Parasitic dodder expresses an arsenal of secreted cellulases with multi-substrate specificity during host invasion.

Cuscuta campestris is a common and problematic parasitic plant which relies on haustoria to connect to and siphon nutrients from host plants. Glycoside hydrolase family 9 (GH9) cellulases (EC 3.2.1.4) play critical roles in plant cell wall biosynthesis and disassembly, but their roles during Cuscuta host invasion remains underexplored. In this study, we identified 22 full-length GH9 cellulase genes in C. campestris genome, which encoded fifteen secreted and seven membrane-anchored cellulases that showed distinct phylogenetic relationships. Expression profiles suggested that some of the genes are involved in biosynthesis and remodeling of the parasite's cell wall during haustoriogenesis, while other genes encoding secreted B- and C-type cellulases are tentatively associated with degrading host cell walls during invasion. Transcriptomic data in a host-free system and in the presence of susceptible or partially resistant tomato hosts, showed for especially GH9B7, GH9B11 and GH9B12 a shift in expression profiles in the presence of hosts, being more highly expressed during host attachment, indicating that Cuscuta can tune cellulase expression in response to a host. Functional analyses of recombinant B- and C-type cellulases showed endoglucanase activities over wide pH and temperature conditions, and activities towards multiple cellulose and hemicellulose substrates. These findings improve our understanding of host cell wall disassembly by Cuscuta, and cellulase activity towards broad substrate range potentially explain its wide host range. This is the first study to provide a broad biochemical insight into Cuscuta GH9 cellulases, which based on our study may have potential applications in industrial bioprocessing.

Temperature has a major effect on the cuticular wax composition of bilberry (Vaccinium myrtillus L.) fruit.

Cuticle is the first layer protecting plants against external biotic and abiotic factors and is responsive to climatic factors as well as determined by genetic adaptations. In this study, the chemical composition of bilberry fruit cuticular wax was investigated through a latitudinal gradient from Latvia (56°N 24°E) through Finland (65°N 25°E) to northern Norway (69°N 18°E) in two seasons 2018 and 2019. Changes in the major cuticular wax compounds, including triterpenoids, fatty acids, alkanes, aldehydes, ketones, and primary alcohols, were detected by GC-MS analysis. Generally, a decreasing trend in the proportion of triterpenoids from southern to northern latitudes, accompanied with an increase in proportion of fatty acids, aldehydes, and alkanes, in bilberry fruit cuticular wax was observed. A correlation analysis between climatic factors with proportion of wax compounds indicated that temperature was the main factor affecting the cuticular wax composition in bilberries. A controlled phytotron experiment with southern and northern bilberry ecotypes confirmed the major effect of temperature on bilberry fruit cuticular wax load and composition. Elevated temperature increased wax load most in berries of northern ecotypes. The level of triterpenoids was higher, while levels of fatty acids and alkanes were lower, in wax of bilberry fruits ripened at 18°C compared to 12°C in both northern and southern ecotypes. Based on our results, it can be postulated that the predicted increase in temperature due to climate change leads to alterations in fruit cuticular wax load and composition. In northern ecotypes, the alterations were especially evident.

Flavonoid biosynthesis is differentially altered in detached and attached ripening bilberries in response to spectral light quality.

Light spectral quality is known to affect flavonoid biosynthesis during fruit ripening. However, the response of fruits to different light conditions, when ripening autonomously from the parent plant (detached), has been less explored. In this study, we analyzed the effect of light quality on detached and naturally ripening (attached) non-climacteric wild bilberry (Vaccinium myrtillus L.) fruits accumulating high amounts of anthocyanins and flavonols. Our results indicated contrasting responses for the accumulation of phenolic compounds in the berries in response to red and blue light treatments. For detached berries, supplemental blue light resulted in the highest accumulation of anthocyanins, while naturally ripening berries had elevated accumulation under supplemental red light treatment. Both red and blue supplemental light increased the expression levels of all the major structural genes of the flavonoid pathway during ripening. Notably, the key regulatory gene of anthocyanin biosynthesis, VmMYBA1, was found to express fivefold higher under blue light treatment in the detached berries compared to the control. The red light treatment of naturally ripening berries selectively increased the delphinidin branch of anthocyanins, whereas in detached berries, blue light increased other anthocyanin classes along with delphinidins. In addition, red and far-red light had a positive influence on the accumulation of flavonols, especially quercetin and myricetin glycoside derivatives, in both ripening conditions. Our results of differential light effects on attached and detached berries, which lacks signaling from the mother plant, provide new insights in understanding the light-mediated regulatory mechanisms in non-climacteric fruit ripening.

