Time-lapse light dark-shift recordings included sensor tips placed directly above the aggregate ; and inserting the tip into the central core of the aggregate. Recordings of the oxygen signal were taken every second. Light sources were 65 W halogen lamps and experiments were conducted with either 170 µmol photons m−2 s −1 , or 320 µmol photons m−2 s −1 . Dark conditions were realized by switching off the lamps, removing them from the table, and carefully placing a carton box over the entire profiling setup to avoid residual light from the room. Background light intensities under the box were < 1 µmol m−2 s −1 . Theoretical limits of oxygen and DIC flux and whole aggregate O2 flux calculations were calculated from depth concentration profiles according to Ploug et al. , with a diffusion coefficient for O2 in 3.5% saline water of 2.175 × 10−5 cm2 s −1 at 24◦C and 2.3535 × 10−5 cm2 s −1 at 27◦C. Inside the aggregate, the apparent diffusivity of O2 was assumed to be 0.95 . Carbon fixation was estimated based on a photosynthetic quotient of 1.2 . The diffusion of oxygen in agar was not found to be different than in water over a wide range of salinities . In vineyard production systems, canopy management practices are usually employed to control the source-sink balance and improve the cluster microclimate leading to an improved grape composition and resultant wines . Canopy density is usually controlled during the dormant season thought the winter pruning. Additional canopy management practices may be applied during berry development. Fruit-zone leaf removal and especially, shoot thinning have been widely used in order to increase the cluster exposure to solar radiation, reduce crop load as well as decreasing the pest pressures , 25 liter pot increasing flavonoid content and diminishing herbaceous aromas . Nevertheless, when high air temperature and excessive radiation combine, detrimental effects on berry acidity and flavonoid content have been reported in warm climate regions .

Leaf removal consists of removing basal leaves around the clusters in the east or north side during grape development increasing the cluster exposure to solar radiation. It is well known that an early leaf removal increased total soluble solids, anthocyanins, and flavonols . However, some authors reported increases in titratable acidity in Sangiovese and Teran cultivars while other authors found decreases in acidity with basal leaf removal on Tempranillo . Conversely, Sivilotti et al. reported a positive effect of leaf removal applied after flowering on Merlot grapevine by improving cluster integrity by reducing incidence of Botrytis, and lower herbaceous aromas without affecting yield and cluster mass. Contrariwise, Pastore et al. reported that defoliation at veraison reduced the anthocyanin content and increased the impact of sunburn. In fact, these authors found that leaf removal induced a general delay in the transcriptional ripening program, which was particularly apparent for structural and regulatory genes involved in the anthocyanin biosynthesis. Clearly, vineyard location, cultivar , timing of leaf removal , method , and degree of leaf removal , the growing season , among others, are all factors influencing how leaf removal affects grapevine berry composition and integrity. On the other hand, shoot thinning has been related to increased cluster and berry mass and the number of berries per cluster, with a reduction on yield . Conversely, Wang et al. observed that shoot thinning had relatively minor impacts on yield components because of a compensatory effect due to the lower cluster number with concomitant increase in cluster mass. Contrarily, shoot thinning practices on grapevine did not show a great impact on berry primary metabolism , however, secondary metabolites were affected by them . In fact, we recently reported an increase of two-fold in the flavonol content of Merlot berries when leaf or shoot removal was applied mainly by increasing the proportion of quercetin and kaempferol derivatives in detriment of the myricetin derivatives . Berry composition is dependent on a complex balance between compounds derived from primary and secondary metabolism. Between secondary metabolites, flavonoids play an important role in the quality and the antioxidant properties of grapes and are very responsive to environmental factors such as solar exposure .

Anthocyanin compounds are responsive of the berry color, and flavonols act as a UV shields, contribute to the wine antioxidant capacity, color stability, and hue through copigmentation with anthocyanins . On the other hand, the methoxypyrazines are wine key odorants contributing to their herbaceous characteristics and have been related to unripe berries and poor-quality wines when these are not part of the wine typicity . Since they can be present in grape berry and wines at high levels, they may have an important sensorial impact on wine quality . Among methoxypyrazines, the 3-isobutyl-2- methoxypyrazine is considered the most relevant to wine flavor due to its correlation with the intensity of the bell pepper character of wines and its content at harvest seems to be dependent of the solar exposure . The differences found in the literature about the effect of manipulating the canopy architecture on the flavonoid and aromatic content due to different solar exposure of berries in warm climates opens an important field of research. Therefore, we aimed to find the optimal ranges of berry solar exposure estimated as percent of kaempferol for flavonoid synthesis up regulation and the thresholds for their degradation, and to evaluate how canopy management practices such as leaf removal, shoot thinning and a combination of both affect the grapevine yield components, berry composition, flavonoid profile, and herbaceous aromas.Leaf area index was measured on 21 June to characterize grapevine canopy growth and converted into leaf area on by a smartphone based program, VitiCanopy, coupled with an iOS system . The gap fraction threshold was set to 0.75, extinction coefficient was set to 0.7, and sub-divisions were 25. A “selfie-stick” was used for an easy access to place the device about 75 cm underneath the canopy. The device was positioned with the maximum length of the screen being perpendicular to the cordon, raspberry cultivation pot and the cordon being in the middle of the screen according to previous work . In each experimental unit, three images were taken to capture half canopy of each vine, and analyzed by the software. The relationship between leaf dry mass and area was determined on a sub-sample of leaves of different sizes using a leaf area meter . Total leaf area was calculated by defoliating one grapevine per treatment replicate after harvest and using the regressive relationship between leaf dry mass and leaf area. At harvest, clusters were manually removed, counted, and weighed on a top-loading balance. Leaf area to fruit ratio was calculated by dividing leaf area with crop weight.

