Volatile organic compounds (VOCs) detected by GC/MS in Asai et al. [16].
Abstract
Citrus plants are well known as a rich source of functional chemicals; however, metabolites involved in defense responses against environmental stresses are not yet well understood. Among environmental stresses, mechanical wounding is a continuous threat toward the growth and survival of plants. Recent advances in analytical technology and informatics enable comprehensive analysis of primary and secondary metabolites. In this chapter, metabolic profiling of leaf metabolites in seven Citrus species during responses against wound stress as well as defense-related phytohormone treatments was described. Moreover, we discussed current metabolomic techniques, application of these techniques to researches on Citrus defense responses and metabolic profiling-oriented identification of novel compounds.
Keywords
- defense response
- wound stress
- metabolic changes
- GC/MS
- metabolomics
1. Introduction
Wound stresses such as mechanical injury and herbivore feeding are unavoidable and continuous threats to growth and survival of plants. Damaged tissue allows pathogen invasion and leads to spread of disease into whole plant. Higher plants have evolved defense mechanisms against such attack of natural enemies. For instance, plants accumulate wound-healing compounds such as suberin in response to wounding [1] and prepare defense chemicals including repellents and toxins as well as physical defense reaction such as cell wall reinforcement. Moreover, it has been reported that phytoalexins, antimicrobial compounds produced by plants in response to pathogen infections, are accumulated after wounding [2].
2. Metabolomic approach to investigate responses to wound stress
As described above, when higher plants face to wound stresses, plant metabolism is drastically changed in order to defend themselves against stresses. This metabolic change involves accumulation of defense compounds such as phytoalexins and lignin-like compounds, regulation of signaling pathway, up-regulation of substrate supplies and increase or decrease of many other specific compounds. This reconfiguration of metabolic network is highly complex, and therefore, details in plant defense mechanism are still unelucidated. To understand the mechanism, comprehensive perspective of regulation of metabolic network must be needed. Recently, the “omics” technologies have been developed to characterize and quantify all of the molecules leading to the phenotype of an organism in non-targeted and non-biased manner. Among “omics” technologies, the term “metabolomics” has been used to address the analysis of low-molecular metabolites. Recent advances in technologies of mass spectrometry (MS) and nuclear magnetic resonance (NMR) as well as bioinformatics such as multivariate analyses and chemical libraries enable the application of metabolomics to varieties of organisms. Metabolomics has become in the spotlight as a powerful tool to gain comprehensive and collective information of metabolic network and to find out biomarkers related to defense mechanisms [5].
Since
3. Metabolomic analysis of leaf volatiles during wound responses
3.1. Volatile compounds in plant defense responses
Plants induce various defense reactions including phytoalexin and/or pathogenesis-related (PR) protein, hypersensitive reaction (HR) and emission of volatile organic compounds (VOCs) in response to wounding [6–8]. Among these, emission of VOCs is involved not only in direct defenses, such as toxins and repellents against herbivores, but also in indirect defenses that include recruitment of natural enemies against herbivores and elicitation of defense mechanisms in intact receiver plants [9, 10]. Plant VOCs consist of two major classes of compounds, that is, terpenoids and C6 green leaf volatiles (GLVs). Terpenoids are one of the most structurally diverse groups of plant metabolites and synthesized from two biological precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate. It has been demonstrated that several terpenoids play roles as antimicrobial or antifeedant compounds in direct defense responses [11, 12]. GLVs consist of C6 aldehydes, alcohols and their esters and are synthesized from α-linolenic acid through the lipoxygenase pathway. The compounds
3.2. Method of metabolic profiling of VOCs
To compare responses among species, plant materials used for study should be maintained under the same condition to minimize effects of environmental factors. We usually use plants grown in the same field, and at least five leaves per square meter were used for the study. To investigate the responses against stresses, freshly excised leaves were immediately exposed to stresses, and during the treatments, leaves were placed under strictly controlled condition to avoid influence of environmental factors. The treated leaves were immediately frozen in liquid nitrogen and maintained at −80°C before analysis [16].
