Methods applications and concepts of metabolite profiling Secondary metabolism

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Lloyd W. Sumner, David V. Huhman, Ewa Urbanczyk-Wochniak and Zhentian Lei

Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA

Abstract

Plants manufacture a vast array of secondary metabolites/natural products for protection against biotic or abiotic environmental challenges. These compounds provide increased fitness due to their antimicrobial, anti-herbivory, and/or alleopathic activities. Secondary metabolites also serve fundamental roles as key signaling compounds in mutualistic interactions and plant development. Metabolic profiling and integrated functional genomics are advancing the understanding of these intriguing biosynthetic pathways and the response of these pathways to environmental challenges. This chapter provides an overview of the basic methods, select applications, and future directions of metabolic profiling of secondary metabolism. The emphasis of the application section includes the combination of primary and secondary metabolic profiling. The future directions section describes the need for increased chromatographic and mass resolution, as well as the inevitable need and benefit of spatially and temporally resolved metabolic profiling.

Introduction

Secondary metabolites represent a diverse and vast array of compounds that have evolved over time and are found throughout a wide range of terrestrial and marine species [1-8]. Plants contain an especially rich source of natural products and approximately 100,000 unique plant natural products have been identified to date [9]. However, there are still a large number that have not been identified and overall estimates exceeding 200,000 throughout the plant kingdom are common [5, 6]. A representative list of secondary metabolite classes is provided in Table 1. The large number and diversity of plant secondary metabolites can be attributed to the broad substrate specificity and the generation of multiple reactions products that are typical of natural product enzymes. These enzymatic traits enhance the probability of generating chemical diversity and hence beneficial compounds. The selection and retention of chemical diversity is a critical factor in an organism's adaptation and fitness [10-12] and a primary reason for the large number of natural products.

Table 1. Representative secondary metabolite classes

Artemisinins Acetophenones

Alkaloids (imidazole, isoquinoline, piperidine/pyridine, purine, pyrrolizide, quinoline, quinolizidine, terepene, tropane, and tropolone alkaloids) Amines

Anthranoids/Anthraquinones Anthocyanidins Aristolochic acids Aurones

Azoxyglycosides

Benzenoids

Coumarins

Cyanogenic glycosides Condensed tannins Dibenzofurans

Flavonoids (flavanols, flavones, flavanones, etc.) Glucosinolates Hyrdroxybenzoic acid

Hydroxycinnamic acids

Isoflavonoids

Isothiocyanates

Lignins/Lignans

Non protein amino acids

Phenanthrenes

Phenolics

Phenols (phloroglucinols, acylphloro glucinols, etc.) Phenylpropanoids Polyacetylenes Polyines Polyketides

Steroidal and Triterepenoid Saponins

Stilbenes

Taxols

Terepenoids (hemi, mono, sesqui, di, tri, and tetra) Thiosulfinates Xanthones

Plants manufacture a vast array of secondary metabolites/natural products for protection against biotic or abiotic environmental challenges [5]. Thus, these compounds provide increased fitness due to their antimicrobial, anti-herbivory, and/or alleopathic activities. These toxic chemical weapons thwart potential damage by pathogenic viruses/bacteria/fungi/herbivores and/or minimize competition with other plants. For example, select secondary metabolites produce unfavorable responses in targeted plant predators such as bloat (saponins) in cattle and infertility in sheep (isoflavones). Many natural products also have other beneficial biological functions such as flavor/fragrance/color attractants [13-15], UV-protectants, antioxidants, signaling compounds associated with ecological interactions and symbiotic nodulation [16-18], and nutraceutical/pharmacological properties related to human and animal health [16-25]. In fact, natural products account for approximately 30% of all the sales of human therapeutics [26]. The anticancer utility of taxol [27, 28] and the antimalarial properties of artemisinin [29-31] are good examples.

In addition to the large diversity in basic chemical structures, many natural products are further conjugated with a variety of sugars and/or organic acids. The conjugation process is believed to be an import part of the cellular detoxification and storage mechanisms. However, they can also dramatically impact the biological activity of these compounds. Additional derivatives of natural products are achieved through the attachment of chemical moieties, such as acylation or prenylation, which continue to add to the chemical diversity of the metabolome and impact biological activity [32-34].

Methods

The vast numbers of plant secondary metabolites represent an extreme challenge for large-scale metabolite profiling, i.e., metabolomics, and a singular tool for profiling all primary or secondary plant metabolites currently does not exist. Most present strategies involve 'divide and conquer' strategies. This is achieved by employing a series of parallel targeted profiling methods focused on singular or multiple metabolite classes. Natural product classes are selectively extracted through the use of optimized solvents and often analyzed separately or in parallel. If specific natural products are of particular low abundance, enrichment methods such a solid phase extraction may also be employed.