The Coordinated Action of MYB Activators and Repressors Controls Proanthocyanidin and Anthocyanin Biosynthesis in Vaccinium.

Vaccinium berries are regarded as "superfoods" owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (V. corymbosum) and wild bilberry (V. myrtillus), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins.

A dPCR Method for Quantitative Authentication of Wild Lingonberry (Vaccinium vitis-idaea) versus Cultivated American Cranberry (V. macrocarpon).

Berries of the genus Vaccinium are highly valued health-beneficial superfoods, which are commonly subjected to adulteration and mixed with each other, or with other common berry species. A quantitative DNA-based method utilizing a chip-based digital polymerase chain reaction (dPCR) technique was developed for identifying and quantifying wild lingonberry (V. vitis-idaea) and cultivated American cranberry (V. macrocarpon). The dPCR method with species-specific primers for mini-barcoding was designed based on the indel regions found in the trnI-CAU-trnL-CAA locus in the chloroplast genome. The designed primers were able to amplify only target species, enabling to distinguish the two closely related species with good sensitivity. Our results illustrated the ability of the method to identify lingonberry and American cranberry DNA using PCR without the need for probes or further sequencing. The dPCR method could also quantify the DNA copy number in mixed samples. Based on this study, the method provides a basis for a simple, fast, and sensitive quantitative authentication analysis of lingonberry and American cranberry by dPCR. Moreover, it can also provide a platform for authentication analyses of other plant species as well by utilizing the indel regions of chloroplast genomes.

Insights into sugar metabolism during bilberry (Vaccinium myrtillus L.) fruit development.

Bilberry fruit is regarded as one of the best natural sources of anthocyanins and is widely explored for its health-beneficial compounds. Besides anthocyanins, one of the major attributes that determine the berry quality is the accumulation of sugars that provide sweetness and flavor to ripening fruit. In this study, we have identified 25 sugar metabolism-related genes in bilberry, including invertases (INVs), hexokinases (HKs), fructokinases (FKs), sucrose synthases (SSs), sucrose phosphate synthases (SPSs), and sucrose phosphate phosphatases (SPPs). The results indicate that isoforms of the identified genes are expressed differentially during berry development, suggesting specialized functions. The highest sugar content was found in ripe berries, with fructose and glucose dominating accompanied by low sucrose amount. The related enzyme activities during berry development and ripening were further analyzed to understand the molecular mechanism of sugar accumulation. The activity of INVs in the cell wall and vacuole increased toward ripe berries. Amylase activity involved in starch metabolism was not detected in unripe berries but was found in ripe berries. Sucrose resynthesizing SS enzyme activity was detected upon early ripening and had the highest activity in ripe berries. Interestingly, our transcriptome data showed that supplemental irradiation with red and blue light triggered upregulation of several sugar metabolism-related genes, including α- and ß-amylases. Also, differential expression patterns in responses to red and blue light were found across sucrose, galactose, and sugar-alcohol metabolism. Our enzymological and transcriptional data provide new understanding of the bilberry fruit sugar metabolism having major effect on fruit quality.

Hierarchical regulation of MYBPA1 by anthocyanin- and proanthocyanidin-related MYB proteins is conserved in Vaccinium species.

