Accumulation Of Proanthocyanidins Research Articles

Brassinolide inhibits flavonoid biosynthesis and red-flesh coloration via the MdBEH2.2-MdMYB60 complex in apple.

Flavonoid content, which is an important indicator of the nutritional value of fruits and vegetables, directly determines the marketability of many fruit crops, including apple (Malus domestica). Brassinosteroids (BRs) are steroid hormones that affect flavonoid biosynthesis in plants, but the underlying regulatory mechanism remains unclear. In this study, treatments with brassinolide (the most active BR) and brassinazole (a BR biosynthesis inhibitor) decreased and increased, respectively, the flavonoid, anthocyanin, and proanthocyanidin (PA) content in red-fleshed apple seedlings and calli. We subsequently demonstrated that a BZR (BRI1-EMS-suppressor (BES)/brassinazole-resistant) family transcription factor, MdBEH2.2, participates in BR-regulated flavonoid biosynthesis. Specifically, MdBEH2.2 inhibits the accumulation of flavonoids, anthocyanins, and PAs in apple seedlings; however, brassinazole treatment weakens the inhibitory effect. Additionally, we confirmed that a BR-induced MYB TF, MdMYB60, interacts with MdBEH2.2. The resulting MdBEH2.2-MdMYB60 complex further enhances the inhibitory effect of MdBEH2.2 or MdMYB60 on the transcription of flavonoid biosynthesis-related genes. These results indicate that brassinolide decreases flavonoid content through the MdBEH2.2-MdMYB60 regulatory module. Our findings further clarify the molecular mechanism mediating the regulation of flavonoid biosynthesis by BR signals in horticultural crops.

Spectral parameters of Robinia pseudoacacia L. seeds in technogenic conditions of urban environment

Robinia pseudoacacia L. (black locust) is an invasive species with high activity in alien flora of Ukraine. Invasive potential of plants in unfavorable ecological conditions is due to the presence of adaptive mechanisms. One of the criteria for the invasive potential of foreign plants is considered to be seed productivity. For plants of black locust note a high level of seed productivity. However, it remains to be seen what factors ensure the formation of viable seeds in extreme environments. Among the factors that ensure the formation of viable plant seeds, note the barrier function of the seed coat relative to biotic and abiotic environmental factors. The physiological role of this protective barrier is realized due to the specific properties and component composition of the surface tissues of seeds. Among the metabolites with protective properties localized in the seed coat, consider proanthocyanidins (condensed tannins), which are formed during the biosynthesis of phenolic compounds. To objectively assess changes in the accumulation of proanthocyanidins in the seed coat, it is necessary to use non-destructive methods, because the isolation of these compounds destroys their native polymer structure. The influence of technogenic pollution of Dnipro city on the reflectance, colorimetric and fluorescent characteristics of Robinia pseudoacacia L. seeds is investigated in the work. Mature seeds of black locust were collected at monitoring sites in the Botanical Garden of Oles Honchar Dnipro National University (ecologically favorable zone) and at some points of linear roadside plantations of one of the main highways. It was found that the chronic effect of aerogenic pollutants on black locust plants caused an increased accumulation of proanthocyanidins in the seed coat, which may be due to changes in the biosynthesis of these compounds at the stage of oxidative polymerization of flavonoid subunits. Markers of this reaction of plants are the change of position and increase of intensity of maxima in the reflectance spectra of visible region, increase of value of dominant wavelength, decrease of conditional purity of color tone and colorimetric coefficients, increase of intensity of maxima in seed fluorescence spectra. The protective effect of these adaptive changes is associated with the strengthening of the barrier function of the seed coat to adverse environmental factors to maintain the viability of the seeds under the negative impact of technogenic pollution.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit

SUMMARYProanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor‐associated amphiphilic repression‐like motif in the C‐terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non‐astringency in persimmon.

Jasmonate induces biosynthesis of anthocyanin and proanthocyanidin in apple by mediating the JAZ1-TRB1-MYB9 complex.

Jasmonate (JA) induces the biosynthesis of anthocyanin and proanthocyanidin. MdMYB9 is essential for modulating the accumulation of both anthocyanin and proanthocyanidin in apple, but the molecular mechanism for induction of anthocyanin and proanthocyanidin biosynthesis by JA is unclear. In this study, we discovered an apple telomere-binding protein (MdTRB1) to be the interacting protein of MdMYB9. A series of biological assays showed that MdTRB1 acted as a positive modulator of anthocyanin and proanthocyanidin accumulation, and is dependent on MdMYB9. MdTRB1 interacted with MdMYB9 and enhanced the activation activity of MdMYB9 to its downstream genes. In addition, we found that the JA signaling repressor MdJAZ1 interacted with MdTRB1 and interfered with the interaction between MdTRB1 and MdMYB9, therefore negatively modulating MdTRB1-promoted biosynthesis of anthocyanin and proanthocyanidin. These results show that the JAZ1-TRB1-MYB9 module dynamically modulates JA-mediated accumulation of anthocyanin and proanthocyanidin. Taken together, our data further expand the functional study of TRB1 and provide insights for further studies of the modulation of anthocyanin and proanthocyanidin biosynthesis by JA.

