Interactive Effects of Mechanical Leaf Removal Timing and Regulated Deficit Irrigation on Biosynthesis of Methoxypyrazines and Phenolics on a Procumbent Grapevine (Vitis vinifera cv. Merlot) in the San Joaquin Valley of California

An experiment was conducted in the San Joaquin Valley of California on Merlot to determine the interaction of mechanical leaf removal (control, pre-bloom, post-fruit set) and applied water amounts [sustained deficit irrigation (SDI) at (0.8) and regulated deficit irrigation (RDI) at 0.8 (bud break-fruit set) – 0.5 (fruit set-veraison) – 0.8 (veraison-leaf fall) of estimated vineyard evapotranspiration (ETc) on productivity and berry skin anthocyanin content, composition and its unit cost per hectare.  The pre-bloom leaf removal treatment consistently maintained at least 20%of photosynthetically active radiation in the fruit zone in both years of the study, while post-fruit set leaf removal was inconsistent across years.  The RDI treatments reduced berry mass, while the post-fruit set leaf removal treatment reduced berry skin mass. The pre-bloom treatment did not affect yield in either year.  Exposed leaf area and leaf area to fruit ratio (m2/kg) were reduced with leaf removal treatments.  The RDI treatment consistently advanced Brix in juice.  Anthocyanin concentration was improved with pre-bloom leaf removal in both years while irrigation treatments had no effect.  Proportion of acylated and hydroxylated anthocyanins were not affected by leaf removal treatments.  In both years SDI increased di-hydroxylated anthocyanins while RDI increased tri-hydroxylated anthocyanins.  Pre-bloom leaf removal when combined with RDI maximized total skin anthocyanins (TSA) per hectare while no leaf removal and SDI produced the least. The cost to produce one unit of TSA was reduced 35%with the combination of pre-bloom leaf removal and RDI treatments when compared to no leaf removal and SDI.  This study provides information to red wine grape growers in warm regions on how to manage fruit to enhance anthocyanin concentration and proportion of anthocyanin hydroxylation while reducing input costs through mechanization and reduced irrigation.

The majority of wine grapes grown in the San Joaquin Valley (SJV) of California are used for bulk wine production.  Fruit used to make red wines from this region are characterized by low anthocyanin accumulation, and receive the lowest price per ton compared to other growing regions in the state. Approximately 34%of the Merlot grapes crushed in the state were grown in the SJV with an average grower return of $ 443/ton compared to $753/ton state average (Cal. Dept. Food. Agric. 2013).  In recent years more efforts have been directed towards applying principles of canopy management with the aid of vineyard mechanization and deficit irrigation practices (Kurtural et al. 2013; Terry and Kurtural 2011; Wessner and Kurtural 2013; Williams et al. 2012) to improve berry composition and grower returns per ton through enhancing the color profile of red wine grapes grown in the region.

Anthocyanins are synthesized via the flavonoid pathway in grapevine cultivars that harbor the wild-type VvmybA1 transcription factor for the expression of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) (Kobayashi et al. 2004).  The encoded enzyme UFGT catalyzes the glycosylation of unstable anthocyanidin aglycones into pigmented anthocyanins.  Two primary anthocyanins (cyanidin, delphinidin) are synthesized in the cytosol of the berry epidermis.  Cyanidin has a B-ring, di-hydroxylated at the 3’ and 4’ positions and delphinidin has a tri-hydroxylated B-ring because of an additional hydroxyl group at the 5’ position.  Flavonoid precursors are initially recruited from the phenylpropanoid pathway by chalcone synthases (CHS1, CHS2, and CHS3) and enter the flavonoid pathway.  Two parallel pathways downstream of flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’5’H) (Castellarin et al. 2007) produce either cyanidin or delphinidin.  The 3’ position of cyanidin and delphinidin and sequentially the 5’ position of delphinidin are methoxylated by o-methyl-transferase (OMT) that generates peonidin, petunidin and malvidin, respectively (Castellarin et al. 2007).

The concentration and relative abundance of single and total anthocyanins are variable among red winegrape cultivars due to genetic control and developmental regulation.  However, there is general agreement in literature that when amount of diffuse light is increased (Dokoozlian and Kliewer 1995) or when an amelioration of microclimate temperature is associated with a concomitant increase in diffuse light quantity (Spayd et al. 2002), beneficial effects on total skin anthocyanin content of red wine grapes grown in hot climates are observed. Conversely, exposure of clusters to direct sunlight (Berqvist et al. 2001) or low sunlight with concomitant increase in berry temperature (Tarara et al. 2008) was shown to decrease anthocyanin accumulation, increase the proportion of 3’4’ hydroxylated anthocyanidins, and decrease the acylated anthocyanins contributing to total skin anthocyanins.  Cortell and Kennedy (2006) also reported a reduction of tri-hydroxylated anthocyanins in shade grown Pinot noir.  Therefore, the effect of sun exposure results from the interaction of several factors that are hardly uncoupled under vineyard conditions.

