The vineyard used for the experiment is situated in the Austrian federal state of Burgenland, specifically in the Mittelburgenland region, which is internationally known as “Blaufränkischland”. The selected vineyard site is called “Altsatz” and is recognised for its exceptional quality in the winemaking village of Lutzmannsburg. The vineyard, located at 47°27′02″ N, 16°38′45″ E at an elevation of 261 m a.s.l., is characterised by a brown loam soil derived from calcareous fine sediments of Tertiary loess material. It was planted in 2003 with the Blaufränkisch variety (clone: IBY selection; rootstock: Kober 5BB) and was in its 20th year after planting at the time of the sampling. The grapevines were planted at a spacing of 0.8 m, with row spacing of approximately 2.65 m. The total area of the vineyard is 0.24 ha. The vines were trained in a high culture system with standard commercial practices. No leaf removal was applied in the fruit zone, and no grape thinning was carried out, allowing the grapes to develop under natural crop load and shading profiles. Additionally, a conventional plant protection and an alternating soil management system were implemented. The vineyard has a north-south orientation, enabling separate sampling of grapes exposed to morning (east) and afternoon (west) sun.

Sampling was performed from pea size to overripeness. The experimental setup is schematically illustrated in Figure 1. Initially, weekly; later, biweekly intervals were used. To avoid a thinning effect, previously sampled vines were marked and not resampled. Sampling was restricted to healthy Blaufränkisch vines without symptoms of trunk diseases ( e.g., ESCA, Bois noir). Border rows and the first vine section were excluded, and only phenologically representative clusters were selected. To maintain proportionality, basal and distal grapes were sampled in a 1:1 ratio. As indicated earlier, samples were taken separately from the east and west sides. On each sampling date, six clusters from the east and six from the west canopy side were collected and pooled to obtain one representative composite sample per canopy side. Samples were then flash-frozen with solid CO ₂(s) at –70 °C (FA Linde, Vienna, Austria), vacuum-sealed, and stored at –26 °C until chromatographic analysis. The rapid freezing caused pedicels to become brittle, facilitating berry separation and thus improving sample representativeness.

Canopy temperatures were recorded throughout the experiment with iButton loggers (model MF1921G/H/Z, Moritz Fuchs, Weingarten, Germany) placed at three vineyard positions. Details regarding exposure to morning and afternoon sun are presented in Table 1. The supplementary data in Figure S1 show the measured average temperatures.

Precautions were taken to minimise artefact formation and carotenoid degradation due to isomerisation and oxidation (Oliveira et al., 2003; Oliveira et al., 2004; Oliveira et al., 2006). All extraction steps were conducted under red light in a nitrogen atmosphere; freeze-drying was avoided (Lashbrooke et al., 2010). Frozen grapes were de-stemmed, embrittled with liquid nitrogen (two cycles), and supplemented with 20 µL BHA solution (120 mg/10 mL ethanol) prior to pulverisation in a laboratory mill (IKA-Werke, Staufen, Germany). Approximately 5 g of berry powder was weighed into 25 mL Eppendorf tubes, and 50 µL of the internal standard trans-β-apo-8′-carotenal (313 µg/mL in ethanol) was added. Extraction was carried out with 5 mL dichloromethane containing 0.1 % BHT. Samples were vortexed for 1 min, shaken for 30 min, and centrifuged (5000 rpm, 5 min, 4 °C). The extraction was repeated twice, and the combined organic phases were evaporated to dryness under nitrogen. Extracts were stored at −24 °C and reconstituted in 300 µL tBME/MeOH (90:10; v/ v) and filtered through a 0.2 µm PTFE syringe filter (Agilent Technologies, Waldbronn, Germany) into amber vials prior to analysis by UHPLC-DAD or LC-APCI-MS/MS ⁿ. All samples were extracted in triplicate.

For identification, samples were analysed on an Agilent 1100/1200 series (Waldbronn, Germany) HPLC system, consisted of a binary pump (G1312A), autosampler (G1329A), column oven (G1316A), and diode array detector (G1315B), and was coupled with an ion-trap mass spectrometer (HCT Ultra ETD II, Bruker Daltonics, Bremen, Germany) using an APCI ionization source. The column temperature was set to 22 °C. The mobile phases consisted of methanol/water (85:15, v/ v; eluent A) and methanol/tBME (10:90, v/ v; eluent B), using a gradient program as follows: 0 min, 2.0 % B; 1.8 min 14.5 % B; 2 min, 14.5 % B; 12 min, 21 % B; 12.5 min, 21 % B; 17 min, 25 % B; 32 min, 69 % B; 33 min 95 % B; 36 min 95 % B; 37 min 2 % and isocratic at 2 % B for 5.0 min. The flow rate was 0.25 mL/min, and the injection volume was 5 µL. The diode array detector was operated in an acquisition range of λ 200–700 nm. The HPLC-MS runs were additionally monitored at λ 450 nm. The APCI source was operated in positive mode using nitrogen as nebuliser gas at a pressure of 45 psi, with a vaporiser temperature of 350 °C. The scan range was set between m/ z 125 and 1250 using the “ultra-scan” mode with a mass scanning range of 26,000 m/ z per second. Ionisation voltage at high-voltage (HV) capillary −3500 V, HV end plate offset −500 V, trap drive 64.0, octopole radiofrequency (RF) amplitude 187.1 Vpp, lens 2 −60.0 V, capillary exit −115.0 V, target mass 500 m/z, max. accumulation time 200 ms, ion charge control (ICC) target 100,000 and average of four spectra. Data were processed and evaluated with Data Analysis 4.0 software (Bruker Daltonics, Bremen, Germany).