Postharvest Dehydration Temperature Modulates the Transcriptomic Programme and Flavonoid Profile of Grape Berries.

Raisins are a popular and nutritious snack that is produced through the dehydration of postharvest grape berries under high temperature (HT). However, the response of the endogenous metabolism of white grape varieties to postharvest dehydration under different temperature have not been fully elucidated to date. In this study, the white grape cultivar ‘Xiangfei’ was chosen to investigate the effect of dehydration at 50 °C, 40 °C, and 30 °C on the transcriptomic programme and metabolite profiles of grape berries. Postharvest dehydration promoted the accumulation of soluble sugar components and organic acids in berries. The content of gallic acid and its derivatives increased during the dehydration process and the temperature of 40 °C was the optimal for flavonoids and proanthocyanidins accumulation. High-temperature dehydration stress might promote the accumulation of gallic acid by increasing the expression levels of their biosynthesis related genes and regulating the production of NADP+ and NADPH. Compared with that at 30 °C, dehydration at 40 °C accelerated the transcription programme of 7654 genes and induced the continuous upregulation of genes related to the heat stress response and redox homeostasis in each stage. The results of this study indicate that an appropriate dehydration temperature should be selected and applied when producing polyphenols-rich raisins.

Cork cells in cork oak periderms undergo programmed cell death and proanthocyanidin deposition.

Vascular plants with secondary growth develop a periderm mostly composed of dead suberized cork cells to face environmental hostile conditions. Cork oak has a highly active and long-living phellogen forming a remarkably thick periderm that is periodically debarked for industrial purposes. This wounding originates the quick formation of a new traumatic periderm, making cork oak an exceptional model to study the first periderm differentiation during normal development in young sprigs and traumatic (wound) periderm formation after debarking. Here, we studied the poorly known first periderm differentiation steps that involve cell wall suberization, polyphenolic accumulation and programmed cell death (PCD) by combining transmission electron microscopy, histochemical and molecular methods in periderms from young sprigs. These processes were further compared with traumatic periderms formed after wounding using molecular and histochemical techniques, such as the polyphenolic accumulation. In the first periderms from young sprigs, four distinct differentiation stages were defined according to the presence of PCD morphological features. First young and traumatic periderms showed an upregulation of genes related to suberin biosynthesis, proanthocyanidins biosynthesis and transport, autophagy, and PCD. Traumatic periderms revealed an overall upregulation of these genes, likely resulting from ontogeny differences and distinct phellogen origin associated with a faster metabolism, highlighting the impact of wounding on phellogen activity after debarking. First periderms from young sprigs showed gradual accumulation of proanthocyanidins in the vacuoles throughout PCD stages until total filled lumens, whereas in traumatic periderms, these compounds were found cell wall linked in already empty cells. This work enabled a comprehensive overview of the cork cells differentiation processes contributing to deepening the knowledge of the fundamental ontogenic program of this protective tissue, which is also a unique forest product, constituting the basis of a sustainable and profitable industry.

The MdHY5-MdWRKY41-MdMYB transcription factor cascade regulates the anthocyanin and proanthocyanidin biosynthesis in red-fleshed apple

Red-fleshed apple fruits are popular because of their high flavonoid content. Although MdMYB10 and its homologs have been identified as crucial regulators of the fruit coloring process, other transcription factors (TFs) contributing to the differences in flesh coloration have not been fully characterized. In this study, we investigated the regulatory effects of MdWRKY41 on anthocyanin and proanthocyanidin (PA) synthesis in red-fleshed apples. The overexpression of MdWRKY41 in red-fleshed apple calli inhibited anthocyanin and PA accumulation by downregulating the expression of a MYB TF gene (MdMYB12) and specific structural genes (MdLAR, MdUFGT, and MdANR). Furthermore, MdWRKY41 was shown to interact with MdMYB16 to form a complex that can further suppress MdANR and MdUFGT expression. Interestingly, MdWRKY41 was targeted by the photoresponse factor MdHY5 and inhibited its transcription. Overall, our findings provide insights into a novel MdHY5-MdWRKY41-MdMYB regulatory module influencing anthocyanin and PA synthesis in red-fleshed apple fruits.