Members of the Vaccinium genus bear fruits rich in anthocyanins, a class of red-purple flavonoid pigments that provide human health benefits, although the localization and concentrations of anthocyanins differ between species: blueberry (V. corymbosum) has white flesh, while bilberry (V. myrtillus) has red flesh. Comparative transcriptomics between blueberry and bilberry revealed that MYBPA1.1 and MYBA1 strongly correlated with the presence of anthocyanins, but were absent or weakly expressed in blueberry flesh. MYBPA1.1 had a biphasic expression profile, correlating with both proanthocyanidin biosynthesis early during fruit development and anthocyanin biosynthesis during berry ripening. MYBPA1.1 was unable to induce anthocyanin or proanthocyanidin accumulation in Nicotiana benthamiana, but activated promoters of flavonoid biosynthesis genes. The MYBPA1.1 promoter is directly activated by MYBA1 and MYBPA2 proteins, which regulate anthocyanins and proanthocyanidins, respectively. Our findings suggest that the lack of VcMYBA1 expression in blueberry flesh results in an absence of VcMYBPA1.1 expression, which are both required for anthocyanin regulation. In contrast, VmMYBA1 is well expressed in bilberry flesh, up-regulating VmMYBPA1.1, allowing coordinated regulation of flavonoid biosynthesis genes and anthocyanin accumulation. The hierarchal model described here for Vaccinium may also occur in a wider group of plants as a means to co-regulate different branches of the flavonoid pathway.

A chromosome-scale assembly of the bilberry genome identifies a complex locus controlling berry anthocyanin composition.

Bilberry (Vaccinium myrtillus L.) belongs to the Vaccinium genus, which includes blueberries (Vaccinium spp.) and cranberry (V. macrocarpon). Unlike its cultivated relatives, bilberry remains largely undomesticated, with berry harvesting almost entirely from the wild. As such, it represents an ideal target for genomic analysis, providing comparisons with the domesticated Vaccinium species. Bilberry is prized for its taste and health properties and has provided essential nutrition for Northern European indigenous populations. It contains high concentrations of phytonutrients, with perhaps the most important being the purple colored anthocyanins, found in both skin and flesh. Here, we present the first bilberry genome assembly, comprising 12 pseudochromosomes assembled using Oxford Nanopore (ONT) and Hi-C Technologies. The pseudochromosomes represent 96.6% complete BUSCO genes with an assessed LAI score of 16.3, showing a high conservation of synteny against the blueberry genome. Kmer analysis showed an unusual third peak, indicating the sequenced samples may have been from two individuals. The alternate alleles were purged so that the final assembly represents only one haplotype. A total of 36,404 genes were annotated after nearly 48% of the assembly was masked to remove repeats. To illustrate the genome quality, we describe the complex MYBA locus, and identify the key regulating MYB genes that determine anthocyanin production. The new bilberry genome builds on the genomic resources and knowledge of Vaccinium species, to help understand the genetics underpinning some of the quality attributes that breeding programs aspire to improve. The high conservation of synteny between bilberry and blueberry genomes means that comparative genome mapping can be applied to transfer knowledge about marker-trait association between these two species, as the loci involved in key characters are orthologous.

Resolving the developmental distribution patterns of polyphenols and related primary metabolites in bilberry (Vaccinium myrtillus) fruit.

Bilberry (Vaccinium myrtillus) is a commercially important wild berry species, which accumulates high amounts of polyphenols, particularly anthocyanins, in the skin and flesh. Whilst a number of studies have quantified these phytochemicals in intact ripe bilberry fruit, we extend the current knowledge by investigating the spatial distribution of anthocyanin-associated polyphenols in fruit tissue, and study their links with primary metabolism during ripening. To address this, we used LC-MS and mass spectrometry imaging to measure and map primary and secondary metabolites in fruit. Correlation analysis showed that five sugars displayed strong positive correlations with anthocyanin accumulation, whereas all amino acids were negatively correlated. The accumulation patterns of polyphenols correlated in fruit skin and flesh, but altered with development. Finally, spatial segmentation analysis revealed that the chemical signatures of ripening first appear at defined regions under the skin and rapidly expand to encompass the entire fruit at the eating-ripe stage.

Red and blue light treatments of ripening bilberry fruits reveal differences in signalling through abscisic acid-regulated anthocyanin biosynthesis.