Leaf removal is a practice that can improve light transmittance into fruiting zone of the canopy (Diago et al. 2012; Poni et al. 2006; Wessner and Kurtural 2013; Williams 2012).  When leaf removal was applied pre-bloom it was shown to decrease berry set and hence grapevine yield, but improved total skin anthocyanin concentration of red wine grapes (Diago et al. 2012).  The results were used as a means of crop control with the increase in relative skin mass and reduction in yield per cluster being interpreted as the causal increase in anthocyanin concentration.  Therefore, since growers in SJV are paid in tons produced per hectare, previous work in the hot climate of SJV focused on post-fruit set, but was conducted pre-veraison in order to not adversely affect yield (Wessner and Kurtural 2013; Williams 2012). The leaf removal studies conducted in SJV resulted in improved photosynthetically active radiation exposure to canopy interior but no physiological gain for the cultivars studied, some deleterious effects were noted due to overexposure of clusters to direct solar radiation or vegetative compensation response (Geller and Kurtural 2012; Kurtural et al. 2013; Williams 2012).Water deficits were shown to consistently promote higher concentrations of anthocyanins in red wine grapes (Kennedy et al. 2002; Romero et al. 2010; Terry and Kurtural 2011).  However, there were conflicting results as to whether or not there were any direct effects on berry metabolism other than inhibition of berry growth.  It remained unclear if water deficits altered the biosynthetic pathway or if high anthocyanin concentrations resulted from elevated sensitivity of berry growth to water deficits.  Matthews and Anderson (1989) reported that growth of berries was inhibited more and concentrations of anthocyanin in berry skin and wine increased when water deficits were imposed before veraison rather than after veraison.  Similarly, Terry and Kurtural (2011) reported water deficits imposed one-week post-fruit set until veraison resulted in a 25%amelioration of total skin anthocyanins in central SJV.  Based on the observation of similar anthocyanin content per berry, Kennedy et al. (2002) and Terry and Kurtural (2011) concluded that post-veraison water deficits only inhibited fruit growth.

Gene expression studies investigating the regulation of anthocyanin biosynthesis in the grapevine concluded that both pre and post-veraison water deficits increased anthocyanin accumulation.  What is more, water deficits can progressively modify the canopy microclimate by defoliating the basal leaves subtending the fruiting zone, with greater exposure to solar radiation (Terry and Kurtural 2011; Williams 2012).  However, the increase in anthocyanin concentration observed in Merlot grapevines exposed to water deficits was determined not to be due to basal leaf defoliation and exposed solar radiation but was the direct result of water deficit.  Castellarin et al. (2007) reported that the increase in anthocyanin concentration in Merlot was not due to overexpression of FLS1 gene that is strongly correlated with cluster light exposure for flavonoid biosynthesis in the grapevine, but was due to up-regulation of flavonoid synthesis genes, in particular UFGT, CHS2, CHS3, and F3H.While canopy and crop load management studies (Geller and Kurtural 2012, Kurtural 2013; Terry and Kurtural 2011, Wessner and Kurtural 2013) and irrigation studies, particularly those implementing deficit irrigation, have been conducted in the coastal grape growing regions of California (Matthews and Anderson 1988; Williams 2010; 2014), no such studies have combined both factors on wine grapes cultivated in the hot climate of the SJV of California. The overachieving objective of this trial was to quantitatively increase the concentration of total skin anthocyanins of Merlot by investigating the interactive effects of manipulating solar radiation and water amounts applied in this hot climate. The specific objectives of the trial were to improve the light microclimate without adversely affecting yield components while simultaneously reducing applied water amounts to quantitatively and qualitatively improve the skin anthocyanin composition of Merlot in a resource limited environment.

The experiment was a three (leaf removal) × two (deficit irrigation) factorial with a split-plot design with four replicated blocks.  Three rows of 190 vines each comprised one block and four guard rows separated each block.  The three leaf removal treatments were randomly applied as main plot to three rows each.  Each main plot of three rows was split into two deficit irrigation treatments as sub-plot at random, in the geographic middle on the East-West axis of the vineyard.  Each experimental unit consisted of 285 vines of which 48 were sampled from an equi-distant grid per treatment-replicate.