Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries

agronomy Article Influence of Foliar Kaolin Application and Irrigation on Photosynthetic Activity of Grape Berries Andreia Garrido 1, João Serôdio 2, Ric De Vos 3, Artur Conde 1,4 and Ana Cunha 1,4,5,* 1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (A.G.); [email protected] (A.C.) 2Centre for Environmental and Marine Studies (CESAM), University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal; jser[email protected] 3Wageningen Plant Research, Wageningen University and Research Centre (Wageningen-UR), PO Box 16, 6700 Wageningen, The Netherlands; [email protected] 4Centre of Molecular and Environmental Biology (CBMA), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 5 Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal *Correspondence: [email protected]; Tel.: +351253-604-046 Received: 30 September 2019; Accepted: 24 October 2019; Published: 27 October 2019   Abstract: Climate changes may cause severe impacts both on grapevine and berry development. Foliar application of kaolin has been suggested as a mitigation strategy to cope with stress caused by excessive heat/radiation absorbed by leaves and grape berry clusters. However, its effect on the light micro-environment inside the canopy and clusters, as well as on the acclimation status and physiological responses of the grape berries, is unclear. The main objective of this work was to evaluate the effect of foliar kaolin application on the photosynthetic activity of the exocarp and seeds, which are the main photosynthetically active berry tissues. For this purpose, berries from high light (HL) and low light (LL) microclimates in the canopy, from kaolin-treated and non-treated, irrigated and non-irrigated plants, were collected at three developmental stages. Photochemical and non-photochemical efficiencies of both tissues were obtained by a pulse amplitude modulated chlorophyll fluorescence imaging analysis. The maximum quantum efficiency (F v /F m ) data for green HL-grown berries suggest that kaolin application can protect the berry exocarp from light stress. At the mature stage, exocarps of LL grapes from irrigated plants treated with kaolin presented higher F v /F m and relative electron transport rates (rETR 200 ) than those without kaolin. However, for the seeds, a negative interaction between kaolin and irrigation were observed especially in HL grapes. These results highlight the impact of foliar kaolin application on the photosynthetic performance of grape berries growing under different light microclimates and irrigation regimes, throughout the season. This provides insights for a more case-oriented application of this mitigation strategy on grapevines. Keywords: light micro-climates; mitigation strategies; kaolin; irrigation; Vitis vinifera L.; grape berry tissues; pulse amplitude modulated (PAM) fluorometry; photosynthesis; photosynthetic pigments 1. Introduction Viticulture is a historically important agronomic and socio-economic sector in Portugal. According to the last report from the International Organization of Vine and Wine (OIV), Portugal is the 11th world and 5th European wine producer [ 1 ]. With 14 winemaking regions distributed throughout the country, the Vinhos Verdes or Minho region, as well as the Douro and Alentejo, are the major contributors for national exports and growth of this sector [2]. Agronomy 2019,9, 685; doi:10.3390/agronomy9110685 www.mdpi.com/journal/agronomy Agronomy 2019,9, 685 2 of 18 Grapevine is influenced by a complex and interacting system commonly called terroir, which, according to the OIV [ 3 ], includes specific soil, topography, climate, landscape characteristics and biodiversity features, and interaction with applied vitivini-cultural practices. This complex system influences the canopy microclimate and grapevine physiology and development and, consequently, grape berry quality and the organoleptic properties of its wine, which is typical of each region. Currently, climate change projections point to a particularly pronounced temperature variation, with an overall increase of up to 3.7 ◦ C by the end of this century, compared to the 1985–2005 reference period [ 4 ]. These temperature changes will have great impacts in the Mediterranean wine regions [ 5 ]. According to recent investigations using very high resolution bioclimatic zoning, both temperature and dryness are predicted to increase in several economically important Portuguese viticulture regions, including the Vinhos Verdes region [ 6 ]. Therefore, Portuguese vineyards will be subject to increased stress due to the interaction of the existing high radiation levels with the foreseen elevated air temperature and drought, which, all together, can have high impact on grapevine phenology, physiology, and productivity. Several of these climate impacts have already been reported, such as: earlier phenological timings and shortenings of the grapevine growing season [ 7 ], sunburns in leaves and grape berries [ 8 ], reduction of stomatal conductance and decrease of photosynthetic rates, either by stomatal and non-stomatal limitations [ 9 ], appearance and/or intensification of grapevine-related pests and diseases [ 10 , 11 ], increased grape sugar concentrations that lead to higher wine alcohol levels, lower acidities, and modification of varietal aroma compounds [ 12 ], and higher inter-annual yield and wine production variability [13]. In order to mitigate these adverse climate effects, new short-term measures have recently been implemented in Portuguese vineyards, such as smart irrigation [ 14 , 15 ] and foliar application of kaolin [ 16 ]. Vineyards are not traditionally irrigated and there are even restrictions on this practice in some regions, such as the Douro region [ 15 ]. However, according to a recent projection model, a 10% reduction in grapevine yield is expected in the Minho region if irrigation is not applied [ 14 ]. Kaolin is a white, chemically inert, and non-toxic clay material (Al 2 Si 2 O 5 (OH) 4 ) that can reflect radiation, including photosynthetically active (PAR), ultraviolet (UV), and infrared radiation (IR) [ 16 ]. Foliar application of this mineral has become a cost-efficient mitigation strategy to cope with water stress and excessive heat/radiation absorbed by leaves and grape berry clusters, which also proves effective in alleviating negative impacts on grapevines [ 17 – 21 ]. However, the amount and spectral quality of light intercepted by leaves and transmitted/reflected into the canopy, crucial factors for leaf, and fruit physiology and development [ 22 ] are also important aspects to consider when mitigation practices are used. Previous work done by our group, using pulse amplitude modulated (PAM) chlorophyll fluorescence imaging, has mapped grape berry photosynthesis at a histological level, and revealed both the exocarp and the seed outer integument as the main photosynthetically competent tissues [ 23 ]. More recently, we have studied the photosynthetic performance of grape