MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Combined effects of sulfur dioxide, glutathione and light exposure on the conservation of bottled Sauvignon blanc.

Oxygen exposure may trigger a series of changes that could be detrimental to the quality white wines. This study evaluated the combined effects of sulfur dioxide, glutathione and light exposure on the chemistry and sensory perception of bottled Sauvignon blanc. The wines were manually bottled into clear bottles, closed with low oxygen transfer rate stoppers, and stored for three months, either exposed or protected from light. The wines exposed to artificial light showed higher rates of sulfite loss and oxygen consumption, were significantly darker in color, exhibited significant changes in the concentration of phenolics and volatile compounds, were perceived as less fruity/floral, and had higher nuances of solvent, earthy and honey aromas than the ones protected from light. The treatments with higher amounts of initial sulfites and glutathione were able to delay some of these changes but were less significant than protecting the wines from artificial light.

'White gold' guano fertilizer drove agricultural intensification in the Atacama Desert from AD 1000.

The archaeological record shows that large pre-Inca agricultural systems supported settlements for centuries around the ravines and oases of northern Chile's hyperarid Atacama Desert. This raises questions about how such productivity was achieved and sustained, and its social implications. Using isotopic data of well-preserved ancient plant remains from Atacama sites, we show a dramatic increase in crop nitrogen isotope values (δ15N) from around AD 1000. Maize was most affected, with δ15N values as high as +30‰, and human bone collagen following a similar trend; moreover, their carbon isotope values (δ13C) indicate a considerable increase in the consumption of maize at the same time. We attribute the shift to extremely high δ15N values-the highest in the world for archaeological plants-to the use of seabird guano to fertilize crops. Guano-'white gold' as it came to be called-thus sustained agricultural intensification, supporting a substantial population in an otherwise extreme environment.

CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons.

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of 'Thompson Seedless' somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

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.

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.

Short-term UV-B radiation affects photosynthetic performance and antioxidant gene expression in highbush blueberry leaves.

The impact of increased artificial UV-B radiation on photosynthetic performance, antioxidant and SOD activities and molecular antioxidant metabolism responses in leaves of two highbush blueberry (Vaccinium corymbosum L. cv. Brigitta and Bluegold) genotypes was studied. Plants were grown in a solid substrate and exposed to 0, 0.07, 0.12 and 0.19 W m(-2) of biologically-effective UV-B irradiance for 0-72 h. Our findings show that net photosynthesis (Pn) decreased significantly in Bluegold, accompanied by a reduction in the effective quantum yield (ФPSII) and electron transport rate (ETR), especially at the highest UV-B irradiation. On the other hand, Brigitta showed a better photosynthetic performance, as well as a clear increment in the antioxidant activity response that could be associated with increased superoxide dismutase activity (SOD) in the early hours of induced UV-B stress in all treatments. At the molecular level, the expression of the three antioxidant genes evaluated in both genotypes had a similar tendency. However, ascorbate peroxidase (APX) expression was significantly increased (6-fold) in Bluegold compared to Brigitta. Thus, the reduction of Pn concomitant with a lower photochemical performance and a reduced response of antioxidant metabolism suggest that the Bluegold genotype is more sensitive to UV-B radiation, while Brigitta appears to tolerate better moderate UV-B irradiance in a short-term experiment.

The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine.

Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of transcriptional activators.

Effects of UV-B radiation on anatomical characteristics, phenolic compounds and gene expression of the phenylpropanoid pathway in highbush blueberry leaves.

The effects of increased doses of UV-B radiation on anatomical, biochemical and molecular features of leaves of two highbush blueberry (Vaccinium corymbosum L. cv. Brigitta and Bluegold) genotypes were investigated. Plants were grown in a solid substrate and exposed to 0, 0.07, 0.12 and 0.19 Wm(-2) of biologically effective UV-B radiation for up to 72 h. Leaf thickness and the adaxial epidermis thickness fell more than 3-fold in both genotypes at the highest UV-B dose. Moreover, in Bluegold an evident disorganization in the different cell layers was observed at the highest UV-B radiation. A significant decrease in chlorophyll a/b after 6 h in Brigitta under the greater UV-B doses was observed. Anthocyanin and total phenolics were increased, especially at 0.19 Wm(-2), when compared to the control in both genotypes.Chlorogenic acid was the most abundant hydroxycinnamic acid in Brigitta, and was significantly higher (P ≤ 0.05) than in Bluegold leaves. Regarding the expression of phenylpropanoid genes, only the transcription factor VcMYBPA1 showed a significant and sustained induction at higher doses of UV-B radiation in both genotypes compared to the controls. Thus, the reduction of leaf thickness concomitant with a lower lipid peroxidation and rapid enhancement of secondary metabolites, accompanied by a stable induction of the VcMYBPA1 transcription factor suggest a better performance against UV-B radiation of the Brigitta genotype.

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.

Phytoplasma and virus detection in commercial plantings of Vitis vinifera cv. Merlot exhibiting premature berry dehydration

A new and devastating physiological disorder of Vitis vinifera cv. Merlot was recently reported, known as premature berry dehydration (PBD), which is characterized by plant growth reduction, induction of general senescence and pedicel necrosis in the fruit, causing significant reductions in vineyard production. The causes of this disease remain unclear and previous reports suggest that it may be associated with phloem disruption and water provision. For this reason, any factor causing phloem disturbances could cause an important change in the berry water status. As some micro-organisms have been reported to disrupt phloem flow, we analyzed the occurrence of phytoplasma and viruses in commercial vineyards presenting PBD. In this study, a phytoplasma was detected by electron microscopy and nested PCR while virus infections were diagnosed by RT-PCR in samples collected during two growing seasons. The presence of phytoplasma only in samples from grape plants with PBD suggests that this pathogen may be one of the causal agents of this disorder. We suggest that the influence of other factors, such as virus infections, agronomic handling and environmental conditions also modulate berry dehydration. This is the first study at the microscopic and molecular levels that correlates phytoplasma presence with PBD.