As a result of teaching many undergraduate and graduate students about plant natural products in a wide range of plant biology courses, the need for a comprehensive yet thorough collecti
S Bhinu, Kothandarman Narasimhan, and Sanjay Swarup
Mass Spectrometry
Mass spectrometry (MS) complements NMR as a critical technique for elucidating organic and natural product structures Its exceptional sensitivity far exceeds NMR's capabilities However, MS spectra interpretation is more intricate, and extracting connectivity information, essential for structural determination, requires meticulous analysis of fragmentation spectra.
Mass spectrometers generate charged particles (ions) from analyzed substances These ions fragment under their high energy, releasing smaller charged particles whose masses can be measured using electric and magnetic fields The fragmentation process, governed by the stability of ions, provides insights into the underlying molecular structures However, the difficulty in generating molecular ions limits the direct determination of molecular formulas for unknown compounds.
There are many different techniques in mass spectrometry that can be divided according to their ion formation and according to the process of how we sort out the originally generated ions and the ions resulting from fragmentation reactions.
Gas-phase ionization methods rely upon ionizing samples that are in the gas phase Gas-phase ionization is limited to volatile samples, which are usually introduced into the mass spectrometer through a heated batch inlet, heated direct insertion probe, or, most commonly, a gas chromatograph.
Electron ionization or electron impact ionization (EI) is the oldest and best characterized of all the ionization methods A beam of electrons passes through the gas-phase sample and collides with the neutral analyte molecule This collision can knock off an electron from the analyte molecule, resulting in a positively charged radical ion The ionization process can produce, in favorable cases, molecular ions that will have the same molecular weight and elemental composition as the starting analyte, or it can produce fragment ions that correspond to smaller pieces of the analyte molecules.
Electron impact (EI) mass spectrometry commonly employs electrons with 70 eV energy, but reducing this energy minimizes fragmentation while diminishing ion formation EI mass spectra are well-established and applicable to most volatile compounds, yielding reproducible spectra that provide structural information through fragmentation patterns These reproducible spectra can be stored in searchable libraries, allowing identification based on "fingerprints" However, some high-molecular-weight compounds may lack a molecular ion peak, and EI's requirement for sample volatility limits its mass range to typically less than 1000 Da, which is generally not a significant limitation for natural products.
Alternatively, chemical ionization (CI) can be used This is a two-step process, where in a first step a reagent gas, typically methane, isobutene, or ammonia is ionized through electron impact In a second step, these reagent gas ions are then reacted with the analytes, which, in turn, produce the analyte ions to be analyzed by the spectrometer.
The advantage of chemical ionization is the occurrence of much less fragmentation, which typically means that molecular ions are easier to produce, and the spectra obtained are less complex It can also be interfaced with liquid chromatography (LC-MS).
Field desorption and ionization are gentle techniques for ionization They commonly generate mass spectra with minimal or no fragment ions These methods utilize electron tunneling from an emitter possessing a high electrical potential.
The sample is deposited onto the emitter, the emitter is biased to a high potential (several kilovolts), and a current is passed through the emitter to heat up the filament Mass spectra are acquired as the emitter current is gradually increased, and the sample is evaporated from the emitter into the gas phase. Characteristic positive ions produced are radical molecular ions and cation-attached species such as [M+Na] + The latter are probably produced during desorption by the attachment of trace alkali metal ions present in the analyte FD leads to simple mass spectra, typically one molecular or molecular-like ionic species per compound It works well for small organic molecules The mass range in which this technique can be applied is less than about 2000 to 3000 Da; however, it is very sample dependent.
The sample is evaporated from a direct insertion probe, gas chromatograph, or gas inlet As the gas molecules pass near the emitter, they are ionized by electron tunneling This again leads to very simple mass spectra, typically one molecular or molecular-like ionic species per compound The sample must be thermally volatile and is introduced in the same way as for electron ionization This limits the mass range to typically less than 1000 Da.
In these methods, the sample is deposited on a target that is bombarded with neutral or ionic atoms The most common approach for organic mass spectrometry is to dissolve the analyte in a liquid matrix with low volatility and to use a relatively high current of bombarding particles (fast atom bombardment [FAB] or dynamic secondary ion mass spectrometry [SIMS])
The analyte is dissolved in a liquid matrix such as glycerol, thioglycerol, or m-nitrobenzyl alcohol, and a small amount is placed on a target The target is bombarded with a fast (neutral) atom beam (for example, 6 keV xenon atoms) that desorbs molecular-like ions and fragments from the analyte Cluster ions from the liquid matrix are also desorbed, and they produce a chemical background that varies with the matrix used It provides a rapid and simple technique that can be applied to a large number of compounds and is often used for high-resolution measurements The typical mass range for this technique is 300 Da to about 6000 Da FAB has been used for many years to obtain high-resolution mass data of especially higher masses from easy-to-fragment molecules.
9.3.2.2 Secondary Ion Mass Spectrometry (SIMS)
Dynamic SIMS is a technique similar to FAB, utilizing an ion beam instead of a neutral beam This approach allows for higher kinetic energy and enhanced sensitivity, particularly for heavier masses Notably, this technique was widely employed for analyzing protein masses below 13,000 Da but has since been predominantly replaced by electrospray ionization methods.
Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) are two ionization techniques used in mass spectrometry In these methods, the analyte is introduced as a solution into the mass spectrometer at atmospheric pressure The sample is desolvated to ions as they enter the ion source using these methods They widely use flow-injection and LC-MS techniques.
UV-Vis, IR Spectroscopy
The use of UV-Vis (ultraviolet visible) spectroscopy in the structure elucidation process is limited. Nevertheless, UV-Vis spectroscopy plays an important role as probably the most often used tool for detection in separations It also finds wide applications in quantitative analysis, not only in the context of separations, but also, for a large number of assay techniques, where chromophores are used to assess biochemical reactions Quantitative applications are based on Beer–Lambert’s law:
A = log(I 0 /I) = εlC where A = absorbance, an optical parameter measured with a spectrophotometer, I = intensity of light leaving sample cell, I 0 = intensity of light incident upon sample cell, l = length of the sample cell, C molar concentration, and ε = molar absorptivity.
The typical wavelength range of a spectrometer covers 190 to 800 nm UV spectra are typically recorded as a plot of absorbance versus wavelength; however, only very few are reproduced in chemical literature Typically, wavelengths of band maxima are reported along with their respective absorptivities All organic compounds absorb UV radiation; thus, solvents also have UV absorption When measuring
UV spectra or intensities, solvent cutoffs must be taken into consideration Some cutoffs are provided in Table 9.6.
For most of the natural products we deal with, there are only a few types of chromophores that we use to probe our samples in assays or HPLC separations Especially useful in this context are chro- mophores that have one or more double bonds If these double bonds are conjugated, we can easily reach absorption maxima in the range of 280 to 350 nm or even larger Mostly, however, the maxima of chromophores fall in the range up to 220 nm (see Table 9.7).
In the case of conjugated double-bond systems, such as dienes, enones, and some benzene derivatives,
Woodward–Fieser rules are commonly used to estimate the UV maxima of compounds.
Analytical infrared (IR) spectroscopy utilizes several techniques based on the absorption of electromagnetic radiation in the range of 1-1000 μm Within this range, near-IR (1-2.5 μm), mid-IR (2.5-25 μm), and far-IR (>25 μm) regions are distinguished Mid-IR, abundant in structural data, is notably accessible This region is not just valuable for identifying functional groups but also offers distinct molecular fingerprints, providing valuable insights into molecular structure.
Solvent Cutoff [nm] Solvent Cutoff [nm] Solvent Cutoff [nm]
Chromophore λ (nm) Chromophore λ (nm) Chromophore λ (nm)
R-COOH 205 R-COOR 205 regions that can be used to uniquely identify compounds For IR measurements, it is common to report wavelengths in terms of wave numbers ν (cm –1 or kaysers) All observable IR bands are due to the interaction of the electrical vector of the electromagnetic radiation with the electric dipole of nonsym- metrical bonds It turns out that IR spectroscopy can easily be used as a semiempirical method for structural analysis because it was observed that there is a good correlation between the position of band maxima and organic functional groups or structural characteristics.