berry tissues from clusters growing at three distinct light microclimates in the canopy and observed microclimate-related differences in their photosynthetic capacity and acclimation status [ 24 ]. This led to the hypothesis that, if a specific viticulture practice changes the light reaching the clusters, and alters its light microclimate, it may impact the photosynthetic activity of berry tissues and associated tissue-specific biochemical processes. In fact, foliar kaolin application may have direct implications on light distribution at the whole canopy level, and irrigation is an indirect one, through increased vegetative growth. For instance, it has already been shown that kaolin application generally reduces the photosynthetic rates of individual leaves in other agricultural crops (e.g., apple, almond, and walnut canopies) [ 25 , 26 ], due to a 20%–40% increase in the reflection of PAR [ 27 ]. However, the photosynthesis of the whole canopy remained unaffected or even increased (9%), because of the better light distribution within the canopy [ 28 – 30 ]. In another study, decreased photosynthesis was observed in the inner leaves of irrigated grapevines due to higher vegetative growth [ 31 ]. While the function of photosynthesis in fruits is still poorly Agronomy 2019,9, 685 3 of 18 understood, it can be linked with primary and secondary metabolomic pathways [ 32 , 33 ]. Therefore, any effect on photosynthesis may impact grape berry development and composition. Therefore, the main objective of the present work was to evaluate the effects of foliar kaolin application on the photosynthetic activity of grape berry tissues from clusters growing at two distinct microclimates, which include high light (HL) and low light (LL) microclimates, of irrigated and non-irrigated grapevines, during the season. 2. Materials and Methods 2.1. Site Description, Applied Treatments, and Sampling Grape berry samples were collected in 2018 from field-grown ‘Alvarinho’ cultivar grapevines (Vitis vinifera L.) in the commercial vineyard Quinta Cova da Raposa in the Demarcated Region of Vinho Verde, Braga, Portugal (41 ◦ 34’16.4” N, 8 ◦ 23’42.0” W). The vineyard is managed by following standard cultural practices applied in organic farming, and is arranged in terraces along a granitic hillside with high drainage. The vine training system applied for this cultivar follows the settings of Sylvoz (Simple Ascending and Recumbent Cord). The sector selected for the trial was located on a hill with NW-SE orientation and the vineyard rows with a NE-SW orientation. The treatments applied were: kaolin (K) and non-kaolin (NK) application on leaves, and irrigation (I) and non-irrigation (NI), in a complete factorial design (four treatment combinations) with two blocks, each with three to four vines per combination treatment (Figure 1B). A suspension of 5% (w/v in water) kaolin (EPAGRO ® , Sunprotect, Alverca do Ribatejo, Portugal) was applied twice on leaves on both sides of the rows. On July 6 and 27, corresponding to four weeks after anthesis (WAA) or BBCH-73 (BBCH-scale used for grapes by Lorenz et al. [ 34 ]) and seven WAA or BBCH-77, respectively. Irrigation of half of the plants, started on July 26 (seven WAA, BBCH-77), (Figure 1A,B). Water was applied by drip irrigation with one dripper per vine and a drip line placed approximately 80 cm above the soil. Irrigation occurred every three days, once a day either early in the morning or late in the afternoon, for 2 h, with an average dripper capacity of 5.5 ± 1.6 L h −1 (n=12 randomly selected drippers, ± SD). Clusters with contrasting light exposure were also selected to harvest grape berries during their development. These were called low light (LL) and high light (HL) clusters. LL clusters grew in the shaded inner zones of the canopy, which were exposed only to diffuse, reflected, and transmitted light, while HL clusters were exposed to direct or reflected sunlight most of the day. Six independent sub-clusters (three per block), each containing 15–20 grape berries, were collected randomly from clusters growing at each of the experimental conditions (four treatments × 2 microclimates) from the southeast side of rows. Berries were harvested in the morning (9–10 a.m.) at three distinct developmental stages: Green (16 July, 6 WAA, BBCH-75), V é raison (29 August, 12 WAA, BBCH-83), and Mature (17 September, 15 WAA, BBCH-89). The material was transported in refrigerated boxes to the Center for Environmental and Marine Studies (CESAM) laboratory and used within 2–6 h for imaging fluorometry experiments. For other assays, berries were immediately frozen in liquid nitrogen and stored at −80 ◦C. Agronomy 2019,9, 685 4 of 18 Agronomy 2019, 9, x FOR PEER REVIEW 3 of 18 Therefore, the main objective of the present work was to evaluate the effects of foliar kaolin application on the photosynthetic activity of grape berry tissues from clusters growing at two distinct microclimates, which include high light (HL) and low light (LL) microclimates, of irrigated and nonirrigated grapevines, during the season. 2. Materials and Methods 2.1. Site Description, Applied Treatments, and Sampling Grape berry samples were collected in 2018 from field-grown ‘Alvarinho’ cultivar grapevines (Vitis vinifera L.) in the commercial vineyard Quinta Cova da Raposa in the Demarcated Region of Vinho Verde, Braga, Portugal (41°34’16.4” N 8°23’42.0” W). The vineyard is managed by following standard cultural practices applied in organic farming, and is arranged in terraces along a granitic hillside with high drainage. The vine training system applied for this cultivar follows the settings of Sylvoz (Simple Ascending and Recumbent Cord). The sector selected for the trial was located on a hill with NW-SE orientation and the vineyard rows with a NE-SW orientation. The treatments applied were: kaolin (K) and non-kaolin (NK) application on leaves, and irrigation (I) and non-irrigation (NI), in a complete factorial design (four treatment combinations) with two blocks, each with three to four vines per combination treatment (Figure 1B). A suspension of 5% (w/v in water) kaolin (EPAGRO ® , Sunprotect, Alverca do Ribatejo, Portugal) was applied twice on leaves on both sides of the rows. On July 6 and 27, corresponding to four weeks after anthesis (WAA) or BBCH-73 (BBCH-scale used for grapes by Lorenz et al. [34]) and seven WAA or BBCH-77, respectively. Irrigation of half of the plants, started on July 26 (seven WAA, BBCH-77), (Figure 1A,B). Water was applied by drip irrigation with one dripper per vine and a drip line placed approximately 80 cm above the soil. Irrigation occurred every three days, once a day either early in the morning or late in the afternoon, for 2 h, with an average dripper capacity of 5.5 ± 1.6 L h −1 (n = 12 randomly selected drippers, ± SD). Clusters with contrasting light exposure were also selected to harvest grape berries during their development. These were called low light (LL) and high light (HL) clusters. LL clusters grew in the shaded inner zones