The Effect of Canopy Position on the Fruit Quality Parameters and Contents of Bioactive Compounds and Minerals in ‘Braeburn’ Apples

: This study attempts to clarify the effect of canopy position on the physico-chemical parameters of apples cv. Braeburn. The experiments were carried out on fruit from the inner and outer part of the canopy in two growing seasons and at two harvest dates. Light measurements revealed that the average value of photo active radiation (PAR) for the inside and outside canopy amounted to 30.3 µ mol/m 2 /s and 133.7 µ mol/m 2 /s, respectively. Production year and canopy position signiﬁ-cantly inﬂuenced ground color parameters a* , b* , C* , and h ◦ , while the harvest date inﬂuenced all color parameters studied. For additional (red blush) coloration, the production year signiﬁcantly inﬂuenced only the L* parameter, harvest date inﬂuenced all color parameters, and canopy position inﬂuenced L, a* , and C* . Only the fruits of the second harvest date showed more intense additional (red blush) coloration. The production year signiﬁcantly affected fruit mass, ﬁrmness, total soluble solids (SSC), titratable acidity (TA), SSC/TA ratio, DPPH radical scavenging assay (AOP), total phenolic content (TPC), and total ﬂavonoid content (TFC). The harvest date signiﬁcantly inﬂuenced fruit mass, SSC, TA, SSC/TA, AOP, TPC, and TFC. The canopy position signiﬁcantly inﬂuenced SSC, TA, AOP, TPC, and TFC. Regarding mineral content, the production year signiﬁcantly affected the content of Fe, Ni, Cu, and Ca and the K/Ca ratio. The harvest date signiﬁcantly affected Fe, Cu, Sr, K and K/Ca. The canopy position affected Fe, Ni, Zn, Sr, Ca, and K/Ca ratio, with a clear signiﬁcant trend regarding the effect of canopy position only for Ca content (ﬁrst and second year of the second harvest date) and K/Ca ratio (ﬁrst year of both harvest dates). PCA analyses identiﬁed distinguishing features between apples, with differences deﬁned speciﬁcally by AOP, TPC, TFC, Rb, Sr, Ca, and K/Ca on the PC 1 and Mn, Fe, Ni, Cu, and Zn on PC 2.


Introduction
In addition to all other environmental factors, the fruit quality of any plant species (including apple) is strongly influenced by light.As reported in many research papers [1][2][3][4][5][6][7][8], fruits exposed to sunlight may differ in quality from shaded fruits.Besides light, temperature can also have a strong influence on some fruit quality characteristics [9].
Therefore, fruit position in the tree canopy has a strong influence on fruit quality, as it correlates with the fruit's exposure to light and ultimately to temperature.This phenomenon has been observed in various fruit crops.For example, Fouché et al. [10] found that apples (cv.'Granny Smith') in the outer part of the canopy were exposed to 54% of full sunlight, while fruit in the inner part of canopy received only 2% of full sunlight during an average day.The appearance of the fruit provides the first general impression or attraction and plays and important role in consumer acceptance or rejection [11].There are numerous studies available reporting the impact of fruit position in the canopy on fruit appearance and quality attributes such as color [8,12] and fruit size [13,14].In addition, fruit position in the canopy can also affect the soluble solids content (SSC) [1,5,8,15] and titratable acidity (TA) [3] that determines the taste of the fruit, which is a major factor in the consumption of apples [16].The effect of canopy position on different bioactive compounds in fruit has been reported in several studies, e.g., antioxidant activity [4,7,8] and polyphenols content [6,7,17,18].However, there are few studies addressing the effect of fruit canopy position on mineral content in fruit.A few studies [8,19,20] reported that fruit position in the canopy can affect mineral content, but they obtained opposite results for some elements.This aspect is very important, because the content of some elements in the fruits can have an impact on the quality of the fruits.For example, calcium deficiency in apple fruit is often associated with the post-harvest disorder bitter pit [21].
The novelty of the present study is that it deals with the mineral contents of apples from two harvest dates in two producing seasons.Most other available studies dealt mainly with fruit from only one harvest date.In addition, there is no published research on the effects of fruit position in the canopy on fruit quality parameters of 'Braeburn' apples.Therefore, the aim of this study is to investigate the effect of fruit position in the canopy from two different harvest dates on fruit quality characteristics, with an emphasis on mineral content, of 'Braeburn' apples.

Plant Material and Experiment Set Up
The fruit samples of apple 'Braeburn' were collected in 2011 and 2012 from a commercial apple orchard near Krapina city, Croatia (latitude 46 • 09 N, longitude 15 • 53 E).The experiments were carried out on adult trees (8 years old), planted with 3 m (between rows) ×1 m (inside rows) distances and grafted onto M9 rootstock with training system of spindle bush.Standard cultivation practices (fertilization, irrigation, pruning, etc.) were applied uniformly in all treatments.Fruits were harvested in both years in early October on two different harvest dates (10 days apart).Harvest dates were determined by continuous monitoring of fruit maturity through specific analyses.Three replicates were conducted with 5 trees each (15 trees in total).The selected apple trees had similar yields in both years.Fifteen fruits were randomly picked from the internal/inside and 15 from external/outside part of canopy of apple trees.The analytical work was performed in the laboratory of Department of Pomology, Unit of Horticulture and Landscape Architecture, Faculty of Agriculture, University of Zagreb; Dept. of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Slovenia, and the Jožef Stefan Institute, Ljubljana, Slovenia.Weather data were taken from the Croatian Meteorological Service from a weather station located about 500 m from the experimental orchard.Light measurements were carried out by Laboratory of Lighting and Photometry, Faculty of Electrical Engineering, University of Ljubljana, Slovenia.

