Variations in Essential Oil Compositions and Changes in Oil Cells during Leaf Development of Citral Chemotype of Camphora officinarum Nees ex Wall.

: The citral chemotype of Camphora officinarum Nees ex Wall. is a promising industrial plant that contains an abundance of citral, which is widely used in medical, chemical, food, and other fields. For a more in-depth exploration, the dynamic characteristics of its essential oil (EO), oil compositions (OCs), and oil cells during leaf development were determined in the present study. The leaf phenotype changed rapidly from the 1st to the 4th week after leaf bud germination. The oil yield (OY), obtained via supercritical carbon dioxide extraction, reached the highest value of 2.82% ± 0.20% in the 12th week. Leaf development is a prerequisite for the production of EO, and the difference in the OY was not significant after leaf maturation. The OCs, analyzed using gas chromatography–mass spectrometry (GC-MS), mainly included aldehydes, alcohols, and hydrocarbons. Different types of compounds accumulated differently during leaf development: the highest relative content of alcohol in the OCs was 30.18% in the 2nd week, while that of aldehyde was 76.11% in the 6th week. In total, 130 OCs were detected, and two isomers of citral, namely, geranial and neral, had the highest relative levels of 51.12% (12th week) and 28.63% (6th week), respectively. The OY was closely related to the developmental stage of the oil cells. In the 1st–2nd weeks, the oil cells were mostly in the non-essential oil stage and essential oil-forming stage, with a lower OY; oil cells reached saturation in the 12–24th weeks, with a higher OY. Transmission electron microscopy showed that osmium droplets were present in large quantities during leaf development and gradually integrated into the vacuoles, finally making the vacuoles become oil bladders for oil storage. In conclusion, EO may have new uses due to the different OCs in leaf development; additionally, the microscopic changes in C. officinarum provide a reference for the cellular mechanism of EO accumulation.


Introduction
Camphora officinarum Nees ex Wall., belonging to the Camphora of the Lauraneae family, is the main landscaping and economic species, and it is widely distributed in tropical and subtropical Asia.Chemical polymorphism has been discovered in C. officinarum, resulting in the identification of various types, including linalool, camphor, eucalyptol, isonerol, and borneol types [1].In C. officinarum, the citral type is a newly discovered valuable chemotype, with its leaf EO being rich in citral.Citral has a strong lemon-like flavor and contains two isomers, namely, neral and geranial [2].Citral can be used as a raw material to synthesize lonones, damascenone, isopulegol, and other important chemical compositions.Due to their antioxidant [3] and antibacterial [4] effects, as well as in the treatment of cardiovascular diseases and leukemia [5], citral and its derivatives have been widely used in the fields of healthcare, chemicals, and food.In the past decade, the demand for citral has outrun the supply.As a result, the citral chemotype, C. officinarum, was widely cultivated in Jiangxi, Guangxi, Guangdong, Hunan, Yunnan, and other places in China to alleviate the Three clonal progenies of 3-year-old citral-type C. officinarum were used as the materials for this study.The progenies were cultivated in the asexual propagation garden of NanChang Institute of Technology (latitude: 28 • 41 ′ 47 ′′ N, longitude: 116 • 1 ′ 49 ′′ E), and three healthy samples were randomly selected from each clonal progeny.The leaves sprouting from buds served as the first collection stage, followed by collection stages at 1, 2, 3, 4, 6, 8, 10, 12, and 24 weeks.At each stage, leaves were collected from the east, south, west, and north of the canopy and mixed for the experiment [1].The samples were identified according to the species and chemotype levels by Professor Jin Zhinong.Voucher specimens were deposited in the Botanical Herbarium of Jiangxi Provincial Engineering Research Center for Seed-Breeding and Utilization of Camphor Trees, with the voucher numbers ZS003 (C1 species), ZS032 (C2 species), and NGN01(C3 species).

Determination of Leaf Size
Six pieces were randomly selected from each sample.The leaf shape was determined using a 1241 leaf area meter (Beijing YaXin LiYi Technology Co., Ltd., Beijing, China).