Typical group frequencies often found in natural products are listed in Table 9.8.
Hyphenated Techniques
The combination of gas chromatography (GC) and mass spectrometry (MS) for the detection and identification of constituents of essential oils has become a powerful analytical tool in phytochemical analysis The sample to be analyzed is injected into the GC, where it is swept through a capillary column by an inert gas stream The components of the sample are separated based on their differential adsorptive interactions with the liquid phase of the GC column The separated components, then, individually pass through the mass spectrometer, where ionization, fragmentation, and mass detection take place The GC-
MS combination allows for the separation of essential oil components and the acquisition of mass spectra of the separated components Utilization of GC retention data along with MS fragmentation and com- parison with spectral libraries allows for compound identification.
In the following two examples, goldenrod (Solidago canadensis) leaf essential oil and Randia matudae floral essential oil were analyzed by GC-MS In these studies, the essential oils were analyzed using an Agilent 6890 GC with Agilent 5973 (Agilent Technologies, Palo Alto, CA) mass selective detector, a fused silica capillary column (HP-5ms, 30 m × 0.25 mm), helium as the carrier gas, 1 ml/min flow rate,
Important Group Frequencies for IR Spectroscopy
Alkyl Aryl. sat unsat. sat unsat. cyclic sat unsat.
C-N N-HAlkylAryl and splitless injection The injector temperature was 200°C, and the oven temperature was programmed as follows: 40°C initial temperature, hold for 10 min; increased at 3°/min to 200°C; increased 2°/min to 220°C The MS detector temperature was 280°C
Retention indices (RIs) of the essential oil components were determined by reference to a homologous series of normal alkanes Thus, a mixture of alkanes (n-octane through n-triacontane) is injected into the GC-MS system and analyzed using the temperature program above The retention indices of the alkanes are defined as n-octane = 800, n-nonane = 900, n-decane = 1000, and so on A plot of RI versus retention time for the homologous alkanes is used as a standard curve to determine the RIs of the components of the essential oils RIs for essential oil components can then be compared with published RIs An excellent compilation of GC RIs along with MS fragmentation patterns can be found in the literature (Adams, 1995) Mass spectral fragmentations of the individual essential oil components are compared with the NIST library of mass spectra (through the ChemStation data system of the instrument) as well as mass spectra compiled in Adams (1995).
9.5.1.1 Solidago canadensis (Goldenrod) Leaf Essential Oil
Goldenrod leaf oil, derived from Solidago canadensis, has been traditionally used for its therapeutic properties Chemical analysis via GC-MS reveals its composition, primarily consisting of monoterpene (42.1%) and sesquiterpene (51.2%) hydrocarbons, with smaller amounts of oxygenated monoterpenoids (5.3%) and sesquiterpenoids (1.4%) The major components include germacrene D (34.4%), αααα-pinene (13.3%), limonene (11.0%), sabinene (8.0%), and myrcene (6.3%) These compounds contribute to the oil's potential antihypertensive, antiseptic, and anti-inflammatory properties.
D to be the most abundant component in agreement with this work However, Schmidt and co-workers (1999) found cyclocolorenone to be a major component (38%) in goldenrod from northern Germany, and Kasali and co-workers (2002) found 6- epi -ββββ-cubebene to be a major component (21%) in gold- enrod oil from Poland Interestingly, neither of these compounds was detected in our sample of goldenrod leaf oil.
FIGURE 9.69 Total ion current (TIC) chromatogram of Solidago canadensis leaf essential oil.
Chemical Composition of Solidago canadensis Leaf Essential Oil
Note: RT = Retention time; RI = retention index; TIC = total ion count; Area = % based on TIC; and QI = quality index based on agreement with NIST reference spectrum.
9.5.1.2 Randia matudae Floral Essential Oil
Randia matudae (Rubiaceae) is a subcanopy tree, 10 to 20 m tall, found in Mexico and Costa Rica
(Haber et al., 2000) The flowers of this tree produce a strong fragrance at night that serves to attract hawk moths (Sphingidae) that feed on nectar as well as pollinate this species The GC of R matudae floral essential oil is shown in Figure 9.70, and the floral essential oil composition is compiled in Table 9.10.
Mixture analysis using chromatographic techniques such as GC-MS has a long history in natural products chemistry, but many of the earlier investigations were hampered by the low volatility of a large number of compounds, such as polyphenols With the introduction of APCI and ESI interfaces, the chromatographic process could be extended to liquid chromatography applications that allow for the analyses of compounds regardless of their volatility Whereas GC-MS investigations provide some structural information (EI MS fragmentation), ESI and APCI tend to give only molecular weight information To enhance structural information, tandem mass spectrometry (MS-MS) experiments can be performed Wide use of these techniques led to affordable benchtop instruments, and LC-MS has grown into one of the most important and most widely used analytical techniques in natural products analysis
The n-hexane extract of Ligusticum chuanxiong could be clearly separated by reversed-phase HPLC analysis (Zschocke et al., 2005) Figure 9.71 shows the HPLC chromatogram that is the basis by which to analyze four of the apparent six peaks.
The peaks labeled 1 through 4 in the chromatogram give the APCI mass spectra shown in Figure 9.72. These spectra, which commonly show a [M+H] + ion and a [M+CH3CN+H] + ion, are consistent with the structures shown in Figure 9.73
FIGURE 9.70 Total ion current chromatogram of Randia matudae floral essential oil.
Sample Name: Randia matudae essoilTIC: RAMAEO.D
Chemical Composition of Randia matudae Floral Essential Oil
8.89 995 — 6-Methyl-5-hepten-2-ol Trace Trace 95
13.22 1088 1088 trans-Linalool oxide Trace Trace 83
14.31 1111 1111 cis-Rose oxide Trace Trace 90
44.72 1863 — cis-11-Hexadecen-1-ol Trace Trace 91
FIGURE 9.71 High-performance liquid chromatography (HPLC) chromatogram of Ligusticum chuangxiong extract with ultraviolet (UV) detection at 235 nm.
FIGURE 9.72 Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) of peaks 1 to 4 of Ligusticum chuangxiong extract.
FIGURE 9.73 Structures identified in Ligusticum chuangxiong by liquid chromatography/mass spectrometry (LC-MS). time [min]
An example from an investigation of Vernonia fastigiata (Vogler et al., 1997) is presented, which illustrates the use of MS-MS spectra, as well as the use of single-ion monitoring In this example, we deal with pairs of isomeric compounds (m/z = 421 or 423), which nicely show up when using single- ion monitoring (see Figure 9.74) Furthermore, it was demonstrated that by monitoring the two ions at m/z = 275 and m/z = 257, all but one compound belong to the same skeleton (see Figure 9.75, Figure 9.76, Figure 9.77, and Table 9.11).
FIGURE 9.74 Single-ion monitoring of Vernonia fastigiata extract.
Oper vernoroh Vernonia EE-rohextrakt MEOH / H2O 25%/80M/55% 90M/100%/100M 650 uL APCI +Q1MS LMR UP LR
FIGURE 9.77 m/z = 437, structure I, Table 9.11 All spectra taken under CID conditions using 2.5 mTorr argon, 18 V, vaporizer set at 200 ° C, nebulizer capillary at 70 ° C.
Summary of MS Results for Vernonia fastigiata
Results from APCI-LC-MS- Messungen
R 2 Methac i-Bu Methac i-Bu Ang Methac Methac i-Bu Methac
R 3 H H Ac Ac Ac Ac Ac Ac Ac
Note: Methac = methylacryloyl, i-Bu = isobutyroyl, Ang = angeloyl, Ac = acyl.