There exist a growing number of successful technical methods that are employed in metabolic profiling of secondary metabolites [35, 36] and the selection of any specific method is usually a compromise between sensitivity, selectivity and speed [37]. GC/MS is capable of profiling many of the smaller and volatile secondary metabolites including the isoprenoids [38], triterepenoids such as P-amyrin [39], and phenylpropanoid aglycones such as ferulic acid [39]. However, a large number of secondary metabolites are conjugated with sugars as described above and are not amenable to GC/MS even following derivatization. Therefore, high performance liquid chromatography (HPLC) coupled to ultraviolet (UV) and mass spectrometry (MS) detection [40, 41], capillary electrophoresis-MS [42-44], NMR [45], and/or HPLC-NMR [46-49] are heavily relied upon in most approaches for metabolic profiling of secondary metabolism. The use of various established metabolomics technologies have been reviewed previously [35] and will not be replicated here. However, a detailed discussion of emerging technologies that offer significant enhancements in metabolic profiling of secondary metabolites will be discussed in the 'Future directions' section below.

Applications

Functional genomics and systems biology approaches based upon high density microarray analyses have traditionally been pursued in a limited number of model plant species such as Arabidopsis, rice, andMedicago as these species offer the major genomic and transcript sequence resources. Fortunately, the quantity of sequence information in the form of genomic or expressed sequence tags (ESTs) is growing exponentially for a vast number of plant species (http://www.tigr.org/tdb/tgi/plant. shtml) which is making cDNA or oligonucleotide arrays for these species possible. However, these resources are coming at additional costs. Metabolomics and/or metabolic profiling on the other hand are less species dependent as most primary and some secondary metabolites such as flavonoids are observed across major por tions of the plant kingdom. Thus, metabolomics offers greater diversity in its application to various plant species relative to transcriptomics and proteomics platforms without the additional costs. Accordingly, metabolic profiling has been significantly utilized in the study of primary metabolism of model species [13, 50-54] and also in many other crop plants such as potato [55-58], tomato [59], and cucurbits [60]. However, the study of secondary metabolism in model species has been less actively pursued [61, 62].

Metabolic profiling as a tool to study secondary metabolism has traditionally been focused on two major areas. First, it was traditionally a phytochemical tool for the rigorous separation, isolation, and identification of individual and unknown secondary metabolites [63]. For example, LC/MS might be used to obtain a nominal or accurate mass of a highly purified unknown metabolite to aid in structural determination. Secondly, metabolic profiling has been used as a tool to study the molecular aspects of secondary metabolism [15, 64, 65]. These efforts often focus upon a limited number of secondary metabolites related to the specific pathway being studied and less attention is directed toward the cumulative differential profiles. More recently, the scale and scope of metabolic profiling related to secondary metabolism have dramatically broadened towards a larger-scale and more comprehensive nature [39, 41, 44, 66, 67]. However, these larger-scale functional genomics applications are still somewhat limited.

The most exciting applications of metabolomics are not focused solely on specific natural product classes, but are bridging the gap by profiling both primary and secondary metabolites to better understand the interrelationship between these two important areas. For example, von Roepenack-Lahaye and colleagues have developed a capillary HPLC coupled to quadrupole time-of-flight mass spectrometry (LC-QtofMS) method for profiling both primary and secondary metabolites and used it to evaluate chalcone synthase deficient tt4 mutants in Arabidopsis [68]. Hirai and colleagues have also used an integrated approach composed of multiple technologies to show that sulfur and nitrogen metabolism were coordinately modulated with the secondary metabolism of glucosinolates and anthocyanins [42, 69, 70]. Further, these pioneers also integrated metabolomic and mRNA expression data to render gene-to-metabolite networks used in the identification of gene function and subsequent improvement in the production of useful compounds in plants. Similarly, Nikiforova and colleagues determined the impact of sulfur deprivation on primary metabolism and flavonoid levels and used this information to reconstruct the coordinating network of their mutual influences [71].

Colleagues at The Noble Foundation are currently applying metabolic profiling in both genomic and functional genomic approaches for discovery of new genes and for new insight into the biosynthetic mechanisms related to secondary metabolism. A major area of focus includes triterpene saponins. Although the biosynthetic pathway is poorly understood, these compounds have a large diversity of important biological activities including anti-herbivory (i.e., hemolytic and cause bloat), antifungal, antimicrobial, alleopathic, lowering of cholesterol, anticancer, and utility as adjuvants. Recently, Achnine and coworkers utilized EST mining, in vitro assays, and metabolic profiling to identify putative glycosyltransferases (GTs) involved in triterpenoid

Glufosinate Metabolism

Figure 1. A proposed mechanistic model of the metabolic response ofMedicago truncatula cell suspension cultures to methyl jasmonate elicitation [39]. The data suggest a major reprogramming of metabolism in which as carbon normally destined for sucrose is redirected towards secondary metabolism (triterpene saponin).

