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Methoxypyrazine accumulation and O-methyltransferase gene expression in Sauvignon Blanc grapes: the role of leaf removal, light exposure and berry development. Scott Gregan, and Brian Jordan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05806 • Publication Date (Web): 28 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016
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Journal of Agricultural and Food Chemistry
Methoxypyrazine Accumulation and O-methyltransferase Gene Expression in Sauvignon Blanc Grapes: The Role of Leaf Removal, Light Exposure and Berry Development.
Scott M Gregan and Brian Jordan
Department of Food, Wine and Molecular Biology, Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch 7647, New Zealand.
Corresponding author contact: +6434230611
[email protected] 1 ACS Paragon Plus Environment
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Abstract
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Methoxypyrazines are present in the grapes of certain Vitis vinifera varieties including
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Sauvignon Blanc and contribute herbaceous/green aromas to wine. Environmental factors such
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as light exposure and temperature can influence methoxypyrazine levels and viticultural
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interventions such as canopy manipulation have the ability to reduce methoxypyrazine
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accumulation in grapes. We assessed methoxypyrazine levels and showed that leaf removal
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significantly reduces accumulation in Sauvignon Blanc grapes. The main effect of reducing
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methoxypyrazines was pre-veraison, as post-veraison treatments had no effect on
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concentrations at harvest. Methoxypyrazine concentrations in controls peaked pre-veraison
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and decreased through to harvest. Dilution due to an increase in berry weight was found to be
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the major driver of decreasing concentrations, as methoxypyrazine levels on a per berry basis
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were found to increase through development in two out of three seasons. In the one year of
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our study which showed contrasting results, analyses of weather data indicates that warmer
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than average temperatures appear to be the principal factor affecting the berries ability to
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accumulate and retain methoxypyrazines. We also explored the expression of potential
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biosynthetic O-methyltransferase genes VvOMT1, VvOMT2 and VvOMT3, no significant
17
differences were observed with respect to effect of leaf removal and light exposure.
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Keywords
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Methoxypyrazine, O-methyltransferase, grapes, Vitis vinifera, leaf removal, light exposure,
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temperature, aroma, wine
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Introduction
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Methoxypyrazines (MPs) are a strongly odorant class of compounds that occur widely in the
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plant kingdom and contribute herbaceous and vegetal sensory characteristics to fruits and
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vegetables.1,2 In grapes (Vitis vinifera), they are particularly important as aromatic components
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in the wine matrix, in part due to their low sensory detection threshold.3 The most abundant
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MP in grapes is 3-isobutyl-2-methoxypyrazine (IBMP) and is responsible for the green capsicum
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character in wine.4 3-Isopropyl-2-methoxypyrazine (IPMP) and 3-sec-butyl-2-methoxypyrazine
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are generally present at lower concentrations in grapes5,with IPMP contributing a green pea
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character to wine.6 MPs are considered as positive varietal aromas in white Sauvignon Blanc
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wines,7 whereas the opposite is generally true for red wine varieties such as Cabernet
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Sauvignon where high levels of MPs are seen as detrimental for wine quality.8,9 The
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concentration of MPs in wine therefore, is an important consideration for winemakers, as
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excessive levels can be undesirable whatever the variety. MPs are distinct in that they exist in
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grape berries as volatile, free compounds and their final concentration in grapes is highly
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correlated to MP concentration in wine.10 Therefore the potential to influence MP levels at
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harvest using viticultural techniques is of high interest for the wine industry. Understanding the
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factors that control MP accumulation in grapes is needed to achieve the optimal outcome with
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respect to the desired wine style.
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Canopy manipulation, canopy microclimate and climatic and environmental aspects including
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the role of light exposure, temperature and development, have all been implicated in
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influencing the MP composition in grapes.9,11–18 There have been many studies to characterize
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the factors involved in determining the levels of MPs and while considerable progress has been
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made in understanding their formation and degradation, the mechanisms of either are far from 3 ACS Paragon Plus Environment
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resolved. Furthermore, there is very little understanding of the biosynthetic pathway that exists
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in plants to form MPs. Dunlevy et al.19 described two O-methyltransferase (OMT) genes
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(VvOMT1 and VvOMT2) and while implicated in MP regulation, VvOMT1 and VvOMT2 had a
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high Km for the hydroxypyrazine precursors of MPs and preferred to methylate the flavonol
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quercetin. QTL mapping approaches led to the identification of two additional OMTs, VvOMT3
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and VvOMT4,17,20 VvOMT3 in particular, had a high affinity and specificity for hydroxypyrazine
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methylation and was highly expressed at the time of IBMP accumulation in grapes. These
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studies suggest that VvOMT3 seems likely to play a major role in MP biosynthesis.