Physico-Chemical Measurements 2.2.1. Fruit Mass, Firmness, and Color
The average value of fruit mass of each fruit was calculated using a digital analytical balance (OHAUS Adventurer AX2202, Ohaus Corporation, Parsippani, NJ, USA) with an accuracy of 0.01 g and expressed in g.On each fruit, ground and additional fruit skin color parameters were measured separately using a colorimeter (ColorTec PCM; ColorTec Associates Inc., Clinton, NJ, USA) according to the CIE L*a*b* and CIE L*C*h • (Commission Internationale d'eclairage) systems.In the CIE L*a*b* color space, the L* value corresponds to a dark-bright scale and represents the relative lightness of colors in a range from 0 to 100 (0 = black, 100 = white) [22].The a* and b* scales extend from −60 to 60, where a* is negative for green and positive for red and b* is negative for blue and positive for yellow [22].According to Carreño et al. [23], the hue angle (h • ) and chroma (C*) are calculated as follows: where a* and b*-variables in the CIE L*a*b system.Hue angle (h • ) describes the relative amount of redness and yellowness, where 0 • /360 • is defined for red/magenta, 90 • for yellow, 180 • for green, and 270 • for blue color [23].C* indicates the color intensity [24].
The firmness was measured using PCE PTR-200 (PCE Instruments, Jupiter/Palm Beach, FL, USA) fitted with 11 mm diameter plunger and expressed in kg•cm −2 .Measurements were made at four equatorial positions on each fruit at 90 • .2.2.2.Soluble Solids Content (SSC), Titratable Acidity (TA), and SSC/TA Ratio SSC was measured with a hand digital refractometer (Atago, PAL-1, Tokyo, Japan) and expressed as %.TA was determined by titration with 0.1 N NaOH, expressed as g•L −1 (as malic acid) according to AOAC 954.07 [25].The SSC/TA ratio was calculated from the corresponding values of SSC and TA for each fruit.

DPPH Radical Scavenging Assay (AOP), Determination of Total Polyphenols (TPC), and Determination of Total Flavonoids (TFC)
Ten grams of joint apple sample crushed in liquid nitrogen was extracted in 10 mL of 3% metaphosphoric acid.The homogenate was centrifuged at 1700× g for 5 min (Centrifuge 5415c; Eppendorf, Germany) and the supernatant was filtered through 0.45 µm filters (17 mm cellulose acetate syringe filter; Sartorius AG, Goettingen, Germany).These extracts were used for the determination of total antioxidant potential (AOP), TPC, and TFC.
The AOP of the apple metaphosphoric acid extracts was determined spectrophotometrically as the 2,2-diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich, Darmstadt, Germany) free radical scavenging capacity, as described by Brand-Williams et al. [26]; then, 1.5 mL of 560 µM DPPH methanolic solution was mixed with 60 µL of apple extract and vortexed.After incubation at room temperature for 15 min, absorbance was measured at 520 nm using a spectrophotometer (Cecil Aurius Series CE 2021 UV/Vis; Cecil Instruments Limited, Cambridge, UK), against methanol as the blank.AOP was quantified via calibration using the Trolox five-point standard curve (1.56 to 10.94 mg/L).Results are expressed as µmol Trolox equivalents (µmol TE•100 g −1 of F.W.).
TPC was measured using a modified Folin-Ciocalteu colorimetric method according to Singleton et al. [27].Aliquots of test samples (200 µL metaphosphoric acid extracts) were incubated with 2540 µL diluted Folin-Ciocalteu reagent (10 mL Folin-Ciocalteu reagent [Merck, Darmstad, Germany] in 20 mL deionized water).After 2 min of incubation, 420 µL of 20% Na 2 CO 3 (Merck) was added to the mixtures.After an additional 30 min of incubation at room temperature, 910 µL of deionized water was added and the absorbance of the mixtures was measured using a spectrophotometer at 765 nm against deionized water as a blank.All samples were processed in triplicate.TPC was quantified via calibration using gallic acid (Fluka, Buchs, Switzerland) as a standard.The eight-point calibration curve ranged from 1.7 mg/L to 13.6 mg/L gallic acid.TPC is expressed in mg gallic acid equivalents (GAE)/100 g FW.TFC was determined as described by Lin and Tang [28].The sample (250 µL) of metaphosphoric acid extract was mixed with 750 µL of 95% ethanol, 50 µL of 10% aluminium chloride (AlCl 3 ), 50 µL of 1 M potassium acetate (CH 3 COOK), and 1400 µL of deionized water in a 15 mL vial.The solution was mixed, and absorbance was measured at 415 nm after 40 min of incubation at room temperature.Measurements were performed in triplicate, and calculations were based on a five-point standard curve ranging from 0.3 to 15.0 mg quercetin/L.Results are expressed in quercetin equivalents (mg QE/100 g Fw).