Approximately 5 g of berry powder, from the same extraction batch as used for carotenoid analysis (Section 3.1), was weighed into a centrifuge tube. Subsequently, 5 mL of methanol/water (80:20, v/v) was added, and the mixture was shaken for 1 min. Extraction was carried out for 4 h on the laboratory shaker, followed by centrifugation for 5 min (5000 rpm, 4 °C). An aliquot of 4 mL of the supernatant was collected and stored at –20 °C until analysis by HS-SPME-GC-MS/MS.

For acid hydrolysis, 100 µL of the methanolic extract was mixed with 4.9 mL of McIlvaine buffer (pH 3.2) and 2 g of sodium chloride. Hydrolysis was carried out at 100 °C for 4 hours in a drying oven. Upon completion of the hydrolysis, the samples were rapidly cooled to –24 °C. Prior to analysis, deuterated C ₁₃-norisoprenoids were added as internal standards.

Post-hydrolysis (total) norisoprenoids in grape samples were analysed by headspace solid-phase microextraction coupled to gas chromatography–tandem mass spectrometry (HS-SPME-GC-MS/MS) using a Thermo Trace 1300 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a TSQ Duo triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), following the method described by Scharf et al. (2026), with modifications regarding the SPME fibre type and extraction conditions to account for differences in sample matrix compared to the original method. HS-SPME extraction was performed using a divinylbenzene–carboxen–polydimethylsiloxane (DVB–CAR–PDMS) fibre (50/30 µm coating thickness; 2 cm length; Supelco, Bellefonte, PA, USA). Samples were pre-incubated for 6 min at 40 °C. Adsorption lasted 45 min at the same temperature. Then desorption took place in the injector in splitless mode for 2 min at 200 °C. The fibre was then reconditioned for 2 min at 260 °C.

Quantification was performed using an internal standard-based assay as described by Scharf et al. (2026). Briefly, TDN, vitispiranes, β-damascenone, and β-ionone were used as representative standards, and their isotopically labelled analogues (d6-TDN, d5-vitispiranes, d4-β-damascenone, and d3-β-ionone) served as internal standards. Calibration curves were established for each of the 4 standard compounds. For norisoprenoids lacking authentic reference standards, concentrations were estimated using the internal standard and the calibration curve of the chemically most similar reference compound. All analyses were carried out as triplicates. Instrument control, data acquisition, and data analysis were carried out using Xcalibur (version 3.0.63).

Compounds present as isomers (actinidols and vitispiranes) were quantitated as one and referred to as one.

(all- E)-lutein (peak 14) and (all- E)-zeaxanthin (peak 15) are the quantitatively most important xanthophylls in grapes across a wide range of red and white cultivars (Guedes de Pinho et al., 2001; Oliveira et al., 2003; Oliveira et al., 2004; Mendes-Pinto et al., 2004; Lashbrooke et al., 2010; Kwasniewski et al., 2010; Kamffer et al., 2010; Bunea et al., 2012). These xanthophylls were also detected in Blaufränkisch grapes. Lutein is an asymmetric dihydroxyl xanthophyll with one β- and one ε-ring. Consequently, it will undergo retro-Diels−Alder (RDA) fragmentation of the ε-ring to produce [M+H−56] ⁺ or eliminate the ε-ring to present [M+H−122] ⁺, which can be used to distinguish lutein from zeaxanthin, in addition to their slightly distinct UV–vis absorption spectra. Cis-isomers of lutein were observed, supported by the small hypsochromic shift (λ 4–6 nm) and the presence of a characteristic cis-peak in the near-UV region. Based on retention times and spectral features, the 9 Z- and 9′ Z-lutein isomers were tentatively assigned ( Aman et al., 2005).

