Assessing Volatiles in Three Chinese Dwarf Cherry Cultivars during Veraison and Maturation Using Headspace-Solid Phase Microextraction with Gas Chromatography-Mass Spectrometry.

Chinese dwarf cherry is a native shrub in northwest China with a rich and unique fruit aroma. This study aims to determine the changes in volatile profiles during the maturation period, which provides a theoretical basis for the optimal harvest times and the breeding of aroma-rich varieties. The variation in the production of 164 volatile compounds from three Chinese dwarf cherry cultivars, namely, "Jing'ou 1", "Jing'ou 2", and "Jing'ou 3", were investigated by headspace-solid phase microextraction (HS-SPME)-GC-MS. These volatiles mainly constituted alcohols, carbonyls, esters, terpenoids, and hydrocarbons. Their maturation process could be divided into three stages, namely prophase, metaphase, and anaphase. Prophase contained an abundance of hydrocarbons and carbonyls, primarily benzaldehyde being dominant among all volatiles. During metaphase, volatiles remained at a low level of abundance and diversity. Anaphase coincided with full maturation and was associated with esters and terpenoids; in particular, "Jing'ou 3" presented more compound diversity and a high level of acetate esters. The periods including the week prior to veraison and the week during maturation were particularly critical in volatile formation in Chinese dwarf cherries. This study reveals that the low level or lack of hexanal might be one of the distinctive characteristics separating Chinese dwarf cherries from other Cerasus or Rosaceae fruits.

Introduction

The Chinese dwarf cherry [Cerasus humilis (Bge.) Sok.] is still a mostly wild species that is endemic to China. It is a perennial deciduous shrub that is particularly valued for its possession of both edible and medicinal characteristics. Chinese dwarf cherries are mainly distributed in North China, as they are particularly suited to the drought-prone, cold environment of the region. Their extensive root systems enable them to survive in dry and barren conditions, and this feature has resulted in them being investigated for their use in windbreaks and sand stabilization.[1] Their fruits contain a relatively high content of calcium (524 mg/kg), which is why they are also referred to as “calcium fruit”.[2] Furthermore, they are rich in bioactive secondary metabolites, such as polyphenols, procyanidins, and various vitamins and amino acids,[3,4] thereby indicating promise as novel nutraceuticals and dietary supplements.[5] Chinese dwarf cherry kernels, known as “Yuliren”, have been used for over 2000 years[6] as Traditional Chinese Medicine (TCM) for the treatment of constipation, edema, and dyspepsia.

Chinese dwarf cherries have a distinctive rich, unique flavor. A total of 85 volatiles were previously identified in the ripe fruit of 30 different germplasms, mainly esters and terpenoids.[7] However, the volatile composition and content of unripe fruits is unknown, and furthermore, the chemical profile changes of these volatile compounds during the maturation process remain to be elucidated.

Changes in volatile composition and content are a key factor in fruit quality control and have thus been investigated in many different types of fruits. There were 32 volatile compounds detected during the strawberry maturation process. The major volatiles in unripe strawberries were C6 terpenoids and alcohols, while furanones, esters, and lactones dominated in the fully mature stages.[8] A total of 56 volatile compounds were detected during the apricot maturation process. The major volatiles in the mature green stage of the “Golden sun” apricot fruit were alcohols, while the ester content was much higher than the other compounds in the commercial-ripe stage and full-ripe stage.[9] A total of 37 volatile compounds were detected during the “Red lantern” cherry maturation process. Aldehydes were the predominant volatiles in “Red lantern” cherries, and their content continuously increased during the mature green stage to veraison, with the C6 and aromatic aldehydes reaching their highest content during that time. However, their content gradually declined after veraison to the full-ripe stage. On the contrary, the alcohol content in “Hongdeng” cherries increased as the maturation process progressed.[10] There were 51 volatile compounds detected during the grape maturation process, with C6 compounds, mostly alcohols and carbonyls, exhibiting a downtrend during the maturation process, while most terpenoids and esters increased with maturation. Veraison is thus a key time point for the formation of volatile compounds in grapes.[11] Volatile formation during Chinese dwarf cherry development, including veraison, is yet to be investigated.

“Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3” are the table fruit cultivars that were selected in previous studies, which began in 1998 and were known as “Chinese dwarf cherries seed selection from wild form to tame type”. In December 2014, the State Forestry Administration cultivar assessment committee in China certified that “Jing’ou 1” and “Jing’ou 2” constitute the only cultivar that can mature in districts with a 100-day frost-free period. “Jing’ou 3” is still under review. These three Chinese dwarf cherry cultivars are rich in polyphenols, minerals, vitamins, γ-aminobutyric acid, nervonic acid, and other components that have significant pharmaceutical value.[3,12,13] Researches have revealed that the sugar-acid ratio, content of calcium, and vitamin C were divergent greatly among the three cultivars.[2−4] Using headspace solid-phase microextraction (HS-SPME) coupled to GC–MS, volatile changes of the three cultivars were determined during the veraison and maturation periods. An understanding of the critical periods of volatile formation in fruits could inform quality control and determine optimal harvest times.

Results and Discussion

Volatile Compound Identification

A total of 164 volatile compounds were identified in the three cultivars of Chinese dwarf cherry during the entire maturation process, including 48 esters, 36 hydrocarbons, 30 terpenoids, 19 carbonyls, 16 alcohols, 3 lactones, and 12 other compounds (for details, refer to Supporting Information Table S1). Among them, only two C6 compounds were found in the samples, including 1-hexanol (10.62 μg/kg FW) and caproic acid (12.91 μg/kg FW), which presented after veraison with small contents, and the results were generally consistent with previous studies in which only a few C6 compounds, 1-hexanol (9.14 μg/kg FW) and hexanal (0.03 μg/kg FW), were detected among 30 different Chinese dwarf cherry germplasms.[7] However, C6 compounds were the dominant volatiles in sweet cherries or other different cherry varieties, and among them, 1-hexanol (2.60–121.01 μg/kg FW), (E)-2-hexen-1-ol (14.64–207.96 μg/kg FW), hexanal (25.75–364.51 μg/kg FW), (E)-2-hexenal (45.53–412.71 μg/kg FW) were the characteristic C6 compounds. This might reflect remarkable breed differentiation among cherry species; moreover, the genetic diversity and the related mechanism at the molecular level should be researched further.[10,14−16]

There were 121, 119, and 123 volatile compounds identified from “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3”, respectively, and 87 of these were shared among the cultivars (for details refer to Supporting Information Table S1). The changes in the total volatile compounds during fruit maturation development in Chinese dwarf cherries are shown in Table .

Table 1

Changes in Total Volatile Compound Numbers and Contents (μg/kg Fresh Weight, FW) in “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3” Fruits during Veraison and Maturationa

	Jing’ou 1		Jing’ou 2		Jing’ou 3
weeks after veraison	volatiles numbers	volatile contents (μg/kg FW)	volatile numbers	volatile contents (μg/kg FW)	volatile numbers	volatile contents (μg/kg FW)
–2	36	3034.79 ± 384.47a	34	3148.10 ± 234.68a	37	2700.83 ± 159.57b
–1	38	2330.41 ± 105.68b	34	2508.76 ± 338.53b	39	2583.37 ± 69.8b
0	33	1379.10 ± 18.88c	32	2439.68 ± 174.05b	32	2639.80 ± 111.48b
1	22	1070.66 ± 143.77c	22	1135.80 ± 160.57c	20	1493.41 ± 29.04c
2	26	614.62 ± 15.55d	29	614.75 ± 28.98d	31	576.76 ± 60.55d
3	44	346.32 ± 25.52d	43	582.81 ± 219.21d	36	495.38 ± 54.54d
4	51	2153.31 ± 65.98b	45	1328.35 ± 438.7c	46	3295.54 ± 487.32a

Different superscripts indicate significant differences between the samples for the same cultivar (P < 0.05).

