A group of grapevine MYBA transcription factors located in chromosome 14 control anthocyanin synthesis in vegetative organs with different specificities compared with the berry color locus.

Grapevine organs accumulate anthocyanins in a cultivar-specific and environmentally induced manner. The MYBA1-A2 genes within the berry color locus in chromosome 2 represent the major genetic determinants of fruit color. The simultaneous occurrence of transposon insertions and point mutations in these genes is responsible for most white-skinned phenotypes; however, the red pigmentation found in vegetative organs suggests the presence of additional regulators. This work describes a genomic region of chromosome 14 containing three closely related R2R3-MYB genes, named MYBA5, MYBA6 and MYBA7. Ectopic expression of the latter two genes in grapevine hairy roots promoted anthocyanin accumulation without affecting other phenylpropanoids. Transcriptomic profiling of hairy roots expressing MYBA1, MYBA6 and MYBA7 showed that these regulators share the activation of late biosynthetic and modification/transport-related genes, but differ in the activation of the FLAVONOID-3'5'-HYDROXYLASE (F3'5'H) family. An alternatively spliced MYBA6 variant was incapable of activating anthocyanin synthesis, however, because of the lack of an MYC1 interaction domain. MYBA1, MYBA6.1 and MYBA7 activated the promoters of UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFGT) and ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (3AT), but only MYBA1 induced F3'5'H in concordance with the low proportion of tri-hydroxylated anthocyanins found in MYBA6-A7 hairy roots. This putative new color locus is related to the red/cyanidic pigmentation of vegetative organs in black- and white-skinned cultivars, and forms part of the UV-B radiation response pathway orchestrated by ELONGATED HYPOCOTYL 5 (HY5). These results demonstrate the involvement of additional anthocyanin regulators in grapevine and suggest an evolutionary divergence between the two grape color loci for controlling additional targets of the flavonoid pathway.

The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment.

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

Characterization of the genetic diversity, structure, and admixture of 7 Chilean chicken breeds.

The Mapuche fowl is an autochthonous breed raised in Chile and represents an important zoogenetic resource for the local economy. This study aimed at investigating the genetic diversity, relationship and population structure of 96 local Chilean chickens derived from 3 ecotype of Mapuche fowl (Kollonka, Ketro, and Kollonka de aretes), 2 ecotype Chilean (Trintre, Cogote pelado) and 2 breeds (Light Brahma and Barred Plymouth Rock) using 12 microsatellite markers. In total, 113 alleles were detected in all populations, with a mean of 7.6 alleles per population. In all population chicken breeds, the observed and expected heterozygosity ranged from 0.91 to 0.98 and from 0.69 to 0.79. Furthermore, all populations showed significant deviations from Hardy-Weinberg expectations. Across each population, the global heterozygosity deficit (FIT) was -0.174, population differentiation index (FST) was 0.073, and the global inbreeding of individuals within breed (FIS) was -0.267. The phylogenetic relationships of chickens were examined using neighbor-joining trees constructed at the level of population. The highest Nei's standard genetic distance value of 0.559 was observed between Barred Plymouth Rock and Light Brahma, whereas the minimum value (0.099) was found between Kollonka and Trintre. The neighbor-joining tree constructed at population level revealed 2 main clusters, with Light Brahma, Barred Plymouth Rock, Ketro and Kollonka de aretes in 1 cluster, and Kollonka, Trintre and Cogote pelado breeds in the second cluster. Based on the results of the STRUCTURE analysis, the most likely number of clustering of the population evaluated was at K = 3, with Light Brahma and Barred Plymouth Rock breeds forming their own distinct clusters, while Kollonka, Ketro, Kollonka de aretes, Trintre and Cogote pelado breeds clustered together. This study represents the first report of genetic diversity in these populations in Chile. These results can be used as baseline genetic information for genetic conservation program, for instance, to control inbreeding and to implement further genetic studies in local Chilean chickens.

Research Note: Characterization of peasant family poultry farming in Southern Chile.