Determination of Moisture Content
The fresh leaves of each sample plant were randomly divided into 3 parts, with 6 pieces for each.The leaves were cut along the middle vein and then cut into small pieces with an area of approximately 1 cm × 1 cm.The moisture content of the leaves was measured with an MA150 rapid moisture meter (Sartorius Stedim Biotech S.A. Co., Ltd., Frankfurt, Germany).

Acquisition of Oil Yield and GC-MS Analysis
The leaf samples were randomly divided into 3 parts, weighing 20 g each.EO was extracted using the supercritical carbon dioxide extraction SFE-2 (Applied Separations Co., Ltd., Allentown, PA, USA), and the detection conditions were consistent with those in the study of Chen TB [12].The EO was weighed using an BSA4202-CW accurate electronic balance (Sartorius Stedim Biotech S.A. Co., Ltd., Frankfurt, Germany), and the samples were stored in the dark at 4 • C. The essential oil samples were separately dissolved in formaldehyde chromatographic pure solution and then centrifuged in a lowtemperature freezing centrifuge, and the supernatants of three C1 samples were mixed in equal volumes.The supernatants were determined on a 7890B-5975C gas chromatography-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with an HP-5MS capillary column (30 m × 250 µm i.d.; film thickness 0.25 µm).The detection conditions were consistent with those in the study of Zhang BH [13].The oil yield (OY) was calculated based on the weight of the EO (W EO ), the weight of the leaf (W L ), and the moisture content (MC), and the formula is shown in Equation ( 1):

Determination of Oil Cell Size
The leaves were cut into small pieces of 5 mm × 5 mm after the main vein was removed, and the pieces were put into 5% NaOH solution and treated in an incubator at 60 • C. The NaOH solution was replaced once after 12 h, and then the pieces were washed with distilled water after 24 h and put in a H 2 O 2 solution for 5~15 min.The treated leaves were observed under a Ni-U+ DS-Ri2 positive microscope (Nikon Co., Ltd., Tokyo, Japan), and the diameter of the oil cells was measured using the NIS-elements D software 4.5.Three leaves were taken from each sample, and six fields were taken from each leaf.The diameters of all oil cells were measured under each field, and the average value was taken as the diameter of the oil cells of the sample.

Microscopic Observation of Oil Cells
The leaves were cut into small pieces of 1 mm × 1 mm after the main vein was removed, and the tissue blocks were put into an electron microscope fixative solution and then stored at room temperature for 2 h and 4 • C for preservation and transportation.The samples were rinsed with 0.1 M PB (pH 7.4) 3 times for 15 min each.The sample processing method was obtained from the study of Liu [14].The cuprum grids were observed under HT7800 transmission electron microscopy (Hitachi Co., Ltd., Tokyo, Japan), and images were taken.

Statistical Analysis
An analysis of the significance of differences was achieved through the SPSS software 22.0; a one-way ANOVA and Duncan's method were utilized for multiple comparisons.

Dynamic Changes in Leaf Size
The leaves of C. officinarum germinated in the middle of March.From the 1st to the 2nd week, the leaves were red and tender (Figure 1).A rapid growth period occurred from the 1st to the 4th week of leaf development (WLD) (Figure 2 and Table 1), with the leaves gradually turning green.The growth rate decreased from the 6th to the 12th WLD, with the leaf size increasing more slowly from the 12th to the 24th weeks.The leaf development was closely related to the growth phases and external environmental conditions of the citral chemotype of C. officinarum.When the leaves developed during the 1st-4th weeks (March to April), the temperature increased significantly, and the leaves grew rapidly.From April to May, C. officinarum entered the flowering stage.To complete reproduction successfully, limited resources were rationally allocated and transferred, and the vegetative growth of the plant changed to reproductive growth.At the same time, the rainy season occurred from April to May, with a decrease in temperature and light, and the leaf size change rate from the 6 to the 12th week was reduced due to the compound effects of its own growth stage and external conditions.During the 12-24th WLDs, the mature leaves tended to be leathery, and the leaf size changed slowly.