Alcohols

There were 16 alcohols, 9 of which were shared in common among the three cultivars. “Jing’ou 1” had three characteristic compounds, 3-phenyl-1-propanol, 2,4a,5,8a-tetramethyl-1,2,3,4,7,8-hexahydronaphthalen-1-ol, and phytol, while “Jing’ou 2” had two, 5-methyl-1-heptanol and dihydro-β-ionol, and cyclooctanol only existed in “Jing’ou 3”.

The content of alcohols increased slightly in the week prior to veraison according to Figure and then proceeded to decline. It reached the lowest point in the week before ripening and then increased rapidly and peaked during the week of full maturation. Volatile alcohol compounds generally have the aroma of green grass and are considered to be important signs of immaturity in many fruits, such as Annona montana and strawberries.[17,18] However, alcohols were the most abundant aroma compounds among the four sweet cherry cultivars grown in Spain, especially linear forms, followed by aromatic and branched compounds.[19] Among them, (E)-2-hexen-1-ol was a predominantly linear alcohol, with fresh green odor, which was considered to be the main aroma compounds in sweet cherries, while it was not detected in these three cultivars of “Jing’ou” series nor in 30 Chinese dwarf cherry germplasms Ye et al. previously reported.

Figure 1

Changes in the volatile contents during veraison and maturation.

In contrast with sweet cherries, aromatic alcohols were the most abundant volatiles in the volatile alcohol profiles among three “Jing’ou” series cultivars during their dynamic changes in the ripening process. Benzyl alcohol (floral note; Figure ), especially, and phenylethyl alcohol (rose-like note; Figure ), were the dominant aromatic alcohol compounds. Many alcohol contents continuously decreased from veraison to maturation, such as 2-ethylhexanol (Figure ); however, the contents of benzyl alcohol and phenylethyl alcohol increased sharply at maturity. They accounted for a large proportion of the total maturity of alcohols (87.8–95.3%); therefore, an upward trend was presented of the total content of alcohol compounds in the mature period. Benzyl alcohol presented significantly in ripe fruits of “Jing’ou 3”. Besides, 2-ethylhexanol, the key linear alcohol compound among three Chinese dwarf cultivars mainly presented in unripe fruits. In addition, four alcohols were only detected when the fruits were nearly or fully ripe, including 1-hexanol, 3-phenyl-1-propanol, dihydro-β-ionol, and 2,4a,5,8a-tetramethyl-1,2,3,4,7,8-hexahydronaphthalen-1-ol.

Figure 2

Changes in the major volatiles during veraison and maturation.

Carbonyls

Carbonyls include aldehydes and ketones. A total of 19 carbonyls were identified. Of these, there are three characteristic compounds, 4-(2,6,6-trimethylcyclohexa-1,3-dienyl)but-3-en-2-one, 4,8-dimethylnon-7-en-2-one, and (2E,4E)-2,4-octadienal, which were detected only in “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3”, respectively. The carbonyl content declined during fruit ripening in all three cultivars. In the week before veraison, the carbonyl content in the fruits of “Jing’ou 2” and “Jing’ou 3” displayed a slight and temporary rise but still exhibited a downward trend thereafter and was lowest during maturity in all three cultivars.

Among these, benzaldehyde (almond note; Figure ) constituted the main carbonyls, the content of which was highest in the immature fruits, around 2000–3000 μg/kg FW. However, its content continued to decrease with the fruit ripening process. “Jing’ou 1” contained only half benzaldehyde (1077.64 μg/kg FW) than other two cultivars, which led to its total carbonyls be at a significantly lower level during veraison. Benzaldehyde is a characteristic volatile compound in cherry fruits, and it is also added to flavorings and liquid foods to make cherry flavors.[20,21] The benzaldehyde content of 12 sweet cherry (Prunus avium L.) cultivars grown in Turkey was 19.32 μg/kg FW. Starks Gold had the highest content of 50.89 μg/kg FW while the content varied from 4.26 to 20.52 μg/kg FW in other 11 cultivars.[15] In a previous study, the average content of benzaldehyde among thirty different Chinese dwarf cherry germplasms was 27.09 μg/kg FW, with a range between 0.96 and 205.12 μg/kg FW.[7] Carbonyls controlled the total aromas in unripe fruits, and although it decreased as the fruit ripened, it would not completely disappear. Benzaldehyde still presented a relatively high content level (43.51–181.95 μg/kg FW) in three “Jing’ou” series cultivars, compared with other cherry varieties or different dwarf cherry germplasms.

