Yuncong Yao

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 CO2 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 CO2, 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 CO2 induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.

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.

The Balance of Expression of Dihydroflavonol 4-reductase and Flavonol Synthase Regulates Flavonoid Biosynthesis and Red Foliage Coloration in Crabapples

Red leaf color is an attractive trait of Malus families, including crabapple (Malus spp.); however, little is known about the molecular mechanisms that regulate the coloration. Dihydroflavonols are intermediates in the production of both colored anthocyanins and colorless flavonols, and this current study focused on the gene expression balance involved in the relative accumulation of these compounds in crabapple leaves. Levels of anthocyanins and the transcript abundances of the anthocyanin biosynthetic gene, dihydroflavonol 4-reductase (McDFR) and the flavonol biosynthetic gene, flavonol synthase (McFLS), were assessed during the leaf development in two crabapple cultivars, ‘Royalty’ and ‘Flame’. The concentrations of anthocyanins and flavonols correlated with leaf color and we propose that the expression of McDFR and McFLS influences their accumulation. Further studies showed that overexpression of McDFR, or silencing of McFLS, increased anthocyanin production, resulting in red-leaf and red fruit peel phenotypes. Conversely, elevated flavonol production and green phenotypes in crabapple leaves and apple peel were observed when McFLS was overexpressed or McDFR was silenced. These results suggest that the relative activities of McDFR and McFLS are important determinants of the red color of crabapple leaves, via the regulation of the metabolic fate of substrates that these enzymes have in common.

A Malus Crabapple Chalcone Synthase Gene, McCHS, Regulates Red Petal Color and Flavonoid Biosynthesis

Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

Low Medium pH Value Enhances Anthocyanin Accumulation in Malus Crabapple Leaves

Anthocyanin is a critical factor involved in coloration of plant tissues, but the mechanism how medium pH values affect anthocyanin accumulation in woody plants is unknown. We analyzed anthocyanin composition and the expression of elements encoding anthocyanin and flavonols biosynthesis underlying different medium pH values by using three different leave color type cultivars. HPLC analysis demonstrated that high medium pH values treatment induced a dramatic decrease in the concentration of cyaniding in crabapple leaves. Conversely, the high medium pH values induced up-regulation of the content of flavones and flavonols, suggesting that low pH treatment-induced anthocyanin accumulation. Quantitative real time PCR experiment showed the expression level of anthocyanidin synthase (McANS) and uridine diphosphate glucose flavonoid 3-O-glucosyltransferase (McUFGT) was up-regulated by low pH values treatment, and high medium pH value treatment up-regulate the transcription level of flavonol synthase (McFLS). Meanwhile, several MYB TFs have been suggested in the regulation of pH responses. These results strongly indicate that the low pH treatment-induced anthocyanin accumulation is mediated by the variation of mRNA transcription of the anthocyanin biosynthetic genes.

Photoperiod and shading regulate coloration and anthocyanin accumulation in the leaves of malus crabapples

In many ornamental plants, anthocyanin accumulation in leaves is a critical determinant of foliage coloration. However, the mechanism by which photoperiod and shading affect coloration and anthocyanin accumulation in the leaves of woody plants is less clear. By examining the leaves and calli of crabapple (Malus spp.) cultivars with different leaf color characteristics, we analyzed the foliage coloration, flavonoid contents, and expression levels of anthocyanin biosynthetic genes and the McMYB10 transcription factor under different photoperiods and shading treatments. The red color parameters, flavonoid contents, and expression levels of some anthocyanin-related genes in both the leaf and callus were generally higher under sun-exposure or long-day treatments compared to shading or short-day treatments. These data indicate that red color formation in cultivars with leaves that are naturally red requires a suitable photoperiod and light intensity; high light intensity or long-day conditions activate anthocyanin biosynthesis-related genes, leading to anthocyanin accumulation. Our results also indicate that the regulation of anthocyanin biosynthetic metabolism in response to light conditions differs between red-leafed and green-leafed cultivars. Taken together, our data reveal that photoperiod and light intensity are important in the regulation of pigmentation and anthocyanin accumulation in crabapple leaves and calli. Moreover, we provide a useful approach for studying and inducing the production of flavonoids by crabapple callus culture under certain photoperiods.

Promotion of flavonoid biosynthesis in leaves and calli of ornamental crabapple (Malus sp.) by high carbon to nitrogen ratios.

Flavonoids are secondary metabolites that play important roles in plant physiology. Despite numerous studies examined the effects of available carbon (C) or nitrogen (N) on flavonoid biosynthesis, the mechanism of C/N interactive effects on flavonoid metabolism is still unclear. In this study, we analyzed the composition of flavonoids and the expression levels of flavonoid-related genes in leaves and calli of crabapple (Malus sp.) cultivars with different leaf colors grown on media with different C/N ratios. Our results show that high C/N ratios induce anthocyanin pigmentation in leaves of the ever-red cultivar ‘Royalty’ and the spring-red cultivar ‘Prairifire,’ as well as in three types of calli derived from the ever-green cultivar ‘Spring Snow,’ but not in the leaves of the ever-green cultivar ‘Flame.’ This phenomenon therefore correlated with anthocyanin content in these different samples. In addition, high C/N ratios in the growth media resulted in an increase in the concentration of flavones and flavonols in the leaves of the three crabapple cultivars. The transcript levels of the general flavonoid pathway genes [from chalcone synthase (CHS) to uridine diphosphat-glucose: flavonoid 3-O-glycosyltransferase (UFGT) and flavonol synthase (FLS)] increased in response to high C/N ratios, and this in turn was correlated with the concentration of anthocyanins, flavones and flavonols in the leaves and calli. Expression of the late flavonoid/anthocyanin biosynthetic genes, anthocyanidin synthase (ANS), UFGT and FLS in particular, was more strongly influenced by C/N ratios than other structural genes, and the increased expression of the structural genes under high C/N ratios coincided with a coordinated increase in transcript levels of a MYB transcription factor, MYB10. These results are likely to be useful for future generation of plants with an optimized flavonoid/anthocyanin content or desirable organ coloration.

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.

McWRI1, a transcription factor of the AP2/SHEN family, regulates the biosynthesis of the cuticular waxes on the apple fruit surface under low temperature

Cuticular waxes of plant and organ surfaces play an important role in protecting plants from biotic and abiotic stress and extending the freshness, storage time and shelf life in the post-harvest agricultural products. WRI1, a transcription factor of AP2/SHEN families, had been found to trigger the related genes taking part in the biosynthesis of seed oil in many plants. But whether WRI1 is involved in the biosynthesis of the cuticular waxes on the Malus fruits surface has been unclear. We investigated the changes of wax composition and structure, the related genes and WRI1 expression on Malus asiatica Nakai and sieversii fruits with the low temperature treatments, found that low temperature induced the up-regulated expression of McWRI1, which promoted gene expression of McKCS, McLACs and McWAX in very-long-chain fatty acid biosynthesis pathway, resulting in the accumulation of alkanes component and alteration of wax structure on the fruit surface. Corresponding results were verified in McWRI1 silenced by VIGS, and WRI1 silenced down-regulated the related genes on two kinds of fruits, it caused the diversity alteration in content of some alkanes, fatty acid and ester component in two kinds of fruits. We further conducted Y1H assay to find that McWRI1 transcription factor activated the promoter of McKCS, McLAC and McWAX to regulate their expression. These results demonstrated that McWRI1 is involved in regulating the genes related synthesis of very long chain fatty acid on surface of apple fruits in storage process, providing a highlight for improvement of the modified atmosphere storage of apple fruits.