Determination of Elements
Multi-element determination of the element concentration (K, Ca, Mn, Fe, Cu, Ni, Zn, Rb, Sr) was performed via nondestructive energy-dispersive X-ray fluorescence spectrometry using a Si(Li) detector (Canberra, Meriden, CT, USA), a spectroscopy amplifier (model M2024, Canberra, Meriden, CT, USA), an analog-to-digital converter (model M8075, Canberra, Meriden, CT, USA), and a PC-based multichannel analyzer (model S-100, Canberra, Meriden, CT, USA).Approximately 0.5 to 1.0 g of the selected sample was weighed to prepare the pellets using in-house-made pellet die and a hydraulic press.The disc radioisotope excitation source of Cd-109 (740 MBq) was used (Eckert and Ziegler, Valencia, CA, USA) as the primary excitation source.The spectral analysis program AXIL (Analysis of X-ray spectra by Iterative Least squares) (IAEA, Vienna, Austria) was used for the analysis of complex X-ray spectra, while the quantification was performed with the inhouse-developed software for quantitative analysis of environmental samples (QAES) [29].Results of elemental concentration are expressed in (µg/g) dry matter.

Light Measurements
Although all experiments were carried out in the years 2011 and 2012, light measurements were conducted in 2023.Light measurements (illuminance and PAR-photo active radiation) were carried out on two apple trees trained as spindle bush training system during June 2023 with twelve wireless light and color sensors (Pasco 3248).The sensors were protected with waterproof transparent bags using a vacuum sealer machine and then fixed to the branches of both trees.Six sensors were used to measure illuminance and PAR at the end of the branches, where there were actually no leaves creating shadows on the fruit, and six were used to measure inside the tree canopies, where leaves create shadows on the fruit.As vertical illuminance and vertical PAR were measured, different orientations of the sensor were used to cover all major orientations of the sky.Measurements were performed every 2 min and recorded in the sensor's internal memory.In cases where it was possible to pair outside and inside sensors with the same orientation, the ratio of PAR was also calculated.

Statistical Analysis
Year (Y), harvest date (H), and canopy position (C) were considered as factors, and three-way ANOVA using a generalized linear model was performed using SAS software program version-9.4(SAS Institute Inc., Cary, NC, USA).The effect of Y, H, and C and their interactions on different physico-chemical characteristics was analyzed using Student's t-test.

PCA Analysis
In order to get better a insight into the bioactive compounds and mineral concentration of the fruits, PCA analyses of these data were performed with prior data standardization.Prior to PCA analysis, Bartlett's sphericity test and Kaiser-Meyer-Olkin measure of sampling adequacy were used to verify that the performance of PCA was correct.Bartlett's sphericity test showed p-value of less than 0.0001, indicating that it is highly unlikely that the observed correlation matrix was obtained from a population with zero correlation.The Kaiser-Meyer-Olkin measure of sampling adequacy gave a value of 0.6, which was acceptable for continuing PCA analysis [30].

Background Color
The ANOVA revealed that the background fruit color was significantly affected by harvest date alone, and for the most part, by year and canopy (except for variable L*).The interactions of year × harvest date (except for variable b*) and harvest date × canopy (except for variable L*) also showed significant effects.However, the main effect of year × harvest date was not significant.The overall interaction between all sources of variables showed a significant difference only for b* and C* (Table 1).The data presented in Table 1 show the influence of canopy position on the basic fruit skin color of 'Braeburn' apple fruit harvested at two different dates within two years.The fruit skin color parameter L* showed no significant difference in the comparisons tested.
As for the color variable a*, the fruits from the first harvest date from the outer part of the canopy had significantly lower values (p ≤ 0.001) than the fruits from the inner part of the canopy in both years.In both years, the fruits of the second harvest date and from the outer part of the canopy had significantly higher a* values than the fruits from the inner part of the canopy (Table 1).
In both years, fruits from the first harvest date from the outside part of the canopy had a significantly higher b* value (first year p ≤ 0.001; second year p ≤ 0.01) than fruits from the inside of the canopy.The first-year fruits from the second harvest date from inside the canopy had a significantly higher (p ≤ 0.05) b* value than fruits from the outside of the canopy, while no significant difference was observed in the second year (Table 1).
Regarding C* value, fruits from both years from the first harvest date from outside the canopy had significantly (p ≤ 0.001) higher values than fruits from inside the canopy.The fruits from the second harvest date from the first year from outside the canopy had a significantly lower C* value (p ≤ 0.05) than the fruits from inside the canopy, while no significant difference was found (Table 1) in the second year.
Regarding color variable h • , the fruits from both years of the first harvest date from outside the canopy had a significantly higher h • value (p ≤ 0.001) compared with fruits from inside the canopy.In contrast, fruits from both years of the second harvest date from outside the canopy had significantly lower h • values (first year p ≤ 0.05; second year p ≤ 0.01) than the fruits from inside the canopy (Table 1).