Since LC-NMR became commercially available around 1997, a large number of applications of LC-
The application of LC-NMR in natural products research has brought new insights, as evidenced by publications from leading European groups Researchers like Albert, Hostettmann, and Bringmann have showcased the utility of LC-NMR, often coupled with LC-MS, in analyzing a wide range of natural products with minimal sample consumption Advancements in NMR technology, including high field instruments, smaller detection cells, and improved RF components, have greatly enhanced sensitivity, allowing for detailed analysis of samples in the center of HPLC peaks where concentration is highest These improvements have made LC-NMR a valuable tool for routine analysis in natural products research.
CD 3 OD), efficient solvent suppression techniques were introduced In addition, the introduction of inverse detection experiments enabled spectroscopists to extend their investigations to the less sensitive elemen- tary nuclei This was further improved by pulsed field gradient probes Improvements in NMR experi- ments, in general, such as selective excitation techniques, opened up new possibilities in obtaining complete structural information Despite all of these improvements, the amount of sample presents a challenge for NMR spectroscopy, which under these circumstances normally reaches the detection limit of the instrument
Using gradient probe technology and detection cell volumes of 60 to 120 μl, compounds with a molecular weight of 450 can be detected in on-flow runs in amounts as little as 10 μg For stop-flow, realistic limits are probably at 1 μg and, in special cases, certainly lower When we consider a typical HPLC peak width, which is most likely something around 500 μl, we can estimate sample amounts to be in that range, the amount typically required for bioassays (see Chapter 10) When implementing the latest available techniques, like LC-SPE-NMR (Godejohann et al., 2004) with cold-probe technology, the amount of sample per HPLC peak being detectable will be dramatically reduced, so that the analytical part is well in the range of typical bioassay procedures Using this technique, detection limits will reach the several nanogram range.
Conclusions
Over the past decades, advancements in structural elucidation have been remarkable NMR spectrometry has flourished with 2D techniques and proton-detected heteronuclear correlations, while mass spectrometry has expanded significantly with ESI and APCI interfaces, enabling the widespread use of LC-MS methods Additionally, the introduction of microprobes and improved sensitivity in NMR spectroscopy have significantly reduced the sample size required for characterization.
Adams, R.P (1995) Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing, Carol Stream, Illinois.
Albert, K (1995) On-line use of NMR detection in separation chemistry J Chromatogr A 703: 123. Bringmann, G., C Guenther, J Schlauer, and M Rueckert (1998) HPLC-NMR on-line coupling including the ROESY technique: direct characterization of naphthylisoquinoline alkaloids in crude plant extracts.
Bringmann, G., K Messer, M Wohlarth, J Kraus, K Dumbuya, and M Rueckert (1999) HPLC-CD on-line coupling in combination with HPLC-NMR and HPLC-MS/MS for the determination of the full absolute stereostructure of new metabolites in plant extracts Anal Chem 71: 2678.
Bringmann, G., M Wohlfarth, H Rischer, J Schlauer, and J Brun (2002) Extract screening by HPLC coupled to MS–MS, NMR, and CD: a dimeric and three monomeric naphthylisoquinoline alkaloids from
De Hoffmann, E and V Stroobant (2002) Mass Spectrometry: Principles and Applications, 2nd ed Wiley, West Sussex, United Kingdom.
Duarte, I.F., M Godejohann, U Braumann, M Spraul, and A.M Gil (2003) Application of NMR spectroscopy and LC-NMR/MS to the identification of carbohydrates in beer J Agric Food Chem 51: 4847–4852. Glaser, T., A Lienau, D Zeeb, M Krucker, M Dachtler, and K Albert (2003) Qualitative and quantitative determination of carotenoid stereoisomers in a variety of spinach samples by use of MSPD before HPLC-UV, HPLC-APCI-MS, and HPLC-NMR on-line coupling Chromatographia 57: S-19. Godejohann, M., L.H Tseng, U Braumann, J Fuchser, and M Spraul (2004) Characterization of a parac- etamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe J Chromatogr A 1058: 191.
Günther, H (1995) NMR Spectroscopy, 2nd ed John Wiley & Sons, New York.
Haber, W.A., W Zuchowski, and E Bello (2000) An Introduction to Cloud Forest Trees, Monteverde, Costa
Rica Mountain Gem Publications, Monteverde, Costa Rica.
Iossifova, T., I Klaiber, B Vogler, L Evstatieva, I Kostova, and W Kraus (1998) LC-coupled spectroscopic investigation of Fraxinus pallisiae bark In Quality of Medicinal Plants and Herbal Medicinal Products. Hrsg.: Gesellschaft für Arzneimittelforschung 46th Annual Congress of the Society of Medicinal Plant, Wien, 31.08.-04.09 E24 (Abstracts of Plenary Lectures, Short Lectures and Posters)
Kasali, A.A., O Ekundayo, C Paul, and W.A Konig (2002) Epi-Cubebanes from Solidago canadensis.
Krucker, M., A Lienau, K Putzbach, M.D Grynbaum, P Schuler, and K Albert (2004) Hyphenation of capillary HPLC to microcoil 1 H NMR spectroscopy for the determination of tocopherol homologues.
Queiroz, E.F., J.L Wolfender, K.K Atindehou, D Traore, and K Hostettmann (2002) On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spec- trometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation J Chromatogr A 974: 123.
Ramm, M., J.L Wolfender, E.F Queiroz, K Hostettmann, and M Hamburger (2004) Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array de- tection, electrospray ionization mass spectrometry and nuclear magnetic resonance J Chromatogr A 1034: 139.
Sandvoss, M., A Weltring, A Preiss, K Levsen, and G Wuensch (2001) Combination of matrix solid-phase dispersion extraction and direct on-line liquid chromatography-nuclear magnetic resonance spectros- copy-tandem mass spectrometry as a new efficient approach for the rapid screening of natural products: application to the total asterosaponin fraction of the starfish Asterias rubens J Chromatogr A 917: 75–86. Schmidt, C.O., H.J Bouwmeester, N Bulow, and W.A Konig (1999) Isolation, characterization, and mech- anistic studies of (-)-alpha-gurjunene synthase from Solidago canadensis Arch Biochem Biophys 364: 167–177.
Setzer, W.N., M.C Setzer, J.M Schmidt, D.M Moriarity, B Vogler, S Reeb, A.M Holmes, and W.A Haber. (2000) Cytotoxic components from the bark of Stauranthus perforatus from Monteverde, Costa Rica.
Setzer, W.N., B Vogler, R.B Bates, J.M Schmidt, C.W Dicus, P Nakkiew, and W.A Haber (2003) HPLC- NMR/HPLC-MS analysis of the bark extract of Stauranthus perforatus Phytochem Anal 14: 54–59. Sheppard-Hanger, S (1994) The Aromatherapy Practitioner Reference Manual Atlantic Institute of Aroma- therapy, Tampa, Florida.
Smallcombe, S.H., S.L Patt, and P.A Keiffer (1995) WET solvent suppression and its applications to LC NMR and high-resolution NMR spectroscopy J Magnetic Res Ser A 117: 295.
Stout, G.H and L.H Jensen (1989) X-Ray Structure Determination: A Practical Guide, 2nd ed John Wiley
Vogler, B., I Klaiber, G Roos, C.U Walter, W Hiller, P Sandor, and W Kraus (1997) Combination of LC-
MS and LC-NMR as a tool for the structure determination of natural products J Nat Prod 61: 175–178. Vogler, B., J.R Stoehr, I Klaiber, and R Bauer (1999) Online structure elucidation of amides and polyox- igenated cylcohexane derivatives by LC-NMR and LC-MS from crude extracts of Piper species In
2000 Years of Natural Products Research — Past, Present and Future (Joint Meeting of the ASP, AFERP,
GA and PSE, July 26–30Z), T.J.C Luijendijk and R Verpoorte (Eds.) Vrije Univeristei, Amsterdam, p 315.
Waridel, P., J.L Wolfender, J.B Lachavanne, and K Hostettmann (2004) Ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species Phytochemistry 65: 945.