The biosynthesis of anthocyanins has been shown to be influenced by light quality. However, the molecular mechanisms underlying the light-mediated regulation of fruit anthocyanin biosynthesis are not well understood. In this study, we analysed the effects of supplemental red and blue light on the anthocyanin biosynthesis in non-climacteric bilberry (Vaccinium myrtillus L.). After 6 days of continuous irradiation during ripening, both red and blue light elevated concentration of anthocyanins, up to 12- and 4-folds, respectively, compared to the control. Transcriptomic analysis of ripening berries showed that both light treatments up-regulated all the major anthocyanin structural genes, the key regulatory MYB transcription factors and abscisic acid (ABA) biosynthetic genes. However, higher induction of specific genes of anthocyanin and delphinidin biosynthesis alongside ABA signal perception and metabolism were found in red light. The difference in red and blue light signalling was found in 9-cis-epoxycarotenoid dioxygenase (NCED), ABA receptor pyrabactin resistance-like (PYL) and catabolic ABA-8'hydroxylase gene expression. Red light also up-regulated expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) domain transporters, which may indicate involvement of these proteins in vesicular trafficking of anthocyanins during fruit ripening. Our results suggest differential signal transduction and transport mechanisms between red and blue light in ABA-regulated anthocyanin and delphinidin biosynthesis during bilberry fruit ripening.

Authentication of berries and berry-based food products.

Berries represent one of the most important and high-valued group of modern-day health-beneficial "superfoods" whose dietary consumption has been recognized to be beneficial for human health for a long time. In addition to being delicious, berries are rich in nutrients, vitamins, and several bioactive compounds, including carotenoids, flavonoids, phenolic acids, and hydrolysable tannins. However, due to their high value, berries and berry-based products are often subject to fraudulent adulteration, commonly for economical gain, but also unintentionally due to misidentification of species. Deliberate adulteration often comprises the substitution of high-value berries with lower value counterparts and mislabeling of product contents. As adulteration is deceptive toward customers and presents a risk for public health, food authentication through different methods is applied as a countermeasure. Although many authentication methods have been developed in terms of fast, sensitive, reliable, and low-cost analysis and have been applied in the authentication of a myriad of food products and species, their application on berries and berry-based products is still limited. The present review provides an overview of the development and application of analytical chemistry methods, such as isotope ratio analysis, liquid and gas chromatography, spectroscopy, as well as DNA-based methods and electronic sensors, for the authentication of berries and berry-based food products. We provide an overview of the earlier use and recent advances of these methods, as well as discuss the advances and drawbacks related to their application.

MYBA and MYBPA transcription factors co-regulate anthocyanin biosynthesis in blue-coloured berries.

The regulatory network of R2R3 MYB transcription factors in anthocyanin biosynthesis is not fully understood in blue-coloured berries containing delphinidin compounds. We used blue berries of bilberry (Vaccinium myrtillus) to comprehensively characterise flavonoid-regulating R2R3 MYBs, which revealed a new type of co-regulation in anthocyanin biosynthesis between members of MYBA-, MYBPA1- and MYBPA2-subgroups. VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 expression was elevated at berry ripening and by abscisic acid treatment. Additionally, VmMYBA1 and VmMYBPA1.1 expression was strongly downregulated in a white berry mutant. Complementation and transient overexpression assays confirmed VmMYBA1 and VmMYBA2 to induce anthocyanin accumulation. Promoter activation assays showed that VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 had similar activity towards dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), but differential regulation activity for UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and flavonoid 3'5'-hydroxylase (F3'5'H) promoters. Silencing of VmMYBPA1.1 in berries led to the downregulation of key anthocyanin and delphinidin biosynthesis genes. Functional analyses of other MYBPA regulators, and a member of novel MYBPA3 subgroup, associated them with proanthocyanidin biosynthesis and F3'5'H expression. The existence of 18 flavonoid-regulating MYBs indicated gene duplication, which may have enabled functional diversification among MYBA, MYBPA1 and MYBPA2 subgroups. Our results provide new insights into the intricate regulation of the complex anthocyanin profile found in blue-coloured berries involving regulation of both cyanidin and delphinidin branches.

Trace Element Concentration and Stable Isotope Ratio Analysis in Blueberries and Bilberries: A Tool for Quality and Authenticity Control.

