C
arotenoids have recently been the fo-cus of intense interest in both plant biology and medicine. They have im-portant photosynthetic functions (photopro-tection and light-harvesting) and some of their biosynthetic enzymes are targets for bleach-ing herbicides. It has also been suggested that they may provide protection against cancer and heart disease1.The carotenoids are among the most wide-spread of all natural pigments, and are respon-sible for many of the colours appearing in animals, plants and micro-organisms (Fig. 1). These molecules are important in photosynthe-sis because they carry out three major functions. First, they absorb solar energy in the blue-green region of the spectrum and transfer it to the chlorophylls (this is the antenna function or accessory light-harvesting role); second, they are important for the assembly and stability of some of the photosynthetic pigment-protein complexes (a structural role)2,3; and third, they act as photoprotectors by preventing the for-mation of singlet oxygen and by directly quenching this and other harmful free radicals4. The colour of a carotenoid is principally a consequence of the number of conjugated double bonds. As the number of double bonds increases the carotenoid absorbs light further to the red region of the spectrum. Most of the desaturation steps occur in the early stages of the biosynthetic pathway. Subsequent modifi-cations and embellishments, such as the cy-clization of end groups or the introduction of hydroxyl, methoxyl and glucoside groups, can have additional and more subtle spectral conse-quences. The wide diversity of carotenoids seen in nature is a result of the different com-binations of desaturation and modification of the end groups.
Function of carotenoids in photosynthesis
The one essential function of carotenoids in photosynthesis is photoprotection. If either bacteriochlorophyll (Bchl) or chlorophyll (Chl) is illuminated in the presence of oxygen (Fig. 2), triplet excited Bchl (or Chl) will sensitize the formation of singlet oxygen (1DgO
2*). This is a destructive oxidizing agent that can rap-idly kill cells. Carotenoids prevent this harm-ful reaction by rapidly quenching the triplet sensitizer and by direct quenching of singlet oxygen. The energy level of singlet oxygen is 294 kJ mol21, and any carotenoid whose trip-let state energy is less than this will be effective in quenching5. In essence, this necessitates the presence of carotenoids with more than seven conjugated double bonds (neurosporene and beyond).
The second major function of carotenoids is as accessory light-harvesting molecules. They absorb light at wavelengths between 400 and 550 nm and transfer the energy to the (B)chls, thereby extending the spectral range over which light can drive photosynthesis. The pref-erential incorporation of different carotenoids in different parts of the bacterial photosyn-thetic apparatus has been observed6. This may be due to differences in their light-harvesting, photoprotective or structural roles in different locations. For photosynthesis, the two main functions of carotenoids (which reflect the photochemical properties of the carotenoid excited triplet and singlet states respectively) occur efficiently in vivo only because the car-otenoids are packaged, together with the (B)chl, into both the reaction centre and light-harvesting complexes. Specific, short-range (,5Å) contacts are made between carotenoid and (B)chl pigments. Such contacts can now
be observed in atomic detail and have been elucidated by studies on the structure of the light harvesting LH2 complexes from the photo-synthetic bacteria Rhodopseudomonas acido-phila7 and Rhodospirillum molischianum8 (Fig. 3). The structures of the carotenoids rhodopin glucoside and lycopene, respectively, emphasize the point that the photosynthetic prokaryotes display great diversity in their carotenoid content. The colour of cultures of photosynthetic bacteria is almost entirely due to the carotenoids because bacteriochloro-phyll, unlike chlorobacteriochloro-phyll, is a pale blue colour (Fig. 1a). Different species of photosynthetic bacteria display a wide range of different colours – from green through orange, brown and red to deep purple. Although we might ex-pect these colour differences to reflect the fact that each species has a completely distinct set of biosynthetic enzymes, there may be a sim-pler explanation. We propose that this variety is achieved using only a modest genetic out-lay, primarily by adjusting the number of desaturations at the outset of the biosynthetic pathway. This idea has been tested by swap-ping the native phytoene desaturase, which car-ries out three desaturations, with one capable of carrying out four desaturations.
Pathways of carotenoid biosynthesis Carotenoids are synthesized by a specialized branch of the isoprenoid or terpenoid pathway that is also used for the biosynthesis of a wide variety of important compounds, such as chol-esterol, quinones and steroid hormones9. All isoprenoid compounds are built up from the C5isoprene unit, the carbon skeleton of which is clearly seen in the important intermediate isopentenyl diphosphate (IDP). IDP then undergoes a double bond isomerization to give dimethylallyl diphosphate (DMADP). The first condensation between DMADP and IDP gives the C10 compound geranyldiphosphate and further additions of IDP extend the chain to give the C20 compound geranylgeranyldi-phosphate (Fig. 4). Geranylgeranyldigeranylgeranyldi-phosphate gives rise to the carotenoids and to the diter-penes including phytol, which provides the isoprenoid sidechain of the chlorophylls.
