Determination of volatile oil and non-volatile organic compounds contents and ultrastructure changes in the petals of Dendrobium huoshanense upon Zn application
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摘要: 霍山石斛花瓣具有宜人的香气和保健价值, 具有开发功能性食品的潜力。为获得用于功能性食品生产的优质原料, 在大田栽培条件下提高霍山石斛花瓣的食品品质性状越来越受到人们关注。该研究主要关注Zn施用对霍山石斛花瓣中挥发油和非挥发性有机物的影响, 并从花瓣超微结构变化视角探讨其机制。采用气相色谱-质谱联用技术(GC-MS)分析挥发油成分, 化学分析法测定非挥发性有机物(可溶性糖、游离氨基酸和酚类化合物)含量, 透射电镜观察超微结构变化。结果表明, 施Zn提高了霍山石斛花瓣挥发油(主要由萜烯类及其衍生物组成)和非挥发性有机物含量, 不同Zn处理对挥发油和非挥发性有机物生产影响差异明显。挥发油、可溶性糖、自由氨基酸及酚类化合物含量最大增幅分别达28.57%、33.53%、28.89%和58.41%。超微结构变化表明, 施Zn促进花瓣细胞液泡融合, 增加细胞中线粒体、淀粉粒和脂滴的产生。Zn施用能提高霍山石斛花瓣挥发油和非挥发性有机物的含量。 超微结构分析能为挥发油和非挥发性有机物生产提供新的解剖学见解。Abstract: The petals of Dendrobium huoshanense have great potential as functional food because of their pleasant fragrance and health values. To obtain high-quality raw materials for functional food production, more attention is being directed toward improving the food quality traits of D. huoshanense petals under field cultivation conditions. In this study, we investigated the effects of Zn application on the volatile oil and non-volatile organic compounds contents in the petals of D. huoshanense. Furthermore, the mechanism was also studied from the perspective of petal ultrastructural changes. Volatile oil constituents were investigated using gas chromatography-mass spectrometry. The contents of non-volatile organic compounds, including soluble sugar, free amino acids, and phenolic compounds, were investigated using chemical analysis methods. Ultrastructural changes were observed via transmission electron microscopy. The results showed that Zn application increased the yield of volatile oils (primarily terpenes and their derivatives) and non-volatile organic compounds from the petals of D. huoshanense. Different levels of Zn treatments changed the production of the above-mentioned volatiles and non-volatile compounds to varying degrees. The maximum rate of increase in the content of volatile oil, soluble sugars, free amino acids, and phenolic compounds was 28.57%, 33.53%, 28.89%, and 58.41%, respectively. Ultrastructural changes at the cellular level showed that Zn application promoted vacuole fusion and increased the production of mitochondria, starch grains, and lipid droplets. In conclusion, Zn application improves the production of volatile oils and non-volatile organic compounds. Ultrastructural analysis has provided new anatomical insights into the production of volatile oils and non-volatile organic compounds.
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Key words:
- Dendrobium huoshanense /
- Non-volatiles /
- Ultrastructure /
- Volatile oils /
- Zn levels
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Figure 1. Representative samples of petals of Dendrobium hu oshanense treated with different levels of Zn. A (Zn0): 0 mg∙(100 mL)−1 ZnSO4·7H2O; B (Zn 2.9): 2.9 mg∙(100 mL)−1 ZnSO4·7H2O; C (Zn5.8): 5.8 mg∙(100 mL)−1 ZnSO4·7H2O; D (Zn11.6): 11.6 mg∙(100 mL)−1 ZnSO4·7H2O.
Figure 2. Total ion chromatogram of petal volatile oils of Dendrobium huoshanense treated with different levels of Zn application. A: 0 mg∙(100 mL)−1 ZnSO4·7H2O treatment; B: 2.9 mg∙(100 mL)−1 ZnSO4·7H2O treatment; C: 5.8 mg∙(100 mL)−1 ZnSO4·7H2O treatment; D: 11.6 mg∙(100 mL)−1 ZnSO4·7H2O treatment.
Figure 3. Bi-plot (score plot and loading plot) of the PCA of the volatile oil constituents in petals of Dendrobium huoshanense as influenced by different levels of Zn application. Zn0: 0 mg∙(100 mL)−1 ZnSO4·7H2O; Zn2.9: 2.9 mg∙(100 mL)−1 ZnSO4·7H2O; Zn5.8: 5.8 mg∙(100 mL)−1 ZnSO4·7H2O; Zn11.6: 11.6 mg∙(100 mL)−1 ZnSO4·7H2O. No.1–51 represents the corresponding volatile oil components in Table 1.