Wolfender, J.L., L Verotta, L Belvisi, N Fuzzatti, and K Hostettmann (2003) Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MS n Phytochem Anal 14: 290. Xiao, H.B., M Krucker, K Albert, and X.M Liang (2004) Determination and identification of isoflavonoids in Radix astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatog- raphy with photodiode array and mass spectrometric detection J Chromatogr A 1032: 117
Zschocke, S., I Klaiber, R Bauer, and B Vogler (2005) HPLC-coupled spectroscopic techniques (UV, MS,NMR) for the structure elucidation of phthalides in Ligusticum chuanxiong Mol Diversity 9: 33–39.
Phytochemicals: The Chemical Components of Plants
Harry L Brielmann, William N Setzer, Peter B Kaufman, Ara Kirakosyan, and Leland J Cseke
1.1 Introduction 21.2 Lipids and Derivatives 31.2.1 Hydrocarbons 31.2.1.1 Saturated Hydrocarbons 41.2.1.2 Unsaturated Hydrocarbons 41.2.2 Functionalized Hydrocarbons 61.2.2.1 Halogenated Hydrocarbons 61.2.2.2 Alcohols 61.2.2.3 Sulfides and Glucosinolates 71.2.2.4 Aldehydes and Ketones 71.2.2.5 Esters 81.2.2.6 Fatty Acids 81.2.3 Terpenes 101.2.3.1 Hemiterpenes: C 5 121.2.3.2 Monoterpenes: C 10 121.2.3.3 Sesquiterpenes: C15 141.2.3.4 Diterpenes: C 20 141.2.3.5 Triterpenes: C 30 171.2.3.6 Tetraterpenes: C 40 191.3 Aromatics 191.3.1 Tetrapyrroles 191.3.2 Phenols 191.3.2.1 Simple Phenols 201.3.2.2 Phenol Ethers 211.3.2.3 Phenylpropanoids 221.3.2.4 Flavonoids 221.3.2.5 Tannins 251.3.2.6 Quinones 261.4 Carbohydrates 271.4.1 Monosaccharides 271.4.2 Oligosaccharides 281.4.3 Polysaccharides 291.5 Amines and Alkaloids 301.5.1 Amines 301.5.1.1 Aliphatic Monoamines 301.5.1.2 Aliphatic Polyamines 301.5.1.3 Aromatic Amines 301.5.2 Alkaloids 301.6 Amino Acids, Nonprotein Amino Acids, and Proteins 361.6.1 Amino Acids 371.6.2 Nonprotein Amino Acids 37
2 Natural Products from Plants, Second Edition
1.6.3 Proteins 37 1.6.3.1 Storage Proteins, Lectins, and Diet 39 1.7 Nucleic Acids, Nucleotides, and Nucleosides 40 1.8 Conclusions 41 References 42
Phytochemicals, as the word implies, are the individual chemicals from which plants are made In this chapter, we will look at these materials, specifically, the organic components of higher plants Numerous journals, individual books, and encyclopedic series of books have been written on this subject The goal here is to review this area in a concise format that is easily understandable The reader not familiar with chemistry may be somewhat intimidated by the material presented here However, we believe that understanding the chemical composition of plants is a prerequisite to understanding many of the remaining topics of this book. This is especially true for material covered in Chapters 2 and 3 For those interested in reviewing a specific area in greater detail, the references section includes numerous citations for each organic group covered. During the course of this survey, several themes will be emphasized These include (1) the rich diversity of chemical structures known to be synthesized by plants through an amazingly diverse network of metabolic pathways (see Figure 2.1 in Chapter 2); (2) basic differences in the chemical properties of the compounds; (3) adaptive functions of these compounds for plants; (4) uses of the compounds by humans (see essays below); and (5) examples of typical plants (listed by common name and scientific binomial name) that contain the respective types of compounds Often, these will be derived from common plants with which most of us are familiar Some marine algal plants are also included, because they contain many truly unique bioactive molecules.
The general categories of plant natural products are organized very broadly in terms of increasing oxidation state This begins with the lipids, including the simple and functionalized hydrocarbons, as well as the terpenes, which are treated separately Following this are the unsaturated natural products, including the polyacetylene and aromatic compounds We then cross over into the realm of the primarily hydrophilic molecules, including the sugars, and continue with those that can form salts, including the alkaloids, the amino acids, and the nucleosides Overall, this scheme provides a simple organizational pattern for discussing the phytochemicals It is consistent with the way that chemists often categorize organic chemicals in general and is roughly equivalent to a normal-phase chromatographic analysis of a given plant species Like any organizational scheme for this subject, be it taxonomic, phylogenetic, or biochemical, it should only serve as a rough guide.
Essay on Phytochemicals of Medicinal Value in Plants
Phytochemicals, abundant in today's diet, have long been recognized for their medicinal properties While some phytochemicals are classified as secondary metabolites, many are essential for plant growth and development Numerous resources, including journals, books, dictionaries, and databases, document the vast array of plant-derived natural products The American Society of Pharmacognosy recognizes various chemistry journals dedicated to natural products, such as Chemistry of Natural Compounds, Journal of Ethnopharmacology, and Fitoterapia These publications highlight the continued significance of phytochemicals in contemporary medicine and scientific research.
Regulation by Environmental Stresses
A host of environmental factors is involved in the regulation of metabolite biosynthesis in plants The need for this control of synthesis stems from the fact that plants must be able to adjust the production of metabolites according to changing factors if they are to survive Light is obviously a key factor in the ultimate production of many compounds, because it supplies the energy needed to fix carbon It is also more directly necessary for the biosynthesis of compounds, such as chlorophylls, as mentioned in Chapter 2 Here, photons trigger the enzymatic conversion of protochlorophyllide and phytol to chloro- phylls a and b and, hence, to chlorophyll–protein complexes in chloroplasts (Mohr and Schopfer, 1995). Light also catalyzes the synthesis of anthocyanin pigment, via the plant pigment phytochrome, in many tissues of many plants, such as cotyledon (seed leaf) epidermal cells and hypocotyl (stem portion below the cotyledons) subepidermal cells in mustard seedlings (Mohr and Schopfer, 1995) Light intensity plays an important role in the biosynthesis of medicinally important metabolites An excellent case in point is the tree of joy (Camptotheca accuminata) (Figure 3.1), where levels of the antiprostate cancer drug, camptothecin (an alkaloid metabolite), significantly increase as the amount of light reaching the tops of the plants decreases.
Temperature is another important factor that regulates plant metabolism At reduced temperatures around 0°C, most enzymes are inactive, but as the temperature increases, the rate of enzyme activity increases up to about 40°C, above which most plant enzymes become inactivated and even permanently damaged Many enzymes are always present in plant cells at a certain level, but specific temperatures can trigger a dramatic change in these levels For example, levels of heat shock proteins (HSPs), constitutively present as chaperones, rapidly increase at temperatures of 40°C and above for most organisms At this point, HSPs act to help repair enzymes that may have been damaged due to the excess heat Please note that not all organisms have enzymes that are only active between the temperatures of
0 and 40°C Some thermophilic bacteria, for example, thrive at high temperatures in excess of 95°C (O’Brien, 1996).
Carbon dioxide gas is the fundamental carbon source for all plant metabolites (see Figure 2.1) Its levels can vary depending on the environment, and this variation causes changes in biosynthetic output. For example, elevated carbon dioxide levels in the earth’s atmosphere due to increased burning of fossil fuels and burning of tropical rainforests worldwide, together with elevated temperatures (global warm- ing) due to elevated levels of greenhouse gases are currently causing increases in total photosynthate produced in temperate-zone plants (Teeri, 1997) This is especially true for plants with C-4 photosyn- thesis These plants are adapted to higher temperature regimes and have little or no loss of carbon through
Figure 3.1 depicts author Peter Kaufman amidst a plantation of tree of joy (Camptotheca accuminata) trees established in southern Louisiana at the Citrus Experiment Station near Port Sulfur This research project was supported by the Agricultural Experiment Station of Louisiana State University, Baton Rouge, and Xylo Med Research, Inc The photograph was captured by Tracy Moore, President of Xylo Med Research, Inc.