The carotenoid-specific pathway begins with the synthesis of the C40precursor phytoene, which has three conjugated double bonds. This is then desaturated by phytoene desaturase to produce neurosporene (three desaturations; nine conjugated double bonds) in Rhodo-bacter13, or lycopene (four desaturations; 11 conjugated double bonds) in Erwinia14, plants and other photosynthetic bacteria such as Rhodopseudomonas acidophila and Rhodo-spirillum rubrum. A small number of subse-quent enzymes introduce further double bonds, hydroxy and methoxy groups and, in Erwinia and plants, cyclize the ends of the molecule to produce a range of final products.
Carotenoid diversity:
a modular role for the
phytoene desaturase step
Guillermo Garcia-Asua, Helen P. Lang,
Richard J. Cogdell and C. Neil Hunter
There is structural conservation of early carotenoid biosynthesis enzymes in evolution-arily diverse prokaryotes15. However, phyto-ene desaturases (Pds) in higher plants, algae and cyanobacteria seem to be phylogeneti-cally unrelated to the structurally and func-tionally conserved CrtI-type desaturases in purple bacteria and fungi16. Phytoene de-saturases catalyse the introduction of only two double bonds into phytoene to form z -carotene17, which is further desaturated to lycopene by a second enzyme, z-carotene de-saturase18. CrtI enzymes catalyse four desatu-rations or three in the case of Rhodobacter. Differences between Pds and CrtI provide an excellent target for herbicides and it has been shown that cyanobacteria transformed with crtI acquire resistance to norflurazon19.
Genetic manipulation of carotenoid biosynthetic pathways
The genes encoding the carotenoid biosyn-thetic pathway enzymes have been cloned and sequenced in two species of photosynthetic bacteria (Rhodobacter capsulatus and Rhodo-bacter sphaeroides) and the non-photosynthetic bacterium Erwinia herbicola, which produces plant-type carotenoids. In Rb. sphaeroides, for example, the genes are clustered within a 9.1 kb region of the chromosome20, which is contained within a larger 45 kb cluster of photo-synthesis related genes21. Transposon muta-genesis has been used to map these genes, and to produce a range of strains in which differ-ent carotenoid biosynthesis genes have been inactivated20. The coloured carotenoids – neurosporene (nine conjugated double bonds) and beyond – are essential structural elements during the assembly of the peripheral antenna complex, LH2 (Refs 2, 3). Interestingly, how-ever, both the reaction centre and the core antenna complex (LH1) do not have this re-quirement, and can be successfully assembled in the absence of carotenoids22.
446
perspectives
November 1998, Vol. 3, No. 11
Fig. 2. The role of carotenoids in photoprotection. (a) and (b) Excess light can induce the creation of triplet excited (bacterio)chlorophyll molecules. (c) These triplet excited species can sensitize the formation of singlet oxygen, which is toxic to the cell. (d) Close contact of either 3
(B)chl* or 1D
gO2* with carotenoid molecules results in these harmful species being quenched. (e) Excited carotenoid molecules dissipate energy in the form of heat to return to the ground state.
(B)chl 1(B)chl*
(a)
(b)
(c)
(d)
(e)
hv hv
(Singlet excited)
(Triplet excited)
(Singlet oxygen)
1(B)chl* 3(B)chl* 3(B)chl*1 O
2 (B)chl 11DgO2* 3(B)chl* or 1DgO
2* 1 Carotenoid (B)chl or O213Carotenoid* 3Carotenoid* Carotenoid 1 heat
Fig. 3. The carotenoids of the light-harvesting LH2 complexes of (a) Rhodopseudomonas
acidophila and (b) Rhodospirillum molischianum shown in atomic detail. The red carotenoid
is rhodopin glucoside; the orange/brown carotenoid is lycopene. The two types of (B)chl molecules, with different absorption maxima at 800 and 850 nm (B800 and B850), are light-green and dark-light-green respectively.
Splicing carotenoid biosynthetic pathways to genetically engineer carotenoid diversity Genetic complementation of transposon mu-tants with homologous genes from other or-ganisms is a valuable tool to investigate the importance of each step in the pathway. The replacement of inactive genes with foreign counterparts also provides an insight into the enzyme specificity of each reaction.
Overexpression of Erwinia carotenoid bio-synthesis genes in Escherichia coli cells re-sults in their acquiring a yellow pigmentation23.