of the canopy, which were exposed only to diffuse, reflected, and transmitted light, while HL clusters were exposed to direct or reflected sunlight most of the day. Six independent sub-clusters (three per block), each containing 15–20 grape berries, were collected randomly from clusters growing at each of the experimental conditions (four treatments × 2 microclimates) from the southeast side of rows. Berries were harvested in the morning (9–10 a.m.) at three distinct developmental stages: Green (16 July, 6 WAA, BBCH-75), Véraison (29 August, 12 WAA, BBCH-83), and Mature (17 September, 15 WAA, BBCH-89). The material was transported in refrigerated boxes to the Center for Environmental and Marine Studies (CESAM) laboratory and used within 2–6 h for imaging fluorometry experiments. For other assays, berries were immediately frozen in liquid nitrogen and stored at −80 °C. Figure 1. ( A ) Timeline of the grape growing season depicting the sampling times, foliar kaolin application dates, and the onset of irrigation. ( B ) Scheme of treatment combinations applied in the field: irrigation (I)/non-irrigation (NI) ×kaolin (K)/non-kaolin (NK). (WAA - weeks after anthesis). 2.2. Light Intensity and Temperature Measurements for Microclimate Characterization In order to characterize the microclimates in the vicinity of the growing clusters of all experimental conditions, light intensities and temperatures were registered on cloudless days (mean of 1500 ± 300 µ mol photons m −2 s −1 ), between 15 h and 17 h, at green and mature stages, as described by Garrido et al. [24] . The light intensity ( µ mol photons m −2 s −1 ) was measured with a radiometer (LI-COR, LI-250 Light Meter, Lincoln, NE, USA) and the temperature ( ◦ C) was measured with an infrared digital thermometer (Infrared, DT8380, Beijing, China). Both parameters were determined in the frontal region of the clusters (LL and HL), at the southeast side of the row, and in full sun-exposed leaves. The devices were placed perpendicularly to the plant organ (cluster or leaf). The light sensor was placed on the organ surface facing outward, which registered the light intensity reaching at this point, and the thermometer was pointing to the organ at a distance of about 15 cm, which registered an average organ temperature. Sixteen replicate measurements per treatment were considered on randomly selected vines. 2.3. Kaolin Film Transmittance and Reflectance Transmittance and reflectance spectra were obtained using a spectrometer (USB2000-VIS-NIR, grating #3, Ocean Optics, Duiven, The Netherlands), connected to a 400 mm-diameter fiber optic (QP400-2-VIS/NIR-BX; Ocean Optics), and recorded using the spectral acquisition software Spectra Suite (Ocean Optics, https://oceanoptics.com/). The transmittance spectrum was obtained by spreading a 5% (w/v) kaolin suspension prepared in 70% ethanol (allowing fast evaporation to prevent solvent interference), over a glass plate, which simulated particle distributions similar to those observed in the field. A halogen lamp was placed underneath to illuminate the spectrometer sensor positioned 3 cm above the glass plate. Different spectra were obtained from different areas randomly (n=3). The reflectance spectrum was obtained, according to Dinis et al. [ 18 ], by pointing the fiber optics perpendicularly to the surface of collected leaves illuminated by the same halogen lamp. Three independent spectra were obtained from different leaf regions of both kaolin-treated and non-treated leaves. Transmittance and reflectance spectra were recorded for the 370–900 nm spectral range, with a spectral resolution of 0.33 nm. The transmittance spectra were expressed as a percentage of the controls (glass). Reflectance spectra were normalized to the spectrum reflected from a reference white panel (WS-1-SL Spectralon Reference Standard, Ocean Optics). Agronomy 2019,9, 685 5 of 18 2.4. Chlorophyll Fluorescence Analysis The photosynthetic activity of grape berry tissues was assessed as described by Garrido et al. [ 24 ]. For this, an imaging chlorophyll fluorometer was used (Open FluorCAM 800 MF; Photon Systems Instruments, Dr á sov, Czech Republic), which was comprised of four 13 × 13 cm LED panels emitting red light (emission peak at 621 nm, 40-nm bandwidth) and a 2/3 inch CCD camera (CCD381, Beijing, China) with a F1.2 (2.8–6 mm) objective. Two of the LED panels provided modulated measuring light (<0.1 µ mol m −2 s −1 ) and the other two provided saturating pulses (>7500 µ mol m −2 s −1 , 0.8 s). Chlorophyll fluorescence images were captured and processed using FluorCam7 software (Photon Systems Instruments, Drásov, Czech Republic). In a dark cabinet, exocarps and seeds were separated from dark-adapted berries and disposed in 8 × 8-well plates filled with water. Two independent plates were prepared for each microclimate (LL and HL), with each comprising all treatments and tissues. Exocarps and seeds were placed alternately in three rows each, using two columns per treatment, in a total of 12 biological replicates per condition and tissue (n=3×2×2=12). Each plate was subjected to the experiments described below. The maximum quantum efficiency of photosystem II [F v /F m =(F m− F 0 )/F m ], which is a chlorophyll fluorescence parameter that reflects the probability of electrons being transferred from the PSII reaction center for the transport chain of electrons by quanta absorbed [ 35 , 36 ], was computed following a saturation pulse (SP). The isolated tissues were then acclimated to an actinic light (AL) of 200 µ mol photons m −2 s −1 for 15 min, and, after a new SP, the effective quantum yield of PSII [ ΦII =(F 0m− F s )/F 0m ] was computed. This parameter correlated with the quantum yield of CO 2 fixation in a wide range of physiological conditions [ 37 ]. From ΦII and PFR (photosynthetic photon fluence rates) (200 µ mol photons m −2 s −1 ), the relative electron transport rate through PSII (rETR 200 = ΦII × PFR) was calculated. Then, tissues were exposed to 1500 µ mol photons m −2 s −1 for 15 min, with an SP being applied every 3 min. The last F’ m values (at 15 min) were used to calculate the non-photochemical quenching [NPQ =(Fm−F’m)/F’m]. 2.5. Analysis of Chlorophylls and Carotenoids by High Performance Liquid Chromatography Coupled to A Photodiode Array Detector (HPLC-PDA) The extraction procedure was adapted from Fraser et al. [ 38 ]. Freeze-dried material (20 mg) of grape berry tissues, which includes exocarp and seed, was extracted in 1.8 mL of chloroform/methanol (1:1) (chloroform - Emsure ® , Darmstadt, Germany, methanol - Biosolve ® , Dieuze, France) with both 0.1% (w/v) butylated hydroxytoluene (BHT, Sigma ® , Zwijndrecht, The Netherlands) as an antioxidant and Sudan 1 (0.5 µ g mL −1 ) as the internal standard (IS). The samples were vortexed (10 s), kept on ice for 30 min (vortexed in between), and then sonicated for 15 min (Branson ® , 3510 Ultrasonic Cleaner, Danbury, CT, USA). These steps were performed twice. After that, the samples were centrifuged at 16,100 × g(Eppendorf ® , Centrifuge 5415 R, Hamburg, Germany) and the supernatant (approx. 