Responses of Lotus corniculatus to environmental change. 4: Root carbohydrate levels at defoliation and regrowth climatic conditions are major drivers of phenolic content and forage quality.

Differential accumulation of root carbohydrates at defoliation have a higher impact than regrowth environmental conditions on the phenolic content and feed quality of the perennial forage legume Lotus corniculatus. The unpredictable nature of proanthocyanidin (condensed tannin) accumulation in regrowth vegetation of the perennial forage legume Lotus corniculatus represents a dilemma to the wider use of this species in agriculture, and a potential problem in the nutritional ecology of some terrestrial herbivores, as variable condensed tannin levels can result in either beneficial or detrimental effects on animal nutrition. However, the source of this variation has not been extensively explored. High levels of carbon allocation to roots during low-temperature preconditioning of clonal plants were found to significantly increase condensed tannin and flavonol levels in regrowth foliage, while low levels of carbon allocation to roots during periods of high-temperature preconditioning significantly decreased condensed tannin and flavonol levels. Phenolic accumulation and tissue digestibility were also differentially affected by regrowth of these defoliated plants at high CO2 concentrations and by drought. Lower rates of digestion generally paralleled increases in tannin levels in regrowth leaves under the different environmental conditions, with rates of digestion falling in high tannin plants, despite correspondingly higher levels of leaf carbohydrates. Differential accumulation of root carbohydrates between seasons and years may therefore explain some of the variability found in the nutritional quality of the forage of this species.

Transcription Factor VviMYB86 Oppositely Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Grape Berries.

Proanthocyanidins (PAs) and anthocyanins are two vital groups of flavonoid compounds for grape berries and red wines. Several transcription factors (TFs) have been identified to be involved in regulating PA and anthocyanin biosynthesis in grape berries. However, research on TFs with different regulatory mechanisms for these two biosynthesis branches in grapes remains limited. In this study, we identified an R2R3-MYB TF, VviMYB86, whose spatiotemporal gene expression pattern in grape berries coincided well with PA accumulation but contrasted with anthocyanin synthesis. Both in vivo and in vitro experiments verified that VviMYB86 positively regulated PA biosynthesis, primarily by upregulating the expression of the two leucoanthocyanidin reductase (LAR) genes in the Arabidopsis protoplast system, as well as in VviMYB86-overexpressing grape callus cultured under 24 h of darkness. Moreover, VviMYB86 was observed to repress the anthocyanin biosynthesis branch in grapes by downregulating the transcript levels of VviANS and VviUFGT. Overall, VviMYB86 is indicated to have a broad effect on flavonoid synthesis in grape berries. The results of this study will help elucidate the regulatory mechanism governing the expression of the two LAR genes in grape berries and provide new insights into the regulation of PA and anthocyanin biosynthesis in grape berries.

MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon.

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

Comprehensive Analysis of Metabolic Fluxes from Leucoanthocyanins to Anthocyanins and Proanthocyanidins (PAs).

Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo. The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro. Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana, EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa. In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.

Transcription Factors Rc and OsVP1 Coordinately Regulate Preharvest Sprouting Tolerance in Red Pericarp Rice.

Red pericarp associates with seed dormancy or preharvest sprouting (PHS) tolerance in crops. To identify this association's molecular mechanism, a PHS mutant Osviviparous1 (Osvp1) was characterized in rice and crossed with Kasalath, a red pericarp cultivar with Rc (red coleoptiles) genotype. Among the dehulled seeds of F2 progenies, RcRcvp1vp1 seeds performed a lower PHS rate than rcrcvp1vp1 seeds and showed shallower pigmentation than RcRcVP1VP1 seeds. Kasalath and SL9 (an RcRcVP1VP1 substitution line with Nipponbare background) showed more ABA sensitivity than the Nipponbare (rcrcVP1VP1) by the germination assay, and the transcriptional abundance of ABA signal genes OsABI2, OsSnRK2, OsVP1, ABI5, and especially OsVP1 increased in the red pericarp line SL9. Moreover, OsVP1 can directly bind Rc (bHLH) promoter by yeast one-hybrid, which activates Rc and OsLAR expression in red pericarp rice. Furthermore, a luciferase complementation imaging assay showed that OsVP1 interacts with transcriptions factors Rc and OsC1. These results indicate that OsVP1 promotes proanthocyanidin accumulation through the interaction among OsVP1, Rc, and OsC1 and then increases the plant's ABA sensitivity and PHS resistance.

Advances in Biosynthesis and Biological Functions of Proanthocyanidins in Horticultural Plants.