Additional (Red Blush) Color
The ANOVA revealed that fruit color was significantly affected by harvest date alone and by its interaction with year (year × harvest date) and with canopy position (harvest date × canopy), except in the case of the variable CIE lightness (L*).However, the main effect of the year was significant only for the variable L*.The main effect of the canopy proved to be significant in relation to L*, a*, and chroma value (C*) and in relation to its interaction effect with the year, with significant values in relation to L*, a*, and h • , and finally, in its interaction with the harvest date in all CIE variables except L*.The overall interaction between all sources of variables showed a significant difference only for variables b* and C* (Table 2).The data presented in Table 2 show the influence of canopy position on the fruit red blush color of 'Braeburn' apple fruit harvested at two different dates within two years.The fruit color parameter L* was significantly higher in the first year of study for fruit harvested from inside the canopy at both harvest dates (p ≤ 0.001).However, in the second year, the same trend was observed, but only the fruits from the second harvest showed significant differences (p ≤ 0.001).
Regarding the color variable a*, in both years, the fruits of the first harvest date from the inner part of the canopy had significantly (p ≤ 0.001) higher values compared with fruits from the outer part of the canopy.The opposite was found for fruits from the second harvest date, where significantly higher values were recorded for the outside position (first year p ≤ 0.001; second year p ≤ 0.05) (Table 2).
In both years, fruits of the first harvest date from outside the canopy had significantly higher b* values (first year p ≤ 0.001; second year p ≤ 0.01) compared with fruits from the inside of the canopy.On the contrary, fruits of the second harvest from the outside of the canopy had a lower b* value compared with the inside position, with a significant difference observed only in the first year (p ≤ 0.001) (Table 2).
Regarding the color parameter C*, the fruits from both years from the first harvest date from outside the canopy had higher values compared with fruits from inside the canopy, with a significant difference (p ≤ 0.001) found only for the first year.The opposite situation was observed for the fruits of the second year from the second harvest date, where the fruits from outside the canopy had a significantly lower C* value (p ≤ 0.001), while again, no significant difference was observed in the second year (Table 2).
Regarding the color variable h • , the fruits from both years of the first harvest date from the outside of the canopy had a significantly (p ≤ 0.001) higher value compared with fruits from the inside of the canopy.On the other hand, fruits from both years of the second harvest date from the outside of the canopy had a significantly lower h • value (first year p ≤ 0.001, second year p ≤ 0.05) (Table 2).

Effect of Different Canopy Positions on Physico-Chemical Properties of 'Braeburn' Apples
The results of ANOVA show that the variable of year has a significant effect on all physico-chemical properties of 'Braeburn' apples.However, the interaction effect with all variable sources on each parameter was not significant.Similarly, the main effect of the harvest date showed significance on all parameters except firmness, while its interaction with year and canopy showed significance only to some extent.The main effect of canopy, on the other hand, showed no significant differences, and was significant only with SSC and TA.The interaction effect with year showed significance only for fruit mass and SSC, while the interaction effect with harvest date was significant for all parameters except fruit mass.The overall interaction of all variable sources showed significant values for firmness, SSC, and SSC/TA ratio (Table 3).The mass of fruits from the first year was significantly higher (p ≤ 0.05) for fruit from the inner canopy position compared with the outer position from the first harvest date.No significant differences were observed at the second harvest date, although fruit from the inside of the canopy tended to have a higher average mass than fruit from the outside of the canopy.In the second year, fruit from the first harvest date from the outside of the canopy had significantly higher mass (p ≤ 0.05) than fruit from the inside of the canopy.No significant differences were found at the second harvest date, although fruit from the outside of the canopy tended to have a higher average mass than fruit from the inside of the canopy (Table 3).
Regarding fruit firmness, only fruits from the second year of the first harvest date from the outside of the canopy had significantly higher firmness (p ≤ 0.01) than fruits from the inside of the canopy.Although no significant differences were found, it should be noted that in the first year, the fruits of the first harvest date from outside the canopy had higher average values than the fruits from inside the canopy.Also, in the second year, the fruits of the second harvest date from inside the canopy had higher average values than the fruits from outside the canopy (Table 3).
On the other hand, the SSC values of the fruits from the first year of the second harvest date from outside the canopy were significantly higher (p ≤ 0.001) than those of the fruits from inside the canopy.Although the differences were not significant, a nonsignificant trend can be seen for fruits from the first year and the first harvest date and for fruits from the second year from the first and second harvest dates, where the fruits from outside the canopy tended to have a higher average SSC value than those from inside the canopy (Table 3).
Regarding the TA values, only the fruits from the first year from the first harvest date from the outside of the canopy had a significantly higher (p ≤ 0.05) TA value than fruits from the inside of the canopy.Although no significant differences were found, it should be noted that among the fruits from the second year, there was a slightly higher average value in the fruits from the inside of the canopy from both harvest dates compared with the fruits from outside the canopy (Table 3).
Regarding the SSC/TA ratio in the first year on the first harvest date, the fruits from inside the canopy had a significantly higher ratio (p ≤ 0.05) than the fruits from outside the canopy.On the second harvest date, fruit from outside the canopy had a significantly higher ratio (p ≤ 0.05) than fruit from inside the canopy.No significant differences were found in the second year, but on the first harvest date, fruits from the inner part of the canopy had a higher average ratio than fruits from outside the canopy (Table 3).

Effect of Different Canopy Positions on Bioactive Compounds of 'Braeburn' Apples
The results of ANOVA indicated that the main effects of all variables (year, harvest date, and canopy) showed significant values in relation to DPPH antioxidant activity, TPC, and TFC of 'Braeburn' apples.The interaction between year and harvest date (Y × H) showed significant effects on AOP and TPC.The interaction between year and canopy (Y × C) showed a significant effect on TPC.The interaction between harvest date and canopy (H × C) showed a significant effect on TFC.However, the overall interaction between them (Y × H × C) did not show significant values for all bioactive compounds of 'Braeburn' apples (Table 4).
The AOP was significantly higher in fruit harvested from outside the canopy in both years and at both harvest dates (first year p ≤ 0.01, second year p ≤ 0.001) (Table 4).
In the first year, the TPC was higher in fruits harvested from outside the canopy, but a significant difference (p ≤ 0.01) was observed only in fruits of the second harvest date.In contrast, in the second year, fruits harvested from outside the canopy had significantly higher values (p ≤ 0.001) for both harvest dates (Table 4).
In terms of TFC, fruits harvested from the outer part of the canopy had significantly higher values (p ≤ 0.001) than fruits from the inside position (Table 4).All numbers in the first part of the table present average value ± standard deviation, while in the ANOVA part of the table, F value is presented.DPPH-DPPH antioxidant activity, TPC-total polyphenolic contents, TFC-total flavonoid contents; significance in results of bioactive compounds relates to differences between inside and outside position.n.s., * , ** , *** nonsignificant, or significant at p ≤ 0.05, p ≤ 0.01 or ≤0.001, respectively.