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Recent investigations have shown that sunlight exposure and berry shading, generally has a
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greater influence when MPs are accumulating pre-veraison 14,15,18,21, rather than through a
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post-veraison photodegradation mechanism as previously hypothesised.22 Less well studied is
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the significance of leaves as a source and the effects on MP levels in grapes,11,21,23 which is
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relevant because grapevine canopies are generally subject to considerable manipulation. Leaf
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removal techniques may provide effective approaches to influence MP accumulation through
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increased light exposure, source/sink relationships or temperature effects. A clear integration
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of both the timing of accumulation of MPs and the effect that environmental factors has on
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berry composition is necessary to understand the role of the leaf canopy and a better
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understanding on how their genetic regulation reacts to a changing light environment is
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essential.
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In a recent publication and as part of a broader study examining berry composition in
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Sauvignon Blanc grapes,11 we monitored MPs in one season (in 2010) through development
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with respect to leaf removal, light and UV-B exposure; removal of leaves significantly reduced
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MP concentrations. In this follow up research, IBMP and IPMP concentrations and OMT gene 4 ACS Paragon Plus Environment
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expression were monitored over two subsequent seasons (2011-2012), in response to pre-
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veraison and post-veraison leaf removal and light exposure. We re-analyzed our previous study
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(2010 season) in the current context and correlated temperature to differences in seasonal MP
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accumulation. Finally, we present data to evaluate IBMP and IPMP absolute amounts on a per
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berry basis and relate decreasing concentrations post-veraison to dilution effects due to berry
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enlargement.
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Materials and Methods
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Chemical Reagents
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Tris base, beta-mercaptoethanol, IBMP standard and IPMP standard were purchased from
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Sigma–Aldrich (St Louis, MO). EDTA and HCl were purchased from Scharlau Chemicals
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(Barcelona, Spain). NaOH was purchased from VWR International (West Chester, PA). UltraPure
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Distilled Water was purchased from Thermo Fisher Scientific (Waltham, MA).
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Vineyard Leaf Removal Experiments
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A previous study in 2010 demonstrated the effectiveness of leaf removal on modulating MP
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levels in Sauvignon Blanc grapes.11 The leaf removal experiments of this current study were
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conducted during the 2 subsequent growing seasons 2011-2012 in the Lincoln University
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vineyard on the same Sauvignon Blanc vines as described in Gregan et al.11
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Control treatments were vines untouched with a complete maintenance of all canopy leaves.
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Leaf removal treatments were equivalent to those used previously11 and involved removing
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leaves from the basal 600 mm of the canopy to fully expose the fruit, with newly grown leaves
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also removed to maintain the exposure of the fruiting zone. Leaf removal treatments were
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applied to the vines with respect to the onset of veraison and berry phenology. In 2010, a single
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pre-veraison (LR-pre) treatment was applied at -33 days post-veraison (DPV) with berry
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phenology at an average berry weight of 0.6 g (data not shown). In 2011, pre-veraison leaf
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removal (LR-pre) was performed at -29 DPV, at an equivalent berry phenology to 2010 (average
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berry weight of 0.6 g). Date of veraison and berry phenology dictated the post-veraison leaf
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removal treatment timing which was applied post-veraison (LR-post) at 13 DPV (average berry
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weight, 1.5 g). The date of pre-veraison leaf removal in 2012 was chosen in order to capture the
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full progression of MP accumulation through development. To achieve this, leaf removal 6 ACS Paragon Plus Environment
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needed to be at an earlier stage in berry phenology (compared to 2010 and 2011) and was
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applied pre-veraison (LR-pre) at -44 DPV (average berry weight, 0.2 g). The post-veraison
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treatment in 2012 (LR-post) was applied post-veraison at 23 DPV (average berry weight, 1.6 g).
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Also in 2012, additional treatments pre-veraison (LRpartial-pre, -44 DPV) and post-veraison
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(LRpartial-post, 23 DPV) were included. These partial leaf removal treatments consisted of
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removal of shoot laterals around the fruiting zone and then removing 30% of the remaining
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leaves. This enabled us to allow an intermediate level of light exposure on bunches while still
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maintaining a portion of the leaf canopy. The proportion of leaf canopy retained in these
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treatments was not quantitated. Each treatment was replicated three times and the position of
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treatments randomized in blocks within the rows.
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Sample Collection and Monitoring Developmental Parameters
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In 2010, berry samples were collected as described in Gregan et al.,11 at -26, -4, 22 and 41 DPV.
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In 2011, whole grape berry samples were collected at -29, -22, -13, 0, 13, 20, 35, 41 and 48
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DPV.In 2012, samples were collected at -44, -30, -8, 23 and 58 DPV. All samples were taken in
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triplicate, one from each of the replicated vines, with fifteen to twenty-five berries randomly
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selected per replicate. Berries were sampled from both sides of the vine with no more than one
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to two berries taken per bunch. Samples were immediately frozen in liquid nitrogen in the field
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and stored at -80°C prior to further processing for the appropriate analysis.
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Total soluble solids and berry weight were measured throughout the trials to monitor grape
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development. Total soluble solids was measured as degrees Brix (°Bx) using a PAL-1 Digital
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Hand-held ‘Pocket’ Refractometer (Atago, Tokyo, Japan). A total soluble solids concentration of
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8°Bx was used as a measure of veraison to standardize treatments and results between
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seasons, veraison dates being on Day of the Year (DOY) 68 in 2010, DOY 48 in 2011 and DOY 70
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in 2012 (Table 1). A measurement of 8°Bx has been previously used as a measure of the mid-
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point of veraison and has been shown to correspond to maturation onset in Sauvignon Blanc.24
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Temperature data was obtained from the Lincoln Broadfield weather station
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(http://www.cliflo.niwa.co.nz) located approximately 5 km from the Lincoln University
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vineyard. Accumulated degree days (DD) were calculated using a base temperature of 10°C
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(Tb=10°C) and were calculated using the average of daily minimum and maximum temperatures
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minus ten,25 where no negative values are considered. DD were accumulated on a daily basis
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starting on July 1 (Southern hemisphere winter) and finishing on June 30 the following year. DD
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differences from the long-term average (LTA) were calculated using the DD on any given day
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minus the DD for the LTA. The LTA is the average DD calculated daily over the past 82 years
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(1930-2012).
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Methoxypyrazine Analysis
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Analysis of 3-isobutyl-2-methoxypyrazine (IBMP, Figure 1 compound 1) and 3-isopropyl-2-
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methoxypyrazine (IPMP, Figure 1 compound 2) in whole berries was determined using an
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automated HS-SPME (Headspace Solid-Phase Micro-Extraction) technique using a synthetic
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deuterated internal standard, 3-isobutyl-2 (D3)-methoxypyrazine (D3-IBMP). The deuterated
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standard was synthesized in a 5-step procedure using L-leucine as the starting material
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according to the methods of Brenner et al.26 and Murray et al.1 Along with the synthesized D3-
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IBMP, non-deuterated IBMP and IPMP were used to generate standard curves for quantitative
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analysis and were obtained commercially (Sigma–Aldrich, MO, USA). The full protocol is
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described in detail in Gregan et al.11 The entire sample of frozen berries were firstly ground to a
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fine powder in liquid nitrogen using an A11 Basic Analytical Mill (IKA-Werke GmbH & Co. KG,
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Staufen, Germany). IBMP and IPMP analysis was performed on approximately 1 g of
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homogenized frozen berry tissue. Results were reported on both a per weight basis of ng/kg
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(fresh weight) and a per berry basis of pg/berry (average berry fresh weight).
