Patent Application
20210333257
In wine grape growing, canopy density is controlled either during the dormant period through pruning, or during the growing season through fruit-zone leaf removal and shoot thinning. These techniques aim mainly to control fruit load, but also to expose grapes to a higher air circulation and solar radiation. This has been associated to a reduction in fungal diseases (Chellemi and Marois, 1992), but also some desirable effects for specific winemaking targets, such as green aroma removal in Cabernet Sauvignon (Koch et al., 2012) or higher content in phenolic compounds in full bodied red wine grapes (Matus et al., 2009). However, this is not a general rule and some winemaking frameworks, such as sparkling rose, do not pursue a great phenolic content but a higher acidity and acidic fruit aromas, which may be affected by exposure due to increasing temperature (Sweetman et al., 2014). Even when moderate exposure is desired, the impact of the excess of solar radiation can be negative, resulting in flavonoid and organic acid degradation (Martínez-Lüscher et al. 2017).
Canopy size is also the focus of vineyard management due to the need of balance with fruit load. The spatial assessment of vine vigour (i.e., vegetative growth) is a widely used tool in the industry and research, especially for precision agriculture due to its relation to grape composition (Baluja et al., 2012; Cortell et al., 2007). Uneven vine vigour can be an indication of nutrient deficiency, different soil profiles, source/sink balances, different mesoclimates, or pathogens. These factors may lead to a suboptimal use of a plot (Steyn et al., 2016). Therefore, there is a strong need for tools that are able to quantify and optimize the exposure of red grapes and the assessment of canopy size.
In one aspect, the disclosure features a method for determining whether a grape is under exposed or overexposed to solar radiation. In some embodiments, the method comprises:
(a) extracting and purifying flavonols comprising 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonol from grape skins;
(b) determining percentages of 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH in the extracted flavonols using a spectrophotometer and a Partial Least Squares (PLS) regression model, wherein:
if the percentage of 4′-OH flavonol content is below 4%, then the grapes are underexposed and canopy will need pruning; or
if the percentage of 4′-OH flavonol is above 10.5%, then the grapes are overexposed and need cover; or
if 3′4′5′-OH flavonol content is above 45%, then the grapes are underexposed and canopy will need pruning; or
if the percentage of 3′4′5′-OH flavonol is below 30%, then the grapes are overexposed and need cover. In some embodiments, in view of the method, grapes are covered or the canopy is pruned.
4′-OH flavonol is also referred to as kaempferol glycosides herein. 3′4′-OH flavonol is also referred to as quercetin and isorhamnetin glycosides herein. 3′4′5′-OH flavonol is also referred to as myricetin, laricitin and syringetin glycosides herein.
In some embodiments, the PLS regression model comprises an equation using the absorbance of a purified flavonol mixture at multiple wavelengths between 220 and 800 nm. In some embodiments, the PLS model calculates the concentration of each of 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonol, calculates their percentage of total flavonol and plots the percentages in a ternary plot for the comparison to grapes with different exposures. In some embodiments, pruning the grape canopy comprises shoot thinning and/or leaf removal to increase exposure.
Also provided is a computer program product comprising a computer-readable storage medium containing computer program code for:
receiving a data set representing the absorbance of a mixture of flavonols measured at all wavelengths between 220 and 800 nm,
applying a Partial Least Squares (PLS) regression precalibrated model to generate percentage of the one or more flavonols selected from the group consisting of 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH, and comparing the percentage of the flavonol to a cut-off percentage.
In some embodiments, the flavonol is 4′-OH flavonol and the cut-off percentage is 4% out of total flavonol content. In some embodiments, the flavonol is 4′-OH flavonol and the cut-off percentage is 10.5% out of total flavonol content. In some embodiments, the flavonol is 3′4′5′-OH flavonol and the cut-off percentage is 30% out of total flavonol content In some embodiments, the flavonol is 3′4′5′-OH flavonol and the cut-off percentage is 45% out of total flavonol content.
In some embodiments, the computer program product further comprises code for indicating whether pruning is suggested or not based on the comparing.
Exposure to solar radiation is a big driver for grape composition. Flavonols are an important part of acclimation to high solar radiation in plant tissues. Their synthesis is unregulated by UV-B radiation, leaving an unmistakable fingerprint on their diversification according to their substituents in the B-ring of the flavonoid skeleton.
The disclosure is directed to using the flavonol profile of a grape (e.g., red grape plant) as a biomarker (i.e. indicator) to assess the overall exposure of red wine grapes to solar radiation. The percentage of each flavonol in the flavonol profile can be used as an indication of whether the red grape canopy needed to be pruned (leaf removal or shoot thinning) in order to optimize grape composition, or alternatively, whether the grape canopy did not protect grapes and corrective action needs to be taken next season. Changing trellis or row direction or use shade nets are options to reduce the exposure of grapes each with different costs and implications. As discussed herein, experiments were conducted to relate canopy porosity with flavonol profile, to follow flavonol profile development under solar radiation exclusion treatments, and to relate flavonol profile to natural and induced variability in commercial vineyards. Results show a strong relationship between exposure and the three components of flavonol profile, increasing the proportion of 4′, 3′4′ in detriment and 3′4′5′ hydroxylated (—OH) flavonols. However, whereas the proportion of 3′4′ and 3′4′5′-OH flavonols were sensitive to the progress of ripening, the proportion of 4′-OH flavonols remained stable for a wide period and range of total soluble solids. The proportion of 4′-OH flavonols was inversely related to canopy size (i.e., pruning weight) and was responsive to cultural practices such as shoot thinning and leaf removal. In addition, this biomarker was not sensitive to water status or different soluble solids levels, which typically have a strong effect on anthocyanin profile. Together, these results demonstrate the reliability and the usefulness of the proportion of 4′-OH flavonols as a biomarker to assess the effectivity of vineyard practices or spatial variability in relation to canopy size or grape exposure to solar radiation. The proportion of 3′4′5′-OH flavonols can be useful also due to their negative correlation to exposure. Even though 3′4′5′-OH flavonols are not as reliable as 4′-OH flavonols, 3′4′5′-OH flavonols are present in higher proportions and are easier to determine by either HPLC or spectrometry. The possibility of assessing grape solar radiation interception of pools or single grape berries opens the possibility of establishing the relationship between solar radiation and grape quality traits by future studies reducing the need of taking as many light measurements or canopy porosity.