Figure 4. Transmission electron microscopic images showing the submicroscopic structure characteristic of petal cells of Dendrobium huoshanense treated with different levels of Zn. There is an obvious difference in vacuole morphology (A-D), starch and lipid accumulation (E-H), and the number of mitochondria (I-L) in petal cells among the Zn0-Zn11.6 treatments, respectively. Fig. A, E, and I show the Zn0 treatment [0 mg∙(100 mL)−1 ZnSO4·7H2O]; fig. B, F, and J show Zn2.9 treatment [2.9 mg∙(100 mL)−1 ZnSO4·7H2O]; fig. C, G, and K show Zn5.8 treatment [5.8 mg∙(100 mL)−1 ZnSO4·7H2O]; fig. D, H, and L show Zn11.6 treatment [11.6 mg∙(100 mL)−1 ZnSO4·7H2O]. “CW” cell wall, “CN” cell nucleus, “V” vacuole, “SG” starch grain, “P” plastid, “M” mitochondria, “→” lipid droplet.
Table 1. Constituents of volatile oils from the petals
of Dendrobium huoshanense treated with different levels of Zn Compound code Compound name LRI exp LRI lit Treatment Zn0 Zn2.9 Zn5.8 Zn11.6 No.1 2-hexanol 801 803 0.35 — 0.15 0.56 No.2 Acetic acid, butyl ester 809 812 0.12 0.39 0.35 0.76 No.3 2-methylbutanoic acid ethyl ester 838 846 — 0.15 0.48 0.41 No.4 (Z)-hex-3-en-1-ol 847 851 0.25 0.45 — 0.38 No.5 1-hexanol 863 867 5.20 7.68 7.76 7.59 No.6 Acetic acid, 3-methylbutyl ester 876 876 0.41 0.45 0.53 0.75 No.7 α-pinene 938 939 0.22 0.34 0.15 0.77 No.8 Tiglic acid ethyl ester 947 949 6.12 5.76 5.59 5.47 No.9 1-heptanol 968 970 0.89 1.08 2.56 — No.10 β-pinene 973 974 1.11 0.12 0.21 0.75 No.11 1-octen-3-ol 984 986 0.73 0.34 1.16 1.78 No.12 3-octanone 988 987 5.84 4.65 4.60 2.36 No.13 3-octanol 1001 995 0.19 0.11 0.27 0.14 No.14 1,8-cineol 1012 1015 0.51 — 0.47 — No.15 α-ocimene 1021 1018 0.23 0.35 0.77 0.56 No.16 Limonene 1026 1025 0.21 0.32 0.56 0.64 No.17 β-ocimene 1039 1044 15.90 17.48 17.64 18.68 No.18 β-trans-ocimene 1052 1050 0.31 0.45 0.63 0.56 No.19 (E)-2-octen-1-ol 1066 1067 0.19 0.38 0.42 0.50 No.20 Terpinolene 1082 1088 0.11 0.15 0.31 0.26 No.21 Nonanal 1095 1089 1.21 0.12 0.33 0.55 No.22 α-cyclocitral 1103 1102 — 0.18 0.19 0.32 No.23 Linalool 1107 1104 0.38 0.62 0.59 0.43 No.24 (E,E)-2,6-Dimethyl-2,4,6-octatriene 1139 1143.5 — — 0.35 0.41 No.25 3-nonen-2-one 1142 1136 0.25 0.31 0.6 0.52 No.26 1-ethenyl-4-methoxybenzene 1153 1151.6 0.41 0.34 0.87 0.58 No.27 β-terpineol 1159 1160 0.54 1.21 — 0.31 No.28 1,4-dimethoxybenzene 1169 1165 0.34 0.52 0.77 0.79 No.29 (E)-isopentyl 2-methylbut-2-enoate 1184 1195.8 0.23 0.35 0.46 0.32 No.30 β-cyclocitral 1213 1214 — 0.39 0.28 0.42 No.31 4-(2-propenyl) phenol 1242 1254 — 0.84 0.98 — No.32 Geraniol 1252 1254 1.02 0.89 0.43 0.42 No.33 1,3-dimethoxy-5-methylbenzene 1265 1260 0.12 — 0.45 — No.34 β-geranial 1268 1269 0.65 0.57 1.03 0.98 No.35 Safrole 1282 1287 1.12 1.08 1.24 1.09 No.36 δ-elemene 1320 1324 0.53 0.41 1.08 2.30 No.37 3-methoxy-5-methylphenol 1341 1342 0.54 0.65 — 0.98 No.38 Eugenol 1356 1359 0.21 0.32 0.54 0.67 No.39 β-elemene 1368 1373 0.23 0.32 0.54 0.62 No.40 β-caryophyllene 1423 1417 19.48 20.41 21.36 21.94 No.41 α-ionone 1429 1426 0.18 0.31 0.21 0.03 No.42 α,β-dihydro-β-ionone 1436 1433 3.72 2.02 2.32 2.83 No.43 Geranyl acetone 1461 1455 4.72 2.23 2.79 2.56 No.44 α-farnesene 