Philip R. Young

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.

BackgroundCarotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine.ResultsComparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries.ConclusionsThe carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.

Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries

A high performance liquid chromatographic method was developed to profile major sugars and organic acids in grapevine berries. Sugars and organic acids in grapevine berries were extracted by chloroform/polyvinylpolypyrrolidone purification. The extracts were chromatographed on an Aminex HPX-87H ion-exchange HPLC column with 5mM sulphuric acid as mobile phase. Chromatography was visualised via a diode array detector combined with a refractive index detector. The analysis was calibrated using external standard calibration and a novel equation was used to calculate the concentrations of malic acid and fructose from unresolved separation. For the method to be utilised for analysing a large numbers of berry samples, each sample was directly injected after sample extraction and the extraction step was downscaled to allow the use of small amounts of sample material. The concentrations of sugars and organic acids in grapevine berry samples were normalised to the internal standard concentrations obtained after extraction of an internal standard mixture. The analysis method exhibits a good precision and a high analyte recovery from samples spiked with the standard mixture and is suitable for the profiling of major sugars and organic acids in grapevine berry samples at different stages of berry development. This is the first report on the combined profiling of the major sugars and organic acids in grapevine berries using milligram amounts of plant material with direct injection after sample extraction.

Grapevine Plasticity in Response to an Altered Microclimate: Sauvignon Blanc Modulates Specific Metabolites in Response to Increased Berry Exposure

Grapevine responds to increased exposure in the bunch zone by up-regulating photoprotective carotenoids in the early developmental stages and volatile terpenoids in the later ripening stages of the berries in a proposed mechanism of antioxidant homeostasis maintenance. In this study, the metabolic and physiological impacts of an altered microclimate on quality-associated primary and secondary metabolites in grape (Vitis vinifera) ‘Sauvignon Blanc’ berries was determined in a high-altitude vineyard. The leaf and lateral shoot removal in the bunch zones altered the microclimate by increasing the exposure of the berries. The physical parameters (berry diameter and weight), primary metabolites (sugars and organic acids), as well as bunch temperature and leaf water potential were predominantly not affected by the treatment. The increased exposure led to higher levels of specific carotenoids and volatile terpenoids in the exposed berries, with earlier berry stages reacting distinctly from the later developmental stages. Plastic/nonplastic metabolite responses could be further classified to identify metabolites that were developmentally controlled and/or responded to the treatment in a predictable fashion (assessed over two consecutive vintages). The study demonstrates that grapevine berries exhibit a degree of plasticity within their secondary metabolites and respond physiologically to the increased exposure by increasing metabolites with potential antioxidant activity. Taken together, the data provide evidence that the underlying physiological responses relate to the maintenance of stress pathways by modulating antioxidant molecules in the berries.

A rapid qualitative and quantitative evaluation of grape berries at various stages of development using Fourier-transform infrared spectroscopy and multivariate data analysis

Fourier transform (FT) near-infrared (NIR) and attenuated total reflection (ATR) FT mid-infrared (MIR) spectroscopy were used to qualitatively and quantitatively analyse Vitis vinifera L. cv Sauvignon blanc grape berries. FT-NIR and ATR FT-MIR spectroscopy, coupled with spectral preprocessing and multivariate data analysis (MVDA), provided reliable methods to qualitatively assess berry samples at five distinct developmental stages: green, pre-véraison, véraison, post-véraison and ripe (harvest), without any prior metabolite extraction. Compared to NIR spectra, MIR spectra provided more reliable discrimination between the berry samples from the different developmental stages. Interestingly, ATR FT-MIR spectra from fresh homogenized berry samples proved more discriminatory than spectra from frozen homogenized berry samples. Different developmental stages were discriminated by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). In order to generate partial least squares (PLS) models from the MIR/NIR spectral datasets; the major sugars (glucose and fructose) and organic acids (malic acid, succinic acid and tartaric acid) were separated and quantified by high performance liquid chromatography (HPLC) and the data used as a reference dataset. PLS regression was used to develop calibration models to predict the concentration of the major sugars and organic acids in the berry samples from different developmental stages. Our data show that infrared (IR) spectroscopy could provide a rapid, reproducible and cost-effective alternative to the chromatographic analysis of the sugar and organic acid composition of grape berries at various developmental stages, using small sample volumes and requiring limited sample preparation. This provides scope and support for the possible development of hand-held devices to assess quality parameters in field-settings in real-time and non-destructively using IR technologies.

Field-Grown Grapevine Berries Use Carotenoids and the Associated Xanthophyll Cycles to Acclimate to UV Exposure Differentially in High and Low Light (Shade) Conditions