The poultry industry produces most of the meat and eggs for human consumption worldwide. However, family poultry farming still plays an important role in developing countries providing high quality animal products including eggs and poultry meat for family and local consumption. A field survey was taken to 145 family poultry farmers off the commune of Maullin, Los Lagos Region of Southern Chile, to describe their husbandry and breeding practices, and provide information for future development and conservation priorities. Egg production in these poultry systems of the Maullín commune is a family tradition, run mostly by women, provides an extra income from the sale of extra eggs and chicken meat during autumn and winter months. Flocks of 15 to 30 native, creole or indigenous hens, reach point of lay at 5 or 6 months old. Egg production with a mean rate of 40%, peaks during September. Brown eggs are the most frequent, followed by blue-greenish eggs derived from Mapuche fowl ancestry. A ratio of 10 to 20 females per rooster results in ca. 60% hatching rate from natural incubation. While males are kept for two seasons only, females are kept longer, some until old. Diet is based on locally available or self-produced grains, complemented by pasture browsing, scavenging, and kitchen waste. Sanitary management is low or none and technical knowledge derives from ancestral tradition. Investment in accommodation and feeding is low. Results provide information on these systems in non-tropical areas of developing countries where it is scarce, and highlights how these systems can respond to the challenges of future poultry production, considering both climate change and consumers demand for more wholesome, human and sustainable products.

Phyllotaxis transition over the lifespan of a palm tree using Magnetic Resonance Imaging (MRI) and Terrestrial Laser Scanning (TLS): the case of Jubaea chilensis.

BACKGROUND: Jubaea chilensis (Molina) Baillon, is a uniquely large palm species endemic to Chile. It is under threatened status despite its use as an ornamental species throughout the world. This research seeks to identify the phyllotaxis of the species based on an original combination of non-destructive data acquisition technologies, namely Magnetic Resonance Imaging (MRI) in saplings and young individuals and Terrestrial Laser Scanning (TLS) in standing specimens, and a novel analysis methodology. RESULTS: Two phyllotaxis parameters, parastichy pairs and divergence angle, were determined by analyzing specimens at different developmental stages. Spiral phyllotaxis patterns of J. chilensis progressed in complexity from parastichy pairs (3,2) and (3,5) in juvenile specimens and (5,3), (8,5) and (8,13) for adult specimens. Divergence angle was invariable and averaged 136.9°, close to the golden angle. Phyllotactic pattern changes associated with establishment phase, the adult vegetative and the adult reproductive phases were observed. Both technologies, MRI and TLS proved to be adequate for the proposed analysis. CONCLUSIONS: Understanding phyllotactic transitions may assist identification of developmental stages of wild J. chilensis specimens. The proposed methodology may also be useful for the study of other palm species.

Consumer preferences and sensory characteristics of eggs from family farms.

The objective of this study was to evaluate consumer habits as well as the sensory perception and characteristics of farm eggs produced in Los Ríos, Chile. Data were collected from an online survey of 197 respondents and a sensory evaluation carried out by 30 untrained panelists of 4 types of eggs (brown-shell and blue-shell eggs acquired from family farms, free-range eggs acquired from large, industrial systems, and white-shell cage eggs from industrial, cage systems.) To evaluate differences and preferences, data were analyzed in a GLM. In addition, sensory evaluation was analyzed using principal component analysis. In accordance with the survey, 99% of the participants eat eggs (P < 0.001), 58% eat 1 to 3 eggs/wk, and 84% declared to consume eggs at home (<0.0001). Surveyed participants reported that price and size are the determining factors (31%) when purchasing eggs. Among the physical characteristic for consumers, yolk color was the most important attribute rather than white color, egg appearance, texture, flavor, or odor. In the consumer acceptability test, farm eggs (either brown or blue shell) received the most favorable sensory evaluation by the panel and were preferred to both free-range and white-shell cage eggs. Yolk color was the most influential parameter in making this difference. Brown farm eggs were predominately selected for greatest general satisfaction by participants in both the sensory evaluation (P = 0.008) and in the survey (40%; P = 0.026). There were no differences between farm eggs (brown and blue shell, P > 0.05) in the evaluated parameters. There was a consequence in the information given from surveyed consumers and the sensory panel with the yolk color.

Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera.

Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.

The Role of UV-B light on Small RNA Activity During Grapevine Berry Development.