Volatile carbonyl compounds associated with green, grass-like odors, were reported to be negatively correlated with the fruit maturity, and they were abundantly present in unripe fruits, such as Annona montana, strawberries, and Prunus fruits, which were consistent with changes in the dynamic development of total carbonyl compounds among three Chinese dwarf cultivars, while carbonyls were also the most abundant volatiles detected in mature fruits of four sweet cherry cultivars grown in Greece.[17,18,22,23]

Although carbonyls presented significant differences among fruit species and cultivars, hexanal (green grass note) generally was detected as a key carbonyl compound. Interestingly, it was not identified in this study of dynamic changes in volatile profiles among three “Jing’ou” series cultivars. Moreover, only tiny amounts of hexanal (0–0.99 μg/kg FW) were reported in 30 Chinese dwarf cherry germplasms, which provided a further indication that hexanal might present a very low level in Chinese dwarf cherries.[7] This is one of the distinctive characteristics that separates Chinese dwarf cherries from other Cerasus fruits, such as sour or sweet cherries, or other Rosaceae fruits, including peaches, plums, and apricots.[22,24−27] (2E)-2-Decenal was reported to be a potential volatile marker of “Showtime”, a plum cultivar, and it also found a cultivar-dependent relationship among “Jing’ou” series, which was only identified in “Jing’ou 3” at maturity.[22]

Esters

There were 48 esters detected in the three cultivars; 24 of these were shared among the cultivars. Four compounds were detected only in “Jing’ou 1” (isoprenyl acetate, hexyl pivalate, 6-butyloxan-2-one, and linalyl isovalerate), while there was one in “Jing’ou 2” (sec-butyl benzoate) and nine in “Jing’ou 3” (amyl acetate, heptyl dichloroacetate, methyl salicylate, decyl acetate, benzyl propionate, geranyl acetate, geranyl formate, hexyl benzoate, and ethyl behenate). Esters in Chinese dwarf cherries, in contrast with the gradual decline of carbonyls during the ripening process, were lower in the fruits before veraison, increasing significantly in the week prior to ripening and then peaking at maturity.

Esters, associated with sweet and fruit notes contributed to the major aroma of plums, strawberries, and highly aromatic melon varieties at maturity, which especially accounted for 90% of all volatiles in ripe strawberries.[18,28,29] However, it still presented distinctions on species and cultivar levels. For instance, only a low amount esters was present in three Japanese plum cultivars and in sweet cherries grown in Greece and Spain.[19,23,26]

Ester compound development in Chinese dwarf cherries significantly depends on their maturity, which contributed to 33–36% of the total volatiles in mature fruits. Thus, in total, 13 acetate esters were the most abundant, mainly present at maturity, which accounted for 20–28% of the overall volatile profile and 55–78% of total esters, and the most abundant acetate esters were hexyl acetate, benzyl acetate, prenyl acetate, and phenethyl acetate (Figure ). “Jing’ou 3” possessed the highest level of esters among three cultivars at maturity, and hexyl acetate, ethyl benzoate, and benzyl acetate were its characteristic compounds, which were much higher than in the other two cultivars. Prenyl acetate, only detected in “Jing’ou 1” and “Jing’ou 2”, was the highest of the ester compounds in fully matured “Jing’ou 1” (100.19 μg/kg FW), around 16.5% of its total ester content at that time. Great olfactory sensation diversity may be present among different fruit cultivars, depending on the abundance and diversity of volatiles or their interactions. Therefore, specific volatile compounds could be developed as cultivar differentiation markers. In addition, acetate esters also play an important role in other fruit maturation progresses, including plums, grapes, melons, and strawberries.[11,18,28−30] It is worth stating that ester abundance in “Jing’ou 3” was beyond other two “Jing’ou” series cultivars, which coincided with its intense sweet and fruit fragrance in ripening.