1511 1507 2.48 4.13 5.05 6.04 No.45 Butylated Hydroxytoluene 1515 1511 0.13 — 1.20 0.19 No.46 δ-cadinene 1537 1541 1.35 0.36 0.39 0.98 No.47 Trans-nerolidol 1549 1565 0.65 0.37 0.21 0.32 No.48 α-Cedrene epoxide 1562 1570 0.42 0.65 0.09 0.55 No.49 Caryophyllene oxide 1613 1593 0.12 0.23 0.51 0.39 No.50 Methyl jasmonate 1639 1655 0.21 0.34 0.17 0.32 No.51 (E,E)-farnesol 1697 1722 3.12 4.32 2.35 2.87 Total identified (%) 83.25 85.14 91.99 93.65 Yield (v/w, %) 0.21 0.22 0.25 0.27 Grouped compounds (%) Terpenes 42.16 44.84 49.04 54.51 Alcohols 14.02 17.45 16.37 15.30 Ketones 14.71 9.52 10.52 8.30 Esters 7.09 7.44 7.58 8.03 Benzenes 2.87 3.75 6.05 4.30 Aldehydes 1.86 1.26 1.83 2.27 Others 0.54 0.88 0.60 0.94 “—” means not detected. LRIexp: experimental linear retention indices; LRIlit: literature linear retention indices (from NIST Standard Reference Database Number 69). Zn0, Zn2.9, Zn5.8 and Zn11.6 are treatments of application of 0, 2.9, 5.8 and 11.6 mg∙(100 mL)−1 ZnSO4·7H2O. 
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Table 2. Soluble sugar contents (mean±SE) in the petals of Dendrobium huoshanense treated with different levels of Zn
mg∙g−1(FW) Soluble sugar Zn level [mg∙(100 mL)−1(ZnSO4·7H2O)] 0 2.9 5.8 11.6 Fructose 2.96±0.13a 3.32±0.13ab 3.66±0.06bc 3.92±0.05c Glucose 1.25±0.11a 1.53±0.07ab 1.75±0.06bc 2.07±0.12c Galactose 2.41±0.12a 2.53±0.11a 2.81±0.05a 2.88±0.26a Sucrose 1.76±0.07a 2.00±0.06ab 2.06±0.06ab 2.32±0.12b Total contents 8.38±0.42a 9.38±0.24ab 10.28±0.14bc 11.19±0.39c Different lowercase letters in the same row indicate significant differences at P<0.05 level.
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Table 3. Free amino acid contents (mean±SE) in the petals of Dendrobium huoshanense treated with different levels of Zn
μg∙g−1(FW) Free amino acid Zn level [mg∙(100 mL)−1(ZnSO4·7H2O)] 0 2.9 5.8 11.6 Glutamic acid 27.43±0.48a 29.23±0.42a 33.75±0.93b 36.47±0.49c Lysine 2.49±0.06a 2.43±0.09a 2.30±0.05a 2.25±0.06a Leucine 1.34±0.04c 1.25±0.01bc 1.17±0.02b 1.07±0.01a Asparagine 2.33±0.03a 2.54±0.04ab 2.84±0.05b 3.05±0.17bc Alanine 13.24±0.39a 15.08±0.46b 17.66±0.17c 18.74±0.28c Valine 12.89±0.14c 12.04±0.13b 11.20±0.09a 10.80±0.11a Proline 18.69±0.71a 19.25±0.41a 22.98±0.32b 23.44±0.31b Threonine 8.01±0.05a 8.37±0.07a 12.94±0.34b 15.67±0.22c Total contents 86.65±0.45a 90.11±0.40b 104.87±0.41c 111.69±0.35d Different lowercase letters in the same row indicate significant differences at P<0.05 level.
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Table 4. Phenolic compound contents (mean±SE) in the petals of Dendrobium huoshanense treated with different levels of Zn g∙kg−1 (FW)
Phenolic compound Zn level [mg∙(100 mL)−1(ZnSO4·7H2O)] 0 2.9 5.8 11.6 Gallic acid 1.09±0.02a 1.28±0.02b 1.56±0.02c 1.67±0.03c Gallocatechin 0.51±0.01a 0.59±0.01b 0.83±0.01c 0.88±0.02c Catechin 1.80±0.01a 2.13±0.03b 2.47±0.02c 2.88±0.03d p-Coumaric acid 1.12±0.01a 1.41±0.02b 1.54±0.01c 1.73±0.03d Total contents 4.52±0.04a 5.41±0.03b 6.40±0.02c 7.16±0.03d Different lowercase letters in the same row indicate significant differences at P<0.05 level.
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