Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in the premature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAs and reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plant height.

Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses.

The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promoting pentacyclic triterpenes is here evaluated by means of a targeted gene expression and metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies.

The ripening disorder berry shrivel affects anthocyanin biosynthesis and sugar metabolism in Zweigelt grape berries.

MAIN CONCLUSION: Timescale analyses suggest the berry shrivel (BS) disorder is induced before veraison with strong effects on anthocyanin biosynthesis, and minor effects on sugar transport and metabolism. Berry shrivel (BS)-affected grapes have low sugar contents, high acidity, less anthocyanins and flaccid berries. To date no pathogenic causes are known, and studies to elucidate the molecular basis leading to symptom induction and development are limited. Here we present a study on pre-symptomatic as well as symptomatic BS berries to characterize early metabolic changes, with focus on anthocyanin biosynthesis and sugars metabolism. Healthy and BS berries from six sampling time points were used (BBCH79-BBCH89). Our objectives are (1) to search for the beginning of BS-related physiological processes; (2) to search for key enzymes and sugar transporters involved in BS induction and development and (3) to understand the consequences on polyphenol biosynthesis. We employed high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for sugar and polyphenol analyses, respectively. Additionally we conducted expression analyses (qPCR) of key genes and enzymatic activity assays. Our results show that BS-related processes start before veraison, as determined by slightly reduced hexose contents and reduced expression levels of a vacuolar invertase (VviGIN1), two monosaccharide transporters (VviTMT2, VviTMT3) and the anthocyanin biosynthesis (VviUFGT, VviMYBA1/2) genes. Lower amounts of delphinidin and cyanidin glycosidic forms were determined, while caftaric acid, quercetin-3-O-glucuronide and (+)-catechin were increased in BS berries. Although not all results were conclusive, especially for the sugar metabolism, our data provide important knowledge to improve the understanding of the highly complex berry shrivel ripening disorder.

Antifungal Activity of Saponins from the Fruit Pericarp of Sapindus mukorossi against Venturia inaequalis and Botrytis cinerea.

The antifungal activity of an aqueous extract (AE) and the solid fraction of a chloroform-methanol fruit pericarp extract (CME) of Sapindus mukorossi resolved in water was tested for the first time against Venturia inaequalis and Botrytis cinerea-two important fungal pathogens worldwide. In the greenhouse, a CME (1% vol/vol) spray significantly reduced V. inaequalis symptoms and sporulation (99%) on apple seedling leaves (P ≤ 0.05). In field trials, applications of AE (1% vol/vol) reduced the disease severity of B. cinerea on grape, on average, by 63%. Extracts were fractionated by high-performance liquid chromatography and the bioefficacy of the fractions was tested in vitro. Some components of the most fungicidal fraction were identified by liquid chromatography-high resolution mass spectrometry as saponins: sapindoside B (accounting for ≥98% of the total constituents), hederagenin-pentosylhexoside, and oleanolic acid-hexosyl-deoxyhexosyl-hexoside. This fraction inhibited the mycelial growth of V. inaequalis and B. cinerea by 45 and 43%, respectively.

Pentacyclic Triterpene Bioavailability: An Overview of In Vitro and In Vivo Studies.

Pentacyclic triterpenes are naturally found in a great variety of fruits, vegetables and medicinal plants and are therefore part of the human diet. The beneficial health effects of edible and medicinal plants have partly been associated with their triterpene content, but the in vivo efficacy in humans depends on many factors, including absorption and metabolism. This review presents an overview of in vitro and in vivo studies that were carried out to determine the bioavailability of pentacyclic triterpenes and highlights the efforts that have been performed to improve the dissolution properties and absorption of these compounds. As plant matrices play a critical role in triterpene bioaccessibility, this review covers literature data on the bioavailability of pentacyclic triterpenes ingested either from foods and medicinal plants or in their free form.

MdMyb93 is a regulator of suberin deposition in russeted apple fruit skins.

A comparison of the transcriptomes of russeted vs nonrusseted apple skins previously highlighted a tight relationship between a gene encoding an MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. Ultraperformance liquid chromatography-triple time-of-flight (UPLC-TripleTOF) and GC-MS were used to investigate alterations in phenylpropanoid, soluble-free lipid and lipid polyester contents. A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agroinfiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall.

Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as ß-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and ß-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, ß-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

Apple russeting as seen through the RNA-seq lens: strong alterations in the exocarp cell wall.