Terpenoids

There were 30 terpenoids detected in the three cultivars; 18 of these were shared among the cultivars. Nerol was detected only in “Jing’ou 1”, while there were four characteristic terpenoids detected in “Jing’ou 2” (neral, estragole, perillene, and geranylgeraniol) and four in “Jing’ou 3” (α-ocimene, (3E)-, trans-linalool oxide, 2-butynol, and carvacrol). Similar trends were observed with terpenoids and esters, which both at the initial stage before full maturation was followed by a sharp increase at maturity.

Generally, a small amount of terpenoid compounds was present in fruits; while, it could strongly stimulate and influence the overall olfactory sensation, such as geranylacetone, which was related to the sweet and tropical fruit flavors and geranial, a major component of lemongrass oil.[31] These two compounds were present in all three Chinese dwarf cherry cultivars, while the later was only identified near or at fruit maturity.

The distribution of terpenoids showed a great diversity among cultivars and species. Less than 1% terpenoids among total volatiles were present in sweet cherries grown in Spain; in addition, it was also reported to occur as glycosides in cherries.[14,19] However, terpenoids were dominant over all other volatiles in Annona reticulata fruits, and α-pinene, β-pinene, myrcene, and linalool were the most abundant compounds, which presented custard-like and overall fruity aromas.[17]

Linalool (sweet, plum-like note; Figure ) was one of the major terpenoids of fresh plum (0–34.80 μg/kg FW) according to Chai et al.’s research at different germplasm levels, while 30 Chinese dwarf cherry cultivars presented significantly higher linalool contents (6.16–259.46 μg/kg FW), even though in unripe fruit (30.09–66.96 μg/kg FW, before/at veraison) of three cultivars of the “Jing’ou” series.[7,28] Moreover, linalool was the dominant terpenoid among cultivars at maturity, which reached 32.0–50.0% of total terpenoid content.

Linalool oxides have been reported in ripe plums and peaches, and (E)-linalool oxide was found to be the candidate marker for “Showtime”, a red-purple skin plum cultivar.[22,26] Interestingly, (E)-linalool oxide was also detected in “Jing’ou 3” at full ripening, which might also have the potential to be considered as a volatile marker.

The content of major terpenoids , such as β-pinene (only detected in “Jing’ou 1” and “Jing’ou 3”), linalool, terpineol, geraniol in “Jing’ou 2” during the maturity period was less obvious than in other cultivars (Figure ) and then, so was the total terpenoid content. At maturation, dihydropseudoionone in “Jing’ou 3” increased more than that in “Jing’ou 1” and “Jing’ou 2”. During the maturation process in plums, terpineol (lilacs note) presented a steep decrease; it decreased in veraison and then increased rapidly and peaked during the week of full maturation in Chinese dwarf cherries, where “Jing’ou 1” had higher contents than other two cultivars.[26]

Hydrocarbons

There were 36 hydrocarbons detected, including 18 alkanes, 9 arenes, and 9 alkenes, among which 14 were shared among the three cultivars. The hydrocarbon content remained at a stable and lower level before maturation. The content increased significantly in “Jing’ou 3” during the week preceding full maturity, contributed by (1S,2S)-1,2-dimethylcyclopentane and naphthalene (Figure ); the hydrocarbon components were only detected in “Jing’ou 3”, which reached 65.2% of the total hydrocarbons of “Jing’ou 3” at maturity. Hydrocarbon compounds were reported to be present at a relatively low level during fruit ripening, less than 1% among total volatile profiles in four sweet cherry cultivars.[19]