Proanthocyanidins are colorless flavonoid polymers condensed from flavan-3-ol units. They are essential secondary plant metabolites that contribute to the nutritional value and sensory quality of many fruits and the related processed products. Mounting evidence has shown that the accumulation of proanthocyanidins is associated with the resistance of plants against a broad spectrum of abiotic and biotic stress conditions. The biosynthesis of proanthocyanidins has been examined extensively, allowing for identifying and characterizing the key regulators controlling the biosynthetic pathway in many plants. New findings revealed that these specific regulators were involved in the proanthocyanidins biosynthetic network in response to various environmental conditions. This paper reviews the current knowledge regarding the control of key regulators in the underlying proanthocyanidins biosynthetic and molecular mechanisms in response to environmental stress. Furthermore, it discusses the directions for future research on the metabolic engineering of proanthocyanidins production to improve food and fruit crop quality.

FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat.

FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.

Postharvest seed coat darkening in pinto bean (Phaseolus vulgaris) is regulated by Psd , an allele of the basic helix-loop-helix transcription factor P.

Postharvest seed coat darkening in pinto bean is an undesirable trait that reduces the market value of the stored crop. Regular darkening (RD) pintos darken faster after harvest and accumulate higher level of proanthocyanidins (PAs) compared to slow darkening (SD) cultivars. Although the markers cosegregating with the SD trait have been known for some time, the SLOW DARKENING (Sd) gene identity had not been proven.Here, we identified Psd as a candidate for controlling the trait. Genetic complementation, transcript abundance, metabolite analysis, and inheritance study confirmed that Psd is the Sd gene. Psd is another allele of the P (Pigment) gene, whose loss-of-function alleles result in a white seed coat. Psd encodes a bHLH transcription factor with two transcript variants but only one is involved in PA biosynthesis. An additional glutamate residue in the activation domain, and/or an arginine to histidine substitution in the bHLH domain of the Psd-1 transcript in the SD cultivar is likely responsible for the reduced activity of this allele compared to the allele in a RD cultivar, leading to reduced PA accumulation.Overall, we demonstrate that a novel allele of P, Psd , is responsible for the SD phenotype, and describe the development of new, gene-specific, markers that could be utilized in breeding to resolve an economically important issue for bean producers.

LbNR-Derived Nitric Oxide Delays Lycium Fruit Coloration by Transcriptionally Modifying Flavonoid Biosynthetic Pathway.

Anthocyanin-derived fleshy fruit pigmentation has become an excellent system for studying the regulatory network underlying fruit ripening and quality. The transcriptional control of anthocyanin biosynthesis by MYB–bHLH–WDR complexes has been well established, but the intermediate signals through which the environmental or developmental cues regulate these transcription factors remain poorly understood. Here we found that nitric oxide (NO) production during Lycium fruit ripening decreased progressively presenting a negative relationship with anthocyanins. After cloning of the nitric reductase (NR) gene from Lycium barbarum (LbNR) plants, we demonstrated that LbNR-derived NO partially inhibited anthocyanin biosynthesis but enhanced proanthocyanidin (PA) accumulation, and delayed fruit coloration. Application of the NO donor, sodium nitroprusside (SNP), produced a similar effect. The endogenous or exogenous NO downregulated the transcripts both of the regulatory genes and the structural genes that related to anthocyanin biosynthesis, while upregulated both of those genes that related to PA biosynthesis. Given there is a significant negative relationship between the levels of anthocyanins and PAs during Lycium fruit ripening, NO not only inhibited anthocyanin de novo biosynthesis but redirected the flavonoid biosynthetic pathway from anthocyanins to PA production. Two types of LrMYB transcription factors of opposite nature, namely anthocyanin-specific and PA-specific, which belong to the R2R3-MYB subfamily and 1R-MYB subfamily, respectively, were identified from L. ruthenicum fruits. It was further found that NO acts by antagonizing the ABA signaling, a phytohormone we have previously shown playing a positive role in Lycium fruit coloration. Our results provided particularly novel information about NO–ABA–anthocyanin interplay during Lycium fruit development and ripening, which may fill a gap between the developmental cues and the transcriptional regulation of anthocyanin biosynthesis.

Changes in the Proanthocyanidin Composition and Related Gene Expression in Bilberry (Vaccinium myrtillus L.) Tissues.