Dynamic Accumulation of EO
There was a significant difference in the OY of the leaf development stage (p < 0.05).EO accumulated rapidly during the pre-mature stage of the leaf and reached 2.82% ± 0.20%, 1.41% ± 0.04%, and 1.71% ± 0.04% at 10 weeks for C1, C2, and C3, respectively.The OY increased significantly during the 1st-4th weeks of leaf development, depending on the rapid growth of the leaves (Figure 3 and Table 2).In addition to the factors of leaf structure development and photosynthesis ability, the increase in temperature in March also promoted the accumulation of secondary metabolites.Meanwhile, the rainy season from April to May resulted in insufficient light and reduced the OY.Leaf development was basically mature in the 10th week, and the OY in the 10th and 12th weeks was insignificant; however, C1 was an exception to this rule, possibly due to the fact that the leaves of C1 were smaller than those of the other two clonal progenies, and leaf keratinization was not conducive to EO extraction, resulting in a slight decrease in the OY.
growth stages.

Dynamic Accumulation of EO
There was a significant difference in the OY of the leaf development stage (p < 0.05).EO accumulated rapidly during the pre-mature stage of the leaf and reached 2.82% ± 0.20%, 1.41% ± 0.04%, and 1.71% ± 0.04% at 10 weeks for C1, C2, and C3, respectively.The OY increased significantly during the 1st-4th weeks of leaf development, depending on the rapid growth of the leaves (Figure 3 and Table 2).In addition to the factors of leaf structure development and photosynthesis ability, the increase in temperature in March also promoted the accumulation of secondary metabolites.Meanwhile, the rainy season from April to May resulted in insufficient light and reduced the OY.Leaf development was basically mature in the 10th week, and the OY in the 10th and 12th weeks was insignificant; however, C1 was an exception to this rule, possibly due to the fact that the leaves of C1 were smaller than those of the other two clonal progenies, and leaf keratinization was not conducive to EO extraction, resulting in a slight decrease in the OY.

Dynamic Changes in the OCs of EO
A total of 130 compounds were detected at all the stages of leaf development (Table 3).Eucalyptol was represented by the components present at every developmental stage, and α-pinene was represented by the components detected at only one developmental stage.The OCs were categorized into 14 major groups, namely, alcohols, aromatic hydrocarbons, ethylene oxide, ethers, aldehydes, acids, hydrocarbons, ketones, alkenes, alkynes, olefine, esters, phenols, and amides."-" indicates that the OCs were not detected.
The quantities of various OCs are shown in Figure 4A.The category with the highest number of compounds was alcohols, and the numbers of alcohol compositions were 21,22,22,21,19,17,22,20, and 16 at the 1st-24th weeks of leaf development.The relative contents of various OCs varied with the leaf development as shown in Figure 4B and Table 3.The aldehydes increased and then stabilized with the leaf development, with the relative contents of 20.26%, 20.85%, 12.44%, 48.52%, 76.11%, 66.06%, 65.51%, 73.51%, and 50.19% in the 1st-24th weeks; alcohols had a high relative content in young leaves and tended to decrease with leaf development, with the relative contents of 20.88%, 30.18%, 15.06%, 17.57%, 10.79%,9.47%,14.44%, 10.11%, and 12.78% in the 1st-24th weeks."-" indicates that the OCs were not detected.
The quantities of various OCs are shown in Figure 4A.The category with the highest number of compounds was alcohols, and the numbers of alcohol compositions were 21,22,22,21,19,17,22,20, and 16 at the 1st-24th weeks of leaf development.The relative contents of various OCs varied with the leaf development as shown in Figure 4B and Table 3.The aldehydes increased and then stabilized with the leaf development, with the relative contents of 20.26%, 20.85%, 12.44%, 48.52%, 76.11%, 66.06%, 65.51%, 73.51%, and 50.19% in the 1st-24th weeks; alcohols had a high relative content in young leaves and tended to decrease with leaf development, with the relative contents of 20.88%, 30.18%, 15.06%, 17.57%, 10.79%,9.47%,14.44%, 10.11%, and 12.78% in the 1st-24th weeks.
(A) (B) The chemical compound citral is the most relatively abundant compound in this essential oil, including its isomers geranial and neral (Figure 5).During leaf development, geranial and neral rapidly accumulated from the 3rd to the 6th week.During leaf growth, geranial and nerolidol accumulated rapidly from weeks 3 to 6; however, the two isomers did not follow the same accumulation pattern; the relative content of neral increased first The chemical compound citral is the most relatively abundant compound in this essential oil, including its isomers geranial and neral (Figure 5).During leaf development, geranial and neral rapidly accumulated from the 3rd to the 6th week.During leaf growth, geranial and nerolidol accumulated rapidly from weeks 3 to 6; however, the two isomers did not follow the same accumulation pattern; the relative content of neral increased first and then tended to be stable, and it reached its highest value of 28.63% in the 6th week, while geranial reached its highest value of 51.12% in the 12th week in the leaves.
and then tended to be stable, and it reached its highest value of 28.63% in the 6th week, while geranial reached its highest value of 51.12% in the 12th week in the leaves.