Effect of Different Canopy Positions on Mineral Concentration of 'Braeburn' Apples
The results of the ANOVA presented in Table 5 show that all sources of variables show significant levels in relation to the mineral concentration of 'Braeburn' apples.The main effect of the variable year showed significant values for the minerals Fe, Ni, Cu, and Ca, and for the K/Ca ratio.The main effect of the harvest date showed significant values for Fe, Cu, Sr, K, and K/Ca.The main effect of canopy position showed significant values for Fe, Ni, Zn, Sr, Ca, and K/Ca.The interaction between year and harvest date (Y × H) showed significant values for Fe, Ni, Cu, Sr, and K/Ca.The interaction between year and canopy (Y × C) showed significant values for Mn, Fe, Ni, Rb, Sr, and K/Ca.The interaction between harvest date and canopy (H × C) showed significant values Fe and K.The interaction between all variables (Y × H × C) showed significant values Fe and Ca.All numbers present F value.n.s., * , ** , *** -nonsignificant, or significant at p ≤ 0.05, p ≤ 0.01, or ≤0.001, respectively; significance in results of mineral concentration relates to differences between inside and outside position.
Iron (Fe) was significantly higher (p ≤ 0.01) in fruits from the outer part of the canopy than fruits from the inner part in the first year at the second harvest date, while in the second year at the second harvest date, fruits from the inner part of the canopy had a significantly higher value (p ≤ 0.01).Copper (Cu) was significantly higher in fruits from the second year from the outer part of the canopy (p ≤ 0.05) at the second harvest date.Rubidium (Rb) reached a significantly higher value in fruits from the first year at the first harvest date from the inner part of the canopy (p ≤ 0.01) compared with the fruits from outside the canopy.For strontium (Sr), the fruits from the first year at the first harvest date from the outer part of the canopy had a significantly higher value (p ≤ 0.05) than fruits from the inner of the canopy.Regarding potassium (K), fruits from the first year from the first harvest date from the inner part of canopy had a significantly higher value (p ≤ 0.05) than fruits from the outer part of the canopy.Calcium (Ca) reached a higher value (p ≤ 0.001) in fruits from the first year from the second harvest date from outer part of the canopy compared with fruits from the inside part of the canopy and also from the second year at the second harvest date (p ≤ 0.05).Regarding the K/Ca ratio, fruits from the inner part of the canopy had a significantly higher ratio than fruits from the outer part of the canopy in the first year at the first (p ≤ 0.05) and second (p ≤ 0.01) harvest dates.In addition, no significant differences were found between the fruits from the inner and outer part of the canopy in both years and at both harvest dates in terms of Mn, Ni, and Zn (Table 6).

Weather Data
Weather data parameters (temperature, relative humidity, precipitation, and insolation) for the vegetation period (from April to September) are presented for 2011 (Figure 1) and 2012 (Figure 2) and provided by the Croatian Meteorological and Hydrological Service [31].More precipitation (mm) was recorded in September and October of 2012 compared with 2011.
Figures 1 and 2 show the meteorological data for 2011 and 2012, respectively.The average temperature was lower in April, September, and October, while it was higher from May to August in 2012 than in 2011.In all measured months, especially from August to October and with the exception of July, more precipitation was recorded in 2012 than in 2011.However, humidity was lower in most cases from April to August and higher from September to October in 2012 than in 2011.In June, July, and August, insolation was higher, and in April, May, September, and October, it was lower in 2012 than in 2011.and 2012 (Figure 2) and provided by the Croatian Meteorological and Hydrological Service [31].More precipitation (mm) was recorded in September and October of 2012 compared with 2011.

Light Measurements Outside Inside of Canopy
The maximum measured values were up to 96 klx and up to almost 1800 µmol/m 2 /s.The average PAR value for the inside sensors was 30.3 µmol/m 2 /s (with SD of 5.8 µmol/m 2 /s) and 133.7 µmol/m 2 /s (with SD of 52 µmol/m 2 /s) for the outside sensors.

Insolation and Total Irradiance Data
Insolation data for June 2011, 2012, and 2023 amounted to 261.3, 281.1, and 269.5 h, respectively [31].For the same years, total irradiance on horizontal surfaces as recorded in June in the years of 2011, 2012, and 2023 was 269.1, 276.7, and 269.1 W/m 2 , respectively [32].

PCA Analysis and Biplot
PCA (Table 7) revealed three significant principal components (PC) with eigenvalues greater than 1, accounting for 76.31% of the total variability.PC1 (37.99% of the total variability) correlated positively with AOP, TPC, TFC, Rb, SR, Ca, and K/Ca.PC2 (27.51% of the total variability) correlated positively with Mn, Fe, Ni, Cu, and Zn, while PC3 (10.81% of the total variability) correlated negatively with TPC and positively with Sr and K.
The PCA biplot (Figure 3) showed that the fruits from outside the canopy harvested at the second harvest date in both years were clearly distinguished from the rest of the fruits, indicating that their chemical composition was different compared with fruits harvested on the first harvest date and also with those harvested from the inner canopy.