Neoxanthin isomers were detected, with (all- E)-neoxanthin identified based on λ _max values (398, 422, 450 nm) and MS values [M+H] 601.4 (Crupi et al., 2010b; Rivera et al., 2014). Both neochrome a and b were also tentatively identified using absorption maxima and mass spectrometric data ( Kamffer et al., 2010; Mendes-Pinto et al., 2004). Luteoxanthin, auroxanthin, lutein-5,6-epoxide, flavoxanthin, and violaxanthin were tentatively assigned based on characteristic λ _max and retention times, as reported in previous studies (Crupi et al., 2010a; Fariña et al., 2010; Guedes de Pinho et al., 2001; Lashbrooke et al., 2010; Kamffer et al., 2010). Other minor xanthophylls such as antheraxanthin, cryptoxanthin, chrysanthemaxanthin, and echinenone were not detected (Guedes de Pinho et al., 2001; Lashbrooke et al., 2010; Zepka & Mercadante, 2009).

In contrast to the diverse xanthophyll profile described above, (all -E)-β-carotene (peak 24) was the only carotene detected in quantitatively relevant amounts, consistent with literature reports (Guedes de Pinho et al., 2001; Oliveira et al., 2003; Oliveira et al., 2004; Lashbrooke et al., 2010; Kwasniewski et al., 2010). Two additional minor peaks potentially corresponding to β-carotene isomers were observed, but their concentrations were below the limit of quantification (LOQ) of the UHPLC-DAD method (Kwasniewski et al., 2010; Zepka & Mercadante, 2009).

			λ max [nm] c					APCI(+)MS n
peak a	compound	Rt [min] b	cis	I	II	III	III/II [%]	m/z [M+H] +	m/z fragment
1	neochrome a	6.43	-	402	422	448	102.0	601.4	583.4, 565.4
2	unknown 1	6.58	-	-	-	-	-	-	-
3	(all- E)-neoxanthin	7.17	-	414	438	466	83.9	601.4	583.4, 565.4
4	neochrome b	7.76	-	398	422	448	88.0	601.4	583.4, 565.4
5	luteoxanthin	8.36	-	398	422	448	109.1	-	-
6	unknown 2	8.66	-	400	422	450	84.6	-	-
7	auroxanthin	9.22	-	382	402	426	91.2	-	-
8	lutein-5,6-epoxid	9.51	-	418	440	470	85.7	585.4	567.1, 493.1
9	unknown 3	9.78	-	420	446	474	66.7	585.4	567.1, 549, 493.1
10	unknown 4	10.98	-	400	422	448	70.6	-	-
11	flavoxanthin	11.59	-	402	424	450	87.2	-	-
12	unknown 5	12.56	-	-	426	452	-	-	-
13	unknown 6	12.98	-	404	428	456	57.9	569.4	551.4
14	(all- E)-lutein	13.58	-	422	446	474	69.2	569.4	551.4
15	(all- E)-zeaxanthin	14.14	-	428	452	480	40.3	569.4	551.4
16	(9 Z)-lutein	15.57	332	420	440	468	28.6	569.4	551.4
17	(9´ Z)-lutein	15.97	332	420	440	468	47.4	569.4	551.4
18	unknown 7	19.19	-	420	452	482		-	-
IS	β-Apo-8´-carotenal (IS)	20.77			466			417	-
19	chlorophyll b	22.84		316, 344, 466, 598, 648				907.5	-
20	pheophytin b	27.53		436, 528				885.5	-
21	pheophytin a	28.87		410, 506, 536				871.5	-
22	unknown 8	31.91	-					-	-
23	(13 -Z)-β-carotene	32.10	338	422	448	474		-	-
24	(all -E)-β-carotene	32.31	-	428	454	480		-	-
aPeak number bRetention time on the C 30-column. cIdentification by comparison with UV-vis spectrum of the standard compound. dIdentification by comparison with the MS spectrum of the standard compound ( Rosso & Mercadante, 2007; Crupi et al., 2010; Zepka & Mercadante, 2009; Aman et al., 2005; van Breemen et al., 2012; Kamffer et al., 2010)

The temporal evolution of individual carotenoids and total carotenoid concentrations throughout the nine developmental stages is illustrated in Figure 3, whereas a heatmap representation summarising the relative concentration changes across ripening stages and canopy sides is provided in the Table S1. The evolution of carotenoid levels in Blaufränkisch berries followed a pattern comparable to that described for other Vitis vinifera cultivars. Across all developmental stages, β-carotene and lutein represented the predominant carotenoids, accounting for the major proportion of total carotenoids throughout ripening (Razungles et al., 1996; Kwasniewski et al., 2010; Aubert & Chalot, 2018; Asproudi et al., 2020). Other carotenoids, including zeaxanthin, neoxanthin, and neochromes a/b, contributed to a lesser extent but displayed distinct temporal variations.