Light quantity and quality modulate grapevine development and influence berry metabolic processes. Here we studied light as an information signal for developing and ripening grape berries. A Vitis vinifera Sauvignon Blanc field experiment was used to identify the impacts of UVB on core metabolic processes in the berries under both high light (HL) and low light (LL) microclimates. The primary objective was therefore to identify UVB-specific responses on berry processes and metabolites and distinguish them from those responses elicited by variations in light incidence. Canopy manipulation at the bunch zone via early leaf removal, combined with UVB-excluding acrylic sheets installed over the bunch zones resulted in four bunch microclimates: (1) HL (control); (2) LL (control); (3) HL with UVB attenuation and (4) LL with UVB attenuation. Metabolite profiles of three berry developmental stages showed predictable changes to known UV-responsive compound classes in a typical UV acclimation (versus UV damage) response. Interestingly, the berries employed carotenoids and the associated xanthophyll cycles to acclimate to UV exposure and the berry responses differed between HL and LL conditions, particularly in the developmental stages where berries are still photosynthetically active. The developmental stage of the berries was an important factor to consider in interpreting the data. The green berries responded to the different exposure and/or UVB attenuation signals with metabolites that indicate that the berries actively managed its metabolism in relation to the exposure levels, displaying metabolic plasticity in the photosynthesis-related metabolites. Core processes such as photosynthesis, photo-inhibition and acclimation were maintained by differentially modulating metabolites under the four treatments. Ripe berries also responded metabolically to the light quality and quantity, but mostly formed compounds (volatiles and polyphenols) that have direct antioxidant and/or “sunscreening” abilities. The data presented for the green berries and those for the ripe berries conform to what is known for UVB and/or light stress in young, active leaves and older, senescing tissues respectively and provide scope for further evaluation of the sink/source status of fruits in relation to photosignalling and/or stress management.

Regulation of endo‐polygalacturonase activity in Saccharomyces cerevisiae

Pectolytic activity in Saccharomyces cerevisiae is due to the secretion of an endo-polygalacturonase encoded by the PGU1 gene. The ability to degrade polygalacturonic acid has been shown to vary between different strains. In this study, we attempted to elucidate how pectolytic activity is regulated in S. cerevisiae and to determine whether the means of regulation differ between strains. Saccharomyces cerevisiae strains from different genetic backgrounds, with varying ability to degrade pectin, were compared. Activity was found not to be regulated by sequence differences in the PGU1 gene, but by the transcription level of the gene. Expression of PGU1 was found to be determined by the transcription level of its two transcription factors TEC1 and STE12. The activation of PGU1 transcription by galactose was found to be strain specific, independent of the strain being an industrial or a domesticated one. The EUROSCARF yeast deletion library was screened for genes encoding inhibitors and activators of polygalacturonase activity. Fourteen strains were identified, in which deletion of a specific gene resulted in a recovery of polygalacturonase activity; these genes were identified as encoding inhibitors of polygalacturonase activity, and two activators were identified.

Epigenetic regulation of PGU1 transcription in Saccharomyces cerevisiae

The PGU1 gene of Saccharomyces cerevisiae has been shown to encode a polygalacturonase. The polygalacturonase activity in S. cerevisiae is strain specific. There are no significant differences in the PGU1 promoter regions of strains with and without polygalacturonase activity. The PGU1 gene is subtelomeric because it is located within 25 kb of the right telomere of chromosome X. Expressions of genes located in subtelomeric regions in the yeast S. cerevisiae are inhibited compared with the rest of the genome. In this study, we showed that the deletion of genes involved in telomere silencing enhances polygalacturonase activity. PGU1 transcription and polygalacturonase activity are increased when PGU1 is shifted to a different location in the genome, away from the telomere located close to this gene, and the depletion of the histone H4 leads to an increase in PGU1 transcription. We concluded that PGU1 is silenced in strains without polygalacturonase activity due to an epigenetic effect. The results of this study suggest that PGU1 is silenced by being folded into a heterochromatin-like structure at its subtelomeric position on chromosome X. Formation of this silent structure is dependent on the Isw2p chromatin remodeling complex, its histone fold motif containing subunit Dls1p and the N-terminal tail of the H4 histone.

The Brassicaceae species Heliophila coronopifolia produces root border-like cells that protect the root tip and secrete defensin peptides

Background and Aims Root border cells and border‐like cells (BLCs), the latter originally described in Arabidopsis thaliana, have been described as cells released at the root tips of the species in which they occur. BLCs are thought to provide protection to root meristems similar to classical root border cells. In addition, four defensin peptides (Hc‐AFP1‐4) have previously been characterized from Heliophila coronopifolia, a South African semi‐desert flower, and found to be strongly antifungal. This provided an opportunity to evaluate if the BLCs of H. coronopifolia indeed produce these defensins, which would provide evidence towards a defence role for BLCs. Methods Fluorescence microscopy, using live‐cell‐imaging technology, was used to characterize the BLCs of H. coronopifolia. Quantitative real‐time PCR (qRT‐PCR) analysis and immunofluorescence microscopy was used to characterize these defensin peptides. Key Results BLCs originated at the root apical meristem and formed a protective sheath at the tip and along the sides as the root elongated in solid medium. BLCs have a cellulose‐enriched cell wall, intact nuclei and are embedded in a layer of pectin‐rich mucilage. Pectinase treatments led to the dissolution of the sheath and dissociation of the root BLCs. Hc‐AFP1‐4 genes were all expressed in root tissues, but Hc‐AFP3 transcripts were the most abundant in these tissues as measured by qRT‐PCR. A polyclonal antibody that was cross‐reactive with all four defensins, and probably recognizing a general plant defensin epitope, was used in fluorescence microscopy analysis to examine the presence of the peptides in the root tip and BLCs. Data confirmed the peptides present in the root tip tissues, the mucilage sheath and the BLCs. Conclusions This study provides a link between defensin peptides and BLCs, both embedded in a protective pectin mucilage sheath, during normal plant growth and development. The presence of the Hc‐AFP3 defensin peptides in the BLCs suggests a role for these cells in root protection.

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome

Main conclusionTranscriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis.Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275–12280, https://doi.org/10.1073/pnas.96.22.12275, 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene–auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

The Transcriptional Responses and Metabolic Consequences of Acclimation to Elevated Light Exposure in Grapevine Berries

An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening.