Partial Solar Radiation Exclusion with Color Shade Nets Reduces the

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Partial solar radiation exclusion with color shade nets reduce the degradation of organic acids and flavonoids of grape berry (Vitis vinifera L.). Johan Martínez-Lüscher, Christopher Cody Lee Chen, Luca Brillante, and Sahap Kaan Kurtural J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04163 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Journal of Agricultural and Food Chemistry

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Partial solar radiation exclusion with color shade nets reduce the degradation

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of organic acids and flavonoids of grape berry (Vitis vinifera L.).

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Johann Martínez-Lüscher1 Christopher Cody Lee Chen1, Luca Brillante1, Sahap Kaan

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Kurtural1*

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Department of Viticulture and Enology, University of California Davis, 1 Shields Avenue

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Davis, CA 95616 USA.

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*corresponding authors email: [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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The incidence of solar radiation on red-skinned grapes can promote the synthesis of flavonoid

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desirable for wine production, elevated temperature may impair their accumulation. We

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performed a shade cloth trial covering the fruit zone (from pepper-corn size to maturity) with

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four polyethylene 1-meter curtains with different optical properties (20% shading factor Pearl

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colored and 40% shading factor Aluminet, Blue and Black colored) and a Control with no cover.

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Cluster temperature was 3.7°C lower on the Southwest side in Black-40% clusters during the

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warmest part of the day compared to Control. Results indicated a lower berry weight under the

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Aluminet-40%. Berries under the nets had often significantly lower pH and higher TA than

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Control, but only the Black-40% were significant at harvest. Black-40% had higher values of

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anthocyanins than Control towards the last weeks of development. Berry skin flavonol and

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anthocyanin composition and concentration were measured by C18 reversed-phased HPLC; and

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proanthocyanidin isolates were characterized by acid-catalysis in the presence of excess

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phloroglucinol followed by reversed-phase HPLC. Proanthocyanidins and flavonol contents

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were lower in Black-40% before veraison and the first part of ripening, respectively. However,

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their contents in Control decreased towards the end of ripening to a point where any net was

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different from Control. Anthocyanin and flavonol profiles were richer in 3‘4’5’ hydroxylated

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forms. Proanthocyanidin chain length was not affected while small changes were observed in the

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proportion of terminal catechin/epicatechin and in seed galloylation in response to treatments.

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Results show that, shade cloths may efficiently palliate temperature spikes, especially the last

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weeks before harvest, while transmitting enough radiation into the fruit zone to achieve a better

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grape composition compared to uncovered grapes.


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Keywords: Light; temperature; abiotic stress; climate change; flavonols; anthocyanins;

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tannins

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INTRODUCTION

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Wine grapes are grown in different climates with a great range of variation in rainfall,

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temperature and solar radiation. Part of this wide distribution is achieved by using of different

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genotypes (cultivars, clones and rootstocks) adapted to the different conditions.1 In addition,

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further adaptation to the environment is achieved through cultural practices. Grapevine needs a

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suite of canopy management practices to ensure consistent cropping between years and balance

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the leaf/fruit ratio in order to improve grape composition. Winter pruning is one of the most

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common practices, although it is only a rough regulator of yield,2 and does not ensure consistent

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berry composition due to compensating responses from the vegetative growth of the latent buds.3

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Therefore, green pruning, such as shoot 4 or leaf removal 5 among others, are used to finely tune

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the yield and/or reduce the leaf layers around the clusters to ameliorate the amount of solar

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radiation reaching the grape berry.6 An increase in solar radiation through these practices may

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result in higher grape anthocyanin, flavonol and proanthocyanidin content7-9. However, with

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increasing temperatures, these and other metabolites such as malic acid may be degraded more

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rapidly.10

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The up regulation of certain steps of the flavonoid pathway biosynthesis under solar

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radiation exposure has been described fundamentally. For instance, UV-B radiation triggers

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UVR8 photoreceptor signaling cascade leading to the gene expression related to light-grown

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phenotype, including flavonol biosynthesis.11,

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synthetic genes shared by phenolic compounds, such as phenylalanine ammonia lyase; others

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specific to flavonoids, such as flavonoid 3 hydroxylase; the specific genes for flavonol

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This response includes the up-regulation of



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biosynthesis, flavonol synthase and MybF1 transcription factor; and finally, UDP-glucosyl

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transferases, which give place to the end-products of both flavonols and anthocyanins.13 The

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mechanism of anthocyanins and proanthocyanidins synthesis up regulation in response to light

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has yet not been fully elucidated. Transcription factors involved in their regulation have also

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been reported as light-inducible in many fruits, including grapes.14 The role of flavonoids in

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photo protection has been under review several times.14 Flavonols are strongly induced by UV

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radiation, and in fact, they have a strong extinction coefficient at that wavelength range, making

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them ideal to be accumulated in the external cell layers of plant tissues and screen harmful

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radiation.15 Complementarily to the radiation screening effect, flavonoids are present even at the

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chloroplast level and they are able to scavenge oxygen singlet, inhibit enzymatic reactive oxygen

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formation and quench free radical reaction cascades in lipid peroxidation.16,

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oxidized, flavonoids such as proanthocyanidins are able to improve their photoprotectant

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capacity.18 In the case of fruits, the role of flavonoids acquires a new dimension as they may

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serve as attractants as in the case of anthocyanins, which announce the progression of ripening,

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and as repellent, as in the case of proanthocyanidins, which is to a great extent responsible for

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the astringency and bitterness of unripe fruits.14

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Even when

Shifts in the light spectrum, may also have an effect in flavonoid production. González, et

71 19

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al.

displayed how irradiation of grapes with wavelengths in the red and blue waveband

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increased the concentration of phenolic compounds, including anthocyanins, whereas green or

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far red waveband radiation did not induce these effect. Cryptochromes and phytochromes have

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peaks in their absorption spectrum in blue and red light wavelengths regions.14 Therefore, the

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increase in phenolic compounds in response to light is presumably mediated by the cascades of

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signaling related to photoreceptors.19


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The consequence of exposure of fruit to the sun is also an increase in temperature above

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ambient temperature and this is especially pronounced during the last period of ripening, when

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clusters can be tight and dark colored. The effect of temperature on grape coloration (i.e.

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anthocyanins) and acidity of grapes has been extensively studied. Kliewer and Torres 20 reported

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a total lack of coloration of table grapes with night temperatures above 20°C. In a later study,

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with night temperatures fixed at 20°C, an increase in day temperature from 25°C to 35°C was

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enough to decrease anthocyanin concentration more than one fold change in Cabernet Sauvignon

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berries 10. In that case, the authors did not find any down regulation of anthocyanin biosynthesis-

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related genes or enzyme activities, attributing loss of anthocyanins to degradation. Cohen, et al.

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21

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proanthocyanidin content or structure. However, changes in proanthocyanidins have been often

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described in relation to phenology, i.e. progress of ripening,22 which may be highly temperature-

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dependent.23 Lower acidity is also a characteristic of grapes grown in warmer years.24 This is in

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part due to the sensitivity of malate to thermal degradation,25 whereas tartrate, the more abundant

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organic acid in grapes, is rather stable across temperature regimes.26

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studied the effect of temperature regimes at the fruit zone and reported few changes in skin

There have been few efforts to decouple the effects of light (photoreceptor-based 27

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responses) and temperature-based responses to solar radiation. Azuma, et al.

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separate effects of light and temperature on excised berries from vines of ‘Pione’ (V. vinifera x V.

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labrusca) under controlled conditions; and suggested that light and maintaining berry

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temperature under a threshold as equally important to reach a good grape coloration. Spayd

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showed that anthocyanin accumulation in grape skins were greatly reduced by heat gain when

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grapes are exposed to the sun. Furthermore, when heat gain was mimicked with heaters in

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absence of solar radiation, anthocyanin concentrations were further reduced. Therefore, the


, tested the

28


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balance between positive and negative effects of sun exposure resides in achieving enough

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radiation reaching the fruit while avoiding certain temperature threshold. Current changes in

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climate, forecasting decrease in cloud coverage in most viticultural regions coupled with an

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increase in ambient temperature,29 may lead to an exacerbation of problems related to fruit

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overexposure. Thus, there is a growing need of practices in horticulture to avoid overheating of

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fruits. For this purpose several temperature mitigation strategies are currently researched, such as

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the use of reflective materials as kaolin 30 or shade nets .31

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Shade nets have been used for growing fruits, vegetables and ornamental crops for a long

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time.32 In wine grape, nets are mainly used to protect fruits from birds or hail while the use for

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shading purpose has been less common. Shade nets may offer a homogenized and optimized

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dose of solar radiation. Thus, shade nets added to canopy management practices may have the

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potential of combining the desirable effects of less dense canopies, such as increased solar

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exposure and reduced the relative humidity at the fruit zone, without the deleterious effects of

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overexposure. The aim of this study was to test the use of polyethylene shading nets of different

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color to reduce the transmittance of solar radiation into the fruit zone, reducing berry day

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temperature to ameliorate grape composition.

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MATERIAL AND METHODS

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Meteorological variables. Growing degree days (GDD) were calculated as: (Daily

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maximum – Daily minimum) / 2 -10; not accounting for negative values. Clear sky days were

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accounted as the days with at least 75% of the maximum radiation recorded on the 7 days before

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and after each day of the year.