1200 µ L) was transferred to a new Eppendorf tube with a perforated lid. The samples were dried for 1 h in a Speedvac (Savant ® , SC100, Schiphol, The Netherlands) and then stored at − 80 ◦ C until the next steps. Prior to high performance liquid chromatography (HPLC) analysis, samples were dissolved in 200 µ L ethylacetate solution containing 0.1% (w/v) BHT, sonicated (10 min), and then centrifuged as above. Samples were protected from light and kept on ice during all of these procedures. The supernatant (180 µL) was transferred to amber-colored 2 mL HPLC vials with a glass insert and sealed. The HPLC-PDA procedure was adapted from Mokochinski et al. [ 39 ]. The samples (20 µ L) were analyzed using an HPLC (Waters Alliance e2695 Separations Module, Milford, MA, USA) coupled to a photodiode array detector (PDA) (Waters 2996) over the 240 to 700 nm UV/Vis range. Separation was performed on a reverse-phase C 30 column (250 × 4.6 mm i.d., S-5 µ m - YMC Carotenoid, Komatsu, Japan) kept at 35 ◦ C with a flow rate of 1.0 mL min −1 . The compounds were identified based on comparisons of retention times and absorption spectra (240 to 700 nm) with authentic standards. Agronomy 2019,9, 685 6 of 18 2.6. Statistical Analysis Results were statistically analyzed using Analysis of Variance tests (two-way ANOVA), followed by post hoc multiple comparisons using the Bonferroni test whenever the factors had significant effects (GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla, CA, USA). Significant differences (p ≤ 0.05) between sample groups are indicated with different letters. Notation with an asterisk means that only one factor (kaolin or irrigation) was significant. 3. Results and Discussion 3.1. Climatic Conditions and Microclimate Characteristics In order to characterize the climate during the growing season at the study site (Braga), we used the official information available from the Instituto Portugu ê s do Mar e da Atmosfera (IPMA) [ 40 ], to determine the temperatures and total precipitation during 2018 (Figure S1). This growing season was atypical from a climatic point of view, with a relatively cold and extremely dry winter, which caused a delay of sprouting/flowering for two to three weeks [ 41 ], and a relatively cold spring with rainy periods during the vegetative growth of the grapevines. To characterize the microclimates for the LL and HL berry clusters (two a priori selected distinct light microclimates within the canopy), measurements of light intensities and temperatures were performed at the cluster level, at two time points during the growing season, i.e., when the berries were still green (green stage) and, two months later, when the berries were at their mature stage of ripening (Figures 2and 3). Figure 2depicts the average light intensities at LL and HL clusters growing under the different experimental conditions: i.e., irrigation/non irrigation (Figure 2a,c) and with kaolin/without kaolin (Figure 2b,d). The two microclimates were clearly distinct at both ripening stages, with HL clusters receiving about three-fold more light than LL clusters. At the green stage, i.e., before the onset of irrigation (Figure 1A), no significant differences were detected between the two sets of plots assigned to the subsequent irrigation experiment (four plots for irrigated (I) plants, i.e., 2 × NK-I and 2×K-I ) and four plots for non-irrigated (NI) control plants (2 × NK-NI and 2 × K-NI), see Figure 1B) (Figure 2a), which reveals that there were no plot-related effects on microclimate light intensity. At this early ripening stage, and with the adopted measurement procedure, no differences were detected with respect to light intensities reaching the berry clusters due to foliar kaolin application (Figure 2b). However, at a mature stage, both irrigation and kaolin had a small but significant effect on the light intensity reaching the HL clusters (Figure 2c,d). Irrigation slightly reduced the light intensity, likely due to the better vegetative growth of the plants, while foliar kaolin application increased it, likely due to an increased light reflection to both the interior and lower levels of the canopy. In the LL clusters, these effects of irrigation and kaolin on light intensity were not observed, at this time of day. HL grapes consistently experienced higher temperatures than LL ones (Figure 3), and both I and K treatments exerted significant and contrasting effects on this microclimate parameter, mainly at the mature stage. Again, and consistent with what was observed and discussed above for light intensity, no effect was detected for I treatment on the grape berry temperature at the green stage (before the onset of irrigation) (Figure 3a). Agronomy 2019,9, 685 7 of 18 Agronomy 2019, 9, x FOR PEER REVIEW 6 of 18 performed at the cluster level, at two time points during the growing season, i.e., when the berries were still green (green stage) and, two months later, when the berries were at their mature stage of ripening (Figures 2 and 3). Figure 2 depicts the average light intensities at LL and HL clusters growing under the different experimental conditions: i.e., irrigation/non irrigation (Figure 2a,c) and with kaolin/without kaolin (Figure 2b,d). The two microclimates were clearly distinct at both ripening stages, with HL clusters receiving about three-fold more light than LL clusters. At the green stage, i.e., before the onset of irrigation (Figure 1A), no significant differences were detected between the two sets of plots assigned to the subsequent irrigation experiment (four plots for irrigated (I) plants, i.e., 2 × NK-I and 2 × K-I) and four plots for non-irrigated (NI) control plants (2 × NK-NI and 2 × KNI), see Figure 1B) (Figure 2a), which reveals that there were no plot-related effects on microclimate light intensity. At this early ripening stage, and with the adopted measurement procedure, no differences were detected with respect to light intensities reaching the berry clusters due to foliar kaolin application (Figure 2b). However, at a mature stage, both irrigation and kaolin had a small but significant effect on the light intensity reaching the HL clusters (Figure 2c,d). Irrigation slightly reduced the light intensity, likely due to the better vegetative growth of the plants, while foliar kaolin application increased it, likely due to an increased light reflection to both the interior and lower levels of the canopy. In the LL clusters, these effects of irrigation and kaolin on light intensity were not observed, at this time of day. Kaolin treatment LL HL 0 50 100 150 200 B A B A (b) Light intensity (μmol photons m -2 s -1 ) Irrigation treatment LL HL 0 50 100 150 200 B A B A (a) Light intensity (μmol m -2 s -1 ) LL HL 0 50 100 150 200 B A,a B A,b (c) Light intensity (μmol m -2 s -1 ) LL HL 0 50 100 150 200 B A,b B A,a (d) Light intensity (μmol photons m -2 s -1 ) Green Mature Figure 2. Light intensities received by LL and HL clusters at the green stage (a,b) and the mature stage (c,d), for plants with irrigation (blue columns, note: the textured blue columns at green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with a standard deviation (n = 16 plants). Statistical notation: per ripening stage, different capital letters refer to significant differences (two-way ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters for differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the lowercase letters were omitted. HL grapes consistently experienced higher temperatures than LL ones (Figure 3), and both I and K treatments exerted significant and contrasting effects on this microclimate parameter, mainly at the mature stage. Again, and consistent with what was observed and discussed above for light intensity, no effect was detected for I treatment on the grape berry temperature at the green stage (before the onset of irrigation) (Figure 3a). Figure 2. Light intensities received by LL and HL clusters at the green stage ( a , b ) and the mature stage ( c , d ), for plants with irrigation (blue columns, note: the textured blue columns at green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with a standard deviation (n=16 plants). Statistical notation: per ripening stage, different capital letters refer to significant differences (two-way ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters for differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the lowercase letters were omitted. Agronomy 2019, 9, x FOR PEER REVIEW 7 of 18 Irrigation treatment LL HL 20 25 30 35 40 B A B A (a) Temperature (ºC) Kaolin treatment LL HL 20 25 30 35 40 B A,a B A,b (b) Temperature (ºC) LL HL 20 25 30 35 40 B,b A,b B,a A,a (c) Temperature (ºC) LL HL 20 25 30 35 40 B,a A,a A,b A,b (d) Temperature (ºC) Green Mature Figure 3. Temperatures of LL and HL clusters at the green stage (a,b) and the mature stage (c,d), for plants with irrigation (blue columns, note: the textured blue columns at the green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with standard deviation (n = 16 plants). Statistical notation: per ripening stage, different capital letters refer to significant differences (two-way ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters for differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the lowercase letters were omitted. The Kaolin application led to a significant decrease in the temperature of the HL clusters at both green and mature stages (e.g., at this latter stage—from 31 °C to 28.7 °C), and of the LL clusters at the mature stage only (e.g., from 29.5 °C to 28 °C) (Figure 3b,d). The fact that LL clusters’ temperature at the green stage were not affected by kaolin was likely related to the relatively low air temperature at this time of the growing season (early July, Figure S1). Thus, kaolin application on the leaves may increase the incident light by increasing the light reflection inside the canopy (Figure 2d), while maintaining a cooler microclimate for the growing berries, especially during the hot summer days, independently of the irrigation regime. This demonstrates one of the advantages of this mitigation strategy at the grape berry level. The kaolin solution sprayed on the leaves also resulted in leaf temperature reduction (Figure S2d). The present results are in agreement with previous studies in both grapevine leaves and berries [42,43], as well as other crops [26,27]. Therefore, it is likely that kaolin applied to leaves provides cooler temperatures throughout the grapevine by reducing the total amount of radiation transmitted into the canopy. This is also shown by thermal imaging in apple trees [30]. Furthermore, different training systems of the vineyard might influence the light intensities and temperatures inside the canopy [44,45]. For instance, the vine canopy was denser in our previous study in 2015 [24], which resulted in an LL microclimate characterized by much lower light reaching the clusters compared to that in the present study, with major impacts on grape berry photosynthetic competence. At the mature stage, plant irrigation resulted in a significant increase in the grape temperature of both LL and HL clusters (Figure 3c). This increase was unexpected, since a previous study reported lower berry temperature as a result of irrigation, rather than a higher temperature [46]. This response is very interesting and clearly, additional studies, which are controlling/measuring the soil temperature in the rhizosphere, are required to determine the effect of irrigation and the irrigation procedure on the temperature of grape clusters. Figure 3. Temperatures of LL and HL clusters at the green stage ( a , b ) and the mature stage ( c , d ), for plants with irrigation (blue columns, note: the textured blue columns at the green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with standard deviation (n=16 plants). Statistical notation: per ripening stage, different capital letters refer to significant differences (two-way ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters for differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the lowercase letters were omitted. Agronomy 2019,9, 685 8 of 18 The Kaolin application led to a significant decrease in the temperature of the HL clusters at both green and mature stages (e.g., at this latter stage—from 31 ◦ C to 28.7 ◦ C), and of the LL clusters at the mature stage only (e.g., from 29.5 ◦ C to 28 ◦ C) (Figure 3b,d). The fact that LL clusters’ temperature at the green stage were not affected by kaolin was likely related to the relatively low air temperature at this time of the growing season (early July, Figure S1). Thus, kaolin application on the leaves may increase the incident light by increasing the light reflection inside the canopy (Figure 2d), while maintaining a cooler microclimate for the growing berries, especially during the hot summer days, independently of the irrigation regime. This demonstrates one of the advantages of this mitigation strategy at the grape berry level. The kaolin solution sprayed on the leaves also resulted in leaf temperature reduction (Figure S2d). The present results are in agreement with previous studies in both grapevine leaves and berries [ 42 , 43 ], as well as other crops [ 26 , 27 ]. Therefore, it is likely that kaolin applied to leaves provides cooler temperatures throughout the grapevine by reducing the total amount of radiation transmitted into the canopy. This is also shown by thermal imaging in apple trees [ 30 ]. Furthermore, different training systems of the vineyard might influence the light intensities and temperatures inside the canopy [ 44 , 45 ]. For instance, the vine canopy was denser in our previous study in 2015 [ 24 ], which resulted in an LL microclimate characterized by much lower light reaching the clusters compared to that in the present study, with major impacts on grape berry photosynthetic competence. At the mature stage, plant irrigation resulted in a significant increase in the grape temperature of both LL and HL clusters (Figure 3c). This increase was unexpected, since a previous study reported lower berry temperature as a result of irrigation, rather than a higher temperature [ 46 ]. This response is very interesting and clearly, additional studies, which are controlling/measuring the soil temperature in the rhizosphere, are required to determine the effect of irrigation and the irrigation procedure on the temperature of grape clusters. 