Lactones

Lactone compounds (sweet and fruit note) were reported to be key compounds that matter the most to peach volatile profiles, which mainly were present in γ- and δ-forms with an even number of carbon atoms from C6 to C12.[32] γ-Decalactone was one of the key aroma compounds in strawberries, although it tended to be cultivar-specific, and it was also the major lactone compound identified in plums, although only a small amount of lactones was present.[18,28]

Three lactones, γ-octanolactone, γ-decalactone, and dihydroactinidiolide (Figure ), were found in three “Jing’ou” series cultivars. The two γ-lactones appeared when the fruits were almost or fully matured, which was consistent with former studies, and γ-octanolactone was only detected in “Jing’ou 1”.[7] However, the other four previously reported lactones, such as undecane-4-olide, 6-pentylpyran-2-one, 5-decanolide, and γ-nonanolactone, were not detected among the three “Jing’ou” series cultivars.[7] On the other hand, the total lactone content of all three cultivars was only 1/13 of that was detected in the previous 30 Chinese dwarf cherry germplasm. Dihydroactinidiolide was first reported to be present in Chinese dwarf cherries, appearing before veraison of all three cultivars, and it was detected again when “Jing’ou 2” and “Jing’ou 3” fully matured. The lactone compound discrepancy among “Jing’ou” series cultivars and 30 other germplasms might be one of the signs of Chinese dwarf cherry breed differentiations.

Other Compounds

A total of 12 other compounds were detected, including five phenols, five acids, and two oxygenated hydrocarbons. Among these, 2-methylbutanoic acid and benzoic acid (Figure ) had relative high contents before veraison in all three cultivars. Benzoic acid is one of the key precursors of odorous compounds such as eugenol and benzoate esters, and it was also present in cherries.[14]

Principal Component Analysis

Principal component analysis (PCA) was used to examine the patterns in 164 volatiles detected in the three cultivars. The cumulative contribution of the variances of the first two PCs was 98.82%. PC1 accounted for 95.60% of the total variance, while PC2 accounted for 3.22% (Figure A). The biplot of the first two PCs presented the distinctions among the different samples (Figure A), and the corresponding loading plot (Figure B) established the relative importance of each volatile component. Based on this, the “Jing’ou” series cultivars could be divided into three groups (Figure A).

Figure 3

PCA of volatile compounds in “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3” during veraison and maturation. (A) Score scatter plot and (B) loading plot. Notes: J1, J2, and J3 in panel (A) represent the cultivars “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3”, respectively. The symbols, “+” and “–” indicate the week before and after veraison, respectively, and the number corresponds to the weeks. In panel (B), the numbers correspond to the volatile code in Table S1.

Group I: the first group located at the bottom of the second quadrant (J1–2, J1–1, J2–2, J2–1, J2–0, J3–2, J3–1, J3–0) corresponded to the prophase of the maturation process (−2 to 0 weeks after veraison). According to the corresponding loading plot (Figure B), hydrocarbons, carbonyls, and a few alcohols were the main types of volatiles detected at this stage. Characteristic compounds included a relatively high content of 1-octen-3-ol (2), 2-ethylhexanol (4), benzyl alcohol (8), benzaldehyde (21), ethyl benzoate (49), hotrienol (133), 2-methylbutanoic acid (153), and benzoic acid (157).

Group II: the samples in this group were concentrated in the left corner of the forth quadrant (J1–0, J1+1, J1+2, J1+3, J2+1, J2+2, J2+3, J3+1, J3+2, J3+3) and corresponded to the metaphase of the maturation process (1–3 weeks after veraison). Hydrocarbons and esters were the primary types of volatiles observed. The characteristic components of this group were nonanal (25), 1,5-di(methoxycarbonyloxy)pentane (52), 1-ethyl-3-methylcyclopentane (86), and β-ocimene, (3Z)-(127).