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RNA Extractions and cDNA Synthesis
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Total RNA for qPCR analysis was extracted from whole berry tissue using the Spectrum™ Plant
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Total RNA kit (Sigma-Aldrich). Approximately 150 mg of frozen powdered tissue (as ground for
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MP analysis) was transferred to a 2 mL microcentrifuge tube containing 700 μL lysis buffer (+ β-
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mercaptoethanol) and further disrupted using a TissueLyser II™ (Qiagen, Valencia, CA), before
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continuing the RNA extraction protocol according to the manufacturer’s instructions. Total RNA
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was DNA digested using the Ambion TURBO DNA-free™ Kit (Thermo Fisher Scientific) and RNA
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quantity and quality were determined using the Qubit™ 1.0 Fluorometer (Thermo Fisher
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Scientific) and a DS-11 Spectrophotometer (DeNovix, Wilmington, DE). cDNA synthesis of total
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RNA (300 ng) was performed using the PrimeScript™ RT Reagent Kit (Perfect Real Time)
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(Clontech, Mountain View, CA) according to the manufacturer’s protocol. cDNA for qPCR was
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diluted 1:25 in distilled water.
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Analysis of VvOMT1, VvOMT2 and VvOMT3 mRNA Expression by Quantitative Real-Time PCR
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(qPCR)
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Vector constructs were generated for use as qPCR standards by cloning partial cDNA fragments
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into the pGEM®-T Easy Vector System (Promega, Madison, WI), then transforming into One
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Shot® TOP10 Chemically Competent E. coli cells (Thermo Fisher Scientific) and purifying using
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the ISOLATE II Plasmid Mini Kit (Bioline, London, UK), all according the manufacturer’s
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instructions. The primers used to amplify the partial cDNA fragments were; for VvOMT1,
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forward 5’-GGTTCGGCAGCATTAGAACA-3’, reverse 5’-CATTCCACCTCGCTTCTCTC-3’; for
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VvOMT2, forward 5’-TCCGCATATCAAGGGCATTA-3’, reverse 5’-GCCTCGATTATCATCTGCAA-3’; 9 ACS Paragon Plus Environment
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for VvOMT3, forward 5’-ATGGAGAAAGTGGTAAAAATCATGG-3’, reverse 5’-
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TGCCTAATTTCGTGTCCTAATGAC-3’;17 for VvGAPDH (Glyceraldehyde 3-P Dehydrogenase),
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forward 5’-GGCAAAGTGTTGCCTTCATT-3’, reverse 5’-TCTGGGGAGTAAACCTCACCT-3’; and for
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VvActin, forward 5’-TCTCTCATTGGGATGGAAGC-3’, reverse 5’-TGAATCATATCGTGCGAAGG-3’.
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Primers were designed using the Primer3Plus software.27
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Expression of VvOMT1, VvOMT2 and VvOMT3 genes were analyzed by qPCR using the Eco™
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Real Time PCR System and Eco/EcoStudy software v4.0 (Illumina, San Diego, CA). Each reaction
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was prepared in 15 μL, which consisted of 7.5 μL of SYBR® Premix Ex Taq™ II (Clontech), 0.2 μM
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of each primer, 5 μL of diluted cDNA and the appropriate volume of distilled water. Negative
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controls which contained distilled water substituted for template were included in each run.
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Samples were run in duplicate and the thermal cycling conditions were: an initial denaturation
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at 95°C (30 s) for polymerase activation; followed by 40 cycles of 95°C for 10 s, 56°C for 30 s and
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72°C for 30 s.
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Relative quantitation of VvOMT1, VvOMT2 and VvOMT3 expression were performed using the
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standard curve method, normalized to the internal reference genes VvGAPDH and VvActin.
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Reference genes were initially analyzed separately for their expression stability through
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development using geNorm software.28 Standard curves were generated using serial dilutions
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of vector constructs (pGEM-T Easy plus target gene fragment) of known concentration. Relative
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quantitation was obtained by graphing the cycle threshold (Ct) values against the standard
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curves and then normalizing to the average values for both reference genes in each respective
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sample. Results are given as relative to the lowest levels of expression detected.