We explored the effects of ultraviolet B radiation (UV-B) on the developmental dynamics of microRNAs and phased small-interfering-RNA (phasi-RNAs)-producing loci by sequencing small RNAs in vegetative and reproductive organs of grapevine (Vitis vinifera L.). In particular, we tested different UV-B conditions in in vitro-grown plantlets (high-fluence exposition) and in berries from field-grown (radiation filtering) and greenhouse-grown (low- and high-fluence expositions) adult plants throughout fruit development and ripening. The functional significance of the observed UV-coordinated miRNA responses was supported by degradome evidences of ARGONAUTE (AGO)-programmed slicing of mRNAs. Co-expression patterns of the up-regulated miRNAs miR156, miR482, miR530, and miR828 with cognate target gene expressions in response to high-fluence UV-B was tested by q-RT-PCR. The observed UV-response relationships were also interrogated against two published UV-stress and developmental transcriptome datasets. Together, the dynamics observed between miRNAs and targets suggest that changes in target abundance are mediated transcriptionally and, in some cases, modulated post-transcriptionally by miRNAs. Despite the major changes in target abundance are being controlled primarily by those developmental effects that are similar between treatments, we show evidence for novel miRNA-regulatory networks in grape. A model is proposed where high-fluence UV-B increases miR168 and miR530 that target ARGONAUTE 1 (AGO1) and a Plus-3 domain mRNA, respectively, while decreasing miR403 that targets AGO2, thereby coordinating post-transcriptional gene silencing activities by different AGOs. Up-regulation of miR3627/4376 could facilitate anthocyanin accumulation by antagonizing a calcium effector, whereas miR395 and miR399, induced by micronutrient deficiencies known to trigger anthocyanin accumulation, respond positively to UV-B radiation. Finally, increases in the abundance of an anthocyanin-regulatory MYB-bHLH-WD40 complex elucidated in Arabidopsis, mediated by UV-B-induced changes in miR156/miR535, could contribute to the observed up-regulation of miR828. In turn, miR828 would regulate the AtMYB113-ortologues MYBA5, A6 and A7 (and thereby anthocyanins) via a widely conserved and previously validated auto-regulatory loop involving miR828 and phasi TAS4abc RNAs.

Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1.

Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes.

Exogenous application of pectin-derived oligosaccharides to grape berries modifies anthocyanin accumulation, composition and gene expression.

Anthocyanins are secondary metabolites synthesized in grape berry skins via the phenylpropanoid pathway, with functions ranging from skin coloration to protection against pathogens or UV light. Accumulation of these compounds is highly variable depending on genetics, environmental factors and viticultural practices. Besides their biological functions, anthocyanins improve wine quality, as a high anthocyanin content in berries has a positive impact on the color, total phenolic concentration and, ultimately, the price of wine. The present work studies the effect of the pre-veraison application of pectin derived oligosaccharides (PDO) on the synthesis and accumulation of these compounds, and associates the changes observed with the expression of key genes in the phenylpropanoid pathways. To this end, pre-veraison Cabernet Sauvignon bunches were treated with PDO to subsequently determine total anthocyanin content, the anthocyanin profile (by HPLC-DAD) and gene expression (by qRT-PCR), using Ethrel and water treatments for comparison. The results show that PDO were as efficient as Ethrel in generating a significant rise in total anthocyanin content at 30 days after treatment (dat), compared with water treatments (1.32, 1.48 and 1.02 mg e.Mv-3G/g FW respectively) without any undesirable effect on berry size, soluble solids, tartaric acid concentration or pH. In addition, a significant alteration in the anthocyanin profile was observed. Specifically, a significant increase in the relative concentration of malvidin was observed for both PDO and Ethrel treatments, compared with water controls (52.8; 55.0 and 48.3%, respectively), with a significant rise in tri-hydroxylated forms and a fall in di-hydroxylated anthocyanins. The results of gene expression analyses suggest that the increment in total anthocyanin content is related to a short term increase in phenylalanine ammonia-lyase (PAL) expression, mediated by a decrease in MYB4A expression. A longer term increase in UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) expression, probably mediated by a rise in MYBA1 was also observed. Regarding the anthocyanin profile, despite the increase observed in MYB5A expression in PDO and Ethrel treatments, no changes in flavonoid 3'-hydroxylase (F-3'-H); flavonoid 3'5'-hydroxylase (F-3'5'-H) or O-methyltransferase (OMT) could be related with the profile modifications described. Overall, this study highlights that application of PDO is a novel means of altering specific grape berry anthocyanins, and could be a means of positively influencing wine quality without the addition of agrochemicals.