Regulation of Metabolite Synthesis in Plants 103 photorespiration (ca 30%) However, the ultimate impact of such climatic perturbations on the bio- synthesis of compounds other than photosynthetically produced sugars is unknown.
Flooding of plant root systems for variable periods of time is another kind of environmental stress. The stress imposed here is mainly due to oxygen deprivation to the roots For terrestrial plants, too much water results in the stunting of shoot growth, reduced chlorophyll biosynthesis in the leaves, and enhanced ethylene biosynthesis However, aquatic plants, such as rice (Oryza sativa) and cattail (Typha spp.), can tolerate continuous flooding, because they have air passages in the root and shoot systems that allow atmospheric oxygen to permeate into the cells of their flooded roots Where nonaquatic plants are periodically flooded by irrigation, after the soil has dried out, plant growth and chlorophyll biosynthesis are not impaired but, rather, are stimulated In the case of the tree of joy, Camptotheca accuminata, such periodic flooding episodes result in greatly enhanced growth of new shoots that have significantly higher levels of camptothecin (CPT) than the shoots of plants that were not irrigated and have only old-growth shoots (Liu et al., 1997).
It is also known that the acidity (pH), salinity, and nutrient conditions of the plant environment have a huge impact on the growth of plants For example, the dependence of the structure and ionization states of many molecular constituents of the cell ensure that cellular processes are sensitive to pH. Different plant species differ in their responses to pH conditions Most plants grow well in soil that is neutral, mildly acidic, or mildly basic However, acidic stress usually induces changes in the cellular biochemistry and physiology of the whole plant (Gerendas and Raticliffe, 2000) The biological effects often include visible symptoms of injury, including chlorosis, necrosis, or reduction in root and shoot growth Other effects are invisible, such as the presence of high concentrations of H + and Al + ions, effects on membrane and ion transport systems, reduced photosynthesis, altered water balance, and variation in enzyme activities (Velikova et al., 2000) In addition, acid stress is accompanied by changes in endogenous hormones that, in turn, cause changes in related physiological processes Similarly, many plants develop severe chlorosis when grown in alkaline soils due to the reduced availability of iron and manganese at high pH Other species, however, are well adapted to such conditions at the extremes of pH Thus, the biological effects are numerous and complex, and similar effects occur under conditions of high salinity or low levels of both macro- and micronutrients Many research efforts are under way to characterize how certain plants are able to tolerate such environmental stresses while others are not.
Essay on the Effects of Different Light Intensities on the Production of
Camptothecin in the Tree of Joy ( Camptotheca accuminata )
In the following experimental example, University of Michigan biology students, Atul Rustgi, Ashish Goyal, and Kathryn Timberlake provide an essay on their bach- elor’s degree research project covering the effects of different light intensities on camptothecin levels in tree of joy plantlets.
In previous experiments, it was shown that a decrease in light intensity will increase the production of camptothecin (CPT) (Liu and Adams, 1996) The objective of the following example was to test such effects of light intensity on the production of CPT in tree of joy plants.
Three trays containing seedlings of Camptotheca accuminata were grown in a green- house Each tray contained plants of the same age and height Each tray of plants was exposed to a particular light intensity different from that of the other two trays In each tray, the seedlings were arranged in two rows The first tray received no shading and had a light intensity at the top of the plants of 3000 μEãm –2 ãs –1 The second received
104 Natural Products from Plants, Second Edition
1× shading by means of a thin wire screen that was held above the plants by four posts at each corner of the tray The light intensity measured at the top of this set of plants was 750 μEãm –2 ãs –1 The third tray received 2ì shading by means of two wire screens The light intensity measured at the top of this set of plants was 300 to 400 μEãm –2 ãs –1 (see Figure 3.2 and Figure 3.3).
At the time of setup, a random sampling of the largest top leaves of the plants was taken This was done in order to get a measurement of the initial concentration (T0) of CPT in these seedlings before any experimental variables were induced The fol- lowing procedure was used to determine the concentration of CPT in the T0 samples and in successive samples:
1 Freeze leaves in liquid nitrogen
2 Crush to a powder using mortar and pestle
3 Add 1 g of crushed leaves to a beaker containing 50 ml of methanol (MeOH)
6 Transfer liquid portion to a clean beaker
FIGURE 3.2 Photograph of students Ashish Goyan, Kathryn Timberlake, and Atul Rustgi measuring light intensity with a photo flux density meter (Ly-Cor, Inc.) in their shading experiment with seedlings of tree of joy, Camptotheca accuminata (Photo courtesy of David Bay.)
FIGURE 3.3 Shading experiment with tree of joy ( Camptotheca accuminata ) seedlings grown at three different light intensities in the greenhouse at the University of Michigan (Photo courtesy of David Bay.)
Regulation of Metabolite Synthesis in Plants 105
8 Add 0.01 g of dried filtrate to 400 μl of refrigerated MeOH in order to avoid evaporation
10 Use a sonicator to fully dissolve the residue
11 Analyze 10 μl of sample by high-performance liquid chromatograpy (HPLC) (Liu et al., 1997)
For HPLC analysis, 10 μl injections were run on a C-18 column using a gradient of
A reversed-phase HPLC method was developed to analyze Camptothecin (CPT) in extracts of Nothapodytes nimmoniana Graham (Icacinaceae) for quality control purposes The mobile phase consisted of 20 to 80% acetonitrile (ACN) over 60 minutes, with a detection wavelength of 347 nm, temperature of 40°C, and flow rate of 1 ml/min Peak identification was confirmed by spiking a T0 sample with additional CPT.
500 µl of MeOH was added to the sample The graph for this run showed a noticeably larger peak compared to a run without the extra CPT (Figure 3.5), indicating the peak represents CPT This peak's area corresponds to the amount of CPT in the injection.
At T1 (week 1), six leaves were taken from each of the three trays The six leaves were a collection of the largest top three leaves of two different plants in the same row of a particular tray For the next 4 weeks, leaves were taken from the tops of the plants of a new row so as to avoid getting young buds from a plant with leaves that were removed the preceding week The six leaves were then used in the procedure described above in order to obtain data The CPT peaks for the chromatographs of these successive trials could be identified by comparing the new chromatographs to that of T0 and searching for similarities in the shape of and the time of elution of the CPT peak Standard curves using purified CPT were also run during each analysis.
FIGURE 3.4 High-pressure liquid chromatography trace illustrating camptothecin peak from nonspiked sample extract from tree of joy ( Camptotheca accuminata ) seedlings.
106 Natural Products from Plants, Second Edition
With a standard curve, the amount of CPT in unknown samples can be determined.
A sample chromatogram for 2.87E-03M sample is shown in Figure 3.4 The standard curve results and a graphical representation are shown in Table 3.1 and Figure 3.6, respectively.
The results for the three different amounts of shading are shown in Table 3.2, Table 3.3, and Table 3.4.
A sample chromatograph of the first run (T1) is presented in Figure 3.5 A graphical representation of a comparison of all three runs is shown in Figure 3.7.
The data show that Run 1, which had no shading, had a slow decrease in the amount of CPT production up to week 1 Thereafter, there was a continuous slow rise in the
FIGURE 3.5 High-pressure liquid chromatography trace illustrating camptothecin peak from a camptothecin-spiked sample extract from tree of joy ( Camptotheca accuminata ) seedlings.
Data on Areas under Curves for Respective Camptothecin Concentrations
Note : Also used for the calculation of the standard curve for camptothecin in Figure 3.6.
Regulation of Metabolite Synthesis in Plants 107
FIGURE 3.6 Standard curve for concentration of camptothecin plotted against areas under the high-pressure liquid chromatography peaks.
Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown without Artificial Shading
No Shading (Run 1) Time (days) Area under the Curve Amount of Camptothecin (moles)
Note : Simulated full sunlight conditions.