Group III: samples in this group were widely spread in the first quadrant (J1+4, J2+4, and J3+4) and corresponded to the anaphase (4 weeks after veraison) period of maturation. Figure A indicated that there were differences in volatile composition among the three cultivars at maturity. Furthermore, group III could be divided into two subgroups, group III (A) and group III (B).

Group III (A): this group included “Jing’ou 1” and “Jing’ou 2” at the fully ripe stage. Esters were the main type of compounds observed. Characteristic compounds included a relatively high content of phenylethyl alcohol (10), prenyl acetate (51), phenethyl acetate (60), linalool (131), and geraniol (142).

Group III (B): the types of aroma compounds in “Jing’ou 3” were much more abundant than group III (A). Terpenoids played quite a part of role in “Jing’ou 3” at maturity, so did some esters, especially acetate esters and hydrocarbons and alcohols. The characteristic compounds of this group were isoamyl acetate (36), hexyl acetate (41), benzyl acetate (50), decyl acetate (54), geranyl acetate (66), (1S,2S)-1,2-dimethylcyclopentane (90), naphthalene (98), limonene, (+)-(125), α-ocimene, (3e)-(128), linalool oxide, trans-(130), and geranic acid (148).

Combination of both Figures and 3 indicated that the three Chinese dwarf cherry cultivars basically had similar volatile changing trend during veraison and maturation. Carbonyls dominated at the very beginning of prophase (2 weeks before veraison) and then decreased sharply during prophase and metaphase of the maturation process, while alcohols, esters, and terpenoids accumulated. All three cultivars had their highest alcohols, esters terpenoids, and hydrocarbons at maturity. However, it is worth noting that J1–0 (“Jing’ou 1” at veraison) and J3–4 (“Jing’ou 3” at maturity) are the two exceptions in Figure A. According to the grouping above, unlike other two cultivars (J2–0 and J3–0), J1–0 was at Group II, the metaphase. Combined with Table and Figure , volatile compounds of “Jing’ou 1” before veraison were basically the lowest compared with other two cultivars, and carbonyls in “Jing’ou 1”, the dominant compound before veraison, were also the lowest among the “Jing’ou” series cultivars. Likewise, according to Figure B, J3–4 does not group closely with the other two. Volatile types of “Jing’ou 3” had much more diversity, such as terpenoids, esters, hydrocarbons, and alcohols, while esters were dominant in the other two cultivars, which contributed to the differences at maturity and coincided with the intense and complex fragrance of “Jing’ou 3” fruits in ripening.

Volatile compounds could be both the direct products of a particular metabolic pathway and the result of interactions among various pathways or different end products.[23] The fruit aroma can be quantified and intuitively evaluated according to the chemical profiles of volatile compounds, which could help the breeding of varieties with different fragrances. A high proportion of terpenoid compounds could present more fresh and light fragrance, while mature fruits with a relatively larger proportion of esters have more intense and stronger aroma. However, not all volatile compounds promote the overall aroma at the same level. High concentrations of esters, carbonyls, and alcohols, admittedly, show strong influences on fragrances, while the low level of volatiles in fruits also needs to be considered, particularly, some terpenoids and hydrocarbons of low sensory thresholds.[23,33]

Conclusions

A dynamic volatile compound evaluation of three Chinese dwarf cherry cultivars with different flavors during their maturation process was conducted, and a total of 164 volatile compounds were detected. In the early stage of the ripening process, the immature fruit contained an abundance of hydrocarbons and carbonyls, primarily benzaldehyde. Then, ester and terpenoid compounds were dominant along with maturation progression. This study showed that the week prior to veraison and the week of full maturity were considered as key points in volatile formation in Chinese dwarf cherry fruit, during which the volatiles changed rapidly. Therefore, the characteristic aroma components in these two periods could be used to effectively monitor the maturation degree of the Chinese dwarf cherry fruits, and it also helps local orchards to reasonably predict the harvest time. Moreover, there are some differences in the content and quantity of the aromatic substances among the different cultivars. These differences became more obvious at maturation, which provides a theoretical basis for the breeding of aroma-rich varieties.