In the literature, a clear change in the carotenoid profile has already been shown in berries during the ripening process (Asproudi et al., 2020). Previous findings showed β-carotene and some xanthophylls (neochrome, neoxanthin, flavoxanthin, and lutein) are already abundant before veraison, although their concentrations decreased sharply thereafter (Razungles et al., 1988; Marais et al., 1991; Razungles et al., 1996). Other xanthophylls, including violaxanthin, luteoxanthin, and lutein-5,6-epoxide, accumulated later in the ripening process. This observation also applies to the Blaufränkisch grape variety, with the highest levels of nearly all carotenoids detected at the initial sampling date (Jul 23), which then decreased until maturity and some carotenoids ( e.g., neochrome b) are no longer detectable at ripeness.

For both canopy sides, total carotenoid concentration decreased significantly during ripening, although the magnitude and timing of these changes differed between the two sun-exposed sides. Two-way ANOVA confirmed ripening stage as the dominant factor affecting carotenoid concentrations, with significant effects observed for nearly all quantified compounds ( p < 0.0001; Table 3). In addition, canopy side significantly influenced the concentrations of most major carotenoids, including (all- E)-β-carotene, lutein, zeaxanthin, neoxanthin, flavoxanthin, and lutein-5,6-epoxide, indicating a strong microclimatic effect of cluster exposure within the canopy. Significant interactions of ripening stage and canopy side further demonstrated that carotenoid degradation and accumulation patterns differed between morning- and afternoon-exposed berries throughout ripening, suggesting that canopy microclimate modulates carotenoid accumulation and degradation dynamics in a stage-dependent manner. On the afternoon sun (west) side, the most pronounced decline occurred within two weeks after veraison, likely associated with increased expression of carotenoid cleavage dioxygenases (CCDs) VvCCD1 and VvCCD4 from the time of veraison, as previously reported (Mathieu et al., 2005; Young et al., 2012). In contrast, berries from the morning sun (east) side exhibited only a slight short-term decrease after veraison, followed by a significant increase approximately 4 weeks after veraison. Although carotenoid concentrations generally decline after veraison due to plastid transition processes and enhanced carotenoid cleavage activity (Razungles et al., 1988; Young et al., 2012), delayed declines or temporary increases of individual carotenoids during ripening have also been reported in grape berries under specific environmental conditions (Asproudi et al., 2020). In the present study, this behaviour was predominantly observed in berries from the morning-sun side and coincided with significant ripening time × canopy side interaction effects identified by two-way ANOVA, suggesting that canopy microclimate influenced carotenoid degradation dynamics during ripening. Reduced thermal and photooxidative stress on the morning-sun side may therefore have delayed carotenoid degradation and cleavage processes, resulting in temporarily elevated concentrations during mid-ripening. A further decline was observed as berries approached normal ripeness. Between ripeness (Sep 29) and overripeness (Oct 13), carotenoid concentration remained stable on the west side, whereas a significant decrease was observed on the east side, and finally the lowest concentration was reached.

Considering individual carotenoids, a consistent pattern emerged: most carotenoids peaked prior to veraison and declined thereafter, though at compound-specific rates. β-carotene decreased by 51 % at the morning-sun side and by 63 % at the west side until veraison. Thereafter, concentrations at the east side increased again, while they remained nearly constant on the west side. Initial β-carotene levels (Jul 23) were comparable between both sides (2765 µg/kg FW for the morning-sun and 2357 µg/kg FW for the west side). At maturity (Sep 29), however, the east-side berries contained substantially higher levels (1400 µg/kg FW) than those from the west side (839 µg/kg FW). Lutein showed a similar trend, with a pronounced increase about four weeks after veraison at the east side, whereas concentrations continuously declined on the west side, a pattern that agrees with findings in Vitis vinifera cv. Nebbiolo (Asproudi et al., 2020). Among the xanthophylls, zeaxanthin was initially higher at the morning-sun side (1418 µg/kg FW) than at the west side (838 µg/kg FW), but levels dropped sharply at both locations, with a minor transient rise around veraison. The comparatively high initial zeaxanthin concentrations observed on the morning-sun side may reflect transient microclimatic differences within the canopy. Zeaxanthin is part of the dynamic xanthophyll cycle, which enables rapid photoprotective adjustment to fluctuating irradiance and temperature conditions through reversible interconversion with violaxanthin and antheraxanthin. Under the warm and dry conditions prevailing prior to sampling, short-term differences in light exposure and thermal stress may therefore have contributed to the pronounced positional differences observed for zeaxanthin. At maturity, zeaxanthin contents were reduced by 76 % and 72 % at the east and west sides, respectively, relative to initial values. Neoxanthin exhibited the highest concentrations at the onset of sampling, followed by a temporary increase several weeks after veraison and a continuous decline thereafter. Neochrome b remained nearly constant until veraison but decreased sharply afterwards and was no longer detectable at full ripeness in berries from either side.