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Experimental site and plant material. The experiment was conducted at University of

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California Oakville Experimental Station (38.428, -122.409; Oakville, CA) during season 2016.

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Plants were 7-year-old 'Cabernet Sauvignon' clone FPS#7 grafted on 110 Richter (V. berlandieri

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Planch. x V. rupestris Scheele) rootstock. Vines were pruned as a bilateral cordon with 24 single-

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bud spurs and trained in an open vertical shoot positon system (VSP) with shoots positioned

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upwards with a 68º angle and tucked between catch-wires. Plant spacing was 2 by 2.4 m within

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and between rows, respectively, in rows oriented NW to SE. Irrigation was applied by 2 drippers

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per plant. Water amounts to be applied were calculated following the methods of Williams and

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Ayars

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and this maintained leaf water potentials between -0.8 and -1.0 MPa from treatment application

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to harvest.

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. Following industry standards, the target was to apply 65% of crop evapotranspiration

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Treatment application and experimental design. The experiment was performed with

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five treatments in a randomized complete block design with four replications. Four shade nets

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treatments and a Control (uncovered) were installed on 27 May 2016 (31 days after anthesis,

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pepper-corn size berries, stage 29 of modified Eichhorn-Lorenz scale 34). Each experimental unit

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consisted of three grapevines. Shade nets treatments consisted in polyethylene 6 m by 1 m sheets

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(Ginegar, Kibbutz, Israel) of i) Pearl® with a 20% shading factor, ii) Aluminet® with a 40%

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shading factor, iii) Blue with a 40% shading factor and iv) Black with a 40% shading factor. The

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nets were placed vertically, hanging them with zip ties from the trellis wires, applied on both

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sides of the fruiting zone of the canopy.

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Fruit microclimate characterization. With the nets in place, a spectrometer connected

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to a cosine-corrected head was used to measure spectral radiation at the fruit zone (Black Comet-

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SR, StellarNet; Tampa, FL, USA). Readings were taken in all treatment replicates when solar 7 ACS Paragon Plus Environment


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radiation was striking the clusters through the nets (ca. 15:00 h) to determine maximum direct

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radiation. Three additional readings were taken in 120° angle (forming a tetrahedron) with the

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first one to estimate diffuse radiation. The four readings were summed as an estimation of the

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total radiation potentially reaching the clusters.

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Air temperature inside the canopy of each treatment-replicate was monitored using button

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temperature data loggers (B-Series Watchdog, Spectrum technologies; Aurora, IL, USA). On 7

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September 2016, infra-red thermometers (Spectrum technologies; Aurora, IL, USA) were used to

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measure grape cluster temperature. Two clusters from each side of the row in each experimental

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unit were selected and 5 temperature readings were averaged for each cluster 8 times throughout

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the day.

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Canopy density. Leaf layer number was calculated using point quadrat procedure

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adapted to grapevine canopies by Smart 35. Leaf contacts were recorded making 5 insertions per

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plant (i.e. at 0.3 m intervals), across the three plants in each experimental unit.

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Leaf gas exchange. Leaf gas exchange was measured with a Ciras 3 (PP Systems,

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Amesbury, MA) around solar noon (11:30 to 14:30 h), 4 times throughout the season. For each

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date and experimental unit, two mature and healthy leaves were measured under light saturating

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conditions (>1200 µmol m-1 s-1)36 and values were averaged.

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Leaf water potential. Leaf water potential (Ψl) was determined 8 times throughout the

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season with a pressure chamber (model 616, PMS Instrument company, OR, USA). For this

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purpose, two mature and healthy leaves above the nets and facing NE side of the row were

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excised with a razor blade and measured immediately.


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Chemicals. All solvents were of HPLC grade. Acetonitrile, acetone, ammonium

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phosphate monobasic, methanol, ascorbic acid, glacial acetic acid, hydrochloric acid, methanol

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(MeOH), ortho-phosphoric acid and sodium hydroxide were purchased from Fisher Scientific

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(Santa Clara, CA). Phloroglucinol was purchased from VWR (Visalia, CA). (+)-catechin ((+)-

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catechin hydrate, > 98%, C), (-) –epicatechin ((-)-epicatechin, >90%, EC), malvidin-3-o-

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glucoside, quercetin-3-o-rutinoise, myricetin-3-o-glucoside were purchased from Extrasynthese

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(Genay, France). Standards for the identification of

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Quecetin 3-o-glucunoride, Quercetin 3-o-galactoside, Quercetin3-o-glucoside, Kaemferol 3-o-

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glucoside, Isorhamnetin 3-o-glucoside and Syringetin 3-o-glucoside) were purchased from

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Sigma-Aldrich (St. Louis, MO). Other compounds were identified tentatively by changing HLPC

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procedures and comparing with the literature.37, 38

flavonols (Myricetin 3-o-glucoside,

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Sample collection and preparation. Seventy-five berry samples were collected at dates:

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20 Jun, 19 July, 29 July, 9 August, 19 August, 29 August and 9 September. Berry weight was

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determined as the average weight of the 75. Fifty-five berries were crushed and musts were

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filtered. Must total soluble solids (TSS) was determined with a digital refractometer (Palette,

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Atago, Tokyo, Japan). pH and titratable acidity (TA) with an autotitrator (862 Compact

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titrosampler, Herisau, Switzerland). Titration was performed up to pH 8.2 with NaOH and TA

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was expressed as g L-1 of tartaric acid. The remaining 20 berries remaining from each sample

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were gently peeled, skins and seeds were collected and freeze-dried (Centrivap, Labconco,

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Kansas city, MO). Skins and seeds were ground with a tissue lyser (MM400, Retsch,

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Mammelzen, Germany). For the determination of anthocyanins and flavonols, 50 mg of freeze-

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dried skin powders were extracted overnight at 4°C with methanol:water:7M hydrochloric acid

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(70:29:1) (Del-castillo et al., 2016). Samples were centrifuged for 10 min at 14,000 rpm and



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supernatants were filtered (0.45 µm; VWR, Seattle, WA). Clear extracts were then transferred to

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HPLC vials for analyses. To determine concentration and structure of polymeric

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proanthocyanidins, 200 mg of skins and seeds were extracted with acetone:water (2:1). Acetone

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was removed from proanthocyanidin extracts using a concentrator connected to a cold trap

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(Centrivap, Labconco, Kansas City, MO). Proanthocyanidins were purified using a DSC-18 solid

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phase extraction (1 g bed wt, Agilent, Santa Clara, CA). Cartridges were activated passing

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15 mL of methanol and 15 mL of water. Then, 1 mL of sample was pipetted, washed with 15 mL

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of water and then polymeric proanthocyanidins were eluted with 9 mL of methanol. Purified

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extracts were precipitated and resuspended in 1 mL of methanol. Polymeric proanthocyanidins

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cleavage into sub units was performed through acid catalysis presence of phloroglucinol and

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ascorbic acid for 20 min at 50°C. The reaction was stopped adding 40 mM sodium acetate.39

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HPLC procedures. Anthocyanins and flavonols were analyzed by a HPLC-DAD (1260 40

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Series, Agilent, Santa Clara, CA) following the methods in Ritchey and Waterhouse

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mobile phases were, (A) 50 mM dihydrogen ammonium phosphate adjusted to pH 2.6 with

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orthophosphoric acid, (B) 20% of phase A with 80% acetonitrile, and (C) 0.2 M orthophosphoric

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acid adjusted with NaOH to pH 1.5. HPLC gradient started with 100% of mobile phase A,

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followed by 92% A – 8% B at 8 min, 14% B – 86% C at 20 min, 1.5% A- 16.5% B -82% C at 25

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min, 21.5% B -78.5% C at 35 min, 50% B -50% C at 70 min and 100% A from 75 min to 80

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min. Absorbance was recorded at 365 nm and Quercetin 3-o-glucoside was used for the

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quantification of flavonols. Absorbance was recorded at 520 nm and Malvidin 3-o-glucoside was

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used for the quantification of anthocyanins.

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Individual anthocyanins and flavonols were grouped according to their substituents in the 3’, 4’

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and 5’ positions B-ring of the general flavonoid skeleton. Hereby, they were grouped in 10 ACS Paragon Plus Environment

. The

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compounds derived from hydroxylation of 3’ and 4’ positions (i.e. Cyanidin-based and

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Quercetin-based compounds for anthocyanins and flavonols, respectively) and compounds

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derived from hydroxylation of 3’, 4’ and 5’ positions (i.e. Delphinidin-based and Myricetin-

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based compounds for anthocyanins and flavonols, respectively).

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The determination of proanthocyanidin extension and terminal subunits was performed

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through HPLC-DAD (1100 Series, Agilent). The mobile phases used were (A) 1% of acetic acid

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in water and (B) 1% of acetic acid in acetonitrile. The HLPC gradient started at 97% of mobile

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phase A isocratic until 4 min, followed by a gradient up to 82% A – 18% B at 14 min, and two

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isocratic phases with 20% A – 80% B up to 16 min and 97% A – 3% B up to 20 min.

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Absorbance was recorded at 280 nm and Epicatechin (Sigma-Aldrich, St. Louis, MO) was used

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as a quantitative standard. Identification and quantification of extension and terminal sub units

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was performed using retention times and molar relative response factors accessible from the of

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extension-to-terminal subunit on a molar basis.

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Yield components. When grapes matured clusters were detached, counted and weighed.

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In February, grapevines were pruned to 4 buds and pruning wood was weighed. Ravaz index was

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calculated as the ratio of yield-to-pruning weight. All values were averaged for the 3 plants per

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experimental unit. Statistical analyses. Statistical analyses were performed in R language version (v 3.3.1.)

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with a p value for significant differences lower than 0.05 to find differences among the

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treatments. Fisher’s least significant differences (LSD) interval was calculated to be plotted as an

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indicator of variability among the samples with in a sampling point. Percentages were log

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transformed for the statistical analyses. Two-way ANOVA (with sampling date and shade net as

. For each variable and date, one-way ANOVA and Duncan’s post hoc analysis was performed



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factors) was performed on gas exchange derived parameters and leaf water potentials to verify

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the lack of interactions. In that case, mean values per experimental unit were used for one-way

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ANOVA and Duncan’t test.

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RESULTS

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Environmental conditions during the year of study. Compared to the last ten years

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average, the year 2016 (Figure 1) had similar amount of precipitation although these happened

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mainly in March and April. GDD showed a lower heat accumulation than the average at the

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beginning of the growing season, but very similar accumulation of GDD from June onwards. The

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accumulation of clear sky days was high at the study site, as 84.3% of the days had clear sky

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conditions on average during the last 10 years. Similarly, 84.0% of the days had clear sky

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conditions during 2016.

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Gas exchanges and Ψl. Mean net carbon assimilation (Anet), stomatal conductance (gs)

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and intrinsic water use efficiency (Anet /gs) of leaves outside the nets, were not affected by the

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shade nets placed at the fruit zone (Table 1). Anet as well as gs changed across the sampling dates.

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Ψl ranged from –1.30 to –0.86 MPa throughout dates and nets. However, no significant

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differences were found among treatments (Table 1).

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Yield components and Canopy density. Leaf layer number at the fruit zone was not

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significantly different between treatments; however, there was a tendency to have lower leaf

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layer number in Aluminet-40%, Blue-40% and Black-40%, compared to Control and Pearl-20%.


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Cluster counts, yield or pruning weight were not significantly affected by shade net

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treatments. However, control had the highest mean values of these three parameters (Table 2).

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All treatments had similar balance yield-to-pruning weight (Ravaz index).

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Fruit microclimate. The shade nets had different transmission of solar radiation, both

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quantitatively and qualitatively (Fig. 2A). The transmittance of the nets was similar to the values

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reported by the manufacturer, being Aluminet-40% the one with the lower transmittance. Pearl-

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20%, Aluminet-40% and Black-40% nets did not effect a selective screening of any wavelength

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range, whereas Blue-40% had a on average a 10% higher transmittance in the blue region of the

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spectra (ca. 450 nm) and 12% higher in the infra-red range (>750 nm). This transmittance clearly

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determined the spectral radiation received at the fruit zone in under each of the nets (Fig. 2B). It

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was noticeable that radiation under Aluminet-40% was higher than under other nets (Fig. 2A)

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that presented a higher transmittance in Figure 2B. This could be related to the characteristic

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reflecting properties of this material. It is worth mentioning that in Control spectral irradiance

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was different from the open field, where any object other than the ground could reflect radiation

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back to the sensor.

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Cluster temperature (Fig. 3) was rather close to ambient temperatures in the NE side

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clusters. The temperature difference between the cluster and air temperature within the fruit zone

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(∆T) was never higher than 2°C under the nets whereas cluster temperature of controls had a ∆T

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of 4.3°C at 9:00 h, when air temperature was 21.3°C. On the other side, cluster temperature

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decoupled from air temperature, being lower before midday (∆T=-3.4°C at 9:00 am) and much

275

higher after midday. In fact, cluster temperatures of controls were 38.8, 48.0 and 43.7°C at time

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13:00 h, 15:00 h and 17:00 h, respectively, which in turn resulted ∆Ts of 4.1, 10.3 and 7.5°C,

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respectively. Black-40% was the most effective treatment as it had ∆Ts of 2.2, 6.8 and 5.0°C at 13 ACS Paragon Plus Environment


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time 13:00 h, 15:00 h and 17:00 h, respectively. This means that during the warmest part of the

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day (15:00 h), ∆T was 3.7°C lower in Black-40% than control.

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Berry weight and must composition. Berry weight was significantly higher in Black-

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40% than Aluminet-40% on 29 July (Fig. 4A). Although, this was not maintained throughout

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berry development, Aluminet-40% had significantly lower berry weight than Blue-40% and

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Control on 29 August and Blue-40% on 9 September. Must TSS were not significantly different

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at any time point (Fig. 4B). However, a trend was observed on 29 July, where TSS were slightly

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lower in Blue-40% and Black-40% compared to the rest of the treatments. Must pH (Fig. 4C)

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was significantly higher in Control than Black-40%, including harvest point. Blue-40% had a

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significantly higher pH than controls on 29 July but not at any other sampling point. Must TA

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(Fig. 4D) was significantly lower in Control compared to Aluminet-40% on 3 sampling points

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and Black 40% on 2 sampling points, respectively. Although Control was 2.7 g L-1 lower than

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Black-40% on 19 July, Control was only 0.6 g L-1 lower than Black-40% at harvest.

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Grape flavonoids content. Berry skin anthocyanin content (Fig. 4A) increased rapidly

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until 19 August (21.2° Brix), and from that point on Control, Pearl-20%, Alumninet-40% and

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Blue-40% had a trend to decrease, whereas Black-40% continued an increased trend through

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harvest. In fact, Black-40% had higher concentration of anthocyanins than Control in the last two

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sampling points. As for flavonols (Fig. 4B), they started to accumulate before veraison and

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controls already had significantly higher concentration of flavonols than the 40% shading factor

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treatments on 20 June, 24 days after the shade nets were placed. Significant differences were

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maintained between Control and Blue-40% and Black-40% through 19 August, and in fact, for

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the last part of that period, even Pearl-20% and Aluminet-40% had a higher content of flavonols

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than Blue-40% and Black-40%. As grapes approached to their commercial maturity, flavonol 14 ACS Paragon Plus Environment

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content decreased especially in Control, Pearl-20% and Aluminet-40%. This led to have similar

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flavonol content across all treatments at harvest.

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Anthocyanin hydroxylation (3’4’5’/3’4’ -OH; Fig. 5c) tended to be higher in Blue-40%

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and Black-40% than Control in the first two berry samplings. In fact, the difference between

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Control and Blue-40%, the two most different treatments, increased throughout development,

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from 9.9% on 9 August to 18.3% higher on 9 September. Flavonol hydroxylation (Fig. 5d)

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showed to some extent a similar pattern to anthocyanins, with Control and Aluminet-40% having

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the lowest hydroxylation. Although, as it happened in anthocyanins, Blue-40% and Black-40%

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had significantly higher hydroxylation of flavonols than Control, this time Black-40%, and not

310

Blue-40%, had the highest values.

311

Overall, proanthocyanidins content and composition was not greatly affected by partial

312

solar radiation exclusion treatments (Tables 3 and 4). Nevertheless, on 20 June, the only

313

sampling before the onset of veraison, skin proanthocyanidin content was 32% lower in Black-

314

40% compared to Control. No other net had a significantly different content of

315

proanthocyanidins in either skin or seeds at any of the samplings. Among the nets, Aluminet-

316

40% had a higher concentration of seed proanthocyanidins than Blue-40%. Proanthocyanidin

317

mDP remained unaffected by the treatments across tissues and samplings. Compared to Control,

318

the percentage of epigallocatechin was higher in Blue-40% than Control on 29 July. Skin

319

proanthocyanidin galloylation (% of epigallocatechin gallate) was significantly lower in Black-

320

40% on 20 June and Blue-40% on 29 July, Pearl-20% on 19 August, lower in Blue-40% on 9

321

September sampling dates, respectively, and higher in Blue-40% seeds.

322

DISCUSSION



Page 16 of 38

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Canopy development, gas exchange, water potential. Lower CO2 assimilation rates (A)

324

in shaded leaves compared to sun exposed leaves have been reported in previous works.36, 42 At

325

veraíson, measures of saturated net photosynthesis have been shown to be little affected by

326

shading conditions.42 Previous work showed a reduction in canopy development under whole

327

canopy shading, and photosynthesis being reduced up to 40% because of the reduced photon flux

328

density.31, 43 However, in our work these differences were not observed, because the net filtered a

329

maximum of 40% radiation (as opposed to 70% in the cited works). The nets mainly covered the

330

fruit zone and not the full canopy, the trellis system was slightly open in the center thus favoring

331

irradiation of the internal canopy. Gas exchanges and Ψl were performed on leaves out of the net

332

shade, mainly to monitor differences in water status, being this an important driver in the

333

biosynthesis of berry ripening.44 Although carbon fixation and stomatal conductance of leaves at

334

the fruit zone level (i.e. mostly exposed through the nets) were most likely lower due to lower

335

irradiance,36 this was not enough to produce any change in canopy size, plant water status or

336

grape ripening in the shade net treatments. The effect on canopy development could be

337

negligible due to the shade net installation date (27 May), when canopy development was almost

338

complete. Aspects such as canopy size or vine balance, necessary to achieve an adequate grape

339

composition and fruit ripening,45 were therefore maintained and similar between treatments and

340

the effects on grape composition were mainly related to the microclimate at the fruit zone.

341

Berry size, total soluble solids and must acidity. Plant organ size is a function of

342

photomorphogenesis and water balance. Plant cell division and expansion may be altered by sun

343

exposure through photomorphogenic effects at low rates.46 However, in vineyards, where solar

344

radiation is high and processes such as water balance may play a bigger role determining berry

345

growth than photomophogenesis. In previous studies, berry mass was not affected by solar


Page 17 of 38


346

radiation, temperature or specific wave bands.19, 28 Thus, the smaller berry size of Aluminet-40%

347

was not fully expected as these berries were not the one going through the highest nor the lowest

348

temperatures and radiation levels among all treatments. Must acidity was lower (higher pH and lower TA) in Control compared to Black-40%.

349

28

350

Spayd

, where radiation and heat gain effects of sun exposure on grape composition were

351

decoupled, conferred all effect on grape acidity reduction to elevated temperature. Although,

352

there is a long track of studies reporting lower acidity and malate-to-tartrate ratio in grapes

353

grown under elevated temperature, this may be an oversimplification.45, 47-49 Thus, the immediate

354

cause is an increased break down and decreased replenishing of tricarboxylic cycle

355

intermediates.50 However, in the bigger picture, temperature affects timing and speed of events,

356

decoupling the accumulation of sugars and the maturation of the organic acid profile, leading to

357

lower acidity and malate-to-tartrate ratio under elevate temperature.26 In this trial, the few hours

358

a day that temperatures were higher in Control were enough to induce a significant reduction of

359

must acidity. However, this reduction was rather small and it is unlikely that this could have

360

implications for winemaking.

361

Grape skin anthocyanins and flavonols. Solar radiation up regulation of anthocyanin

362

and flavonol biosynthesis has been reported in red wine grapes.7 However, whereas anthocyanins

363

are constitutively high in red wine grape cultivars,51 flavonols are only present in high

364

concentrations as an acclimation response to UV-B radiation.37, 52 In our results, we did not see a

365

significantly higher anthocyanin content in Control at any sampling point. This could be due to

366

the response of anthocyanins accumulation to light, which may increase within a low radiation

367

range but remain similar or even decrease with increasing radiation within a high radiation

368

range.45, 51 On the other hand, we observed a reduction in anthocyanin content in Control few 17 ACS Paragon Plus Environment


Page 18 of 38

369

days before harvest. During the last period of development, anthocyanin degradation usually

370

exceeds biosynthesis.53 It was noteworthy that even flavonols, which are the paradigm of

371

acclimation to high solar radiation,11 decreased in controls during the last days. This has been

372

observed also in other studies, where flavonol accumulation stopped few weeks before

373

commercial harvest and decrease in some cases .7, 54 In previous research, we have shown an

374

enhancement of flavonoid degradation in response to severe water stress.55 The decrease in

375

anthocyanin and flavonol content appears to be a result of berry tissue senescence, which may be

376

modulated by environmental factors.55,

377

anthocyanin and flavonol content in Black-40% nets. Therefore, although flavonoid biosynthesis

378

may be up regulated by increasing sun exposure during the first half of ripening, the same

379

exposure may be deleterious for anthocyanin and flavonol contents during the second half of

380

grape ripening. Hence, maintaining color while waiting for the target astringency or green aroma

381

removal can be a critical issue, especially in warm climate viticulture where there may be an

382

extended ‘hang time’ beyond the target TSS required for fermentations.24, 57-59

56

This would explain the less pronounced decrease in

383

Anthocyanin and flavonols profile hydroxylation has an impact on hue in the case of

384

anthocyanins,38 but also on the antioxidant capacity.60 The redox potential of flavonoids is

385

directly related to their substituents in 3’, 4’ and 5’ positions of the B-ring.60 This, not only has

386

implications for wine making, as higher redox potential of these compounds could make wines

387

more suitable for ageing, but also this property of flavonoids may make 3’4’5;-OH compounds

388

itself more resistant to degradation through the vinification, making them more prone to end up

389

in the wine.61 Thus, the fact that Blue-40% and Black-40% had higher hydroxylation in their

390

anthocyanin and flavonol profiles than Control, can be considered as a positive outcome. This

391

effect is likely due to the up regulation of flavonoid 3’ hydroxylase (F3’H), the enzyme


Page 19 of 38


392

responsible for the synthesis of 3’ 4’ hydroxylated flavonoid precursors, by sun exposure in

393

controls.52

394

Grape skin proanthocyanidins. Although proanthocyanidin content per berry fresh

395

mass (mg g-1 BFM; Table 1) was the highest at the first sampling, content per berry (mg berry-1;

396

data not shown) was the highest on 29 July (around full veraison) and decreased through

397

ripening. Therefore, it can be speculated that most of proanthocyanidin biosynthesis took place

398

before that moment (29 July; 14.9°Brix). The higher proanthocynin content in in Black-40%

399

suggests that proanthocyanidin accumulation may have been down regulated by Black-40% nets.

400

However, proanthocyanidin content across all treatments decreased to a similar level throughout

401

berry development. Koyama, et al. 8 showed an up regulation of flavan-3-ol monomer synthetic

402

enzyme genes in sun-exposed grapes compared to shaded, and this resulted in higher

403

concentration in green berry stages. In fact, several studies found changes in proanthocyanidin

404

accumulation before or around onset of veraison that were not maintained throughout

405

commercial maturity in response to water deficit,62 temperature 21 and sun exposure.63 Therefore,

406

although environmental factors can have a strong effect on proanthocyanidins, the ripening-

407

related degradation often trumps these effects.

408

Proanthocyanidin mDP, which has been related to increase in overall astringency,64 was

409

not different across treatments. However, mDP increased as berries ripened across all treatments.

410

The evolution typical of mean degree of polymerization throughout berry development is not

411

clear as reports include decreases 62, 63 and increases 8, 59 in polymer chain length.

412

As temperatures continue to rise, strategies to reduce berry temperature during ripening

413

may become more necessary. In this study, only Black-40% showed a consistent effectiveness

414

reducing the amount of solar radiation reaching the clusters and reducing berry heat gain during 19 ACS Paragon Plus Environment


415

the warmest part of the day, resulting in a more desirable grape composition. This was likely due

416

to the lower temperature of grapes under in Black-40%. It must be noted that Aluminet-40% had

417

even lower transmittance than Black-40%, but the reflectance of Aluminet-40% appeared to

418

increase the total irradiance at the clusters compared to Black-40%, which results into a higher

419

temperature gain than Black-40%. The use of dyes (as in Blue-40%) and different formulations

420

in plastic manufacturing, have the potential of filtering selectively different wavelengths using

421

plant photomorphogenic responses to induce desirable effects such as flavonoid up regulation,

422

modified canopy architecture or reduce carbon assimilation.19, 32 However, the nets used (Blue-

423

40%), which had a slightly higher transmittance in the blue, far red and infrared wavebands did

424

not have any further beneficial effect compared to Black-40%.

Page 20 of 38

425

In hot climate, wine grape growers have to adopt practices to avoid over exposure to sun,

426

such as letting vegetation sprawl on the afternoon side of the canopies, or trellis that surround the

427

fruit zone with leaves from any direction such as the California sprawl trellis. Shade nets have an

428

installation cost, and may interfere with other practices that require spraying or fruit thinning. In

429

turn, shade nets offer a controlled and homogeneous application of solar radiation when

430

combined with leaf removal to the fruit to promote an adequate fruit composition while avoiding

431

sun damage.

432

Acknowledgements

433

A graduate stipend was provided to Christopher Cody Lee Chen by the Department of

434

Viticulture and Enology and American Society of Enology and Viticulture.


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435

The authors also acknowledge the technical assistance of Andrew Beebe, Cassandra Plank,

436

Runze Yu, Wei-Chao Cheng, and August D’Amato for their help performing the experiment and

437

analyses.

438

Figure Captions

439

Figure 1.

440

experimental year (2016) precipitation, growing degree days, and clear sky days at Oakville, CA

441

USA during the growing season (March-November).

442

Figure 2. Spectral transmittance (A) of shade nets (Pearl-20%, Aluminet-40%, Blue-40% and

443

Black-40%). Total Spectral irradiance (B) received in the open field, in an untreated fruit zone

444

(Control) and under shade nets.

445

Figure 3. Air temperature and Cluster temperature of plants untreated (Control) and under shade

446

nets treatments (Pearl-20%, Aluminet-40%, Blue-40% and Black-40%). Temperatures recorded

447

7 September, 2016 on two clusters (NE and SW side) per experimental unit. Points are means ±

448

standard error (n=4).

449

Figure 4. Berry mass (A) and TSS (B), pH (C) and total acidity (D) throughout berry

450

development of plants untreated (Control) and under shade net treatments (Pearl-20%, Aluminet-

451

40%, Blue-40% and Black-40%). Points are means (n=4) and error bars are minimum significant

452

difference (p

Partial Solar Radiation Exclusion with Color Shade Nets Reduces the

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