This disclosure makes more accessible the determination of flavonols profiles (proportion of each kind of flavonols) in anthocyanin rich tissues either through HPLC-DAD method fine-tuning, or through UV-VIS multi=−wavelength spectrometry and a PLS regression model. We also aimed to propose the proportion of 4′-OH flavonols as a biomarker of the integrated solar radiation received by grapes and test its reliability. In the results, proportion of 4′-OH flavonols was more reliable predictor of canopy porosity than any other predictor variable, including visual assessments of sun damage. This reliability was based on its stability through hang time, when overall flavonols content was decreasing, and the lack of significant effect of water status.
The use of the proportion of 4′-OH flavonols was exemplified by the estimation of grape exposure in management zones resulting from proximal sensing and cultural practices aiming to increase the exposure of the grapes or balancing vine vigor. Given the widely extended use of HPLC-DAD methods to determine anthocyanin profile in research and the use of spectrometry in grape growing industry, we provided the principles to understand and methods to determine flavonol profiles and use them as biomarker of solar radiation interception by grapes.
As described in detail further herein, the disclosure includes methods of determining whether to prune a red grape canopy by determining the percentages of various flavonols (e.g., 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonol) extracted from red grape skins and comparing the determined percentages to threshold values to assist in determination of solar radiation status of grape plants for grape harvest management.
Flavonoids are one of the most versatile metabolites in plants. For instance, they play a key role in promoting sexual reproduction of plants, enabling a normal development of pollen, and making flowers noticeable to pollinators or fruits to animals that contribute to seed dispersal (Koes et al., 1994). They play also an important role in cross-talk between plants and microorganisms, as they are exuded into the rhizosphere (Cooper, 2004; Lagrange et al., 2001). Flavonoids are remarkable for their inhibitory activity on inter-species seed germination and fungal pathogens (Kong et al., 2004). Due to their strong radical scavenging activity, flavonoids are key in the protection of the plant biochemical machinery from oxidative stress. For instance, flavonols and anthocyanins are involved in the mitigation of water stress (Nakabayashi et al., 2014). In the case of flavonols, they are accumulated in epidermal cells of plant tissues in response to light, especially UV, filtering the most novice part of the solar spectrum (Kolb et al., 2001; Martínez-Lüscher et al., 2013b).
Within each class of flavonoids, certain structural variation exist in their basic 15-carbon skeleton, leading to different physicochemical properties. In the case of flavonols and anthocyanins of wine grape (Vitis vinifera), they often present different substituents in their 3 position of the C-ring and the 3′ and 5′ positions of the B-ring. The most frequent substituent in 3 position of C-ring is glucose and in the case of anthocyanins, this substituent is crucial for its stability, color, and increase their hydrophilic characteristic (Vogt and Jones, 2000). In the case of flavonols, aglycones are reported in some cases in grape skin extracts, but often are reported in wines, suggesting that they remain stable after hydrolysis (Castillo-Munoz et al., 2007). In other fruit species, less common sugars and di-glucosides are more frequently found as substituents of in 3 position of the C-ring (Acevedo de la Cruz et al., 2012). In turn, sugar substituents can be acylated, mainly with acetyl or a phenolic hydroxyl groups. This is performed enzymatically and increases the solubility of flavonoids in various media, as well as their stability and their antioxidant capacity (Chebil et al., 2006; Zhao et al., 2017).
Anthocyanins are colorful compounds and hydroxyl and methyl group substituents in 3′ and 5′ positions of the B-ring give place to five kinds of compounds (pelargonidins, cyaniduns, peonidins, delphinidins, petunidins, and malvidins), which by themselves can give place to a range of colors from orange to purple in plant tissues (Dixon et al., 2013). Although flavonol are colorless, their substituents in the B-ring may also lead to different optical properties appreciable in the absorbance in the UV range. For both anthocyanins and flavonols, there is a relationship between the substituents in these positions and the chemical behavior of anthocyanins and flavonols. The addition of hydroxyl groups in 3′ and 5′ positions of the B-ring result in a great increase in oxidation potential as determined by cyclical voltammetry (Arroyo-Currás et al., 2016), and in vitro antioxidant capacity of flavonols, anthocyanins, and flavan-3ols (Tabart et al., 2009).