Overall, carotenoid concentrations were consistently higher in berries from the east (morning sun) side compared to those from the west (afternoon sun) side. This observation agrees with previous studies in different grape varieties showing that berries exposed to lower light and temperature accumulate higher carotenoid levels (Marais et al., 1991; Oliveira et al., 2004; Asproudi et al., 2020). However, the significant interaction of ripening stage and canopy side observed for most major carotenoids indicates that the effect of canopy exposure was not constant throughout ripening, but dynamically changed during berry development. In particular, berries from the east side showed distinct temporal patterns of carotenoid evolution, including delayed declines and transient increases after veraison, whereas west-exposed berries generally exhibited a more continuous decrease. These findings suggest that reduced irradiance and thermal exposure not only promote carotenoid accumulation, but also modulate carotenoid turnover kinetics and degradation dynamics associated with photooxidative stress and CCD activity. In summary, the ripening-dependent decline in carotenoid concentrations in Blaufränkisch grapes appears to be strongly modulated by the interaction between developmental stage and microclimatic conditions within the canopy, jointly determining the balance between carotenoid biosynthesis, photoprotection, and enzymatic cleavage into aroma-active C ₁₃-norisoprenoid precursors.

In this study, twelve C ₁₃-norisoprenoids were quantified in Blaufränkisch grapes using HS-SPME-GC-MS/MS, including, β-damascenone, β-ionone, TPB (4-(2,3,6-trimethylphenyl)buta-1,3-diene), 4-(2,3,6-trimethylphenyl)-butan-2-one, 3,4-didehydro-β-ionone, edulan, TDN, Riesling acetal, isomeric vitispiranes and actinidols. Additionally, two further compounds (unknown_3 MW_172 and unknown_4 MW_172), previously described in the method development study by Scharf et al. (2026) (see Appendix Table A1 for mass spectral data), were included. These compounds exhibit mass spectral characteristics, suggesting a structural and biogenetic relationship to known carotenoid degradation products and pathways, particularly TDN (Winterhalter & Gök, 2013). It should be noted that norisoprenoids were determined after acid hydrolysis of the extracts, thus representing the total (free and glycosidically bound) fraction rather than the native volatile composition of the grapes. Therefore, the observed trends should be interpreted in terms of relative changes in norisoprenoid potential rather than absolute changes in aroma-active compounds. The temporal evolution of individual C ₁₃-norisoprenoids and total C ₁₃-norisoprenoid concentrations throughout the nine developmental stages is illustrated in Figure 4, whereas a heatmap representation summarising the relative concentration changes across ripening stages and canopy sides is provided in Table S2.

Two-way ANOVA identified ripening stage as the dominant factor influencing the concentrations of all quantified C ₁₃-norisoprenoids ( p < 0.0001; Table 4), confirming a strong developmental regulation of norisoprenoid formation during berry maturation. In contrast, canopy side showed compound-specific effects and significantly influenced only selected norisoprenoids, including β-damascenone, actinidols, and 3,4-didehydro-β-ionone. Significant interactions of ripening stage and canopy side were observed for most compounds, indicating that the temporal evolution of norisoprenoid concentrations differed between morning- and afternoon-exposed berries. These findings suggest that microclimatic conditions within the canopy modulate not only the extent, but also the timing and patterns of norisoprenoid formation during ripening.

The concentration of vitispiranes showed a strong overall increase during ripening, reaching the highest levels in the late and overripe stages. This increase was statistically significant across ripening stages for both MS and AS grapes. In MS grapes, vitispirane concentrations increased from 6.70 µg/kg FW at the first sampling date to the highest value of 22.30 µg/kg FW on Aug 25, after which concentrations remained at a comparably high level until the final sampling date. In AS grapes, concentrations also increased significantly during ripening, although the pattern was less linear, reaching the highest value of 28.99 µg/kg FW at the overripe stage on Oct 13.

Similarly, TDN showed an overall increase during the ripening period, with minor fluctuations in the mid-stages but a pronounced rise towards overripe stages, suggesting enhanced formation during late ripening phases. Significant differences among ripening stages were observed for both MS and AS grapes. In MS grapes, TDN increased from 5.92 µg/kg FW on Jul 23 to the highest value of 18.41 µg/kg FW on Sep 09, followed by slightly lower but still elevated concentrations during the later sampling dates. In AS grapes, TDN showed a more fluctuating pattern, including a temporary decrease on Aug 13, but reached its highest concentration at the final sampling date on Oct 13 with 20.55 µg/kg FW.