Effect of Different Canopy Positions on Fruit Skin CIE Color Variables
The color of apples is one of the most important factors determining consumers preference and market price [33].The intensity of light received by the fruit peel has a strong impact on color development [8,34] and can play a fundamental role [33].In general, the outer periphery of the canopy intercepts and reflects a high proportion of the incoming  The color of apples is one of the most important factors determining consumers preference and market price [33].The intensity of light received by the fruit peel has a strong impact on color development [8,34] and can play a fundamental role [33].In general, the outer periphery of the canopy intercepts and reflects a high proportion of the incoming radiation, resulting in different light distribution profiles for different training systems [35].Along a horizontal cross-section of the canopy when trees are grown as central leader trees, light is distributed as a U-shaped pattern [35] according to [36,37].In our spindle training system, the inside sensors registered 30.3 µmol/m 2 /s (with SD of 5.8 µmol/m 2 /s), and the outside sensors registered 133.7 µmol/m 2 /s (with SD of 52 µmol/m 2 /s).The average ratio of PAR between the outside and inside sensors was 4.9, which means that outside fruits received five times more PAR compared with fruits grown inside the canopy.Regarding the insolation variability in Krapina city, data in the literature state 3.6% [31], while total irradiance varies, but is around 1.6% [32].In Lithuania, Kviklys et al. [38] reported that during the growing season, on average, the upper side of the apple canopy received 43% of the available light, the west side 21%, the east side 16%, and the inner side of the canopy received only 12%.Thus, light availability to fruit is significantly affected by canopy position.Four weeks before harvest of 'Fuji' apples, Jakopič et al. [6] found the highest light availability at the top of the canopy (70 µmolm −2 s −1 ), followed by the outer canopy (56 µmolm −2 s −1 ), and then the inner canopy (17 µmolm −2 s −1 ).Similarly, Lin et al. [39] reported that the central part of a large round canopy receives twice as little photosynthetically active radiation compared with the outer canopy.Therefore, different positions of fruits based on their access to light may affect fruit coloration within the canopy.For example, in our study regarding additional color (Section 3.1 and Table 2), the fruits from inside the canopy were found to have a significantly higher L* value in most cases, resulting in a brighter color than fruits from the outside of the canopy, which is consistent with the results of Jakopic et al. [6].Lawes [2] reported that fruits growing under poor light conditions had a less red color than fruits under adequate quantity of and access to light, which is in accordance with our results regarding additional color at the second harvest date in both years (Table 2).Only fruits from the second harvest date showed a more intense additional red color, as evidenced by higher a* and lower h • values.Regarding ground color (Table 1), fruits from the second harvest dates had lower a* value than fruits from the inside of the canopy in both years.Since a negative value of the color value a* means a green color, it can be assumed that this is also in accordance with the results of the additional color, and thus with the above study.However, the reason for the difference at the first harvest is probably that the fruits had not yet developed their full color at the first harvest date, since they were not fully ripe at harvest.A similar situation was found for the color variable b*, where the same significant trend was recorded for the basic and additional colors.However, it is logical that as fruit ripens, it obtains a red color at the expense of yellow color.Jakopic et al. [6] reported statistically significant differences in color values between apples (cv.'Fuji') from the inner part of the canopy and from the outer part of the canopy, but no differences were observed between outer fruits and fruits from the top of the trees.They also found that the fruits from the outer parts and the top of the canopy had darker (lower L* value), less yellow (lower b*), and more red (higher a* value) coloration than fruits from inner parts of the tree canopy, which is mostly in accordance with our results for the second harvest date.Kokalj et al. [40], in their work on postharvest light irradiation, also demonstrated a significant improvement in red coloration due to the accumulation of anthocyanins in three apple cultivars.Regarding the h • value for basic and additional color (Tables 1 and 2), at the first harvest date for both years, fruits from outside of the canopy had significantly higher values, while on the second harvest date, the situation was the opposite.The h • values of Weber et al. [41] for 'Braeburn Mariri Red' apples are consistent with the second harvest date in this study.As the higher h • values (that are further from 0 • and closer to 90 • ) in this study indicate a less red color, it is in accordance with a* and b* values.Regarding chromaticity, the results of the C* value of the background color showed lower values for fruits from the first harvest from the inside position and nearly the same values for fruits from the second harvest (Table 1).A lower C* value indicates less expressed colorfulness.The same tendency was observed for the red blush color, with the exception that fruits from inside of the canopy from the first year, second harvest date, had a higher C* value (Table 2).