Dynamic Changes in Oil Cells
The dynamic changes in the oil cells during leaf development are shown in Figure 6 and Table 4.In general, the oil cells of the three clones were the largest in the 10th week of leaf development.The growth of the oil cells was rapid in the 1st-10th weeks and was slow in the 10th-24th weeks.The diameter of the oil cells in the C3 leaves was the largest in the 10th week, with a value of 48.73 ± 0.77 µm, while the diameter of the oil cells in the C3 leaves was the smallest in the 1st week, with a value of 27.85 ± 2.05 µm.

Dynamic Changes in Oil Cells
The dynamic changes in the oil cells during leaf development are shown in Figure 6 and Table 4.In general, the oil cells of the three clones were the largest in the 10th week of leaf development.The growth of the oil cells was rapid in the 1st-10th weeks and was slow in the 10th-24th weeks.The diameter of the oil cells in the C3 leaves was the largest in the 10th week, with a value of 48.73 ± 0.77 µm, while the diameter of the oil cells in the C 3 leaves was the smallest in the 1st week, with a value of 27.85 ± 2.05 µm.
Horticulturae 2024, 10, x FOR PEER REVIEW 10 of 16 and then tended to be stable, and it reached its highest value of 28.63% in the 6th week, while geranial reached its highest value of 51.12% in the 12th week in the leaves.

Dynamic Changes in Oil Cells
The dynamic changes in the oil cells during leaf development are shown in Figure 6 and Table 4.In general, the oil cells of the three clones were the largest in the 10th week of leaf development.The growth of the oil cells was rapid in the 1st-10th weeks and was slow in the 10th-24th weeks.The diameter of the oil cells in the C3 leaves was the largest in the 10th week, with a value of 48.73 ± 0.77 µm, while the diameter of the oil cells in the C3 leaves was the smallest in the 1st week, with a value of 27.85 ± 2.05 µm.According to the accumulation characteristics of the EO during the development of the oil cells, these cells can be divided into five stages: the nonformation stage, the formation stage, the accumulation stage, the saturation stage, and the disintegration stage [15].We found that the developmental stage of the oil cells seemed to be related to the developmental stage of the leaves.When the leaves had developed in the 1st to 2nd weeks, the oil cells in the leaves were mostly in the essential oil formation stage (Figure 7A,B), and the oil cells in the leaves were mostly in the essential oil accumulation stage during the 4-10th weeks of leaf development (Figure 7C,D).When the leaves reached the 12-24th weeks of development, the oil cells in the leaves were mostly in the saturation period (Figure 7E,F).However, this result was not absolute.Oil cells in different developmental stages were found in the same leaf; for example, the oil cells in the formation and accumulation stages were identified in both the 2-3-week-old leaves (Figure 7G), and the oil cells in the saturation stage and decomposition stage were both found to exist in leaves at the 12-24th weeks of development (Figure 7H).Therefore, it was considered that one developmental stage of oil cells was dominant in leaves at a certain developmental stage, while other stages of oil cells might also exist.According to the accumulation characteristics of the EO during the development of the oil cells, these cells can be divided into five stages: the nonformation stage, the formation stage, the accumulation stage, the saturation stage, and the disintegration stage [15].We found that the developmental stage of the oil cells seemed to be related to the developmental stage of the leaves.When the leaves had developed in the 1st to 2nd weeks, the oil cells in the leaves were mostly in the essential oil formation stage (Figure 7A,B), and the oil cells in the leaves were mostly in the essential oil accumulation stage during the 4-10th weeks of leaf development (Figure 7C,D).When the leaves reached the 12-24th weeks of development, the oil cells in the leaves were mostly in the saturation period (Figure 7E,F).However, this result was not absolute.Oil cells in different developmental stages were found in the same leaf; for example, the oil cells in the formation and accumulation stages were identified in both the 2-3-week-old leaves (Figure 7G), and the oil cells in the saturation stage and decomposition stage were both found to exist in leaves at the 12-24th weeks of development (Figure 7H).Therefore, it was considered that one developmental stage of oil cells was dominant in leaves at a certain developmental stage, while other stages of oil cells might also exist.