Grapes varieties constitutively present a wide range of flavonoids profiles depending their substituents in 3′ and 5′ position (Mattivi et al., 2006). However, most full-colored red wine grapes have a highly 3′ and 5′ substituted profile (rich in anthocyanins and flavonols with hydroxyl or methyl groups in 3′ and 5′ position of the B-ring). For instance, Cabernet Sauvignon, Shiraz, or Tempranillo are examples of highly 3′ 5′ substituted profiles and Sangiovese and Pinot noir are examples of poorly 3′ 5′ substituted profiles, similar to table grapes (Mattivi et al., 2006). Hereby, anthocyanin and flavonol profiles are highly determined genetically and are controlled and inherited as a quantitative loci trait (Fournier-Level et al., 2011; Malacarne et al., 2015). In fact, these profiles have been proposed as an authentication tool for wines (Hermosín-Gutiérrez et al., 2011). Ultimately, profiles are determined by the proportion in the levels of expression of the gene(s) encoding flavonoid 3′ 5′ hydroxylase (F3′5′H) and the synthetic enzyme(s) controlling the synthesis of each flavonoid (i.e., UFGT for anthocyanins and FLS for flavonols) (Castellarin and Di Gaspero, 2007; Martínez-Lüscher et al., 2014a). However, there also a strong environmental regulation of this trait. Water deficit appears to be involved in the regulation of flavonoid 3′ 5′ hydroxylase (F3′5′H), an enzyme responsible for the hydroxylation of dihydrokaempferol and dihydroquercetin into dihydromyricitin (Kaltenbach, 1999;
In previous research, Martínez-Lüscher et al. (2014b) and Martínez-Lüscher et al. (2014a) proposed a mechanistic hypothesis for the constitution of anthocyanin and flavonol profile under UV-B radiation and water deficit. Hereby, grapes from fruit-bearing cuttings grown under natural light filtered through a glasshouse (99.9% and 15% of UV-B and UV-A removal, respectively) responded linearly in exposure time to an artificial source of UV-B. This response corresponded to an increase in flavonol concentration (R2=0.43), decrease in the proportion of 3′4′5′-OH (R2=0.70; negative correlation), and increase in the proportion of 3′4′-OH (R2=0.67) and 4′-OH (R2=0.82) flavonols (Martínez-Lüscher et al., 2014b). The coefficient of determination of 4′-OH flavonols was further improved (R2=0.95) by accounting radiation before and after veraison separately, and post veraison radiation appeared 4.17 times more effective than pre-veraison dose. The study of the interactive effects of UV-B with water deficit revealed that UV-B did not upregulated all genes involved in flavonoid biosynthesis in the same way. Whereas genes encoding for enzymes upstream in the pathway may present a nearly 2-fold induction (e.g., CHS and F3′H). The induction of flavonol synthase (FLS) was 10-fold (Martínez-Lüscher et al., 2014a). This disproportion allegedly resulted in stronger competition of FLS with flavonoid hydroxylases (F3′H and F3′5′H) for flavonol substrates, which reduced their chances of being hydroxylated before being transformed into flavonols (
The disclosure is directed to using the percentages of three flavonols (e.g., 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonol) extracted from red grape skins to determine whether a red grape canopy needs to be pruned, i.e., by shoot removal and/or leaf removal. The inventors have found through experimentation that the proportion of 3′4′ and 3′4′5′-OH flavonols were sensitive to the progress of grape ripening, while the proportion of 4′-OH flavonols remained stable. The three flavonols, 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonols, may be used as markers that indicate, i.e., the progress of red grape ripening, the amount of solar exposure, and the ideal red grape canopy porosity. Once determined and compared to a threshold value, the determined amount of various flavonols can be used to determine whether the grape canopy is pruned, or optionally shaded to sow ripening, or harvested to avoid over ripening.
Flavonols can be extracted from grapes (e.g., grape skins) by either liquid extraction or solid phase extraction. In liquid extraction, a suitable organic solvent (e.g., methanol, ethanol, or ethyl acetate) can be used together with water and a trace amount of acid (e.g., hydrochloric acid). As described in Example 5, a mixture of methanol, water, and hydrochloric acid was used to extract flavonols from grape skins. Liquid extraction can be performed one or more times while adjusting the amounts of organic solvent and acid. A detection method such as mass spectrometry or spectrophotometry can be used during the extraction process to ensure that most of the desired flavonols are in the aqueous phase (e.g., water) and not in the organic phase (e.g., methanol, ethanol, or ethyl acetate). The detection method can also help in adjusting the relative amounts of organic solvent and acid. Following liquid extraction, the extract can be filtered or centrifuged to remove undesired products and further purified using solid extraction, e.g., high-performance liquid chromatography (HPLC). The extract can also be evaporated to obtain a substantially dry, solid composition comprising flavonols. As used herein, “extraction” can include a step of further purification of one or more flavonol.
Solid extraction generally involves the use of a chromatographic resin column, with separation and elution of the differentially enriched fractions from the column. Depending on the technical needs, the particular length and/or particle size of the resin column can be chosen to achieve the ideal separation. Similar to liquid extraction, a mixture of organic solvent, water, and acid can be used to elute and separate the components in the extract from the resin column. Examples of organic solvents that can be used include, but are not limited to, methanol, ethanol, ethyl acetate, chloroform, and dimethyl chloride. Those skilled in the art are familiar with the various parameters that can be adjusted during a chromatographic resin column extraction and purification in order to obtain flavonols and avoid co-elution of flavonols with other components in the extract. For example, as demonstrated in Example 6, the percentage of formic acid, as well as the elution rate, can be adjusted in order to achieve the best separation between flavonols and other phenolic compounds (e.g., anthocyanins). In some embodiments, the percentage of formic acid used to extract the flavonols is between 4-8% (e.g., 5-7%, e.g. about 6%) to achieve separation of the different target flavonols.