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Primers used for analyzing VvOMT1 and VvOMT2,19 VvOMT317 and the reference genes
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VVGAPDH and VvActin29 were as previously described; for VvOMT1, forward 5’-
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GGCCTCAGCGCCGGCGTACG-3’, reverse 5’-TCTCTTTCCCGTTGGAGCT-3’; for VvOMT2, forward 10 ACS Paragon Plus Environment
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5’-TCCGAGAAGATGGCTATGAG-3’, reverse 5’-CTGCAAAGTTGGAATCTTTAA-3’; and for VvOMT3,
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forward 5’-ATGATGGCTCATACTACTAC-3’, reverse 5’-CCTAATTTCGTGTCCTAATG-3’; for
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VvGAPDH, forward 5’- TTCTCGTTGAGGGCTATTCCA-3’, reverse 5’- CCACAGACTTCATCGGTGACA-
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3’; and for VvActin, forward 5’-CTTGCATCCCTCAGCACCTT-3’, reverse 5’-
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TCCTGTGGACAATGGATGGA-3’.
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Statistics
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Statistical analyses were conducted using the GenStat software package (VSN International,
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Hemel Hampstead, UK). Data was analyzed using analysis of variance (ANOVA) and a Fisher’s
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least significant difference (LSD) at the 5% significance level. In Figures 3-7, different lower case
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letters indicate statistical differences through development (time) in control treatments.
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Asterisks (*) indicate statistical differences of pre-veraison leaf removal treatments with
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respect to controls. There was no statistical significance observed with the post-veraison leaf
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removal treatments in any season. Because of large season-to-season variation of the raw data,
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each year was analyzed separately.
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Results
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Grape Development and Vineyard Measurements
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In 2011, the date at which fruit reached any particular Brix was accelerated and DD was the
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greatest (Table 1). This was reflected in veraison being reached at DOY 48 (control), with an DD
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of 740, compared to a veraison date 3 weeks later in 2010 (DOY 68) and 2012 (DOY 70) and an
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DD of 687 and 649, respectively. The transition from veraison to 18°Bx in 2011 was also the
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shortest and this trend of accelerated ripening continued, contributing to earlier berry 11 ACS Paragon Plus Environment
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development and higher TSS at harvest, which were 18.3, 21 and 19.7°Bx in 2010, 2011 and
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2012 respectively. Despite their lower TSS levels, the date of harvest in 2010 and 2012 were 2-4
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weeks later with considerably lower DD (Table 1). The DD difference from the long term
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average (LTA) in 2010 mirrored the results in 2012 (Figure 2), both falling below the LTA before
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flowering and continuing this trend through development to harvest. The main difference
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between 2010 and 2012 was a warm period from veraison to harvest in 2010, which resulted in
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a higher DD at harvest. Conversely, DD in 2011 moved above the LTA pre-flowering and
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continued to track ahead of the LTA to harvest.
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Leaf removal influenced the berries ability to accumulate soluble solids post-veraison,
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significantly reducing TSS levels from 18.3 (control) to 15.2°Bx (LR-pre) at harvest in 2010
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(Figure 3). In 2011, a similar trend was observed, with TSS being reduced in leaf removal
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treatments, but the means were not statistically different. No effect of leaf removal on TSS
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accumulation was observed in 2012.
228 229
Methoxypyrazine Concentrations Throughout Development and in Response to Leaf Removal
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We have previously investigated the effect that leaf removal treatments have on MP
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accumulation in Sauvignon Blanc grapes.11 In 2010, a single pre-veraison leaf removal
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treatment (LR-pre) had a significant influence on both IPMP and IBMP accumulation through
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development (Figure 4). High concentrations of IPMP and IBMP seen in the control treatments
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pre-veraison were maintained at relatively high levels at harvest (20 and 107 ng/g fresh weight
235
respectively). There was also a significant effect of the LR-pre treatments, reducing them to
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10% (IPMP) and 50% (IBMP) of their controls respectively at harvest (41 DPV). Using A-frame
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transparent screens containing UV-excluding materials placed over the fruiting zones of vines 12 ACS Paragon Plus Environment
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demonstrated no differences in IPMP and IBMP composition when compared to leaf removal
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treatments11 (data not shown), which indicated that MP concentrations are not predominantly
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determined by UV-B radiation.