For analysis of VOCs, gas chromatography/mass spectrometry (GC/MS) is suitable analytical platform because it separates majority of the components and mass spectral fragmentation pattern of each compounds makes it easier to identify compounds. Sample preparation methods for GC/MS include essential oil extraction such as solvent extraction or headspace extraction. Among them, headspace extraction method using microfiber solid phase (headspace solid phase microextraction, HS-SPME) is most sensitive and useful method for comprehensive analysis of VOCs. Various SPME fibers are now available for analyses, and among them, divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber has been well used and recommended for VOCs extraction because this fiber consists of triple phases and thus absorbs wide range of volatile compounds [15]. For chromatographic separation of VOCs, nonpolar CP-SIL 8-CB MS capillary column was used, and helium was used as the carrier gas at a liner velocity of 45.0 cm/s. For annotation of VOCs, the mass spectrum data were compared against spectra in the NIST reference library of GC/MS data system, and retention indices (RIs) from the literature were used for identification of VOCs. Identified VOCs in our previous study using seven
Chemical group | Compounds | Retention indices | Molecular weight | Mass spectrum data |
---|---|---|---|---|
GLV aldehydes | Hexanal | 802 | 100 | 41 (100), 44 (97), 56 (89) |
846 | 98 | 41 (100), 83 (97), 55 (95) | ||
Fatty aldehydes | Octanal | 1005 | 128 | 43 (100), 56 (77), 44 (77) |
Nonanal | 1106 | 142 | 57 (100), 41 (66), 56 (66) | |
1162 | 140 | 43 (100), 55 (95), 41 (92) | ||
Fatty alcohol | 1-Octanol | 1075 | 130 | 56 (100), 55 (77), 41 (65) |
Monoterpenenes | α-Thujene | 927 | 136 | 93 (100), 91 (47), 92 (40) |
α-Pinene | 934 | 136 | 93 (100), 92 (40), 91 (33) | |
Camphene | 950 | 136 | 93 (100), 121 (69), 79 (35) | |
Sabinene | 973 | 136 | 93 (100), 91 (33), 77 (28) | |
β-Pinene | 977 | 136 | 93 (100), 41 (43), 69 (41) | |
β-Myrcene | 991 | 136 | 93 (100), 41 (82), 69 (71) | |
α-Phellandrene | 1005 | 136 | 93 (100), 91 (44), 92 (32) | |
3-Carene | 1008 | 136 | 93 (100), 91 (37), 92 (32) | |
α-Terpinene | 1017 | 136 | 121 (100), 93 (98), 136 (61) | |
Cymene | 1025 | 134 | 119 (100), 134 (32), 91 (24) | |
D-Limonene | 1031 | 136 | 68 (100), 93 (100), 67 (66) | |
1039 | 136 | 93 (100), 92 (42), 91 (37) | ||
1050 | 136 | 93 (100), 80 (40), 91 (39) | ||
γ-Terpinene | 1061 | 136 | 93 (100), 136 (59), 90 (58) | |
α-Terpinolen | 1085 | 136 | 93 (100), 121 (86), 136 (82) | |
Monoterpene aldehydes | Citronellal | 1154 | 154 | 41 (100), 69 (95), 95 (54) |
β-Citral | 1241 | 152 | 41 (100), 69 (86), 94 (30) | |
α-Citral | 1271 | 152 | 69 (100), 41 (85), 84 (27) | |
Monoterpene alcohols | Eucalyptol | 1033 | 154 | 43 (100), 81 (56), 84 (51) |
hydrate |
1071 | 154 | 43 (100), 71 (97), 93 (58) | |
Linalool | 1101 | 154 | 71 (100), 93 (75), 55 (60) | |
Terpinen-4-ol | 1180 | 154 | 71 (100), 111 (50), 93 (47) | |
α-Terpineol | 1195 | 154 | 59 (100), 93 (66), 136 (47) | |
Nerol | 1223 | 154 | 69 (100), 41 (78), 68 (24) | |
Geraniol | 1253 | 154 | 69 (100), 41 (67), 68 (24) | |
Thymol | 1293 | 150 | 135 (100), 150 (35), 91 (15) | |