Effect of Different Canopy Positions on Physico-Chemical Properties of 'Braeburn' Apples
Fruit quality is related to the amount of light received in the vicinity of the developing fruit [3], so solar radiation is the key factor in the overall apple fruit quality [42].Normally, the amount of light received by the fruit is influenced by the fruit's position within the canopy.Therefore, it can be assumed that the position of the fruit within the canopy can influence the quality of the apple.
In our study, the effect of the fruit's position in the canopy on the fruit mass and firmness of 'Braeburn' apples (Section 3.2 and Table 3) showed no significant differences.However, it has been reported that light can affect fruit firmness [43] and that fruit under poor light conditions usually have lower firmness [2].When other studies are compared, the influence of fruit's position in the canopy is unclear.For example, Blanpied et al. [44] found that apples from shaded inner canopies were less firm than apples from outer canopies.In another study [45], it was found that the firmness of pears of the cultivar 'Bartlett' was not influenced by canopy position.Laubscher [46] carried out a two-year trial on nectarines cv.'Red Jewel' and found that in first year, there were no significant differences between fruits from the top and bottom of the canopy.In the second year, however, the fruits from the top of the canopy had significantly higher firmness than the fruits from the bottom of the canopy.Lewallen [3] observed that the peaches (cv.Norman) from the outer canopy had lower firmness than peaches from the inner canopy.Lewallen [3] concluded that canopy position or light environment does not have a uniform effect on the flesh firmness of fruit.The results in our study add to the ambiguity on this topic, as no consistent differences or correlations were found in our study.
SSC is strongly influenced by light exposure [47], and thus, by the light distribution within the canopy [3].Light energy is absorbed by chlorophyll to drive photosynthesis, which in turn affects the soluble solids content in fruit [48].Therefore, fruits that receive more sunlight due to different positions in the canopy can be expected to acquire more photoassimilates from nearby leaves and consequently increase SSC, which has already been reported for apples [5,49].In our study (Section 3.2 and Table 3), the significant differences were found only in the first year on the second harvest date, but a similar nonsignificant trend was found all other cases, so the results in this study are in accordance with the literature.It is widely reported that lighted fruits, in contrast to shaded fruits (e.g., fruits from the outer or upper canopy in contrast to fruits from the inner or lower canopy), have higher SSC for apples cvs.'Granny Smith' [1,8], 'Starking' [8], and 'Golden Delicious' [8]; pears [45]; plums cv.'Laetitia' [50]; peaches [51][52][53] and nectarines cv.'Red Jewel ' [46]; kiwifruits [54,55]; mandarins [56]; oranges [57]; grapefruits [58]; and lemons [59].Finally, SSC content is a very important biochemical fruit characteristic because a high level of consumer acceptance has been associated with high levels of SSC, among many other factors [60].
The distribution of light within the tree canopy also influences the TA content in fruits [3].According to the results of a few studies, acid content in fruit is negatively correlated with the amount of light [5,8,45].Due to the low amount of light, the fruits in the inner part of the canopy might have a higher TA content than the fruits from the outer part of the canopy.However, in our study (Section 3.2 and Table 3), no clear effect was found for both years.Furthermore, in contrast to the studies cited above, in the first year and at the first harvest date, the fruits from the outer canopy had a higher TA content than the fruits from the inner part of the canopy.There are some studies that come to the same conclusion.For example, Krishnaprakash et al. [61] reported no significant differences in terms of TA in apple fruit from the top and bottom of canopy.The content of TA in fruit is a very important fruit property, because of consumer acceptance in addition to higher SSC being linked with acidity [60].
As reported by Hamadziripi [8], the ratio of SSC/TA was significantly higher in 'Granny Smith', 'Starking', and 'Golden Delicious' apples in the outer canopy.However, in our study (Section 3.2 and Table 3), no clear effect was found for both years.

Effect of Different Canopy Positions on Bioactive Compounds of 'Braeburn' Apples
In our study (Section 3.3 and Table 4), it is evident that the antioxidant activity of apples was significantly higher in the fruits of the outer part of the canopy than the ones grown in the inner part.Our results are in agreement with Hamadziripi [8], who reported that the flesh of apples from the outer part of the canopy has about 1.4 times higher antioxidant capacity than the flesh of apples from the inner part due to higher light exposure.Similar results were obtained by Drogoudi and Pantelidis [7] and Hagen et al. [4] in apple peels.In addition, two studies [62,63] found lower antioxidant capacity in most cases in kiwifruit and peaches grown under photoselective nets as opposed to natural conditions (without shading).
The results of our study (Section 3.3 and Table 4) show that the fruits from the outer part of the canopy have a significantly higher TPC than those from the inner part of the canopy.Only in the first year at the first harvest date no significant differences were found.However, the nonsignificant trend shows that the fruits from the outer part had a higher average value than the fruits from the inner part.Our results are in agreement with those of Hagen et al. [4], who found that light-exposed apples have a higher content of total phenols in the peel.Similarly, McDonald et al. [17] found that grapefruits from the outer canopy had a higher content of total phenols than fruits from the inner canopy.In addition, two studies [62,63] found that a lower polyphenol content was reported in kiwifruit and peaches grown under photoselective nets as opposed to natural conditions (without shading).
In our study (Section 3.3 and Table 4), fruits from the outer part of the canopy had a significantly higher flavonoid content than fruits from the inner part of the canopy.Our results are in line with several available studies.For example, Awad et al. [64] found that 'Jonagold' apples from the outer part of the canopy had a significantly higher total flavonoid content than fruit from the inner part of the canopy.Similarly, in another study, Awad et al. [65] found that 'Elstar' apples from the top of the canopy had a significantly higher content of flavonoids compared with those from the inner part of the canopy.They [65] also reported that in the skin of 'Jonagold', 'Elstar' and two 'Elstar' mutants, 'Elshof', and 'Red Elstar', a significantly higher content of total flavonoids was found in apples on the sunny side compared with those the shady side of the canopy.In Croatia, it was also reported that 'Granny Smith' apples grown under a photoselective red net had a lower flavonoid content in contrast to those grown under natural conditions [66].
Light intensity and -quality can influence the biosynthesis of phenols, flavonoids, and the antioxidant capacity of fruits [53,[67][68][69].As Kokalj et al. [40] found, postharvest irradiation of apples increased the phenyl alanine amylase activity and phenolic compound content.The foliage has a negative effect on UV radiation within the grape zone [69], so it is clear that the inner parts of the apple canopy are also exposed to less UV light.According to Arakawa et al. [70], the concentration of some polyphenols increases when the fruit is exposed to UV light, as flavonoids can absorb UV radiation and can thus prevent tissue damage.It can be concluded that a reduction in light intensity and UV spectrum probably led to a reduction in the above-mentioned bioactive compounds in apples from the inner part of the apple canopy.

