Painted flowers: Eluta generates pigment patterning in Antirrhinum.

In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays. Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed. Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties.

High-density linkage map construction in an autotetraploid blueberry population and detection of quantitative trait loci for anthocyanin content.

Highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) is the most cultivated type of blueberry, both in New Zealand and overseas. Its perceived nutritional value is conferred by phytonutrients, particularly anthocyanins. Identifying the genetic mechanisms that control the biosynthesis of these metabolites would enable faster development of cultivars with improved fruit qualities. Here, we used recently released tools for genetic mapping in autotetraploids to build a high-density linkage map in highbush blueberry and to detect quantitative trait loci (QTLs) for fruit anthocyanin content. Genotyping was performed by target sequencing, with ∼18,000 single nucleotide polymorphism (SNP) markers being mapped into 12 phased linkage groups (LGs). Fruits were harvested when ripe for two seasons and analyzed with high-performance liquid chromatography-mass spectrometry (HPLC-MS): 25 different anthocyanin compounds were identified and quantified. Two major QTLs that were stable across years were discovered, one on LG2 and one on LG4, and the underlying candidate genes were identified. Interestingly, the presence of anthocyanins containing acylated sugars appeared to be under strong genetic control. Information gained in this study will enable the design of molecular markers for marker-assisted selection and will help build a better understanding of the genetic control of anthocyanin biosynthesis in this crop.

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.

Kiwifruit Metabolomics-An Investigation of within Orchard Metabolite Variability of Two Cultivars of Actinidia chinensis.

Plant metabolomics within field-based food production systems is challenging owing to environmental variability and the complex architecture and metabolic growth cycles of plants. Kiwifruit cultivars of Actinidia chinensis are vigorous perennial vines grown as clones in highly structured orchard environments, intensively managed to maximize fruit yield and quality. To understand the metabolic responses of vines to orchard management practices, we needed to better understand the various sources of metabolic variability encountered in the orchard. Triplicate composite leaf, internode and fruit (mature and immature) samples were collected from each of six Actinidia chinensis var. deliciosa 'Hayward' and A. chinensis var. chinensis 'Zesy002' kiwifruit vines at three times during the growing season and measured by LC-MS. In general, there was more variation in metabolite concentrations within vines than between vines, with 'Hayward' showing a greater percentage of within-vine variability than 'Zesy002' (c. 90 vs. 70% respectively). In specific tissues, the sampler, infection by Pseudomonas syringae var. actinidiae and the rootstock also influenced metabolite variability. A similar pattern of metabolic variability was observed from quantitative analysis of specific carbohydrates and phytohormones. High within-vine metabolic variability indicates that it is more important to obtain sufficient replicate samples than to sample from multiple vines. These data provide an objective basis for optimizing metabolite sampling strategies within kiwifruit orchards.

2-O-ß-d-Glucopyranosyl l-Ascorbic Acid, a Stable Form of Vitamin C, Is Widespread in Crop Plants.

2-O-ß-d-Glucopyranosyl l-ascorbic acid (AA-2ßG) is a stable, bioavailable vitamin C (AA) derivative. We report the distribution and seasonal variation of AA-2ßG in apples and its occurrence in other domesticated crops and in wild harvested Ma̅ori foods. Liquid chromatography-mass spectrometry analyses showed high AA-2ßG concentrations in crab apples (Malus sylvestris) but low concentrations in domesticated apples. Leaves of crab and domesticated apple cultivars contained similar intermediate AA-2ßG concentrations. Fruits and leaves of other crops were analyzed: mainly Rosaceae but also Actinidiaceae and Ericaceae. AA-2ßG was detected in all leaves (0.5-6.1 mg/100 g fr. wt.) but was at lower concentrations in most fruits (0.0-0.5 mg/100 g fr. wt.) except for crab apples (79.4 mg/100 g fr. wt.). Ma̅ori foods from Solanaceae, Piperaceae, Asteraceae, and a fern of Aspleniaceae also contained AA-2ßG. This extensive occurrence suggests a general role in AA metabolism for AA-2ßG.

MYBA From Blueberry (Vaccinium Section Cyanococcus) Is a Subgroup 6 Type R2R3MYB Transcription Factor That Activates Anthocyanin Production.

The Vaccinium genus in the family Ericaceae comprises many species, including the fruit-bearing blueberry, bilberry, cranberry, huckleberry, and lingonberry. Commercially, the most important are the blueberries (Vaccinium section Cyanococcus), such as Vaccinium corymbosum (northern highbush blueberry), Vaccinium virgatum (rabbiteye blueberry), and Vaccinium angustifolium (lowbush blueberry). The rising popularity of blueberries can partly be attributed to their "superfood" status, with an increasing body of evidence around human health benefits resulting from the fruit metabolites, particularly products of the phenylpropanoid pathway such as anthocyanins. Activation of anthocyanin production by R2R3-MYB transcription factors (TFs) has been characterized in many species, but despite recent studies on blueberry, cranberry, and bilberry, no MYB anthocyanin regulators have been reported for Vaccinium. Indeed, there has been conjecture that at least in bilberry, MYB TFs divergent to the usual type are involved. We report identification of MYBA from blueberry, and show through sequence analysis and functional studies that it is homologous to known anthocyanin-promoting R2R3-MYBs of subgroup 6 of the MYB superfamily. In transient assays, MYBA complemented an anthocyanin MYB mutant of Antirrhinum majus and, together with a heterologous bHLH anthocyanin regulator, activated anthocyanin production in Nicotiana benthamiana. Furthermore anthocyanin accumulation and anthocyanin structural gene expression (assayed by qPCR and RNA-seq analyses) correlated with MYBA expression, and MYBA was able to transactivate the DFR promoter from blueberry and other species. The RNA-seq data also revealed a range of other candidate genes involved in the regulation of anthocyanin production in blueberry fruit. The identification of MYBA will help to resolve the regulatory mechanism for anthocyanin pigmentation in the Vaccinium genus. The sequence information should also prove useful in developing tools for the accelerated breeding of new Vaccinium cultivars.

Kiwifruit actinidin digests salivary amylase but not gastric lipase.

Kiwifruit contains the cysteine proteinase actinidin whose strong activity allows kiwifruit to be used as a meat tenderiser. This raises the possibility digestive enzymes, also proteins, are themselves susceptible to degradation by actinidin. Salivary amylase and gastric lipase are exposed to the highest concentrations of actinidin whereas duodenal enzymes are less likely to be inactivated by actinidin due to dilution and inactivation of actinidin by gastric juice. The saliva of six volunteers was exposed to Actinidia deliciosa homogenate and then examined for loss of the starch digesting enzyme, alpha-amylase. In agreement with the known distribution of salivary amylase concentration in saliva, the range of amylase activity within the group of volunteers varied by around 100 fold. Within 5 minutes of incubation of 3 parts saliva to one part green kiwifruit at 37 °C, approximately 85% of the amylase activity was lost. The use of E-64, a selective inhibitor of cysteine proteinases, confirmed that the loss of amylase function was due to actinidin. Amylase protein degradation was followed by SDS-PAGE and western blotting. Recombinant human gastric lipase resisted digestion with kiwifruit even after 30 minutes incubation and remained functionally active after this time period. However, both mountain papaya and pineapple extracts degraded gastric lipase fully during a 30 minutes digestion period. Under conditions where cooked starch is consumed along with kiwifruit it is possible that starch digestion may be retarded whereas lipid digestion in the stomach is unlikely to be affected by kiwifruit consumption.