Vaccinium genus berries-wild bilberries (Vaccinium myrtillus L.) and cultivated highbush blueberries (Vaccinium corymbosum L.)-are consumed worldwide, and their consumption has a trend of stable increase. Thus, considering their wide use in ethnomedicine, for juice and jam production, as functional food, as well as their use in preparations of extracts which have application potential in pharmaceutical and cosmetics industries, studies regarding the composition of these berries are of special importance. The aim of this study is to characterise the elemental and isotopic composition, as well as variation in element concentration in bilberries gathered from different sites in Northern Europe and in commercially available blueberry samples from across the World. Furthermore, our aim was to develop tools for authenticity and quality control of these berries. The elemental composition of berries was analysed using inductively coupled plasma with optical emission detection (ICP-OED), while isotope ratio mass spectrometry (IRMS) was used for the determination of isotope ratio values. The results demonstrated detectable differences between macro- and microelement values in bilberries. IRMS analysis of blueberries revealed significant differences in isotope ratios based on the place of origin, indicating the possibility to use this analytical method for authenticity testing. In none of the samples, pollution was detected, even though there were indications of different growth conditions and geochemical differences affecting bilberry composition.

Analysis of composition, morphology, and biosynthesis of cuticular wax in wild type bilberry (Vaccinium myrtillus L.) and its glossy mutant.

In this study, cuticular wax load, its chemical composition, and biosynthesis, was studied during development of wild type (WT) bilberry fruit and its natural glossy type (GT) mutant. GT fruit cuticular wax load was comparable with WT fruits. In both, the proportion of triterpenoids decreased during fruit development concomitant with increasing proportions of total aliphatic compounds. In GT fruit, a higher proportion of triterpenoids in cuticular wax was accompanied by a lower proportion of fatty acids and ketones compared to WT fruit as well as lower density of crystalloid structures on berry surfaces. Our results suggest that the glossy phenotype could be caused by the absence of rod-like structures in GT fruit associated with reduction in proportions of ketones and fatty acids in the cuticular wax. Especially CER26-like, FAR2, CER3-like, LTP, MIXTA, and BAS genes showed fruit skin preferential expression patterns indicating their role in cuticular wax biosynthesis and secretion.

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.

Compositional and morphological analyses of wax in northern wild berry species.

Aerial surfaces of plants are covered by a waxy cuticle protecting plants from excessive water loss and UV light. In the present study, composition and morphology of cuticular waxes of northern wild berry species bilberry (Vaccinium myrtillus L.), lingonberry (V. vitis-idaea L.), bog bilberry (V. uliginosum L.) and crowberry (Empetrum nigrum L.) were investigated. Scanning electron microscopy (SEM) revealed differences in epicuticular wax morphology, and gas chromatography-mass spectrometry (GC-MS) analysis confirmed variation in chemical composition of cuticular waxes between the berry species. The dominant compounds in bilberry and lingonberry cuticular waxes were triterpenoids, while fatty acids and alkanes were the dominant ones in bog bilberry and crowberry, respectively. Wax extracted by supercritical fluid extraction (SFE) from industrial press cakes of bilberry and lingonberry contained linoleic acid and γ-linolenic acid as the dominant compounds. Furthermore, in vitro sun protection factor (SPF) of berry waxes depicted good UV-B absorbing capacities.

Developmental and Environmental Regulation of Cuticular Wax Biosynthesis in Fleshy Fruits.

The aerial parts of land plants are covered by a hydrophobic layer called cuticle that limits non-stomatal water loss and provides protection against external biotic and abiotic stresses. The cuticle is composed of polymer cutin and wax comprising a mixture of very-long-chain fatty acids and their derivatives, while also bioactive secondary metabolites such as triterpenoids are present. Fleshy fruits are also covered by the cuticle, which has an important protective role during the fruit development and ripening. Research related to the biosynthesis and composition of cuticles on vegetative plant parts has largely promoted the research on cuticular waxes in fruits. The chemical composition of the cuticular wax varies greatly between fruit species and is modified by developmental and environmental cues affecting the protective properties of the wax. This review focuses on the current knowledge of the cuticular wax biosynthesis during fleshy fruits development, and on the effect of environmental factors in regulation of the biosynthesis. Bioactive properties of fruit cuticular waxes are also briefly discussed, as well as the potential for recycling of industrial fruit residues as a valuable raw material for natural wax to be used in food, cosmetics and medicine.

Early growth of Scots pine seedlings is affected by seed origin and light quality.