Following extraction, one, two, or three flavonols selected from 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonols can detected and quantified from grape skins of a grape plant. 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonol can be detected as desired. In some embodiments, the quantity of flavonols is determined using a spectrophotomer to detect one or more wavelengths from a plant sample (e.g., grape skin). Methods of detecting flavonols can include methods involving spectrophotometers are described in, e.g., Lorrain, et al., Molecules 2013, 18, 1076-1100 reviewing some flavonol detection methods.
In some embodiments, the individual fractions within the flavonols group of chemical compounds (i.e. 4′-OH, 3′4′-OH, and 3′4′5′-OH flavonols) are quantified based on a spectra of wavelengths using PLS regression. Examples of quantification based on one wavelength in other contexts can be found in Bouhsain et al., 1997; Richardson et al., 2004. However, in these methods single (or few) wavelength(s) and linear regression are used to quantify the target analytes as a group (i.e. total flavonols), which according to our findings does not correlate well to solar radiation due to degradation. This is a relatively new procedure resulting from the emergence of statistical analyses related to partial least squares (PLS) regression that allows to determine not only total flavonols, but the content of each flavonol group in a mixture. Single (or few) wavelength methods use linear regression and have poor performance with autocorrelared variables such as the overlapping wavelengths of the different flavonols. On the other hand, methods using wide spectra and PLS regression can be calibrated to perform well for auto correlated variables or noisy readings or contaminated samples.
Exemplary wavelengths for generating the spectra include those wavelengths between 220 and 800 nm. In some embodiments, partial least squares (PLS) regression can be applied to spectrophotometric data from the grapes to predict percentage of 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonols. In some embodiments, the PLS regression model is similar to a multivariate regression model with same number of coefficients as predictor variables, for example in the form of y=coef1*x1+coef2*x2+c for 2 variables whereas the PLS regression model for the calculation of flavonol profiles would have 550 predictor variables and 550 coefficients obtained from few dozens of independent observations. The concentration of each flavonol can be generated to calculate for each flavonol group the percentage of total flavonol content of the sample. In some embodiments, the PLS regression model is calibrated and validated from known samples using HPLC-DAD. For example, a model equation can be calibrated using multiple spectra of flavonol samples. In some embodiments, the calibration is performed in R software using a PLS package. Two data frames were constructed, the first, having as many rows as samples used in the calibration (64) and as many columns as predictor variables (800 nm-220 nm=580 variables; 550 variables after applying a Savitzky-Golay filter). The values of the data frame being the absorbance of each sample at each wavelength in the spectrophotometer. The second data frame, has as many rows as samples used in the calibration (64) and as many columns as independent variables (3 variables: concentration of 4′OH, 3′4′OH and 3′4′5′OH flavonols). The values of this data frame are the concentration of each of the 3 flavonol groups determined by HPLC-DAD in the solutions used to measure the spectra in the first data frame. A random selection of 68 samples were used to fit the model and the 14 independent samples left were used to validate the performance of the model. This procedure was repeated 100 times yielding determination coefficients ±SD of R2=0.61±0.18, 0.57±0.19 and 0.68±0.16 for the percentage of 4′OH, 3′4′OH and 3′4′5′OH flavonols, respectively.
In some embodiments, the PLS model calculates percentage of total flavonol and plots the percentages in a ternary plot for the comparison to grapes with different exposures. See, e.g.,
Once the 4′-OH flavonol, 3′4′-OH flavonol, and 3′4′5′-OH flavonols content percentage of the sample(s) have been determined, they can be compared to one or more threshold value(s) to determine the level of solar exposure of the plant. The inventors have discovered that useful thresholds include, for example:
Using the percentage of 4′-OH flavonol,
In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection methods described herein (e.g., the spectrophotometric measurements) into a recommendation for pruning, harvesting, or covering the plant canopy as discussed above.
Any of the analytical steps (converting spectra to flavonol content values or comparing such values to thresholds, or indicating a canopy management step (prune, harvest, cover, etc.) described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps. Thus, embodiments are directed to computer systems configured to perform the steps of any of the methods described herein, potentially with different components performing a respective step or a respective group of steps. Although presented as numbered steps, steps of methods herein can be performed at a same time or in a different order. Additionally, portions of these steps may be used with portions of other steps from other methods. Also, all or portions of a step may be optional. Any of the steps of any of the methods can be performed with modules, circuits, or other means for performing these steps. In some embodiments, a spreadsheet (e.g., Excel™) is used for the regression model.
In some aspects of the present disclosure, a computer product is provided. The computer product can comprise a non-transitory computer readable medium storing a plurality of instructions that when executed determine grape canopy management using the criteria described herein.
Any of the computer systems mentioned herein may utilize any suitable number of subsystems. Examples of such subsystems are shown in
The subsystems shown in
A computer system can include a plurality of the same components or subsystems, e.g., connected together by external interface 181 or by an internal interface. In some embodiments, computer systems, subsystem, or apparatuses can communicate over a network. In such instances, one computer can be considered a client and another computer a server, where each can be part of a same computer system. A client and a server can each include multiple systems, subsystems, or components.