The concentration of β-damascenone also increased, though less linearly; it rose during the mid-ripening phase, followed by a slight decline, and then increased again at the overripe stage. Significant changes during ripening were observed for both MS and AS grapes. In MS grapes, β-damascenone increased from 8.12 µg/kg FW at the first sampling date to the highest value of 15.34 µg/kg FW on Aug 25, followed by lower concentrations during subsequent ripening stages. In AS grapes, the highest value was likewise observed on Aug 25, with 13.02 µg/kg FW, whereas later stages remained at intermediate levels. Compared with vitispiranes and TDN, β-damascenone therefore showed an earlier maximum and a less pronounced late-ripening increase, indicating a compound-specific accumulation pattern.

In contrast, the concentration of β-ionone remained relatively low throughout ripening and generally decreased, indicating a less pronounced tendency for accumulation, a trend that has also been reported in other grape cultivars (Asproudi et al., 2018). Significant differences among ripening stages were nevertheless observed for both MS and AS grapes. The highest β-ionone concentrations occurred at the first sampling date, with 2.92 µg/kg FW in MS grapes and 2.30 µg/kg FW in AS grapes. Thereafter, concentrations declined, particularly in the later ripening stages, reaching 0.57 µg/kg FW in MS grapes at the final sampling date. In AS grapes, β-ionone followed a similar decreasing trend during ripening, although a slight increase was observed again at the overripe stage. Its initially higher concentrations are consistent with the role of VvCCD1, a dioxygenase gene induced at early berry development and implicated in zeaxanthin cleavage (Mathieu et al., 2005).

Notably, the accumulation of vitispiranes, TDN and β-damascenone coincided with the increase in berry weight and sugar concentration, whereas β-ionone followed the opposite trend. The observed trends align with findings in Pinot noir, where a similar accumulation dynamic for carotenoids, norisoprenoids, and other volatile compounds was found (Yuan & Qian, 2016). Similarly, Strauss et al. (1987) documented increases in vitispirane, TDN, and β-damascenone during grape ripening in Riesling. Yuan & Qian (2016) further noted that the accumulation of these three norisoprenoids begins approximately five days before veraison. In Blaufränkisch, however, this process appears to commence significantly earlier, with substantial amounts of these norisoprenoids detected as early as 21 days before veraison. This discrepancy may reflect varietal differences or abiotic factors (Gerdes et al., 2002; Oliveira et al., 2003; Oliveira et al., 2004; Joubert et al., 2016). Overall, the statistically significant differences among ripening stages confirm that C ₁₃-norisoprenoid accumulation in Blaufränkisch is strongly ripening-dependent but follows distinct compound-specific accumulation patterns.

The norisoprenoid TPB exhibited a general increasing trend during ripening, peaking at overripeness. This ripening-related increase was statistically significant for both MS and AS grapes. In MS grapes, TPB remained at comparatively low levels from Jul 23 to Aug 13, ranging from 0.27 to 0.43 µg/kg FW, before increasing significantly from Aug 25 onwards and reaching the highest concentrations on Sep 09 and Oct 13. A similar pattern was observed in AS grapes, where TPB remained low until Aug 13 and then increased significantly during the later ripening stages, with the highest concentration observed at the overripe stage on Oct 13. This sensorially potent norisoprenoid, reported in various grape varieties, such as Sémillon, Chardonnay, and Riesling, contributes vegetal and grassy notes (Janusz et al., 2003; Cox et al., 2005). Reported concentrations in wines range from 0.05 to 2.1 µg/L, with a sensory detection threshold of 0.04 µg/L in white wine (Janusz et al., 2003). This compound may contribute to the aroma potential of Blaufränkisch due to its relatively high content at the overripe stage. No data are currently available on the precise ripening dynamics or agronomic factors influencing the concentration of this norisoprenoid. A structurally related ketone, 4-(2,3,6-trimethylphenyl)-butane-2-one, also exhibited an increasing concentration trend, particularly from the mid-ripening phase onwards. Significant differences among ripening stages were observed for both MS and AS grapes, with the highest concentrations occurring during the later stages, particularly on Sep 09 and Oct 13 in MS grapes and on Oct 13 in AS grapes. However, little is known beyond its first identification in Riesling (Winterhalter, 1991).