Plants have evolved a suite of photoreceptors to perceive information from the surrounding light conditions. The aim of this study was to examine photomorphogenic effects of light quality on the growth of Scots pine (Pinus sylvestris L.) seedlings representing southern (60 °N) and northern (68 °N) origins in Finland. We measured the growth characteristics and the expression of light-responsive genes from seedlings grown under two LED light spectra: (1) Retarder (blue and red wavelengths in ratio 0.7) inducing compact growth, and (2) Booster (moderate in blue, green and far-red wavelengths, and high intensity of red light) promoting shoot elongation. The results show that root elongation, biomass, and branching were reduced under Retarder spectrum in the seedlings representing both origins, while inhibition in seed germination and shoot elongation was mainly detected in the seedlings of northern origin. The expression of ZTL and HY5 was related to Scots pine growth under both light spectra. Moreover, the expression of PHYN correlated with growth when exposed to Retarder, whereas CRY2 expression was associated with growth under Booster. Our data indicates that blue light and the deficiency of far-red light limit the growth of Scots pine seedlings and that northern populations are more sensitive to blue light than southern populations. Furthermore, the data analyses suggest that ZTL and HY5 broadly participate in the light-mediated growth regulation of Scots pine, whereas PHYN responses to direct sunlight and the role of CRY2 is in shade avoidance. Altogether, our study extends the knowledge of light quality and differential gene expression affecting the early growth of Scots pines representing different latitudinal origins.

Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits.

Ripening of non-climacteric bilberry (Vaccinium myrtillus L.) fruit is characterized by a high accumulation of health-beneficial anthocyanins. Plant hormone abscisic acid (ABA) and sucrose have been shown to be among the central signaling molecules coordinating non-climacteric fruit ripening and anthocyanin accumulation in some fruits such as strawberry. Our earlier studies have demonstrated an elevation in endogenous ABA level in bilberry fruit at the onset of ripening indicating a role for ABA in the regulation of bilberry fruit ripening. In the present study, we show that the treatment of unripe green bilberry fruits with exogenous ABA significantly promotes anthocyanin biosynthesis and accumulation both in fruits attached and detached to the plant. In addition, ABA biosynthesis inhibitor, fluridone, delayed anthocyanin accumulation in bilberries. Exogenous ABA also induced the expression of several genes involved in cell wall modification in ripening bilberry fruits. Furthermore, silencing of VmNCED1, the key gene in ABA biosynthesis, was accompanied by the down-regulation in the expression of key anthocyanin biosynthetic genes. In contrast, the treatment of unripe green bilberry fruits with exogenous sucrose or glucose did not lead to an enhancement in the anthocyanin accumulation neither in fruits attached to plant nor in post-harvest fruits. Moreover, sugars failed to induce the expression of genes associated in anthocyanin biosynthesis or ABA biosynthesis while could elevate expression of some genes associated with cell wall modification in post-harvest bilberry fruits. Our results demonstrate that ABA plays a major role in the regulation of ripening-related processes such as anthocyanin biosynthesis and cell wall modification in bilberry fruit, whereas sugars seem to have minor regulatory roles in the processes. The results indicate that the regulation of bilberry fruit ripening differs from strawberry that is currently considered as a model of non-climacteric fruit ripening. In this study, we also identified transcription factors, which expression was enhanced by ABA, as potential regulators of ABA-mediated bilberry fruit ripening processes.

Recognition of candidate transcription factors related to bilberry fruit ripening by de novo transcriptome and qRT-PCR analyses.

Bilberry (Vaccinium myrtillus L.) fruits are an excellent natural resource for human diet because of their special flavor, taste and nutritional value as well as medical properties. Bilberries are recognized for their high anthocyanin content and many of the genes involved in the anthocyanin biosynthesis have been characterized. So far, neither genomic nor RNA-seq data have been available for the species. In the present study, we de novo sequenced two bilberry fruit developmental stages, unripe green (G) and ripening (R). A total of 57,919 unigenes were assembled of which 80.2% were annotated against six public protein databases. The transcriptome served as exploratory data to identify putative transcription factors related to fruit ripening. Differentially expressed genes (DEGs) between G and R stages were prominently upregulated in R stage with the functional annotation indicating their main roles in active metabolism and catalysis. The unigenes encoding putative ripening-related regulatory genes, including members of NAC, WRKY, LOB, ERF, ARF and ABI families, were analysed by qRT-PCR at five bilberry developmental stages. Our de novo transcriptome database contributes to the understanding of the regulatory network associated with the fruit ripening in bilberry and provides the first dataset for wild Vaccinium species acquired by NGS technology.