It should be understood that any of the embodiments of the present disclosure can be implemented in the form of control logic using hardware (e.g. an application specific integrated circuit or field programmable gate array) and/or using computer software with a generally programmable processor in a modular or integrated manner. As user herein, a processor includes a multi-core processor on a same integrated chip, or multiple processing units on a single circuit board or networked. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will know and appreciate other ways and/or methods to implement embodiments of the present disclosure using hardware and a combination of hardware and software.
Any of the software components or functions described in this application may be implemented as software code to be executed by a processor using any suitable computer language such as, for example, Java, C++ or Perl using, for example, conventional or object-oriented techniques. The software code may be stored as a series of instructions or commands on a computer readable medium for storage and/or transmission, suitable media include random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard-drive or a floppy disk, or an optical medium such as a compact disk (CD) or DVD (digital versatile disk), flash memory, and the like. The computer readable medium may be any combination of such storage or transmission devices.
Such programs may also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet. As such, a computer readable medium according to an embodiment of the present invention may be created using a data signal encoded with such programs. Computer readable media encoded with the program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer readable medium may reside on or within a single computer product (e.g. a hard drive, a CD, or an entire computer system), and may be present on or within different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing any of the results mentioned herein to a user.
As noted above, the methods described above indicates to a user how to manage the grape plant canopy for optimal grape fruit maturation. When flavonol content indicates the plant has not received adequate solar radiation, the canopy can be pruned to allow additional light to access the grape fruit. For example, leaves, stems or both can be removed from the grape canopy. Harvesting can be achieved by any appropriate method. The amount of pruning can be adjusted depending on the amount of solar radiation already received and based on, for example, desired harvest date.
Alternatively, if the flavonol content indicates the plant has received sufficient solar radiation, then the user may choose to harvest the grape fruit (e.g., without pruning) or cover the canopy with a material that provides partial or complete shade to the grape plant, thereby slowing further ripening of the fruit.
Effects of Solar Radiation and Berry Development on Flavonol Profile
In previous research, MyBF1 and FLS were upregulated by UV-B radiation in grapevine (cv. Tempranillo) fruit-bearing cuttings, and grape total flavonol content was linearly related with the time of exposure to an artificial source of UV-B radiation. In the study, the increase in flavonol content was confirmed with cv. Cabernet Sauvignon under field conditions up to ripeness. V vinifera grapes typically go through a ripening process where there is a change in color (degradation of chlorophyll and synthesis of anthocyanins), decrease in astringency (decrease in tannin activity related to proanthocyanin degradation), decrease in acidity (break down of organic acids), and sweetening (accumulation of glucose and fructose mainly). During ripening, flavonol as well as anthocyanin accumulation typically increases sharply up to a point (ca. 23 Brix, ripeness). After that point grape growers leave the fruit in the vine for a variable period (hang time), awaiting for a specific flavor and mouthfeel to develop. Although it is considered that sugar translocation into the berries is no longer taking place during hang time, their content in the pulp may increase due to water loss related to tissue senescence (Keller, 2010). During hang time, flavonoids can degrade even faster than water loss, resulting in decreased concentration, and this decrease may be stronger under high sun exposure (Martínez-Lüscher et al., 2017).
In this study, a period of accumulation during ripening and degradation during hang time was observed. While these two processes took place, flavonol profile kept evolving. Flavonol profile was not significantly different between shading factors before the onset of ripening. However, throughout ripening, 40% shading factor had a stronger increase in the proportion of 3′4′5′-OH flavonols. In addition, whereas the proportion of 3′4′-OH and 3′4′5′-OH flavonols kept evolving even passed ripeness, the proportion if 4′-OH flavonols stabilized and remained unchanged for a period of several weeks.
The process leading to flavonol profile constitution under water deficit and solar exposure has been elucidated (Martínez-Lüscher et al., 2014a). The evolution of flavonol profiles through hang time has not been studied before. One plausible reason for profile changing beyond ripeness, when accumulation balance is negative, and synthesis is no longer taking place, is a differential degradation of the flavonols with different levels of hydroxylation. The addition of substituents of the flavonoid B-ring may strongly increase the antioxidant capacity of the compounds (Arroyo-Currás et al., 2016; Tabart et al., 2009). This higher antioxidant capacity results into higher stability, which could explain the differential degradation observed in this study. It is also noteworthy that F3′5′H has been reported to have affinity, not only for dyhidroflavonols, but also flavonols (Kaltenbach et al., 1999). In fact, Kaltenbach et al. (1999) reported that, in vitro, 3′4′-OH substrates may be easily taken by F3′5′H when 4′-OH substrates are depleted. Therefore, although metabolic channeling could avoid this (Winkel, 2004), an active conversion of 3′4′-OH flavonols into 3′4′5′-OH flavonols after ripeness could not be ruled out. There are many uncertainties around how the diversification of flavonoids takes place in vivo beyond levels of expression of genes encoding for their synthesis enzymes, substrate affinities of these, and differential degradation. To what is known, enzymes of the phenylpropanoid pathway have a spatial arrangement in the cell, using channeling between enzymes, which allows the sequestration of toxic intermediates and controlling flux among branch pathways (Winkel, 2004).
