Methoxypyrazine Accumulation and O-Methyltransferase Gene

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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]

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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

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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.

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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

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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

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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

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(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 -

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30 DPV and veraison and this significant reduction continued through to harvest. The LRpartial-

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pre treatment allowed the berries to accumulate more IBMP than the LR-pre treatment,

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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

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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

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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

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fresh weight) (Figure 6). When expressed in this form, the 2010 and 2012 experiments

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produced comparable results. In both years control treatments, IPMP absolute amounts at

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veraison were retained at harvest. In 2010, absolute amounts of IBMP at veraison were also

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retained at harvest, and in 2012, IBMP peaked post-veraison and had decreased from this

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maximum by harvest. In 2010 and 2012, the treatment effects of pre-veraison leaf removal

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significantly reduced absolute amounts of both IPMP and IBMP (as observed when

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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

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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

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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.

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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

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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)

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and -30 DPV (2012), then quickly declined through veraison to be at the limit of detection by 20

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DPV (2011) and 23 DPV (2012). In 2011, the relative expression of VvOMT3 pre-veraison peaked

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at 794-fold 20 DPV levels and was considerably higher than VvOMT1 (106-fold) and VvOMT2

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(133-fold) at equivalent time points. There was no consistent difference in expression of all

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three genes between controls and the LR-pre treatment. In 2012, VvOMT1 and VvOMT3 had

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peak expression of 460-fold and 498-fold 23 DPV levels respectively, VvOMT2 had considerably

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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

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were not statistically different at the conventional P

Methoxypyrazine Accumulation and O-Methyltransferase Gene

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