Berries of genus Vaccinium are rich in flavonoids and proanthocyanidins (PAs). We studied the PA composition and biosynthesis in bilberry (Vaccinium myrtillus L.) tissues and during fruit development. Soluble PAs, analyzed by UHPLC–MS/MS, were most abundant in stem and rhizome with the mean PA polymerization level varying between 4 and 6 in all tissues. Both A- and B-type PAs were present in all tissues. Procyanidin subunits were more common than prodelphinidin subunits in PAs. During fruit ripening, the amount of procyanidin subunits decreased while prodelphinidin subunits and F3′5′H expression increased, indicating a shift in biosynthesis toward the delphinidin branch of the flavonoid pathway. Epicatechin was the most abundant flavan-3-ol in all tissues. Expression of ANR and three isolated LAR genes, analyzed by qRT-PCR, showed connection to accumulation of PAs and flavan-3-ols biosynthesized from different flavonoid branches. Insoluble PAs accumulated during berry development, suggesting that PAs are not recycled after biosynthesis.

Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network, causing differences in pigment composition among mulberry fruits

Mulberry fruits with high concentrations of anthocyanins are favored by consumers because of their good taste, bright color, and high nutritional value. However, neither the regulatory mechanism controlling flavonoid biosynthesis in mulberry nor the molecular basis of different mulberry fruit colors is fully understood. Here, we report that a flavonoid homeostasis network comprising activation and feedback regulation mechanisms determines mulberry fruit color. In vitro and in vivo assays showed that MYBA-bHLH3-TTG1 regulates the biosynthesis of anthocyanins, while TT2L1 and TT2L2 work with bHLH3 or GL3 and form a MYB-bHLH-WD40 (MBW) complex with TTG1 to regulate proanthocyanidin (PA) synthesis. Functional and expression analyses showed that bHLH3 is a key regulator of the regulatory network controlling mulberry fruit coloration and that MYB4 is activated by MBW complexes and participates in negative feedback control of the regulatory network to balance the accumulation of anthocyanins and proanthocyanidins. Our research demonstrates that the interaction between bHLH3 and MYB4 in the homeostasis regulatory network ensures that the fruits accumulate desirable flavonoids and that this network is stable in pigment-rich mulberry fruits. However, the abnormal expression of bHLH3 disrupts the balance of the network and redirects flavonoid metabolic flux in pale-colored fruits, resulting in differences in the levels and proportions of anthocyanins, flavones, and flavonols among differently colored mulberry fruits (red, yellow, and white). The results of our study reveal the molecular basis of the diversity of mulberry fruit colors.

Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.

Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that theBnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.

Methyl Jasmonate Applications From Flowering to Ripe Fruit Stages of Strawberry (Fragaria × ananassa 'Camarosa') Reinforce the Fruit Antioxidant Response at Post-harvest.

Preharvest applications of methyl jasmonate (MeJA) have been shown to improve post-harvest fruit quality in strawberry fruit. However, the effectiveness of consecutive field applications at different phenological stages on the reinforcement of the antioxidant capacity remains to be analyzed. To determine the best antioxidant response of strawberry (Fragaria × ananassa ‘Camarosa’) fruit to different numbers and timing of MeJA applications, we performed three differential preharvest treatments (M1, M2, and M3) consisted of successive field applications of 250 μmol L–1 MeJA at flowering (M3), large green (M2 and M3), and ripe fruit stages (M1, M2, and M3). Then, we analyzed their effects on fruit quality parameters [firmness, skin color, soluble solids content/titratable acidity (SSC/TA) ratio, fruit weight at harvest, and weight loss] along with anthocyanin and proanthocyanidin (PA) accumulation; the antioxidant-related enzymatic activity of catalase (CAT), guaiacol peroxidase (POX), and ascorbate peroxidase (APX); the total flavonoid and phenolic contents, antioxidant capacity, and ascorbic acid content (AAC) during post-harvest storage (0, 24, 48, and 72 h). We also evaluated the effect on lignin, total carbon and nitrogen (%C and N), lipid peroxidation, and C and N isotopes signatures on fruits. Remarkably, the results indicated that MeJA treatment increases anthocyanin and PA contents as well as CAT activity in post-harvest storage, depending on the number of preharvest MeJA applications. Also, M3 fruit showed a higher AAC compared to control at 48 and 72 h. Noticeably, the anthocyanin content and CAT activity were more elevated in M3 treatment comparing with control at all post-harvest times. In turn, APX activity was found higher on all MeJA-treated fruit independent of the number of applications. Unlike, MeJA applications did not generate variations on fruit firmness and weight, lignin contents,% C and N, and in lipid peroxidation and water/nitrogen use efficiency according to C and N isotope discrimination. Finally, we concluded that an increasing number of MeJA applications (M3 treatment) improve anthocyanin, PA, AAC, and CAT activity that could play an essential role against reactive oxygen species, which cause stress that affects fruits during post-harvest storage.