Time-Course Changes in Camptothecin Levels in Tree of Joy Seedlings Grown under 1× (Partial) Shading Conditions
1 × Shading (Run 2) Time (days) Area under the Curve Amount of Camptothecin (moles)
Time-Course Changes in Camptothecin Levels of Tree of Joy Seedlings Grown under 2× (Deep) Shading Conditions
2 × Shading (Run 3) Time (days) Area under the Curve Amount of Camptothecin (moles)
Area under the CurveConcentration of Camptothecin (moles)
Regulation by Biotic Stresses
Unlike environmental stresses, which are predominantly the result of nonliving components of a plant’s environment, biotic stresses are the result of living components of the environment Herbivory (a process where herbivorous animals, insects, and mollusks eat plants as a food source) is one such biotic stress.
According to Larcher (1995), biosynthesis of defense metabolites in plants is often induced or enhanced by herbivory For example, intensively grazed grasses (members of the grass family, Poaceae) frequently contain more biogenic silica than grasses in nongrazed areas Further, damage to plants elicited by herbivores causes an increase in the amounts (per unit dry weight) of polyphenols, tannins, and terpenes.
Such increases occur within the tissues of many plant species, such as birch (Betula spp.) and poplar
Trees (Populus spp.) exhibit increased production of defense metabolites (compounds) in response to herbivory (insect and mollusk attacks) However, this upregulation of defense comes at a cost, as it reduces biomass production Therefore, plants benefit from downregulating defense compound production when not under attack.
Humans can make use of biotic stresses, such as herbivory, to increase yields of desired plant metabolites As cited in Section 3.2, Liu and Adams (1996) showed that bark tissue contains significantly higher amounts of the medicinal metabolite, camptothecin (CPT), than wood tissue by a factor of two in both roots and stems As these trees grow larger in diameter, the proportion of bark tissue decreases substantially Because bark tissue contains significantly more CPT per unit dry weight, Liu and Adams said that it is desirable to grow smaller-diameter trees with many branches present, because the ratio of bark to wood is much greater in such shoots To achieve this condition, simulated herbivory, using coppicing (cutting of trees at ground level to stimulate the development of new, vigorous shoot growth
[“sucker” sprouts”]), will induce the trees to regenerate plants with multiple, small shoots These shoots can then be collected for the extraction of higher yields of CPT.
In Chapter 2, we mentioned that conifers secrete oleoresin (turpentine and rosin) in response to wounding and attack by insects (e.g., bark beetles) and fungal pathogens This is well documented in the classic work by Funk et al (1994) on the occurrence of oleoresinosis in grand fir (Abies grandis) elicited by physical wounding The wounding treatments simulate the wounding that occurs after an attack on stem bark tissues by bark beetles This wounding is achieved by making a series of l mm cuts approximately 3 mm apart along the entire stem on opposite sides of 6-week-old saplings The extent of upregulation of oleoresin biosynthesis by these treatments is substantial Over a 20-day period, one finds an accumulation of a viscous mass of resin acids and the release of volatile monoterpenes at the sites of wounding In response to an attack by the bark beetle (Scolytus ventralis), these oleoresins deter further attack by the beetles and act directly to kill eggs and larvae of the insect as well as to seal its wound (Funk et al., 1994).
There are some very interesting stories dealing with the action of volatile compounds produced in response to herbivory These compounds do not always act directly on the attacking organism For example, during the wounding caused by beet armyworm caterpillars feeding on plant leaves, the insect may produce an oral secretion of a recently discovered fatty-acid-based elicitor/signal called volicitin
(N-(17-hydroxylinolenoyl)-l-glutamine) This elicitor, when applied to damaged leaves of corn (Zea mays) seedlings, induces the seedlings to release a mixture of volatile compounds (octadecanoid-jasmonate signal complex) that attract females of parasitic or predatory wasps (natural enemies) These wasps then kill the feeding caterpillars, thus removing the biotic stress from the plants (Alborn et al., 1997).
There are, of course, many other forms of biotic stresses that plants may encounter Plants need to deal with attack not just from animals and insects, but also, from pathogenic bacteria, fungi, and even some parasitic plant species Under certain circumstances, plants need to deal with the waste products of various organisms, including those from large herds of herbivores, large flocks of birds, and especially, human activity Some of these biotic stresses overlap with the environmental stresses as different living organisms slowly change their environment Changes may occur in pH (as is seen from the acidification activity of sphagnum mosses in bogs) or the nutrient content of the soil (as is seen in developing forests, where generations of trees slowly alter the soil) Plants have developed an elaborate network of biochemical pathways that allow them to respond and deal with all of these changes, whether caused by bacteria or humans.
Biochemical Regulation
Apart from environmental and biotic factors, which influence the synthesis of plant metabolites, there are also factors or conditions acting within the plant that influence the activity of the biochemical pathways An understanding of these factors and how they influence the individual steps of metabolic pathways holds significant benefits for humans Some examples are given in the following sections.
3.4.1 Metabolite Feeds and Radioactive Precursors
One of the traditional ways by which researchers study the pathways for synthesis of plant metabolites is to use 14C-labeled metabolites, especially those that are known precursors in a given metabolic pathway This not only helps one to identify intermediate substrates in a given pathway, but also, helps one to determine the rate-limiting step of that pathway If the rate-limiting step in a pathway that produces a metabolite of interest can be discovered, it is possible to upregulate the synthesis of that metabolite by (1) upregulating gene expression for the enzyme that catalyzes the rate-limiting step (see Section 3.5.2), (2) enhancing enzyme activity (in effect, lowering the Km or affinity of the enzyme for its substrate) by feeding cells with the rate-limiting enzyme’s preferred substrate or by increasing substrate concentration (see Section 3.4.2), and (3) removing the end product of the rate-limiting step to stimulate flux through the pathway (see Section 3.4.6) A study illustrating the use of isotopes to help understand where and how upregulation of metabolite biosynthesis occurs is that of Funk et al (1994). Here, they focused on the oleoresin biosynthetic pathway using in vivo [14C] acetate feeding and analysis of intermediates produced by their respective enzyme activities Two cytochrome P450-dependent diter- penoid hydroxylases involved in the synthesis of (–)-abietic acid (the principal resin acid in grand fir,
Ten days after wounding, Abies grandis trees significantly increase their production of resin acids in wounded stems, ranging from 5- to 100-fold compared to unwounded stems This dramatic increase in resin acid activity effectively deters bark beetle attacks, as described in Section 3.3 of the article.
Substrate concentration elevation is a technique employed to increase end-product biosynthesis in biochemical research For instance, invertase hydrolysis of sucrose to glucose and fructose exemplifies this effect Increased sucrose levels in source cells stimulate invertase activity, leading to the removal or metabolism of end products This action further upregulates other metabolic pathways, such as fructan synthesis in vacuoles, cellulose synthesis in cell walls, and starch synthesis in chloroplasts and amyloplasts.
3.4.3 Enzyme Activity Regulation by Protein Phosphorylation/Dephosphorylation and Cytosolic Calcium in Signal Transduction Pathways
Other biochemical factors that influence the production of metabolites act upon the structures of enzymes. For example, in plant cells, there are enzymes called protein kinases that act to phosphorylate (using a phosphate contained in ATP) other enzymes at particular amino acid residues The additional phosphate group changes the conformation of the enzyme to which it is attached, thus either activating or, in some cases, inhibiting the enzyme The phosphorylated enzyme can return to its original state through the action of other enzymes called phosphatases, which release the inorganic phosphates attached by the kinases Such mechanisms are very important in carbon fixation through photosynthesis via RuP2-Case (Rubisco or ribulose bis phosphate carboxylase) in chloroplasts and dark fixation of carbon dioxide via PEP carboxylase (phosphoenolpyruvate carboxylase) Signal transduction cascades involving calm- odulin-Ca 2+ activation of protein kinases and phosphates, involved in protein phosphorylation/dephos- phorylation reactions downstream in these cascades, are one of the primary mechanisms for enzyme activation (Anderson and Beardall, 1991) One of the key players here is cytosolic calcium (Ca 2+ ) Once it is released from the endoplasmic reticulum (ER), it can bind to the calcium-binding protein, calm- odulin, which in turn, can activate specific protein kinases involved in protein phosphorylation reactions.