Figure 1. A proposed mechanistic model of the metabolic response ofMedicago truncatula cell suspension cultures to methyl jasmonate elicitation [39]. The data suggest a major reprogramming of metabolism in which as carbon normally destined for sucrose is redirected towards secondary metabolism (triterpene saponin).

saponin biosynthesis [41]. In this report, two new uridine diphosphate GTs were identified and characterized that possessed saponin specificity. This project continues with a large number of additional putative GTs under investigation.

In a separate study on biotic stress, Broeckling and colleagues reported a major reprogramming of carbon flow from primary towards secondary saponin metabolism in response to methyl jasmonate elicitation in Medicago truncatula [39, 72]. Based on metabolic profiling of both primary and secondary metabolism, a mechanistic response model was proposed and is presented in Figure 1, which involves a major reprogramming of carbon from primary metabolism towards secondary metabolism (i.e., triterpene saponins). The response includes increased levels of serine/ glycine/threonie metabolism which is believed to result in increased levels of branched chain amino acids suggesting increased hydroxylmethylgluturate (HMG) levels. The increased levels of the polyamine beta-alanine and putrescine imply increased levels of the HMG-CoA ester which serves as the source of carbon for triterpene saponin and sterol production. However, no increase in sterol accumulation was observed supporting carbon flow directed toward saponin production which was confirmed by LC/MS metabolic profiling. Although the HMG-CoA ester was not observed in the metabolic profiles, microarray data (Naoumkina et al., unpublished) reveal increased levels of HMG-CoA synthase and HMG-CoA reduc-tase that further support this model and will be presented in detail elsewhere. Continued efforts are underway that will further integrate transcript, protein, and metabolite data consistent with a systems biology approach.

Future directions

The separation of complex secondary metabolome mixtures is still quite challenging, and there exists a need for greater differentiation and resolution in metabolomics approaches at both the technical and biological levels. We are actively pursuing these needs by increasing chromatographic resolution and by increasing spatially/ temporally resolved biological sampling. These efforts are amplifying the biological context of our metabolic profiling efforts.

Increased chromatographic resolution

Currently, analytical HPLC commonly used in many secondary metabolic profiling approaches has an upper peak capacity (i.e., theoretical number representing the maximum peaks resolvable by the system based on optimum performance) of approximately 300. Based on this estimate, a maximum of 300 components could be resolved in a best case scenario; however in practice, this value is seldom achieved and more realistic peak capacities are between 100 and 200. Thus, current HPLC technologies are limiting the comprehensive scope of metabolomics. Separation efficiencies can be improved by altering selectivity, increasing column lengths, decreasing column diameters, reducing particle sizes, increasing temperature, and/or utilization of alternative column materials. These approaches have been recently reviewed [73] and we are currently evaluating alternative techniques, including capillary/nano-HPLC-QtofMS and ultra-performance liquid chromatog-raphy mass spectrometry (UPLC-MS) in an effort to increase the comprehensive coverage of metabolic profiling. Both methods have yielded increased separation efficiencies. For example, average separation efficiencies exceeding 225,000 plates per meter were obtained by capillary column (300 ^m in diameter) HPLC-QtofMS analysis of a saponin extract from Medicago truncatula (see Fig. 2). This represents an approximate three-fold increase in efficiency as compared to an average efficiency of 87,000 plates per meter for analytical HPLC (4.6 x 250 mm, Agilent 1100) system coupled to a quadrupole ion trap mass spectrometer (LC-QITMS) [40]. All separation gradients and sample loadings were identical. Unfortunately, the standard deviation was higher for the capillary system (16.6%) relative to the analytical system (8.8%). The higher variability was attributed to the passive flow splitting associated with the LC Packings Ultimate HPLC pump; however, active splitting modules are now available that should significantly lower this variability.

We have also completed preliminary evaluations of ultra-performance liquid chromatography mass spectrometry (UPLC-MS) for the analysis of phenolics and saponins. These efforts yielded impressive results as illustrated in Figure 3. The average peak widths were approximately 6 seconds at half height and represent an average separation efficiency of approximately 500,000 plates per meter. These results illustrate that high resolution and separation efficiencies are possible for high pressure liquid chromatography and compare favorably to those obtained by capillary GC/MS. Further, these high efficiencies were reached using faster separa-

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