The formation of 3,4-didehydro-β-ionone increased substantially in samples until mid-ripening, likely driven by increasing concentrations of specific precursor compounds, followed by fluctuations and no consistent further increase towards overripeness. This temporal pattern was statistically significant for both MS and AS grapes. In MS grapes, concentrations increased from 0.11 µg/kg FW at the first sampling date to a maximum of 0.78 µg/kg FW on Aug 25, followed by a decline on Sep 29 and a renewed increase at the overripe stage. In AS grapes, concentrations also increased during ripening, reaching elevated values from Aug 25 onwards and the highest value at the final sampling date on Oct 13. This compound is known to be mechanistically linked to TDN formation but does not occur as a main precursor in grapes. It can be formed via thermal carotenoid degradation or released from glycosidically bound precursors and subsequently rearranges to TDN under acidic conditions (Gueldner & Winterhalter, 1991). In the context of viticulture, no insights are currently available regarding its sensory properties, behaviour during berry ripening, or potential agronomic influencing factors. Similarly limited is the knowledge about the norisoprenoid edulan, whose concentration in this study remained largely constant until the mid-ripening phase and only showed a slight increase at the overripe stage. Significant differences among ripening stages were nevertheless observed for both MS and AS grapes. In MS grapes, edulan reached its highest values from Aug 25 onwards, although concentrations remained very low overall. In AS grapes, the most pronounced increase occurred at the overripe stage, where the highest concentration was observed on Oct 13. Edulan was first identified and described in Vitis vinifera by Strauss et al. (1986).

The concentration of Riesling acetal in Blaufränkisch grapes increases during the early and mid-ripening stages, followed by a slight decline around Sep 09 and a subsequent rise in the overripe stage. Significant differences among ripening stages were observed for both MS and AS grapes. In MS grapes, Riesling acetal increased from 0.01 µg/kg FW at the first sampling date to the highest concentration of 0.15 µg/kg FW on Aug 25, followed by lower but still elevated concentrations during the later ripening stages. In AS grapes, concentrations were generally low during early and mid-ripening, but the highest value was reached at the final sampling date on Oct 13. This norisoprenoid is associated with fruity sensory attributes, and elevated concentrations have been observed in aged Riesling wines (Winterhalter, 1991; Winterhalter & Rouseff, 2002). Riesling acetal has been proposed as a precursor of TDN (Daniel et al., 2009). However, no data are currently available on the behaviour of this compound during berry ripening or on viticultural practices that might influence its concentration.

In the Blaufränkisch grape variety, actinidols exhibit low concentrations during the early ripening stages but show an increase from the mid-ripening phase onwards, reaching their highest levels during later ripening stages. This increase was statistically significant for both MS and AS grapes. In MS grapes, actinidol concentrations were lowest during the first sampling dates and increased markedly from Aug 25 onwards, with the highest concentrations observed on Aug 25, Sep 09, and Oct 13. In AS grapes, concentrations also increased significantly from the mid-ripening phase onwards, with the highest values observed on Aug 25 and Sep 09, followed by slightly lower concentrations during the later stages. Strauss et al. (1986) demonstrated that trans-actinidols can be formed as major products via acid-catalysed degradation of megastigma-4,7-diene-3,6,9-triol, with smaller amounts of TDN being generated under these conditions. Olfactorily, actinidols are described as camphor-like or woody-resinous, similar to the norisoprenoid vitispirane. However, due to their typically very low concentrations, often below the detection threshold, their contribution to wine aroma is considered negligible (Ghiglieno et al., 2023).

The two unidentified compounds (unknown_3 MW_172 and unknown_4 MW_172) exhibited moderate changes over the course of ripening rather than a clear accumulation pattern. Both compounds showed an initial increase towards mid-ripening stages, followed by fluctuations and no consistent further rise towards overripeness. Hydrolysis experiments reported by Scharf et al. (2026) demonstrated that unknown_4 MW_172 exhibits formation trends comparable to TDN. A similar behaviour is observed in Blaufränkisch grapes, where the evolution of its concentration during ripening follows a pattern comparable to that of TDN.

Compound	Ripening stage	Canopy side	Ripening stage × Canopy side
Vitispiranes	˂0.0001	0.4537	˂0.0001
Riesling acetal	˂0.0001	0.3847	0.00060
TDN	˂0.0001	0.54862	˂0.0001
β-damascenone	˂0.0001	0.001	˂0.0001
TPB	˂0.0001	0.08926	0.03514
β-ionone	˂0.0001	0.37053	0.63995
actinidols	˂0.0001	0.00649	0.01127
edulan	˂0.0001	0.99305	0.02037
3,4-didehydro-β-ionone	˂0.0001	0.00313	0.00063
unknown_3 MW_172	˂0.0001	0.99186	˂0.0001
4-(2,3,6-Trimethylphenyl)-butane-2-one	˂0.0001	0.08223	˂0.0001
unknown_4 MW_172	˂0.0001	0.0507	0.50553
p-values obtained from two-way ANOVA evaluating the effects of ripening stage, canopy side, and their interaction on carotenoid concentrations in Blaufränkisch grapes. Significant effects ( p < 0.05) are indicated in bold.

During early ripening, vitispirane concentrations tended to be higher on the afternoon sun (AS) side, whereas during mid- and full ripening, higher concentrations were generally observed on the morning sun (MS) side. Towards overripeness, however, vitispirane concentrations increased markedly on the AS side, reaching 28.99 µg/kg FW compared with 18.79 µg/kg FW on the MS side at the final sampling date. This suggests that the influence of canopy side on vitispirane accumulation was not constant throughout ripening, but shifted during the later ripening phase.