This is of current interest to plant biologists, because the control of cytosolic calcium plays a key role in gravitropic response mechanisms in roots and shoots, where one of the key metabolites is the plant protein, calmodulin.
3.4.4 Regulation by Acetylation, Prenylation, and Glycosylation
Regulation of gene expression at the level of transcription and protein stability are two of the primary consequences of acetylation reactions involving the acetyl group, CH 3 –C=O How does this work? In the case of DNA, histone acetylation increases the access of transcription factors to DNA in the nucleosome by causing weak internucleosomal interactions, whereby histone tails do not constrain DNA.
In contrast, deacetylation reactions bring about strong internucleosomal interactions, whereby histone tails constrain the wrapping of DNA on the nucleosome surface In connection with protein stability, acetylation of the N-terminus of a protein by acetylation of the α-amino group is thought to increase the life of a protein by protecting it from proteolysis (Buchanan et al., 2000) The mechanism by which this occurs is currently unknown.
Regulation by prenylation refers to the addition of the 15-carbon farnesyl group or the 20-carbon geranyl-geranyl group to acceptor proteins, both of which are isoprenoid compounds derived from the cholesterol biosynthetic pathway The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C–S–C) A common consensus sequence at the C-terminus of prenylated proteins was identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine), and X is the C-terminal amino acid In order for the prenylation reaction to occur, the three C-terminal amino acids (AAX) are first removed, and then the cysteine is activated by methylation in a reaction utilizing S-adenosylmethionine as the methyl donor Important examples of prenylated proteins include the oncogenic GTP-binding and hydrolyzing protein Ras and the g-subunit of the visual protein transducin, both of which are farnesylated Numerous GTP-binding and hydrolyzing proteins (termed G-proteins) in the signal transduction cascades have g-subunits modified by geranyl- geranylation In plants, the biosynthesis of the monoterpene olefins and abietic acid constituents of diterpenoid resin (also known as pitch) from grand fir, Abies grandis, also involves prenylation reactions
(see Figure 2.26 and Funk et al., 1994, referenced in Chapter 2).
Glycolipids and glycoproteins are synthesized through glycosylation reactions catalyzed by glycosyl transferases Glycolipids, localized in plastid membranes or the ER, exhibit distinct fatty acid compositions depending on their location Plastid glycolipids contain abundant C16 polyunsaturated fatty acids (e.g., peas), while ER glycolipids are rich in C18 polyunsaturated fatty acids (e.g., spinach).
Many cell surface proteins and secretory proteins carry polysaccharide moieties that are either used as signaling devices within the biosynthetic pathway (e.g., N-linked glycosylation) or are involved in the extracellular matrix (ECM) function of proteins (e.g., O-linked glycosylation) Glycosylation of newly synthesized membrane and secretory proteins is part of the sorting mechanism within the cell and transport to their final destination The cellular locations of glycosylation are the lumen of the ER andGolgi (dictyosome) membrane stacks as well as the grana and intergranal membranes of plastids Glycosylation reactions are also important in IAA (indole-3-acetic acid) metabolism, where glycosyl derivatives include IAA-glucose and myo-inositol-linked IAA Both are considered to be “storage forms” of IAA that release “free IAA” via deglycosylation reactions Another example is found in the seeds of edible legumes In the seeds, most of the isoflavones are stored as glucosyl conjugates, such as genistin and daidzin When the seeds germinate, the respective aglycones, genistein and daidzein, are released via the action of β-glucosidases Genistein is an important receptor molecule in root hairs, where nitrogen fixation by rhizobacteria is initiated It is also very important in deterring attack of legume seedlings by pathogenic fungi (see below) In humans, it is important in preventing the development of colon cancer and osteoporosis (see discussion in Kaufman et al., 1997)
3.4.5 Activation with Fungal Elicitors and Plant Growth Regulators
During the course of evolution, plants evolved intriguing defense strategies against attack by fungal pathogens that cause disease When the fungus attacks the plant, it may synthesize and secrete into the plant’s cells various fungal cell-wall polysaccharides (e.g., chitin, made up of N-acetyl-D-glucosamine) that we call elicitors Such elicitors can act to upregulate the synthesis of specific plant metabolites called phytoalexins (compounds that kill attacking fungal pathogens) Two such phytoalexins are the isoflavonoids, genistein and daidzein In seedlings of soybeans and other members of the bean family (Fabaceae), the levels of these compounds increase dramatically when the plant is attacked by a fungal pathogen They are toxic to the fungal pathogen and act to kill the fungus This has an application with miso and tempeh, both fermented soybean food products Miso is made by culturing soybean curd with the fungus, Aspergillus oryzae The fungus secretes fungal elicitors that cause the soybean to synthesize significantly higher levels of genistein and daidzein This, in turn, produces the food’s distinct flavor It is also of interest that these two isoflavonoids are very important in preventing colon cancer and in treating patients suffering from alcoholism (Duke, 1995).
Naturally occurring or synthetic plant growth regulators were used to upregulate the biosynthesis of enzymes that produce useful metabolites either in intact plants or in plant cell cultures A few of the classic examples are as follows:
• The induction of synthesis and rate of flow of latex (made up mostly of polyterpenes found in the latex of the stems) from wounds in the bark of Brazilian rubber trees (Hevea brasiliensis) by the naturally occurring plant hormone/growth regulator, ethylene (Schery, 1972; Weaver, 1972).
• The induction of synthesis of invertase (β-fructofuranosidase) by the naturally occurring plant hormone/growth regulator, gibberellic acid (GA 3 ), in elongating stems of cereal grasses (Kauf- man and Dayanandan, 1983).
• The induction of synthesis of α-amylase in germinating seeds of cereal grains by the plant hormone, GA 3 , which triggers the hydrolysis of starch to sugar (D-glucose); this action by GA 3 on α-amylase activity is utilized in beer brewing, using modified barley (Hordeum vulgare) substrate (the D-glucose derived from starch stored in the grains) (Jacobsen et al., 1995).
Molecular Regulation
Because the production of every enzyme, along with the enzyme’s location and function within the cells of a given plant, are ultimately controlled by the sequence of nucleotides on strands of DNA, one last category of factors that influence metabolite biosynthesis will be considered These factors interact with the DNA molecules to regulate the activity of the genes that govern the individual enzymes of each pathway.
3.5.1 Regulation of Gene Expression in Plants Occurs on Many Levels
Gene expression involves the production of mRNA, proteins, and final products, with each level regulated by distinct molecular mechanisms These mechanisms vary between genes and are complex, but some examples will be discussed in Chapter 5 This article focuses on the fundamental aspects of gene expression to provide a simplified overview.
The steps of gene expression that take place in the nucleus to produce messenger RNA (DNA → gene
→ primary DNA transcript → mature mRNA) are known as transcription The steps of gene expression that take place in the cytoplasm to form polypeptide chains from this mRNA (mRNA on the ribosomes
→ synthesis of polypeptides → formation of functional protein) are called translation These primary steps are depicted in Figure 3.8, along with a partial list of factors that may influence both transcription and translation at differing times and under differing conditions within differing tissues For example, as depicted in Figure 3.8, degradation of DNA, mRNA, and functional proteins can occur when the appropriate hydrolases are present (DNAases, RNAases, or proteases) Synthesis and degradation of DNA, mRNA, and functional proteins are very important processes in gene regulation and are known as turnover When the rate of synthesis exceeds the rate of degradation, there is a net synthesis of DNA, RNA, or protein; when the converse occurs, there is a net loss of DNA, RNA, or protein This has a direct impact on the amount of production of a given enzyme within a given pathway, as well as the resulting production of a final product.