Microscopic Observation of Oil Cells
With transmission electron microscopy, it was found that the mitochondria and plastids, distributed in the cytoplasm (Figure 8), were more abundant in oil cells than in other cells.Osmium droplets, which were gray or black, varied in size and occurred abundantly in the cytoplasm during the oil cell development (Figure 9).As the oil cells developed, the osmium droplets continued to integrate into the vacuoles, eventually making the vacuoles become oil bladders for oil storage.

Microscopic Observation of Oil Cells
With transmission electron microscopy, it was found that the mitochondria and plastids, distributed in the cytoplasm (Figure 8), were more abundant in oil cells than in other cells.Osmium droplets, which were gray or black, varied in size and occurred abundantly in the cytoplasm during the oil cell development (Figure 9).As the oil cells developed, the osmium droplets continued to integrate into the vacuoles, eventually making the vacuoles become oil bladders for oil storage.Leaf structure is the foundation of secondary metabolic activities and is closely related to changes in OY [16].The OY showed a significant increasing trend with leaf development and stabilized after leaf maturity.The rapid accumulation period of the EO occurred before the leaf maturated (10-12th weeks).For example, the OY of C1 increased significantly from 1.61% ± 0.12% to 2.82% ± 0.20%, and a similar pattern existed in the other EO-forming plants, with the OY rapidly accumulating before the full blooming of flowers [17] and the ripening of fruit [18].Essential oil, a secondary metabolite in plants, is closely related to, but not completely synchronized with, plant growth and development.C. officinarum EO was produced by OCs, and the number and developmental stage of oil cells were closely related to the formation and accumulation of EO [19,20].At the early stage of leaf development (1st-4th weeks), the diameter of the OCs ranged from 30.38 ± 1.42 µm to 35.57± 0.29 µm, and a microscopic observation revealed that the OCs were mainly in the non-essential oil stage and essential oil-forming stage, whereas the diameter of the oil cells in mature leaves ranged from 44.16 ± 0.46 µm to 48.67 ± 0.80 µm, and, at this time, the OCs were mainly in the saturated stage.The OCs were already present in the leaf primordium of the leaf buds, so even young leaves could be extracted to obtain EO, similar to other aromatic plants such as Cyrnbopogon winterianus Jowit [21], Lemongrass [22], and Pelargonium graveolens L'Hérit [23].

