'Flame' and the spring-red leaved cultivar 'Radiant'. RT-PCR analysis showed that the expression of McDFR1 correlated with the expression of a MYB transcription factor, McMYB10, and with anthocyanin accumulation. We isolated five McDFR1 promoter fragments from the three cultivars and identified four different fragments (F1-4) that were present either in several cultivars, or only in one. Yeast one-hybrid and electrophoretic mobility shift assay analyses showed that McMYB10 could bind to all the McDFR1 promoters, except McDFR1-Ra2. The F1, F2 and F3 fragments did not affect McMYB10 binding to the McDFR1 promoters; however, we found evidence that the F4 fragment suppressed binding, and that the MYBGAHV amino-acid sequence maybe an important cis-element for McMYB10 protein binding. This information has potential value for strategies to modify plant color through genetic transformation.

McMYB12 Transcription Factors Co-regulate Proanthocyanidin and Anthocyanin Biosynthesis in Malus Crabapple.

The flavonoid compounds, proanthocyanidins (PAs), protect plants from biotic stresses, contribute to the taste of many fruits, and are beneficial to human health in the form of dietary antioxidants. In this study, we functionally characterized two Malus crabapple R2R3-MYB transcription factors, McMYB12a and McMYB12b, which co-regulate PAs and anthocyanin biosynthesis. McMYB12a was shown to be mainly responsible for upregulating the expression of anthocyanin biosynthetic genes by binding to their promoters, but to be only partially responsible for regulating PAs biosynthetic genes. In contrast, McMYB12b showed preferential binding to the promoters of PAs biosynthetic genes. Overexpression of McMYB12a and McMYB12b in tobacco (Nicotiana tabacum) altered the expression of flavonoid biosynthetic genes and promoted the accumulation of PAs and anthocyanins in tobacco petals. Conversely, transient silencing their expression in crabapple plants, using a conserved gene region, resulted in reduced PAs and anthocyanin production a green leaf phenotype. Meanwhile, transient overexpression of the two genes and silenced McMYB12s in apple (Malus domestica) fruit had a similar effect as overexpression in tobacco and silenced in crabapple. This study reveals a new mechanism for the coordinated regulation of PAs and anthocyanin accumulation in crabapple leaves, which depends on an auto-regulatory balance involving McMYB12a and McMYB12b expression.

Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon.

A hypoxic environment is generally undesirable for most plants and stimulates anaerobic metabolism. It is a beneficial treatment, however, for the removal of astringency from persimmon to improve the fruit quality after harvest. High soluble tannins (SCTs) content is one of most important causes of astringency. High CO2 (95%) treatment effectively reduced SCTs in both "Mopan" and "Gongcheng-shuishi" persimmon fruit by causing increases in acetaldehyde. Using RNA-seq and realtime PCR, twelve ethylene response factor genes (DkERF11-22) were isolated and characterized, to determine those responsive to high CO2 treatment. Only two genes, DkERF19 and DkERF22, showed trans-activation effects on the promoters of deastringency-related genes pyruvate decarboxylase genes (DkPDC2 and DkPDC3) and the transcript levels of these genes was enhanced by hypoxia. Moreover, DkERF19 and the previously isolated DkERF9 had additive effects on activating the DkPDC2 promoter. Taken together, these results provide further evidence that transcriptome changes in the level of DkERF mRNAs regulate deastringency-related genes and their role in the mechanism of persimmon fruit deastringency is discussed.

Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency.

The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO(2) and ethylene treatments efficiently removed the astringency from 'Mopan' persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF-DkADH/DkPDC cascade is involved in regulating persimmon de-astringency.

Expression of ethylene response genes during persimmon fruit astringency removal.

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent 'Yangfeng' and astringent 'Mopan' persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO(2), had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in 'Mopan' persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO(2) induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.