Monoterpene ester | Geranyl acetate | 1380 | 196 | 69 (100), 41 (49), 43 (47) |
Sesquiterpenes | α-Cubebene | 1347 | 204 | 105 (100), 119 (94), 161 (91) |
α-Copaene | 1376 | 204 | 161 (100), 119 (97), 105 (96) | |
β-Elemene | 1390 | 204 | 93 (100), 81 (91), 68 (67) | |
β-Caryophyllene | 1421 | 204 | 93 (100), 69 (87), 133 (81) | |
α-Bergamotene | 1435 | 204 | 119 (100), 93 (98), 41 (41) | |
Aromadendrene | 1439 | 204 | 161 (100), 93 (91), 91 (89) | |
β-Farnesene | 1454 | 204 | 69 (100), 41 (62), 93 (56) | |
Humulene | 1457 | 204 | 93 (100), 80 (32), 121 (23) | |
Alloaromadendrene | 1461 | 204 | 93 (100), 91 (93), 105 (93) | |
γ-Selinene | 1475 | 204 | 189 (100), 133 (64), 204 (49) | |
γ-Muurolene | 1482 | 204 | 161 (100), 105 (57), 81 (42) | |
β-Selinene | 1491 | 204 | 105 (100), 93 (98), 107 (93) | |
α-Selinene | 1498 | 204 | 189 (100), 93 (86), 107 (74) | |
α-Farnesene | 1506 | 204 | 93 (100), 41 (68), 69 (60) | |
β-Bisabolene | 1510 | 204 | 69 (100), 93 (84), 41 (71) | |
δ-Cadinene | 1521 | 204 | 161 (100), 204 (55), 134 (55) | |
β-Sesquiphellandrene | 1526 | 204 | 69 (100), 41 (52), 93 (49) |
Fifty VOCs were identified with our system, and majority of them is terpenoids, that is, monoterpene hydrocarbon and their derivatives and sesquiterpene hydrocarbons. In addition to terpenoids, two GLC aldehydes, three fatty aldehydes and a fatty alcohol were identified. Among VOCs identified, monoterpene hydrocarbons constituted the main part of the leaf VOCs in all species tested according to ratios of each chemical group on the basis of the total ion current peak area measured by GC/MS, but the profiles of VOCs were different among species [16].
3.3. Evaluation of changes in VOCs profiles during wound responses
It has been well known that phytohormones, such as jasmonic acid (JA) and salicylic acid (SA), are involved in the signaling pathway for induction of plant defense mechanisms. Wound stress induces temporal and organ-specific JA accumulation that mediates to activation of defense-related genes and leads to induction of defense responses [17, 18]. In contrast, SA accumulation is caused by insect egg deposition or pathogen infection and results in induction of
For comparison of VOC profiles among species and treatments, it must be necessary to handle large amount of datasets. To evaluate significant changes in VOC profiles, statistical analysis should be employed. For metabolomics, multivariate analysis is typically employed to process datasets. In our previous report, VOC profiling was performed by application of principal component analysis (PCA) [16]. PCA can provide an overview and clustering of all the applied datasets by projecting each sample.
The results of PCA demonstrated that
PCA provides an overview of datasets by unbiased and unsupervised manner, and thus, it shows clear clustering only when the variation within each group is sufficiently less than variation between groups. In contrast, since orthogonal partial least square-discriminant analysis (OPLS-DA) is supervised discriminant analysis that relies on the class membership of each treatment, OPLS-DA should be a powerful tool to evaluate treatment-specific changes in metabolites and thus to find biomarkers in defense responses [25].