Effects of Canopy Size and Water Status on Flavonol Content and Profile
Pruning weights are collected in as a standard practice in viticulture research given its reliability as a proxy of the photosynthetic biomass. The ratio of yield divided by pruning weight (i.e., Ravaz index) is used as an indicator of the balance between sinks and sources, which promotes an adequate ripening (Kliewer and Dokoozlian, 2005). In addition, in training systems where there is a localized fruit zone under the canopy, pruning weights are related to the extinction of light trough the canopy (
In previous research, flavonol profiles were shaped through the interaction of UV-B radiation and water deficit (Martínez-Lüscher et al., 2014a). Although the proportion of 4′, 3′4′, and 3′4′5′-OH were greatly affected by UV-B, the proportion 3′4′ and 3′4′5′-OH flavonols was also affected to some extent by water status (Martínez-Lüscher et al., 2014a). In the data analysed by this study, mean stem water potentials had a wide range of variation (−1.42 to −0.90 MPa) and still the correlation between stem water potentials and the proportion 3′4′ and 3′4′5′-OH flavonols was not significant. One reason for the lack of effect of water status may be the overriding effect of solar radiation on flavonol profiles. Another explanation for this lack of effect is that the datasets used had a significant range of variation in ripening (i.e., TSS). As discussed before, flavonol profiles are highly sensitive to the progress of ripening (
Using the Percentage of 4′-OH Flavonols as Metabolic “Data Logger” of Solar Radiation Incidence on Each Individual Grapes
The percentage of 4′-OH flavonols was highlighted as a great predictor of solar exposure and they had a strong linear correlation (
Other metabolic biomarkers are already widely used in research and are starting to permeate into commercial practices. For example, C13 was first reported by Wickman (1952) and in grape juice by Gaudillere et al. (2002). C13, a stable isotope signal in plant tissues in general, but especially in C3, is an integrator of photosynthetic performance mediated by stomatal conductance. In brief, there is a lower diffusivity of C13 through the stomatal pathway and a fractionation by RUBISCO related to sub stomatal CO2 levels (Farquhar et al., 1989), which makes plants grown under water deficit have a lower proportion of C13 in their carbon pool. Given the importance of maintaining an adequate level of water stress in the production of quality red wine grapes, there is a need for verification of the cultural practices used throughout the season. The analyses of stable isotopes is an affordable option for this purpose, and it has been widely used in studies as a verification for plant water status (de Souza et al., 2003; Gibberd et al., 2001; Koundouras et al., 2008). In a similar way, there is a need for verification of light exposure in relation to canopy management practices. The correlation of 4′-OH flavonol with solar exposure could be useful to verify the changes in solar radiation reaching the fruit in response to treatments aiming to alter fruit microclimate. In the results, a vineyard with presumably an excess of vigor, the amount of solar radiation reaching the grapes needed to be adjusted to reach an optimal grape composition (Haselgrove et al., 2000; Koch et al., 2012). Leaf removal and shoot removal are the most widely used cultural practices for this purpose, the second being more effective in increasing light transmission (
Currently, the ways to estimate solar radiation transmission into the fruit zone are sensors that allow integrating radiation readings either through space (i.e., line quantum sensor) or time (i.e., data logger), but not both, as the determination of the proportion of 4′-OH flavonols of a representative sample of grapes. Although flavonol profiles may be an arbitrary scale, it can be calibrated against values of accumulated radiation (Martínez-Lüscher et al., 2014b). Given the robustness of the response of the proportion 4′-OH flavonols to solar radiation, the calibration of this biomarker for a given genotype should not be influenced by crucial factors in viticulture such as hang time or water deficit, allowing comparisons between different years and plantings.
Plant Water Status
Plant water status was assessed by means of stem water potential (Ψstem) measured around solar noon (12-14:00). Three leaves from each experimental unit were covered with a zip-top plastic bag and aluminium foil for two hours. Leaves were exscinded with a razor blade and Ψstem was determined using a pressure bomb (model 615, PMS).
Canopy Size
Canopy size was estimated by dormant pruning weights. Although pruning weights only include non-photosynthetic tissues and not leaves biomass which most certainly is the most determinant factor, it is a quick and reliable indicator of canopy size used in viticulture. After leaf fall, last year's growth was exscinded from each plant with secateurs and individually weighed. Normalized vegetation difference index (NDVI) was taken using a Crop circle ACS-430 sensor connected to a GPS Geoscout X (Holland Scientific, Lincoln, USA) and a GPS antenna GPS18x (Garmin, Switzerland) mounted onto an all-terrain vehicle; passing through each row. The average of the NDVI recorded through each location was associated to each experimental unit.
Statistical Analyses
Partial least squares regression (PLSR) with a single-response orthogonal scores algorithm was used to evaluate the strength of berry composition and traits as predictor variables of canopy porosity using ‘pls’ package (version 2.6-0) for R programing language (version 3.2.5-6, R core). PLSR model was constructed with two latent factors for all the predictor variables (cluster location, berry traits and composition) and the dependent variable (canopy porosity). PLSR, contrarily to other regression methods, can analyse the effect of a large number of predictor variables (compared to the number of observations) with a high degree of autocorrelation among them, as is the case. PLSR gives two major outputs for each predictor variable: variable importance in the projection (VIP) and model coefficient. VIP is related to the weight on a particular variable used to fit the PLS model. Values above 0.8 are considered to indicate a significant importance of the variable (Wold et al., 2001). Model coefficient, which can be either positive or negative, indicates a positive relationship between variation in the predictor variable and the dependent variable in the case of positive coefficient and a negative relationship in the case of negative relationship.