EO Quality, Leaf Development, and Oil Cells
The relative contents of major OCs in EO were also closely related to leaf development.In the early stages of leaf development, the relative content of alcohols was the highest, reaching a maximum value of 30.18% in the 2nd week; with the growth of leaves, the relative content of aldehydes gradually increased, reaching a maximum value of 76.11% in the 6th week, and then it stabilized after leaf maturity.The same pattern exists in P. graveolens [23].However, not all the OCs of EO showed completely consistent results across the leaf development stages.For example, in this study, the relative content of citral increased first and then tended to be stable, but its two isomers had different accumulation patterns; neral reached its highest value of 28.63% in the 6th week, while geranial reached its highest value of 51.12% in the 12th week in the leaves.That is to say, the changes in the synthesis and relative content of OCs were related to changes in the synthesis pathway; the type of plant metabolism; or the biological function of each chemical, e.g., the gradual formation and accumulation of terpenes at later stages in plant development [24].In a previous study, it was found that the relative content of trans-cinnamaldehyde in Cinnamomum cassia Presl.leaf oil increased and then decreased [25].The relative content of sabinane in the EO of Litsea cubeba was the highest in mature fruits [26].Eugenol and syringa methyl, the main OCs in the EO of Ocimum plants, had the highest relative contents in mature leaves [27].The relative content of L-borneol, the main chemical component of Blumea balsamifera EO, was the highest in mature leaves [28].Many of the 130 ingredients detected in the EO of C. officinarum leaves are well known for their biological activities such as α-terpineol, borneol acetate, limonene oxide, α-pinene, and ylangene.The dynamic determination of the constituents of citral-type C. officinarum EO provides a reference for the isolation of biologically active molecules or the hierarchical utilization of EO.
It is worth noting that the rapid accumulation period of the main OCs of the essential oil lagged behind the period of rapid changes in the leaf shape, and the rapid changes in the leaf shape were concentrated in the 1st-4th weeks; meanwhile, the rapid accumulation of aldehyde compounds represented by citral occurred in the 3rd-6th weeks.Therefore, we believe that the development of leaves and oil cells guarantees the formation, accumulation, and transfer of EO and that the development of leaves and oil cells is closely related to the accumulation of EO.This might be because the production of EO is a part of secondary metabolism, which is controlled by primary metabolism.In the early stages of the growth and development of plants, their metabolic activity is mainly primary metabolism, and the synthetic substrates for secondary metabolism are relatively insufficient, which affects the synthesis of EO [29].

Figure 4 .
Figure 4.The number (A) and relative content (B) of different categories in the oil of the leaves at different developmental stages.

Figure 4 .
Figure 4.The number (A) and relative content (B) of different categories in the oil of the leaves at different developmental stages.

Figure 5 .
Figure 5. Dynamic changes in relative content of neral and geranial.

Figure 5 .
Figure 5. Dynamic changes in relative content of neral and geranial.

Figure 5 .
Figure 5. Dynamic changes in relative content of neral and geranial.

Figure 7 .
Figure 7.The developmental processes of the oil cells at different stages of leaf development.Note: (A): the nonformation stage of essential oil; (B): the formation stage of essential oil; (C,D): the accumulation stage of essential oil; (E,F): the saturation stage of essential oil; (G,H): the oil cells at different developmental stages in the same leaf.The arrows in the pictures point to oil cells.The pictures were taken under 20× magnification.

Figure 8 .
Figure 8. Mitochondria and plastids distributed in the cytoplasm in the oil cells.MT: mitochondria; P: plastid.

Figure 8 .
Figure 8. Mitochondria and plastids distributed in the cytoplasm in the oil cells.MT: mitochondria; P: plastid.

Figure 9 .
Figure 9. Formation of oil sac in oil cells.OS: oil sac; DOD: dark osmium droplets.

1 .
Leaf Development, Oil Cells, and Essential Oil Quantity

Table 1 .
Significance analysis of differences in leaf length, leaf width, and leaf area at different growth stages.

Table 2 .
Significance analysis of differences in oil yield of leaves at different growth stages.Note: C1: citral-type Camphora officinarum Nees ex Wall.cloned progeny ZS003; C2: cloned progeny ZS032; C3: cloned progeny NGN01.The symbols with the same letter in the same column indicate no significant differences (p < 0.05) in the oil yield according to Duncan's one-way ANOVA.

Table 3 .
Oil compositions and category of essential oil from leaves at different growth stages.

Table 4 .
Significance analysis of differences in oil cells of leaves at different growth stages.

Table 4 .
Significance analysis of differences in oil cells of leaves at different growth stages.

Table 4 .
Significance analysis of differences in oil cells of leaves at different growth stages.
Note: C1: citral-type Camphora officinarum Nees ex Wall.cloned progeny ZS003; C2: cloned progeny ZS032; C3: cloned progeny NGN01.Symbols with the same letter in the same column indicate no significant differences (p < 0.05) in the oil cell diameter according to Duncan's one-way ANOVA.