Russeting, a commercially important defect in the exocarp of apple (Malus × domestica), is mainly characterized by the accumulation of suberin on the inner part of the cell wall of the outer epidermal cell layers. However, knowledge on the underlying genetic components triggering this trait remains sketchy. Bulk transcriptomic profiling was performed on the exocarps of three russeted and three waxy apple varieties. This experimental design was chosen to lower the impact of genotype on the obtained results. Validation by qPCR was carried out on representative genes and additional varieties. Gene ontology enrichment revealed a repression of lignin and cuticle biosynthesis genes in russeted exocarps, concomitantly with an enhanced expression of suberin deposition, stress responsive, primary sensing, NAC and MYB-family transcription factors, and specific triterpene biosynthetic genes. Notably, a strong correlation (R(2) = 0.976) between the expression of a MYB93-like transcription factor and key suberin biosynthetic genes was found. Our results suggest that russeting is induced by a decreased expression of cuticle biosynthetic genes, leading to a stress response which not only affects suberin deposition, but also the entire structure of the cell wall. The large number of candidate genes identified in this study provides a solid foundation for further functional studies.

SmuMYB113 is the determinant of fruit color in pepino (Solanum muricatum).

Pepino (Solanum muricatum) is an herbaceous crop phylogenetically related to tomato and potato. Pepino fruit vary in color, size and shape, and are eaten fresh. In this study, we use pepino as a fruit model to understand the transcriptional regulatory mechanisms controlling fruit quality. To identify the key genes involved in anthocyanin biosynthesis in pepino, two genotypes were studied that contrasted in foliar and fruit pigmentation. Anthocyanin profiles were analyzed, as well as the expression of genes that encode enzymes for anthocyanin biosynthesis and transcriptional regulators using both RNA-seq and quantitative PCR. The differential expression of the transcription factor genes R2R3 MYB SmuMYB113 and R3MYB SmuATV suggested their association with purple skin and foliage phenotype. Functional analysis of these genes in both tobacco and pepino showed that SmuMYB113 activates anthocyanins, while SmuATV suppresses anthocyanin accumulation. However, despite elevated expression in all tissues, SmuMYB113 does not significantly elevate flesh pigmentation, suggesting a strong repressive background in fruit flesh tissue. These results will aid understanding of the differential regulation controlling fruit quality aspects between skin and flesh in other fruiting species.

Engineering Nicotiana tabacum trichomes for triterpenic acid production.

In this work, we aimed at implementing the biosynthesis of triterpenic acids in Nicotiana tabacum glandular trichomes. Although endogenous genes coding for enzymes involved in such biosynthetic pathway are found in the Nicotiana tabacum genome, implementing such pathway specifically in glandular trichomes required to boost endogenous enzymatic activities. Five transgenes coding for a farnesyl-diphosphate synthase, a squalene synthase, a squalene epoxidase, a beta-amyrin synthase and a beta-amyrin 28-monooxygenase were introduced in N.tabacum, their expression being driven by pMALD1, a trichome-specific transcriptional promoter. This study aimed at testing whether sinking isoprenoid precursors localized in plastids, by exploiting potential cross-talks allowing the exchange of terpenoid pools from the chloroplast to the cytosol, could be a way to improve overall yield. By analyzing metabolites extracted from entire leaves, a low amount of ursolic acid was detected in plants expressing the five transgenes. Our study shows that the terpene biosynthetic pathway could be, in part, redirected in N.tabacum glandular trichomes with no deleterious phenotype at the whole plant level (chlorosis, dwarfism,…). In light of our results, possible ways to improve the final yield are discussed.

QTL and candidate gene mapping for polyphenolic composition in apple fruit.

BACKGROUND: The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin. RESULTS: Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17. CONCLUSION: We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase.

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.

Transcriptional analysis of apple fruit proanthocyanidin biosynthesis.

Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple.

Identification of Novel Candidate Genes Involved in Apple Cuticle Integrity and Russeting-Associated Triterpene Synthesis Using Metabolomic, Proteomic, and Transcriptomic Data.

Apple russeting develops on the fruit surface when skin integrity has been lost. It induces a modification of fruit wax composition, including its triterpene profile. In the present work, we studied two closely related apple varieties, 'Reinette grise du Canada' and 'Reinette blanche du Canada', which display russeted and non-russeted skin phenotypes, respectively, during fruit development. To better understand the molecular events associated with russeting and the differential triterpene composition, metabolomics data were generated using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and combined with proteomic and transcriptomic data. Our results indicated lower expression of genes linked to cuticle biosynthesis (cutin and wax) in russet apple throughout fruit development, along with an alteration of the specialized metabolism pathways, including triterpene and phenylpropanoid. We identified a lipid transfer protein (LTP3) as a novel player in cuticle formation, possibly involved in the transport of both cutin and wax components in apple skin. Metabolomic data highlighted for the first time a large diversity of triterpene-hydroxycinnamates in russeted tissues, accumulation of which was highly correlated with suberin-related genes, including some enzymes belonging to the BAHD (HXXXD-motif) acyltransferase family. Overall, this study increases our understanding about the crosstalk between triterpene and suberin pathways.

GWAS provides new insights into the genetic mechanisms of phytochemicals production and red skin colour in apple.