Materials and Methods

Materials

Fruits from the third fruiting season of the three Chinese dwarf cherry cultivars, “Jing’ou 1”, “Jing’ou 2”, and “Jing’ou 3”, collected from Zhenglan Banner in Inner Mongolia, China, were used. Located in Hunshadake Sandy Land, the region is featured by its drought, sandy, and the greater diurnal temperature-range environment (Table ). The entire orchard was managed under the same field management practices. According to classical parameters, Chinese dwarf cherries become fully colored at 3 weeks after veraison and then ripen over the next week, during which the color deepens and the pulp softens. Healthy fruits were collected once a week for 7 weeks, starting 2 weeks before veraison (10th August) and continuing until the full maturation stage (21st September) in 2015. For each sample, 200 g of fruits was picked randomly from three trees as one replicate, and three replicates were tested for each sample. All samples were stored at −40 °C before analysis.

Table 2

Local Weekly Meteorological Data during Veraison and Maturation (2015)

time	weeks after veraison	weekly sunshine duration (h)	weekly accumulated temperature (≥10 °C)	weekly precipitation (mm)
4th Aug–10th Aug	–2	79	622	0.00
11th Aug–17th Aug	–1	87	731	3.20
18th Aug–24th Aug	0	46	316	8.80
25th Aug–31st Aug	1	50	339	6.00
1st Sep–7th Sep	2	45	295	39.60
8th Sep–14th Sep	3	48	164	9.20
15th Sep–21st Sep	4	69	275	29.80

HS-SPME Analysis

Before extraction, the sampling fruits were defrosted at 5 °C, while stalks and cores were discarded. The flesh puree was collected after grinding with liquid nitrogen. For each analysis, 3 g of puree and 10 μL of the internal standard (40.5 mg/L 3-octanol ethanolic solution) were transferred into a 20 mL capped vial. Referring to and improving upon the method of Ye et al.,[7] the total amount of extracted volatiles was used as an index to examine the extraction parameters, from which the optimum extraction time (35 min), extraction temperature (55 °C), and NaCl concentration (0.204 mg/mL) were determined. With a split-less mode, the fiber was withdrawn and introduced into an injection port of GC for desorption at 250 °C for 4 min.[7]

GC–MS Analysis

The analysis was performed using Agilent 7890 A GC with an HP-5MS capillary column (0.25 mm × 30 m × 0.25 μm) and a quadrupole mass spectrometer Agilent 5975 C (Agilent, Santa Clara, CA, USA) as the detector. The temperature conditions of the column were referenced by Yang et al.[34] The MS detection proceeded in electron impact mode, and the ionization energy was 70 eV. The mass-scan range was 29–350 m/z at 2.88 scans/s. Helium was the carrier gas at 1 mL/min.

Volatile compound identifications were carried out by matching standard compounds and mass spectral libraries (Wiley6 and NIST 98). A total of 17 standard compounds, including 1-hexanol, 1-octen-3-ol, 2-ethylhexanol, benzaldehyde, benzoic acid, benzyl acetate, benzyl alcohol, ethyl benzoate, geraniol, hexyl acetate, linalool, nonanal, phenethyl acetate, phenylethyl alcohol, prenyl acetate, terpineol, and β-pinene, and the internal standard 3-octanol was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). The retention index values were derived from alkane series (C7–C30) (Sigma, St. Louis, MO, USA). All volatile compounds were quantified as 3-octanol equivalents, and the results were expressed in microgram standard equivalents per kilogram fresh weight (μg/kg FW).

Statistical Analysis

Three parallel repeated tests were performed. Volatile compound differences among seven sampling dates for each cultivar were compared by one-way ANOVA at a significance level of P < 0.05. PCA was used to evaluate the associations between volatile production and sampling time. A covariance matrix was used. SPSS version 22 for Windows (IBM Corp., Armonk, NY, US) and GraphPad Prism version 6.01 for Windows (Graphpad Software, San Diego, CA, US) were used for the statistical analyses.