Linear and non-linear regression was performed with Sigmaplot (13.0) applying a second order function if needed. Correlation was analysed using Spearman rank test. For data arranged by categorical factors (treatments), ANOVA combined with a LSD post hoc was used (R package ‘agricolae’ version 1.2-8).
Four rows of plants (Cabernet Sauvignon Foundation Plant Services clone 7 grafted onto 420A) were pruned, either spur pruned compelling vegetation upwards (vertical shoot positioned; VSP) or cane pruned leaving 50 cm canes and free vegetation (Sprawl). Within these rows, 48 Clusters from 8 plants (4 each of the training systems) were flagged. Pictures from the cluster perspective pointing at the sky were taken using 150° fish-eye lens. These pictures were processed in R (version 3.2.5-6) using package raster (version 2.6). Pictures were cropped with a circle and channel Blue was used to discriminate the pixels capturing the sky or clouds from those capturing the canopy or trellis. Porosity was calculated as the percentage of sky/clouds pixels in the cropped picture. When ripeness was reached (>22° Brix), sun damage and shriveling was visually recorded in a scale from 0 to 3, and 5-berry samples were collected from the top of each cluster. Grapes were weighed and skinned. TSS were determined from the pulps juice and skins were collected and stored at −80° C. for later analyses of anthocyanins and flavonols through HPLC-DAD.
An experiment was conducted in 2016 in a premium vineyard in Oakville, Calif. (38.428° N, 122.409° W). Plants were 7-year-old Cabernet Sauvignon (clone 7) grafted into 110R with a bilateral double cordon trellis with vegetation compelled upwards and a vine spacing of 2.4 by 2 m. A control (uncovered) and two shade nets treatments (20% shade factor and 40% shade factor polyethylene; Ginegar, Kibbutz, Israel) were installed on 27 May 2016 (31 days after anthesis). The uniformity of the nets light transmittance throughout the solar spectrum was verified with a spectrometer (Black Comet-SR, StellarNet; Tampa, Fla., USA). In the range of 300 to 1000 nm, the minimal transmittance from was 77% and 58%, the maximal was 84% and 61%, and the average 81% and 60% for the 20% and 40% shading factor respectively. On dates 20 Jun., 19 Jul., 29 Jul., 9 Aug., 19 Aug., 29 Aug., and 9 Sep. of 2016, 20-berry samples from each experimental unit were collected weighed and stored at −80° C. for later analyses of anthocyanins and flavonols through HPLC-DAD.
Two commercial vineyards were followed during 2016 season across central California (i) Healdsburg (38.66° N; 122.91° W), a 19-year-old Cabernet Sauvignon grafted into 110R (V. berlanderi Planch×V. rupestris Scheele) with quadrilateral (H-shaped) cordon trellis and a vine spacing of 2.14×3.35 m (vine×row); and (ii) Paso Robles (35.58° N 120.63° W), a 14-year-old Merlot grafted into 1103P (V. berlanderi Planch×V. rupestris Scheele) with double cordon trellis VSP and a vine spacing of 1.83×2.44 m (vine×row). Within these vineyards, experimental units consisting in 5 plants each were spread through each plot in a grid. In Paso Robles vineyard, in order to emulate the most common industry practices, leaf removal (North east side), shoot removal (down to 13.6 per meter), and leaf and shoot removal was applied randomly on 4 experimental units each July 27th to 12 randomly distributed experimental units. When commercial maturity was reached, 20-berry samples for each experimental unit were collected, weight and stored at −80° C. for later analyses of anthocyanins and flavonols through HPLC-DAD.
Berries were gently skinned and skins were freeze-dried (Cold Trap 7385020, Labconco, Kansas City, Mo., USA). Dried tissues were ground with a tissue lyser (MM400, Retsch, Germany). Fifty mg of that powder were extracted with methanol:water:7M hydrochloric acid (70:29:1, V:V:V) to determine the concentration anthocyanins and flavonols. Extracts were filtered (0.45 μm, Thermo Scientific) and analysed by a HPLC-DAD (1260 Series, Agilent, Santa Clara, Calif.). HPLC method was adapted from previous methods for flavonols that use formic acid as acid and acetonitrile as organic solvent (Downey and Rochfort, 2008; Hilbert et al., 2015). To quantify flavonols, these methods required anthocyanin removal with a solid phase extraction with a cationic exchange resin (Hilbert et al., 2015) or using a column with the same characteristics (Downey and Rochfort, 2008) to avoid co-elution of flavonols and anthocyanins which interferes the quantification of flavonols given the non-negligible absorbance of anthocyanins at 365 nm.