Also shown in Figure 3.8 are a variety of post-translational modifications that may be required prior to the production of a functional protein (see Section 1.6.3 for a discussion of protein folding) As described in Sections 3.4.3 and 3.4.4, these may include the addition of permanent or temporary chemical modifications through the processes of phosphorylation, acetylation, phenylation, glycosylation, or methylation Each of these processes is also under its own form of molecular regulation Likewise, each protein is only functionally active within a specific location in the cells of specific tissues, and there are many regulatory mechanisms that govern the proper localization of each protein to its appropriate cellular compartment Sometimes, the functional activity of a protein also requires interaction with other protein components, and there are factors that control such interactions in space and time In addition, synthesized mRNA and protein can be stored within the cells for later use, when changing environmental conditions trigger their activation (e.g., long-lived mRNA in seeds and animals eggs; storage proteins in seeds) Thus, the absolute level of gene expression in a cell or tissue is not only dependent on the levels of synthesis, degradation, and storage of DNA, mRNA, and protein, but also, on the timing of chemical modifications, protein–protein interactions, and proper spacial localization Only then can the gene perform its destined function in metabolite production We refer to this complex system of regulation of the steady-state level of such metabolites in cells as homeostasis.
3.5.2 How Plant Genes Are Turned On and Off
As described above, regulation of gene expression can occur at the level of transcription (DNA to RNA), post-transcription (initial RNA transcript to mRNA, translation mRNA to polypeptide), or post-transla- tion (polypeptide to functional protein) These levels of regulation are controlled by a wide range of environmental and developmental signals The mechanisms of regulation are often complex and diverse; so it is a purpose of this section to give the reader an appreciation of this diversity.
What are some of the environmental and developmental signals that regulate gene expression in plants? Basically, they can be any of the environmental, biotic, and biochemical factors that we discussed in Sections 3.2, 3.3, and 3.4 Fundamentally, plants respond to each of these factors at a molecular level by altering the levels of expression of various genes For example, the presence of light may upregulate the synthesis of the mRNA of light-harvesting complexes involved in photosynthesis This is mediated by the phytochrome system involving red and far-red wavelengths of light On the other hand, reduced levels of light may increase the biosynthesis of camptothecin (CPT) due to an increase in the expression of genes within this pathway (see essay in Section 3.2) Other signals are stresses elicited by such factors as ultraviolet light, wounding, or pathogen attack, which can upregulate, at the level of transcription, the synthesis of such enzymes as PAL that leads to synthesis of phenylpropanoid compounds The expression of other enzymes, however, is reduced by the same stresses Still other signals can be attributed to plant hormones that are bound by protein transcription factors within the cell For example, in germinating cereal grass seeds, gibberellins (GAs) can cause de novo synthesis of mRNAs for α-amylase that break down starch to sugar and of proteases that can break down stored proteins in seeds In contrast,
FIGURE 3.8 The primary steps in gene expression and the control points that occur at steps leading from DNA to mRNA and protein synthesis in cells.
Functional Protein proteases Degradation of Protein
Degradation of DNA transcription pore in nuclear membrane exported to cytoplasm translation
*Packing of DNA in chromosome
*Availability of DNA as a template for RNA synthesis.
*Levels or activity of RNA polymerases
*Availability of transcription factors and their interaction with regulatory sequences associated with the gene.
*Targeting (e.g cell wall or organelles).
*Co-factors the plant hormone, abscisic acid (ABA) turns off such gene expression in germinating seeds and is partly responsible for the dormancy of these seeds as well as the dormancy of the buds of temperate-zone trees. The precise mechanisms by which environmental or developmental signals act to control gene expres- sion are not yet completely understood But, research so far has allowed several mechanisms to be promulgated, including the following (some of these examples are expanded upon in Chapter 5):
• The signal (such as GA, gibberellin, or ABA, abscisic acid) could stimulate the synthesis of a protein regulatory factor that binds to particular trans -acting (other proteins) or cis -acting (DNA sequence) elements located upstream in the promoter region of a gene to turn the gene on (as in the case of GA) or off (as in the case of ABA) in gene expression.
• The signal (such as a cytokinin plant hormone that acts to stimulate red-light-induced synthesis of RuP2-Case and light-harvesting complex [LHCP] in greening tissues of duckweed, Lemna gibba) may act to stabilize particular mRNA species, retarding the degradation of the initial
RNA transcripts or mRNA produced from a given gene (Anderson and Beardall, 1991).
Antisense orientation in genetic engineering, such as in the FLAVR SAVR™ tomato, can hinder the function of genes This occurs when the introduced gene of interest is positioned in the reverse orientation, preventing normal RNA function by binding to its complementary RNA molecule In the case of the FLAVR SAVR™ tomato, the antisense orientation of the pectinase gene effectively knocked out its expression, inhibiting the production of pectinases, which are enzymes responsible for fruit ripening.
To produce such transgenic plants as the FLAVR SAVR™ tomato, there is a specific order of questions and answers that must be elucidated In natural products research, one of the first important biochemical questions to ask is “how is the metabolite of interest synthesized?” Another is “what are the enzymes for the respective steps in the pathway?” These are not easy questions to answer, but once these enzymes are isolated and purified, then the molecular biologist can potentially clone the genes that make these enzymes, determine their nucleotide sequences, and characterize their expression patterns within the various plant tissues (see Chapter 5) At this point, the pathway for the metabolite of interest will be well understood, and a new question arises How can the expression of the gene(s) for the rate-limiting enzyme(s) in the biosynthetic pathway be upregulated, or downregulated, so as to make more, or less, of the metabolite of interest through genetic engineering protocols? These protocols include the use of constitutive or super promoters attached upstream of the gene, the use of constructs to suppress gene expression, and the use of genetic transformation to express the gene of interest in organisms that normally do not express this gene If all of the biochemistry is done properly, including (1) the purification of the proteins of interest, (2) the characterization of any isozymes or gene family members for the particular enzyme being studied and their ultimate site(s) of action in the cell, and (3) the elucidation of the function of the enzymes in cell metabolism, then the above-outlined molecular biology work is not only feasible, but also, allows one to turn specific genes on or off in a particular metabolic pathway, thus changing the production of specific metabolites In doing this kind of work, risk assessments are absolutely necessary to determine if a particular transgenic plant can have any detrimental effect on human health or on the environment These are discussed in detail in Redenbaugh et al (1992), Rissler and Mellon (1996), Krimsky and Wrubel (1996), as well as in Chapters 7 and 12.
3.5.3 Transcription Factors Involved in Pathway Regulation
Regulatory proteins called transcription factors function by binding to the promoter of a gene, and in some cases, to additional regions called enhancer and repressor regions Binding to the promoter can have either a stimulatory effect or a suppressive effect on gene activity In addition, enhancer regions may be located at a distance from the gene that they stimulate Many transcription factors are necessary for RNA polymerase to attach just prior to transcription In many cases, transcription begins when the factors at the promoter region bind with the factors at the enhancer region, creating a loop in the DNA.
An example of this looping is depicted in Figure 3.9.
Hundreds of different transcription factors have been discovered; each recognizes and binds with a specific nucleotide sequence in DNA In many cases, a specific combination of transcription factors is necessary to activate each given gene A good example of this is the MADS-box class of transcription factors that control flower development in plants (see below).
Transcription factors are also regulated by signals produced from other molecules For example, hormones can activate transcription factors, and thus, enable the activation and transcription of certain genes In connection with the hormone, indole-3-acetic acid (IAA) or auxin, Nemhauser and Chory (2005) summarized recent work from several labs that led to the discovery of the long-sought-after auxin-binding or receptor protein The scenario goes like this: When IAA is present in zero or low amounts, transcriptional repressor proteins (Aux/IAA) remain bound to an auxin response tran- scription factor (ARF) As a result, target genes of auxin action remain switched off, and the develop- mental process (e.g., embryogenesis in Arabidopsis) does not occur However, if auxin is present in higher amounts, it interacts with leucine-rich repeat F-box proteins called TIR1/AFBs, which are involved in ubiquitin-mediated protein degradation When Aux/IAA proteins bind to auxin-modified TIR1/AFBs, the ARF auxin response transcription factor is no longer repressed As a consequence, the expression target genes for embryogenesis are turned on, and embryogenesis ensues.
Essay on MADS-Box Transcription Factors
Consistent with their function in regulating the expression of other genes, the MADS- box genes encode transcription factors present in animals, fungi, and plants The term