Dormant pruning weight was collected during the dormant season ; and crop load was calculated as the ratio between yield per vine and the pruning mass of each vine. Labor operations costs and gross income per hectare were calculated based on yield and net returns per hectare and methods presented elsewhere . Anthocyanin productivity was calculated as reported by Cook et al. .At each sampling point and experiment, 55 berries were randomly collected from the middle of each treatment-replicate and kept on ice until they were measured. Berries were weighed, and mean berry mass was determined as the average mass of the counted berries. These berries were used to determine the total soluble solids , the pH, and the titratable acidity . TSS was measured as °Brix, with a digital refractometer . The juice pH and TA was determined with an autotitrator using sodium hydroxide to titrate to an end point of pH 8.3, and it was expressed as g•L−1 of tartaric acid.For each sampling point in each experiment, 20 berries were collected, gently peeled, and berry skins were freeze-dried . Dried tissues were ground with a tissue lyser . Fifty mg of the resultant powder was extracted in methanol: water: 7 M hydrochloric acid to simultaneously determine flavonol and anthocyanin concentration and profile as previously described Martınez-Lüscher et al. . Briefly, extracts were filtered and analyzed using an Agilent 1260 series reversedphase high performance liquid chromatography system coupled to a diode array detector. Separation was performed on a reversed-phase C18 column LiChrospher® 100, 250 mm × 4 mm with a 5-µm particle size and a 4-mm guard column of the same material at 25°C with elution at 0.5 ml per minute. The mobile phase was designed to avoid co-elution of anthocyanins and flavonols consisted in a constant 5% of acetic acid and the following gradient of acetonitrile in water: 0 min 8%, at 25 min 12.2%, at 35 min 16.9, at 70 min 35.7%, 65% between 70 and 75 min, and 8% between 80 and 90 min. The identification of flavonoid compounds was conducted by determining the peak area of the absorbance at 280, 365, and 520 nm for flavan-3-ols, flavonols and anthocyanins, respectively. Identification of individual flavan-3-ols, anthocyanins, and flavonols were made by comparison of the commercial standard retention times found in the literature. Commercial standards of epicatechin, malvidin-3-O-glucoside, and quercetin-3-Oglucoside were used for the quantification of flavan-3-ols, anthocyanins, and flavonols, respectively. The determination of proanthocyanidins was performed using an Agilent HPLC-DAD after an acid catalysis in the presence of excess phloroglucinol , with minor modifications described in Martınez-Lüscher et al. .The growing season of 2017 was warmer and drier compared to the reference data for the same period within the last 20 years . Thereby, average daily temperature was 4°C higher and rainfall was 18 mm less. Overexposed berries were the smallest due to overexposure resulting in dehydration thereby reducing berry mass.

Neither total soluble solids nor titratable acidity changed regardless of the degree of exposure to which berries were subjected. However, the juice pH of the Exp+ Deg+ and Exp+ Deg++ berry must was greater compared to Exp− and Exp+ Deg− berries. Berry skin flavonoid content and composition were also affected by the degree of exposure . The berry anthocyanin content of Exp− was similar to Exp+ Deg−. However, overexposed berries resulted in berry anthocyanin content that was 70% and 90% lower when compared to the Exp− berries. Grape berry exposure to solar radiation not only affected the anthocyanin content but also modified the ratio between the tri- and di-substituted anthocyanins leading to a less stable profile in all treatments with exposed berries. Likewise, berry skin flavonol content and composition were strongly affected by the degree of exposure to solar radiation. Therefore, in Exp+ Deg− flavonol content was two-fold greater than Exp−, albeit they abruptly decreased in overexposed grapes where flavonol content was 25% and 50% lower when compared to Exp− berries. Furthermore, in overexposed berries the proportion of kaempferol and quercetin significantly increased while the proportion of myricetin decreased. Regarding proanthocyanidins in berries, mild exposure did not affect their content in Exp+ Deg− compared to Exp− berries. However, greater solar exposure decreased proanthocyanidin content in berries but to a lesser extent compared to Exp−. Finally, the content of flavan-3-ols was severely reduced in Exp+ Deg++ berries .The analyses performed on single berries from two varieties confirmed the obtained response in anthocyanins and flavonols in Cabernet Sauvignon . Thus, exposure affected the accumulation/degradation of these flavonoids. Exposed berries from the East side of the canopy decreased 8%and 36% of the anthocyanin content in Cabernet Sauvignon and Petit Verdot, respectively. Thus, Petit Verdot seemed to be more sensitive to higher level of solar exposure and degraded anthocyanins. Overexposed berries of Cabernet Sauvignon resulted in an 87% decrease of the berry skin anthocyanins when compared to the interior berries . Berry skin anthocyanins and increasing exposure showed a significant trend below the 22% of kaempferol . Conversely, analysis of the segmented regression on Petit Verdot berries did not show a clear trend below the 3.2% of Kaempferol and after the point of inflection, anthocyanins started to degrade . Regarding flavonol content, no differences were observed between cultivars . Conversely, when exposure increased to ca. 60% the content of flavonols in exposed berries of both canopy sides and in both cultivars; the overexposed berries had the lowest flavonol content . Thus, our data revealed a strong positive relationship between the berry skin flavonols and the percentage of kaempferol until 8.6% of kaempferol proportion for Cabernet Sauvignon and 7.2% Petit Verdot .