3.2. Kaolin Film Transmittance and Reflectance Spectral Properties To characterize the potential effects of kaolin applied to the leaves with regard to light intensity and quality, we performed transmittance and reflectance studies. For this purpose, the transmittance spectrum of a film of kaolin solution (5% w/v) on a glass plate was determined (Figure 4a). Although a high percentage of most photosynthetically active radiation (PAR) wavelengths is transmitted by the kaolin film, the blue light range is the least transmitted. The reflectance spectra obtained for leaves of grape plants sprayed with and without kaolin are represented in Figure 4b. Our results showed a relevant percentage of PAR is reflected by this white mineral, as compared to non-spayed (NK) vine leaves, rather than exclusively or mainly reflecting in the ultraviolet (UV) and infrared radiation (IR) ranges [ 47 ]. Additionally, kaolin was more efficient in reflecting UV light than IR light (in the measured ranges). These results are in accordance with previous studies using grapevine leaves [ 18 ] and other crops [ 27 , 28 , 48 , 49 ]. Thus, the beneficial effect of kaolin application is related to the reflection of excess radiation outwards, which reduces the risk of light stress-induced damage to leaves and fruit [ 47 ], while transmitting a very significant proportion of PAR. Agronomy 2019, 9, x FOR PEER REVIEW 8 of 18 3.2. Kaolin Film Transmittance and Reflectance Spectral Properties To characterize the potential effects of kaolin applied to the leaves with regard to light intensity and quality, we performed transmittance and reflectance studies. For this purpose, the transmittance spectrum of a film of kaolin solution (5% w/v) on a glass plate was determined (Figure 4a). Although a high percentage of most photosynthetically active radiation (PAR) wavelengths is transmitted by the kaolin film, the blue light range is the least transmitted. The reflectance spectra obtained for leaves of grape plants sprayed with and without kaolin are represented in Figure 4b. Our results showed a relevant percentage of PAR is reflected by this white mineral, as compared to non-spayed (NK) vine leaves, rather than exclusively or mainly reflecting in the ultraviolet (UV) and infrared radiation (IR) ranges [47]. Additionally, kaolin was more efficient in reflecting UV light than IR light (in the measured ranges). These results are in accordance with previous studies using grapevine leaves [18] and other crops [27,28,48,49]. Thus, the beneficial effect of kaolin application is related to the reflection of excess radiation outwards, which reduces the risk of light stress-induced damage to leaves and fruit [47], while transmitting a very significant proportion of PAR. Together, these results call attention to the fact that foliar kaolin application may directly impact the photosynthesis of the sprayed leaves but also have an indirect effect on the non-sprayed leaves and grape berry clusters inside the canopy. 400 500 600 700 800 900 0 10 20 30 40 50 60 70 Kaolin Control (b) Wavelength (nm) Reflectance (%) 400 500 600 700 800 900 70 80 90 100 (a) Wavelength (nm) Transmittance (% ) Figure 4. Transmittance (%) spectrum (a) of a kaolin suspension (5% w/v) and reflectance (%) spectra (b) of leaves with and without kaolin (control) (n = 3). 3.3. Effects on Berry Photosynthesis and Photosynthetic Pigments 3.3.1. Maximum Quantum Efficiency of PSII The maximum quantum efficiency of PSII (Fv/Fm) was determined ex planta under controlled conditions, using both exocarps and seeds from grape berries grown under the different treatments, microclimates, and three ripening stages (Figure 5). At the green stage, Fv/Fm was similar in both berry tissues (Figure 5a,b). Upon further ripening, the exocarp kept its Fv/Fm values (~ 0.7), while the seeds showed a significant decrease in this parameter, which reached values around 0.4–0.5 at the mature stage, which was in accordance with what was reported by Garrido et al. [24]. Figure 4. Transmittance (%) spectrum ( a ) of a kaolin suspension (5% w/v) and reflectance (%) spectra (b) of leaves with and without kaolin (control) (n=3). Agronomy 2019,9, 685 9 of 18 Together, these results call attention to the fact that foliar kaolin application may directly impact the photosynthesis of the sprayed leaves but also have an indirect effect on the non-sprayed leaves and grape berry clusters inside the canopy. 3.3. Effects on Berry Photosynthesis and Photosynthetic Pigments 3.3.1. Maximum Quantum Efficiency of PSII The maximum quantum efficiency of PSII (F v /F m ) was determined ex planta under controlled conditions, using both exocarps and seeds from grape berries grown under the different treatments, microclimates, and three ripening stages (Figure 5). At the green stage, F v /F m was similar in both berry tissues (Figure 5a,b). Upon further ripening, the exocarp kept its F v /F m values (~0.7), while the seeds showed a significant decrease in this parameter, which reached values around 0.4–0.5 at the mature stage, which was in accordance with what was reported by Garrido et al. [24]. Agronomy 2019, 9, x FOR PEER REVIEW 9 of 18 Figure 5. Maximum quantum efficiency of PSII (Fv/Fm) mean values (n = 12–24 berries, +SD) of exocarps and seed integuments obtained from dark-adapted LL and HL grape berries grown under the four combinations of the two treatments applied: irrigation (I)/ non-irrigation (NI) × kaolin (K)/ non-kaolin (NK). Samples were collected at three development stages (green, véraison, and mature). Statistical notation: for each developmental stage, capital letters refer to differences between light microclimates within the same treatment combination, and lowercase letters refer to differences between treatment combinations within each light microclimate (mean values with a common letter were not significantly different). When capital and lowercase letters are omitted, the respective factor did not have a significant effect (two-way ANOVA p > 0.05). Notation with an asterisk means that only one factor (kaolin or irrigation) was significant. The cluster microclimate (LL vs HL) had a significant effect on the Fv/Fm values in both tissues and all developmental stages, with the exception of seeds at the mature stage, but exocarps and seed integuments responded differently to a light microclimate [24]. At the green stage, the exocarps from berries under control conditions, showed lower Fv/Fm values in HL clusters, while their seeds showed significantly higher values than those from LL berries (Figure 5a,b). This was likely due to their inner location where