HPLC apparatus was an Agilent 1260 series (Agilent, Santa Clara, USA). Starting from the procedures reposted by Downey and Hilbert et al., (2015) conditions were adapted to a reverse phase C18 column LiChrosphere® 100, 250×4 mm with a 5 μm particle size and a 4 mm guard column of the same material. Flow was set to 0.5 mL to avoid exceeding the columns maximum pressure (110 bars) and temperature was set to 25° C. Although higher temperature can reduce the density of the mobile phase sometimes improving chromatography results, it is noteworthy that some phenolic compounds such as anthocyanins are sensitive to thermal degradation (Sadilova et al., 2007). Two mobile phases were designed to always maintain the following proportions (V/V) of Acetonitrile, 0-8 min 8%, at 25 mins 12.2 T, at 35 mins 16.9, at 70 mins 35.7%, 70-75 mins 65% and 80-90 min 8%. For the fine-tuning of the method for the quantification of flavonols, different isocratic concentrations of formic acid were tested from 1.98 to 10% (
Identification of anthocyanins was performed by comparing retention times with previous methods using mass spectrometry (Martínez-Lüscher et al., 2014b). Anthocyanins were quantified by determining the peak area of the absorbance at 520 nm. Malvidin-3-O-Glucoside (Extrasynthese, Genay, France) was used as a quantitative standard for anthocyanins. For the identification of flavonols, standards of myricetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-gluconoide, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, and syringetin-3-O-glucoside (Extrasynthese, Genay, France) were used. Flavonols were quantified by determining the peak area of the absorbance at 365 nm. Quercetin-3-O-glucoside was used as a quantitative standard for all the flavonols. It must be noted that each individual anthocyanin or flavonol have a different molar relative response factors (e.g., absorbance per mol) and even though calculating a response factors for each flavonol would have been possible, this is not the standard practice in the literature and would make comparisons of flavonol profiles harder.
HPLC methods can be fine-tuned to avoid the co-elution of flavonol and other phenolic compounds. The incremental concentrations of formic acid reduced the retention time of all compounds. However, this reduction was differential, especially when comparing anthocyanins and flavonols. For instance, malvidin-3-O-glucoside retention time changed from 46.3 min at 1.8% to 39.6 min at 10%, whereas for quercetin-3-O-glucoside retention time was 50.46 min at 1.8% and 39. min at 10%. This change was rather linear and corresponded to −0.80 and −1.29 minutes (advance) for every 1% increase in formic acid concentration for malvidin-3-O-glucoside and qercetin-3-O-glucside, respectively. This difference results into a shift of flavonols appearing earlier than anthocyanins for increasing formic acid concentrations. In the case of quercetin glucoside and malvidin-3-O-glucoside, this shift was 0.49 minutes for every 1% increase in formic acid concentration in the mobile phases.
The three quercetin glycosides most frequently found in V vinifera grapes and present in the standard used (
At low concentration of formic acid (1.8%), peak sharpness was lower than under any other condition, reducing peak height down to 390 mAU for malvidin-3-O-glucoside (and 159 mAU for quercetin-3-O-glucoside), compared to the 624, 644, and 690 mAU at concentrations of 4, 6, and 10% of formic acid, respectively.
This reduction of sharpness was associated to lower peak area for anthocyanins. For instance, the peak area of malvidin-3-O-glucoside increased from 385 to 422 and 426 mAU in the commercial standard mixture for 1.8, 4, and 5.5% formic acid, respectively (
When sorting variables according to their VIP values, flavonol profile variables %4′-OH, % Myricetin, %3′4′5′-OH, and the ratio 3′4′5′-to-3′4′-OH came in position 1,3,4, and 5 suggest the intimate relationship of flavonol profiles, rather than concentration, and exposure of grapes to solar radiation. Physical determinations such as the visual assessment of fruit damage and relative skin mass came in second and sixth position, respectively. Other variables such as anthocyanin content (either by mg g-1 berry or mg berry-1), total soluble solids (TSS), or cluster position had high VIP values, whereas the trellis system had a low VIP value.
Grapes grown under a shading net had lower flavonol content throughout ripening. This difference was reduced after ripening, and in fact, this was not significant at harvest. Changes in flavonol profile displayed different patterns among the three major groups of flavonols. For instance, % 4′-OH flavonols increased throughout development in all shading conditions until 19th of August (20.9° Brix), and remained similar from that moment to harvest. The proportion of 4′-OH flavonols were not significantly different before veraison but during ripening the significant trend 0%>20%>40% shading factor appeared. 3′4′-OH and 3′4′5′-OH flavonols were the majority of flavonols (>92%), and therefore, the increase in the proportion of one, results in the decrease of the other, and vice versa. The proportion of 3′4′-OH flavonols decreased in all the treatments until harvest. Increasing shading factors resulted in a lower proportion of 3′4′-OH flavonols. In contrast, increasing shading factors resulted in higher proportion of 3′4′5′-OH flavonols and these differences increased throughout development until harvest.
The transmission of light through canopies followed an extinction function as pruning heights increased. The inversion of pruning weights resulted into a linear positive relationship with light transmission for the range of variation of the dataset (
In the two datasets analyzed, the sensitivity of flavonol content and profiles to canopy size, water status, and total soluble solids (TSS) represented two different wine grape production systems (
Modeled accumulated radiation was strongly correlated to the percent of kaempferol (
Total flavonols and canopy porosity did not show a significant trend (
Spatial variability in the proportion of kaempferol in flavonols of Merlot was further studied. Spatial interpolation of the percent of kaempferol displayed a distinct spatial pattern based on semi-variogram investigations (data not shown). Because of the relationship between elevation and the proportion of kaempferol, the latter was kriged using universal block kriging with elevation as a covariate (
After grouping experimental units by their dormant pruning weights into low, medium and high vigor these came as significantly different from each other (
One or more features from any embodiments described herein or in the figures may be combined with one or more features of any other embodiment described herein in the figures without departing from the scope of the disclosure.
All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
The present application claims benefit of priority to U.S. Provisional Patent Application No. 62/720,000, filed Aug. 20, 2018, which is incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/47261 | 8/20/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62720000 | Aug 2018 | US |