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To provide further evidence for the role of leaves in determining MP concentrations in
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Sauvignon Blanc berries, experiments were repeated in 2011 and 2012 (Figure 5) with
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additional sampling time points, post-veraison leaf removal treatments and in 2012, partial leaf
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removal treatments. Interestingly in 2011, the LR-pre and LR-post treatments had little
245
influence on IPMP and IBMP concentrations when compared to controls. The highest
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concentrations of both IPMP and IBMP were pre-veraison with control treatments peaking at
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55 and 120 ng/kg fresh weight, respectively. From these peaks pre-veraison, both IPMP and
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IBMP decreased through development to be 4 and 8 ng/kg fresh weight respectively at harvest
249
(48 DPV), indicating no maintenance of the relatively high levels accumulated pre-veraison and
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as observed in 2010. They were both also equivalent to the LR-pre treatments at harvest. The
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LR-post treatment applied at 13 DPV had little effect on IPMP and IBMP concentrations. The
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results in 2012 were comparable to the results obtained in 2010. At harvest (58 DPV), IPMP and
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IBMP in controls were maintained at 45% and 42% respectively, of their peaks pre-veraison.
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With IBMP specifically, the LR-pre treatment constrained its accumulation in berries between -
255
30 DPV and veraison and this significant reduction continued through to harvest. The LRpartial-
256
pre treatment allowed the berries to accumulate more IBMP than the LR-pre treatment,
257
although still significantly less than the controls, this trend also continuing to harvest. IPMP
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exhibited a small reduction through development with respect to the LR-pre treatment that
259
was significant at harvest (18 ng/kg in control compared to 7 ng/kg in LR-pre). Post-veraison
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leaf removal did not reduce MP concentrations as observed with the pre-veraison leaf removal
261
treatments, IPMP or IBMP levels being equivalent to controls at harvest. 13 ACS Paragon Plus Environment
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In order to evaluate whether the decline in concentration of IPMP and IBMP through
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development from their peaks pre-veraison was related to a degradation or dilution effect,
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absolute amounts of each compound were calculated and expressed as pg/berry (average berry
265
fresh weight) (Figure 6). When expressed in this form, the 2010 and 2012 experiments
266
produced comparable results. In both years control treatments, IPMP absolute amounts at
267
veraison were retained at harvest. In 2010, absolute amounts of IBMP at veraison were also
268
retained at harvest, and in 2012, IBMP peaked post-veraison and had decreased from this
269
maximum by harvest. In 2010 and 2012, the treatment effects of pre-veraison leaf removal
270
significantly reduced absolute amounts of both IPMP and IBMP (as observed when
271
concentrations expressed as ng/g fresh weight). In 2012, post-veraison leaf removal treatments
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had no effect on absolute amounts of IPMP and IBMP when compared to controls. Absolute
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amounts of MPs in 2011 did not show the same trends as observed in 2010 and 2012. Both
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IPMP and IBMP peaked pre-veraison and apart from an increase in controls at 20 DPV, declined
275
significantly to harvest. Pre- and post-veraison leaf removal had no effect on IPMP. Pre-
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veraison leaf removal significantly reduced absolute amounts of IBMP at 20 DPV only, although
277
this significance was not maintained at harvest. Post-veraison leaf removal had no effect of
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absolute amounts on both IPMP and IBMP in 2011.
279 280
VvOMT Gene Expression Throughout Development and in Response to Leaf Removal
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To investigate the impact that leaf removal had on the expression of genes potentially involved
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in MP biosynthesis,17,19,20 the expression patterns of VvOMT1, VvOMT2 and VvOMT3 were
283
investigated in 2011 and 2012 samples using real-time qPCR.
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Similar expression profiles of VvOMT genes were obtained (Figure 7) in both years. The highest
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expression of VvOMT1, VvOMT2 and VvOMT3 were observed before veraison at -22 DPV (2011)
286
and -30 DPV (2012), then quickly declined through veraison to be at the limit of detection by 20
287
DPV (2011) and 23 DPV (2012). In 2011, the relative expression of VvOMT3 pre-veraison peaked
288
at 794-fold 20 DPV levels and was considerably higher than VvOMT1 (106-fold) and VvOMT2
289
(133-fold) at equivalent time points. There was no consistent difference in expression of all
290
three genes between controls and the LR-pre treatment. In 2012, VvOMT1 and VvOMT3 had
291
peak expression of 460-fold and 498-fold 23 DPV levels respectively, VvOMT2 had considerably
292
lower expression (113-fold). In 2012, there were small reductions in expression of VvOMT1 and
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VvOMT3 in the pre-veraison leaf removal treatments compared to controls, but the means
294
were not statistically different at the conventional P