A broadly similar pattern was observed for TDN. At the early sampling dates, TDN concentrations were higher on the AS side, whereas from Aug 13 to Sep 29, MS grapes generally showed higher or comparable concentrations. At overripeness, however, TDN increased considerably on the AS side, reaching 20.55 µg/kg FW compared with 14.48 µg/kg FW on the MS side. These results suggest a partly parallel late-ripening response of vitispiranes and TDN, which is consistent with their interconvertible precursors (Gök, 2015). While Gao et al. (2021) reported higher concentrations of vitispirane, TDN, and TPB on the east-facing (morning sun) side in Chardonnay and Cabernet franc, the present study indicates a shift towards higher vitispiranes and TDN levels on the AS side at overripeness. This discrepancy may be related to cooler late-season conditions (< 20 °C on average) combined with afternoon sun exposure from late September onwards, conditions that have been shown to favour norisoprenoid accumulation (Wang et al., 2020b).

β-damascenone showed a different pattern. Concentrations were slightly higher on the AS side at some early and mid-ripening stages, particularly on Aug 13 and Sep 15, whereas MS grapes showed higher values at Aug 25, Sep 29, and at the final overripe stage. At overripeness, β-damascenone reached 13.39 µg/kg FW on the MS side compared with 10.77 µg/kg FW on the AS side. These observations suggest that β-damascenone did not follow the same late-stage increase on the AS side as observed for vitispiranes and TDN. This is in line with reports of reduced β-damascenone in white wines from grapes exposed to high temperatures and intense sunlight (Marais et al., 1992; Kwasniewski et al., 2010). Similarly, Gao et al. (2021) found no clear differences in β-damascenone concentration between grapes sampled from different vineyard positions. A recent study by Ghiglieno et al. (2023) also confirmed that practices such as shading or defoliation did not result in consistent differences in β-damascenone concentration. In the present study, the higher β-damascenone levels in overripe MS grapes may therefore reflect a compound-specific response to the interaction between canopy side, ripening stage, and late-season temperature conditions.

β-ionone concentrations were generally lower than those of β-damascenone, and canopy side differences were comparatively small throughout most of ripening. During early ripening, β-ionone tended to be higher on the MS side. In the later stages, however, concentrations remained low on both canopy sides, with a slight increase on the AS side at overripeness. At the final sampling date, β-ionone reached 1.09 µg/kg FW on the AS side compared with 0.57 µg/kg FW on the MS side. Among the investigated C ₁₃-norisoprenoids, β-ionone was the only compound that did not exhibit a continuous increase during berry development. Instead, the highest concentrations were observed during the early ripening stages, followed by a gradual decline towards overripeness. Since β-ionone is formed through the oxidative cleavage of β-carotene by carotenoid cleavage dioxygenases (CCDs), its comparatively high concentrations during early ripening may reflect enhanced β-carotene degradation at this stage. The subsequent decline in β-ionone concentration suggests that its accumulation is not solely governed by precursor degradation. Instead, β-ionone may undergo further metabolic transformation, volatilisation, or reduced net formation during ripening due to changes in enzymatic activity. However, the slightly higher β-ionone concentrations observed on the AS side at overripeness may indicate that extended sunlight exposure under cooler late-season conditions favoured β-ionone formation or retention. This interpretation is consistent with reports that extended sunlight can enhance β-ionone formation (Song et al., 2015), and with the observation that lower temperatures during full ripening and overripeness may contribute to norisoprenoid accumulation (Wang et al., 2020a). Gao et al. (2021) also documented significant differences in β-ionone concentrations in grapes sampled from various vineyard positions. The combination of lower temperatures and extended sunlight exposure seems to have a decisive influence on the increase in β-ionone concentrations. Since no glycosylated precursors have been identified, β-ionone is present and accumulates exclusively in its free form, and is, therefore, considered an important contributor to the aroma potential of Blaufränkisch (Pedroza et al., 2010).

When considering the overall changes during berry development, carotenoid concentrations were generally higher in berries exposed to morning sunlight than in those exposed to afternoon sunlight. In contrast, the response of C ₁₃-norisoprenoids was more compound- and stage-dependent. While several norisoprenoids showed higher concentrations on the morning sun side during large parts of berry development, vitispiranes, and TDN increased markedly on the afternoon sun side at overripeness. Notably, the pronounced reduction in carotenoid concentrations on the afternoon sun side during late ripening was not immediately accompanied by a corresponding increase in total C ₁₃-norisoprenoids, although specific compounds such as TDN and vitispiranes increased markedly at overripeness. This observation suggests that canopy-side differences in norisoprenoid accumulation cannot be explained solely by precursor availability.