Effect of Different Canopy Positions on Mineral Concentration of 'Braeburn' Apples
According to Hamadziripi [8], the microclimatic differences due to the different canopy positions may influence the accumulation of mineral nutrients in the fruits.Fruit mineral contents have been associated with some fruit quality parameters, such as ripening, storage life, shelf life including internal disorders, and disease severity in a number of fruit species [71].Therefore, fruit's mineral content is of particular interest to fruit growers.In our study (Section 3.4 and Table 5), significant differences were found only for element Ca in both years at the second harvest date, with fruit from the outer canopy having a significantly higher content.Calcium plays an important role in the structure and function of cell walls and membranes, and it is reported that its deficiency has effects on some postharvest physiological disorders, such as bitterness in apple fruit, according to Jemrić et al. [21].There is also a tendency for the K/Ca ratio to be higher in the fruit from the inner part of the canopy, although no significant differences were found in the second year.For all other elements, no clear and constant effect of canopy position was observed.Hamadziripi [8] studied the effect of canopy position on the mineral content of 'Granny Smith apples' and found that the contents of K, Cu, Mn in the flesh, and Ca in the skin were significantly higher in the fruits from the inner canopy than in those from the outer canopy.
Khalid et al. [72] studied the effect of canopy position on the mineral composition of 'Kinnow' mandarins and found that the K content was significantly higher in the inner part of the canopy than in the outer part of the canopy.However, no significant differences were found for the elements Zn, Cu, and in most cases, Fe.According to Cronje et al. [20] the mandarins from the outer canopy had a higher Ca content, while the fruits of the inner canopy had a higher K content.Montanaro et al. [19] reported that light-exposed kiwifruit that had higher transpiration rates accumulated more Ca than shaded fruit.Our results concerning Ca content are in agreement with those of Montanaro et al. and Cronje et al. [19,20], while they contrast with those of Hamadziripi [8].In our study, only fruits from the inner canopy had a higher K content in the first year of first harvest, which is in agreement with Khalid et al. [72] and Cronje et al. [20].No correlation with canopy position was found for the elements Zn, Fe, and Cu, which is in agreement with Khalid et al. [72], although the outer fruits tend to contain more Ca, Mn, Fe, Ni, and Zn and less Rb in the first year and tend to have a lower K/Ca ratio with no significant differences.

PCA Analysis
The PCA (Section 3.5, Table 7 and Figure 1) showed a significant different in the fruits of the second harvest date from the outer canopy position in terms of the chemical composition.These fruits were distinguished from all other fruits by differences in DPPH, TPC, TFC, Rb, Sr, Ca, and K/Ca (separated by PC1), and there were significant differences between these fruits in each year in terms of Mn, Fe, Ni, Cu, and Zn (separated by PC2).PC3 explained a relatively small proportion of the variability (10.81%) and could be considered less important in explaining differences in chemical composition.Differences in fruit quality parameters in to canopy position have already been mentioned in numerous research papers discussed in this paper [4,7,8,72].

Conclusions
There is a need to better understand the effects of the position of the fruit within the canopy on the physico-chemical parameters of apples, as light plays a crucial role in physiological changes in the fruit cuticle.An area of interest that needs further fundamental research is the effect of canopy position on the accumulation of phenolic compounds and other nutrients and their evolution during fruit growth and ripening.The accumulated phenolics are the main antioxidants that protect fruits from various environmental stressors before and after harvest, but they are also important from a nutritional point of view.The intensity of fruit's ground and red blush color is light dependent and thus determines the consumer's willingness to buy the fruit or not.To some extent, SSC, TA, and their ratio were influenced by canopy position, and they adversely affect subsequent parchment as they are highly associated with consumer product acceptance.For macro-and micro-elements, canopy position showed less effect, although some elements are important to prevent postharvest physiological diseases.In this respect, fruits from the outside position have more Ca, which could make them more resistant to physiological postharvest diseases.In conclusion, the effects of fruit canopy position on macro-and micro-element contents need further clarification, also with regard to other interactions.

Table 1 .
Effect of different canopy positions on CIE variables for background color of 'Braeburn' apples.

Table 2 .
Effect of different canopy positions on CIE variables for additional (red blush) color of 'Braeburn' apples.

Table 3 .
Effect of different canopy positions on physico-chemical properties of 'Braeburn' apples.All numbers in the first part of the table present average value ± standard deviation, while in the ANOVA part of the table, F value is presented.SSC-soluble solids content, TA-titratable acidity, SSC/TA-soluble solids content/titratable acidity.n.s., * , ** , *** -nonsignificant, or significant at p ≤ 0.05, p ≤ 0.01, or ≤0.001, respectively; significance in results of physico-chemical properties relates to differences between inside and outside position.

Table 4 .
Effect of different canopy positions on bioactive compounds of 'Braeburn' apples.

Table 5 .
ANOVA regarding effect of different canopy positions on mineral contents of 'Braeburn' apples.

Table 6 .
Effect of different canopy positions on mineral contents of 'Braeburn' apples.All numbers in the first part of the table present average value ± standard deviation, while in the ANOVA part of the table, F value is represented.n.s., * , ** , *** -nonsignificant, or significant at p ≤ 0.05, p ≤ 0.01, or ≤0.001, respectively; significance in results of mineral concentration relates to differences between inside and outside position.