4. Metabolomic analysis of primary metabolites during wound responses
4.1. Changes in primary metabolism during plant defense responses
Wound stress elicits not only secondary metabolites including VOCs but also whole metabolic network including primary metabolites. Activation of glycolytic pathway in response to wounding leads to energy production as well as substrate production for various defense compounds including defense-related proteins [26]. It has been demonstrated that accumulation of free amino acids was induced in response to environmental stresses. Branched-chain amino acids were induced against drought stress, while aspartate family amino acids were related to the osmotic stress [27, 28]. Moreover, amino acid biosynthetic pathways such as tryptophan pathway have been well known to be involved in biosynthesis of defense compounds [29]. For insight into defense mechanisms in
4.2. Method of metabolomics of primary metabolites
For analysis of primary metabolites, various platforms including capillary-electrophoresis/mass spectrometry, GC/MS and NMR have been developed. Among them, GC/MS has been well used because of its sensitivity and availability for wide range of compounds, although appropriate derivatization should be needed. Extraction and derivatization methods of plant primary metabolites were well developed for validation of food and beverage materials, and data processing software such as peak alignment and peak annotation by mass spectrum is now commercially and non-commercially available. GC/MS-based metabolomics has been frequently and routinely used for many metabolomic studies.
In our previous study, extraction and derivatization were carried out according to the method developed by Fukusaki et al. [30]. In this method, a mixture of methanol/water/chloroform (2.5:1:1 v/v/v) was used as an extraction solvent for a wide range of polar compounds including primary metabolites, and derivatization was carried out by adding methoxyamine hydrochloride, followed by silylation with
4.3. Profiling of primary metabolites in Citrus leaves
Metabolomic analysis of identified primary metabolites in
To assess changes in profiles of metabolites, datasets obtained from wound treated leaves and untreated leaves were statistically analyzed. Hierarchical cluster analysis and OPLS-DA showed that levels in amino acids are high sensitivity to stress treatments, indicating that amino acids are involved in
5. Identification of novel wound-stress related compounds
5.1. Bottleneck in metabolomics
As described above, metabolomics is a strong tool to find characteristic biomarkers related to genetic and/or environmental factors. However, compound annotation and identification are major bottleneck in metabolomics, especially in mass spectrometry-based metabolomics. Currently, several MS databases have been established, and increasing amount of MS data is now available to facilitate metabolite annotation. It has been estimated that there are hundreds of thousands compounds in plant kingdom, and many of them belong to secondary metabolites. Secondary metabolites are well known as functional metabolites that play important roles in plant physiology and/or ecology, and therefore, many of researches on plant physiology and biology have been focused on the secondary metabolism. Despite of the importance of secondary metabolites, identification of many of them is still to be carried out, and many of them are not commercially available. MS databases depend on literatures and analyses of authentic standards, and thus, spectra of secondary metabolites are quite limited. For better annotation in plant metabolomics and understanding of plant physiology, isolation and identification of metabolites are essential.
5.2. Defense-related compounds in Citrus plants
5.3. Isolation and identification of wound-induced compounds in Citurs hassaku leaves
Hassaku (
For detection of wound inducible compounds, hassaku leaves were cut into 5 mm square segments by surgical knife for mechanical wounding. This treatment is unusual in fields, but significantly facilitates the wound responses. Wounded and intact leaves were grounded and extracted with methanol that is a useful solvent to extract a wide range of compounds. Leaf extracts were then subjected to high performance liquid chromatography (HPLC) analysis, and chromatograms obtained from wounded and intact leaf extracts were compared. The HPLC fingerprinting showed that two peaks were occurred only in the chromatogram of wounded leaf extract but not in that of intact leaf extract, suggesting that these two compounds were related to the defense response against mechanical wounding.
To isolate wound-induced compounds, crude extract of wounded leaves was subjected to normal phase open column chromatography, followed by reverse phase preparative HPLC. Isolated compounds were applied to MS analysis and NMR for structural characterization. Spectral analyses revealed that one of wound-induced compounds was hesperetin, a major flavanone in
To investigate physiological roles of the novel compound, antibacterial activities of these compounds were evaluated against plant pathogens,
Although prenylated coumarins have been identified from several
6. Conclusion
In this chapter, metabolic profiling of
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