Understanding the genetic architecture of apple phytochemicals, and their interplay with conventional selection traits, is critical for the development of new apple cultivars with enhanced health benefits. Apple accessions (n = 344) used for this genome-wide association study (GWAS) represented the wide diversity of metabolic profiles in the domesticated and wild Malus genepools. Fruit samples were phenotyped for 34 metabolites, including a stable vitamin C glycoside "ascorbic acid 2-ß-glucoside" (AA-2ßG), and the accessions were genotyped using the Apple 20 K SNP Array. Several fruit quality traits, including red skin over-colour (OCOL), were also assessed. Wild Malus accessions showed at least 2-fold higher average content of several metabolites (e.g. ascorbic acid, chlorogenic acid, phloridzin, and trilobatin) than Malus domestica accessions. Several new genomic regions and potential candidate genes underpinning the genetic diversity of apple phytochemicals were identified. The percentage of phenotypic variance explained by the best SNP ranged between 3% and 21% for the different metabolites. Novel association signals for OCOL in the syntenic regions on chromosomes 13 and 16 suggested that whole genome duplication has played a role in the evolution of apple red skin colour. Genetic correlations between phytochemicals and sensory traits were moderate. This study will assist in the selection of Malus accessions with specific phytochemical profiles to establish innovative genomics-based breeding strategies for the development of apple cultivars with enhanced nutritional value.

Identification of a Strong Anthocyanin Activator, VbMYBA, From Berries of Vaccinium bracteatum Thunb.

Wufanshu (Vaccinium bracteatum Thunb.), which is a wild member of the genus Vaccinium, accumulates high concentration of anthocyanin in its berries. In this study, the accumulated anthocyanins and their derivatives in Wufanshu berries were identified through UHPLC-MS/MS analysis. Candidate anthocyanin biosynthetic genes were identified from the transcriptome of Wufanshu berries. qRT-PCR analyses showed that the expression of anthocyanin structural genes correlated with anthocyanin accumulation in berries. The R2R3-MYB, VbMYBA, which is a homolog of anthocyanin promoting R2R3-MYBs from other Vaccinium species, was also identified. Transient expression of VbMYBA in Nicotiana tabacum leaves confirmed its role as an anthocyanin regulator, and produced a higher anthocyanin concentration when compared with blueberry VcMYBA expression. Dual-luciferase assays further showed that VbMYBA can activate the DFR and UFGT promoters from other Vaccinium species. VbMYBA has an additional 23 aa at the N terminus compared with blueberry VcMYBA, but this was shown not to affect the ability to regulate anthocyanins. Taken together, our results provide important information on the molecular mechanisms responsible for the high anthocyanin content in Wufanshu berries.

Differential regulation of triterpene biosynthesis induced by an early failure in cuticle formation in apple.

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of 'Golden Delicious' (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

Activation of PsMYB10.2 Transcription Causes Anthocyanin Accumulation in Flesh of the Red-Fleshed Mutant of 'Sanyueli' (Prunus salicina Lindl.).

Plum is one of the most important stone fruits in the world and anthocyanin-rich plums are increasingly popular due to their health-promoting potential. In this study, we investigated the mechanisms of anthocyanin accumulation in the flesh of the red-fleshed mutant of the yellow-fleshed plum 'Sanyueli'. RNA-Seq and qRT-PCR showed that anthocyanin biosynthetic genes and the transcription factor PsMYB10.2 were upregulated in the flesh of the mutant. Functional testing in tobacco leaves indicated that PsMYB10.2 was an anthocyanin pathway activator and can activate the promoter of the anthocyanin biosynthetic genes PsUFGT and PsGST. The role of PsMYB10.2 in anthocyanin accumulation in the flesh of plum was further confirmed by virus-induced gene silencing. These results provide information for further elucidating the underlying mechanisms of anthocyanin accumulation in the flesh of plum and for the breeding of new red-fleshed plum cultivars.

Food Quality Assessed by Chemometrics.

Food market globalization, food security as well as increasing consumer demand for safe, minimally processed and healthy food impose the need to establish new approaches for identifying and assessing food quality markers. Nowadays, food industry stakeholders are challenged to assure food quality and safety without compromising several prerequisites such as sustainable and ecologically resilient food production, prolonged shelf life, satisfactory sensory quality, enhanced nutritional value and health-promoting properties. In addition, food fraud related to deliberate product mislabeling or economically intended adulteration is of major concern for both industry and regulatory authorities due to cost and public health implications. Notwithstanding the great number of state-of-the-art analytical tools available for quantifying food quality markers, their implementation results in highly complex and big datasets, which are not easily interpretable. In this context, chemometrics e.g., supervised and unsupervised multivariate exploratory analyses, design-of-experiment methodology, univariate or multivariate regression modelling etc., are commonly implemented as part of food process optimization and food quality assessment. In this Special Issue, we aimed to publish innovative research and perspective papers on chemometric-assisted case studies relating to food quality assessment, food authenticity, mathematical modelling and optimization of processes involved in food manufacturing.