the light transmitted through the skin and flesh tissues reaches values as low as 2% of the incident photon flux density (PFD) [50], which eventually translates a light limitation effect in LL clusters. These microclimate effects were more or less maintained in exocarps upon subsequent ripening (see NK + NI Figure 5a,c,e, while not being significant in véraison), while, in seeds, the difference between LL and HL clusters disappeared (Figure 5b,d,f), likely related to the (large) intrinsic ripening-dependent decrease in photosynthetic competence of this tissue. Both kaolin and irrigation treatments of plants differentially influenced the Fv/Fm values of the two berry tissues, with the seeds globally more responsive than exocarps, particularly to the irrigation Exocarp LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 B,b A,aAA (a) F v /F m Seed LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 AA BA (b) LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 AAAAAABA (c) F v /F m LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 A aB b A a A a AAAA (d) LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 A A AA BAAA (e) F v /F m LL HL 0.3 0.4 0.5 0.6 0.7 0.8 0.9 (f) * Control Kaolin Non-Kaolin, Non-Irrigation (NK,NI) Non-Kaolin, Irrigation (NK,I) Kaolin, Non-Irrigation (K, NI) Kaolin, Irrigation (K, I) Control Kaolin Green Véraison Mature Figure 5. Maximum quantum efficiency of PSII (F v /F m ) mean values (n=12–24 berries, +SD) of exocarps and seed integuments obtained from dark-adapted LL and HL grape berries grown under the four combinations of the two treatments applied: irrigation (I)/non-irrigation (NI) × kaolin (K)/ non-kaolin (NK). Samples were collected at three development stages (green, v é raison, and mature). Statistical notation: for each developmental stage, capital letters refer to differences between light microclimates within the same treatment combination, and lowercase letters refer to differences between treatment combinations within each light microclimate (mean values with a common letter were not significantly different). When capital and lowercase letters are omitted, the respective factor did not have a significant effect (two-way ANOVA p>0.05). Notation with an asterisk means that only one factor (kaolin or irrigation) was significant. Agronomy 2019,9, 685 16 of 18 14. Fraga, H.; Garc í a de Cort á zar Atauri, I.; Santos, J.A. Viticultural irrigation demands under climate change scenarios in Portugal. Agric. Water Manag. 2018,196, 66–74. [CrossRef] 15. Costa, J.M.; Vaz, M.; Escalona, J.; Egipto, R.; Lopes, C.; Medrano, H.; Chaves, M.M. Modern viticulture in southern Europe: Vulnerabilities and strategies for adaptation to water scarcity. Agric. Water Manag. 2016 , 164, 5–18. [CrossRef] 16. Brito, C.; Dinis, L.T.; Moutinho-Pereira, J.; Correia, C. Kaolin, an emerging tool to alleviate the effects of abiotic stresses on crop performance. Sci. Hortic. (Amst.) 2019,250, 310–316. [CrossRef] 17. Dinis, L.T.; Ferreira, H.; Pinto, G.; Bernardo, S.; Correia, C.M.; Moutinho-Pereira, J. Kaolin-based, foliar reflective film protects photosystem II structure and function in grapevine leaves exposed to heat and high solar radiation. Photosynthetica 2016,54, 47–55. [CrossRef] 18. Dinis, L.T.; Malheiro, A.C.; Luzio, A.; Fraga, H.; Ferreira, H.; Gonçalves, I.; Pinto, G.; Correia, C.M.; Moutinho-Pereira, J. Improvement of grapevine physiology and yield under summer stress by kaolin-foliar application: Water relations, photosynthesis and oxidative damage. Photosynthetica 2018 ,56, 641–651. [CrossRef] 19. Conde, A.; Neves, A.; Breia, R.; Pimentel, D.; Dinis, L.T.; Bernardo, S.; Correia, C.M.; Cunha, A.; Ger ó s, H.; Moutinho-Pereira, J. Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves. J. Plant Physiol. 2018,223, 47–56. [CrossRef] [PubMed] 20. Dinis, L.T.; Bernardo, S.; Conde, A.; Pimentel, D.; Ferreira, H.; F é lix, L.; Ger ó s, H.; Correia, C.M.; Moutinho-Pereira, J. Kaolin exogenous application boosts antioxidant capacity and phenolic content in berries and leaves of grapevine under summer stress. J. Plant Physiol. 2016 ,191, 45–53. [CrossRef] [PubMed] 21. Conde, A.; Pimentel, D.; Neves, A.; Dinis, L.-T.; Bernardo, S.; Correia, C.M.; Ger ó s, H.; Moutinho-Pereira, J. Kaolin Foliar Application Has a Stimulatory Effect on Phenylpropanoid and Flavonoid Pathways in Grape Berries. Front. Plant Sci. 2016,7, 1–14. [CrossRef] 22. Poni, S.; Gatti, M.; Palliotti, A.; Dai, Z.; Duch ê ne, E.; Truong, T.T.; Ferrara, G.; Matarrese, A.M.S.; Gallotta, A.; Bellincontro, A.; et al. Grapevine quality: A multiple choice issue. Sci. Hortic. (Amst.) 2018 ,234, 445–462. [CrossRef] 23. Breia, R.; Vieira, S.; Da Silva, J.M.; Ger ó s, H.; Cunha, A. Mapping grape berry photosynthesis by chlorophyll fluorescence imaging: The effect of saturating pulse intensity in different tissues. Photochem. Photobiol. 2013 , 89, 579–585. [CrossRef] 24. Garrido, A.; Breia, R.; Ser ô dio, J.; Cunha, A. Impact of the Light Microclimate on Photosynthetic Activity of Grape Berry (Vitis vinifera): Insights for Radiation Absorption Mitigations’ Measures. In Theory and Practice of Climate Adaptation; Springer: Cham, Switzerland, 2018; pp. 419–441. 25. Le Grange, M.; Wand, S.J.E.; Theron, K.I. Effect of kaolin applications on apple fruit quality and gas exchange of apple leaves. Acta Hortic. 2004,636, 545–550. [CrossRef] 26. Wünsche, J.-N.; Lombardini, L.; Greer, D.H.; Palmer, J.W. “Surround” particle film applications—The effect on whole canopy physiology of apple. Acta Hortic. 2004,636, 565–571. [CrossRef] 27. Rosati, A.; Metcalf, S.G.; Buchner, R.P.; Fulton, A.E.; Lampinen, B.D. Physiological effects of kaolin applications in well-irrigated and water-stressed walnut and almond trees. Ann. Bot. 2006 ,98, 267–275. [CrossRef] [PubMed] 28. Rosati, A.; Metcalf, S.G.; Buchner, R.P.; Fulton, A.E.; Lampinen, B.D. Effects of Kaolin application on light absorption and distribution, radiation use efficiency and photosynthesis of almond and walnut canopies. Ann. Bot. 2007,99, 255–263. [CrossRef] [PubMed] 29. Glenn, D.M.; Puterka, G.J. The use of plastic films and sprayable reflective particle films to increase light penetration in apple canopies and improve apple color and weight. HortScience 2007,42, 91–96. [CrossRef] 30. Glenn, D.M. Particle Film Mechanisms of Action That Reduce the Effect of Environmental Stress in ‘Empire’ Apple. J. Am. Soc. Hortic. Sci. 2009,134, 314–321. [CrossRef] 31. Escalona, J.M.; Flexas, J.; Bota, J.; Medrano, H. Distribution of leaf photosynthesis and transpiration within grapevine canopies under different drought conditions. Vitis 2003,42, 57–64. 32. Obiadalla-Ali, H.; Fernie, A.R.; Lytovchenko, A.; Kossmann, J.; Lloyd, J.R. Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism. Planta 2004,219, 533–540. [CrossRef] Agronomy 2019,9, 685 17 of 18 33. Cocaliadis, M.F.; Fern á ndez-Muñoz, R.; Pons, C.; Orzaez, D.; Granell, A. Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword? J. Exp. Bot. 2014,65, 4589–4598. [CrossRef] 34. Lorenz, D.H.; Eichhorn, K.W.; Bleiholder, H.; Klose, R.; Meier, U.; Weber, E. Growth Stages of the Grapevine: Phenological growth stages of the grapevine (Vitis vinifera L. ssp. vinifera)—Codes and descriptions according to the extended BBCH scale. Austr. J. Grape Wine Res. 1995,1, 100–110. [CrossRef] 35. Baker, N.R. Chlorophyll fluorescence: A probe of photosynthesis in vivo .Annu. Rev. Plant Biol. 2008 ,59, 89–113. [CrossRef] 36. Schreiber, U. Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview. In Chlorophyll a Fluorescence: A Signature of Photosynthesis; Kluwer Academic: Dordrecht, The Netherlands, 2004; pp. 279–319. 37. Genty, B.; Briantais, J.-M.; Baker, N.R. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta Gen. Subj. 1989 ,990, 87–92. [CrossRef] 38. Fraser, P.D.; Pinto, M.E.S.; Holloway, D.E.; Bramley, P.M. Application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J. 2000,24, 551–558. [CrossRef] [PubMed] 39. Mokochinski, J.B.; Mazzafera, P.; Sawaya, A.C.H.F.; Mumm, R.; de Vos, R.C.H.; Hall, R.D. Metabolic responses of Eucalyptus species to different temperature regimes. J. Integr. Plant Biol. 2018,60, 397–411. [CrossRef] 40. IPMA Instituto Portugu ê s do Mar e da Atmosfera. Available online: http://www.ipma.pt/pt/otempo/obs. superficie/index-map-dia-chart.jsp#Braga,Merelim (accessed on 2 June 2019). 41. IVV, Instituto da Vinha e do Vinho, I.P.I. de Mercado. Nota Informativa, No3/2018, 30/07/2018, Previs ã o de Colheita—Campanha 2018/2019. Available online: https://www.ivv.gov.pt/np4/8955.html (accessed on 1 July 2019). 42. Shellie, K.C.; King, B.A. Kaolin particle film and water deficit influence malbec leaf and berry temperature, pigments, and photosynthesis. Am. J. Enol. Vitic. 2013,64, 223–230. [CrossRef] 43. Oliveira, M. Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal. In Grapes and Wines—Advances in Production, Processing, Analysis and Valorization; InTech: Rijeka, Croatia, 2018. 44. Reynolds, A.G.; Heuvel, J.E.V. Influence of grapevine training systems on vine growth and fruit composition: A review. Am. J. Enol. Vitic. 2009,60, 251–268. 45. Kraus, C.; Pennington, T.; Herzog, K.; Hecht, A.; Fischer, M.; Voegele, R.T.; Hoffmann, C.; Töpfer, R.; Kicherer, A. Effects of canopy architecture and microclimate on grapevine health in two training systems. Vitis J. Grapevine Res. 2018,57, 53–60. [CrossRef] 46. Dos Santos, T.P.; Lopes, C.M.; Luc í lia Rodrigues, M.; de Souza, C.R.; Ricardo-da-Silva, J.M.; Maroco, J.P.; Pereira, J.S.; Manuela Chaves, M. Effects of deficit irrigation strategies on cluster microclimate for improving fruit composition of Moscatel field-grown grapevines. Sci. Hortic. (Amst.) 2007,112, 321–330. [CrossRef] 47. Sharma, R.R.; Vijay Rakesh Reddy, S.; Datta, S.C. Particle films and their applications in horticultural crops. Appl. Clay Sci. 2015,116–117, 54–68. [CrossRef] 48. Glenn, D.M.; Prado, E.; Erez, A.; McFerson, J.; Puterka, G.J. A Reflective, Processed-Kaolin Particle Film Affects Fruit Temperature, Radiation Reflection, and Solar Injury in Apple. J. Am. Soc. Hortic. Sci. 2002 ,127, 188–193. [CrossRef] 49. Glenn, M.D. The mechanisms of plant stress mitigation by kaolin-based particle films and applications in horticultural and agricultural crops. HortScience 2012,47, 710–711. [CrossRef] 50. Aschan, G.; Pfanz, H. Non-foliar photosynthesis—A strategy of additional carbon acquisition. Flora 2003 , 198, 81–97. [CrossRef] 51. Kadir, S.; Von Weihe, M.; Al-Khatib, K. Photochemical Efficiency and Recovery of Photosystem II in Grapes After Exposure to Sudden and Gradual Heat Stress. J. Am. Soc. Hortic. Sci. 2007,132, 764–769. [CrossRef] 52. Greer, D.H.; Weston, C. Heat stress affects flowering, berry growth, sugar accumulation and photosynthesis of Vitis vinifera cv. Semillon grapevines grown in a controlled environment. Funct. Plant Biol. 2010 ,37, 206–214. [CrossRef] 53. Luo, H.B.; Ma, L.; Xi, H.F.; Duan, W.; Li, S.H.; Loescher, W.; Wang, J.F.; Wang, L.J. Photosynthetic responses to heat treatments at different temperatures and following recovery in grapevine (Vitis amurensis L.) leaves. PLoS ONE 2011,6. [CrossRef] 54. Xiao, F.; Yang, Z.Q.; Lee, K.W. Photosynthetic and physiological responses to high temperature in grapevine (Vitis vinifera L.) leaves during the seedling stage. J. Hortic. Sci. Biotechnol. 2017,92, 2–10. [CrossRef] Agronomy 2019,9, 685 18 of 18 55. Krause, G.H.; Jahns, P. Non-photochemical Energy Dissipation Determined by Chlorophyll Fluorescence Quenching: Characterization and Function. In Chlorophyll a Fluorescence; Springer: Dordrecht, The Netherlands, 2007. 56. Medrano, H.; Bota, J.; Abad í a, A.; Sampol, B.; Escalona, J.M.; Flexas, J. Effects of drought on light-energy dissipation mechanisms in high-light-acclimated, field-grown grapevines. Funct. Plant Biol. 2002 ,29, 1197–1207. [CrossRef] 57. Szab ó , I.; Bergantino, E.; Giacometti, G.M. Light and oxygenic photosynthesis: Energy dissipation as a protection mechanism against photo-oxidation. EMBO Rep. 2005,6, 629–634. [CrossRef] 58. Joubert, C.; Young, P.R.; Ey é gh é -Bickong, H.A.; Vivier, M.A. Field-Grown Grapevine Berries Use Carotenoids and the Associated Xanthophyll Cycles to Acclimate to UV Exposure Differentially in High and Low Light (Shade) Conditions. Front. Plant Sci. 2016,7, 1–17. [CrossRef] 59. Tikkanen, M.; Grieco, M.; Nurmi, M.; Rantala, M.; Suorsa, M.; Aro, E.M. Regulation of the photosynthetic apparatus under fluctuating growth light. Philos. Trans. R. Soc. B Biol. Sci. 2012 ,367, 3486–3493. [CrossRef] 60. Frioni, T.; Tombesi, S.; Luciani, E.; Sabbatini, P.; Berrios, J.G.; Palliotti, A. Kaolin treatments on Pinot noir grapevines for the control of heat stress damages. Bio Web Conf. 2019,13, 04004. [CrossRef] 61. Brito, C.; Dinis, L.T.; Luzio, A.; Silva, E.; Gonçalves, A.; Meij ó n, M.; Escand ó n, M.; Arrobas, M.; Rodrigues, M. Â .; Moutinho-Pereira, J.; et al. Kaolin and salicylic acid alleviate summer stress in rainfed olive orchards by modulation of distinct physiological and biochemical responses. Sci. Hortic. (Amst.) 2019 , 246, 201–211. [CrossRef] 62. Maxwell, K.; Johnson, G.N. Chlorophyll fluorescence—A practical guide. J. Exp. Bot. 2000 ,51, 659–668. [CrossRef] [PubMed] 63. Cooley, N.M.; Glenn, D.M.; Clingeleffer, P.R.; Walker, R.R. The Effects of Water Deficit and Particle Film Technology Interactions on Cabernet Sauvignon Grape Composition. Acta Hortic. 2008 , 193–200. [CrossRef] 64. Shellie, K.; Glenn, D.M. Wine grape response to foliar kaolin particle film under differing levels of preveraison water stress. HortScience 2008,43, 1392–1397. [CrossRef] 65. Glenn, M.; Cooley, N.; Walker, R.; Clingeleffer, P.; Shellie, K. Impact of kaolin particle film and water deficit on wine grape water use efficiency and plant water relations. HortScience 2010,45, 1178–1187. [CrossRef] 66. Shellie, K.C.; King, B.A. Kaolin particle film and water deficit influence red winegrape color under high solar radiation in an arid climate. Am. J. Enol. Vitic. 2013,64, 214–222. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).