The crtYIBZ genes from E. herbicola were transferred into a range of Rb. sphaeroides car-otenoid biosynthesis mutants by conjugation. In a strain bearing a crtI deletion, these genes enabled the lost function to be replaced by its Erwinia counterpart, and carotenoids charac-teristic of wild-type Rb. sphaeroides were syn-thesized24. This is in spite of the fact that in vitro experiments with Erwinia crtI clearly showed that this enzyme performs four de-saturations14. However when a recipient strain bearing a crtC deletion was used, carotenoids
characteristic of Erwinia were produced. This experiment provided a clear demonstration that ‘foreign’ carotenoid biosynthesis genes het-erologously expressed in Rb. sphaeroides can give rise to a new pathway. The dramatic re-sult of this was to convert Rb. sphaeroides cul-tures from red-brown to a spectacular bright orange colour.
It was important to investigate further the consequences of gene complementation down to the level of a single gene replacement. It is known that many photosynthetic bacteria (for Fig. 4. Carotenoid biosynthesis pathways of Rhodobacter sphaeroides, Rhodospirillum rubrum and Erwinia herbicola. Plants utilize two dif-ferent enzymes that act sequentially to desaturate phytoene. The bacteria Rb. sphaeroides and Rhodospirillum rubrum synthesize acyclic carotenoids, whereas cyclic carotenoids are found in E. herbicola and in plants. Recent in vitro studies10 suggest that the CrtD enzyme of R. sphaeroides is unable to convert methoxyneurosporene to spheroidene (this step is indicated by a broken arrow and a question mark for the CrtD enzyme). The pathway in R. rubrum has not been subjected to a detailed genetic or biochemical analysis and these steps await further studies. However, recent work has revealed the presence of CrtD in R. rubrum11. In the related micro-organism Rubrivivax gelatinosus, CrtC and CrtD have been identified and shown to be involved in both the spheroidene and the spirilloxanthin pathways12.
PP-O
OH
OH
OMe
OMe
OMe
OMe
OMe O
O HO
HO
OH
OH
OMe
OMe
OMe
OMe MeO
HO HO
CrtB
CrtI
CrtI
CrtI
CrtI CrtC
CrtC CrtY
CrtZ
CrtZ CrtD
CrtF?
CrtC
CrtD
CrtF? CrtF
CrtD
CrtC
CrtA
HO
HO
OH
CrtA
CrtF CrtD
b-Carotene
b-Cryptoxanthin
Zeaxanthin Rhodopin
3,4-Didehydrorhodopin
Anhydrorhodovibrin
Rhodovibrin
Monodemethylspirilloxanthin
Spirilloxanthin Demethylspheroidene
Methoxyneurosporene
Spheroidene
Hydroxyspheroidene
Hydroxyspheroidenone
Spheroidenone Hydroxyneurosporene
Phytoene
Phytofluene
z-Carotene (unsymmetrical isomer)
Neurosporene Geranylgeranyl diphosphate
Lycopene
1
2
3
4 Desaturations
example, Rhodopseudomonas acidophila and Rhodospirillum rubrum, which synthesize rhodopin and spirilloxanthin, respectively) can produce carotenoids with a basic complement of four desaturations, rather than the three-desaturations seen in Rb. sphaeroides. A key question was whether this diversity could be achieved simply by having a different phytoene desaturase, followed by a common set of en-zymes to catalyse subsequent steps. This was tested by introducing the four-step phytoene de-saturase from Erwinia into a crtI mutant of Rb. sphaeroides. The result was the synthesis of a high proportion of carotenoids of the spiril-loxanthin series, showing that Rb. sphaeroides could indeed modify lycopene, as well as neu-rosporene. Preliminary identification of the major foreign compound produced (by HPLC analysis, spectral features and NaBH4 reduc-tion) suggested that it was a ketospirilloxanthin-like carotenoid. This indicates that subsequent enzymes in the pathway display enough prom-iscuity with regard to the structure of their sub-strates to act equally well on either neurosporene or lycopene (in the case of CrtC) and spheroid-ene or spirilloxanthin (in the case of CrtA). The fact that these enzymes show specificity to-wards a particular end-group or structure rather than towards a particular substrate molecule has previously been suggested9. The exchange of the phytoene desaturase results in Rb. sphaer-oides producing a new range of carotenoids not naturally present (Fig. 1d and f). Moreover, the fact that some of the carotenoids produced are not naturally present in either Rb. sphaeroides or E. herbicola indicates that the desaturation step is of great relevance to the creation of ex-tensive carotenoid diversity. We can only as-sume that Rb. sphaeroides CrtC, CrtD and CrtF are involved in the synthesis of the spirillox-anthin-like derivative that accumulates. There-fore, similar genes must be present in other closely related photosynthetic bacteria such as Rhodopseudomonas acidophila, Rhodospiril-lum rubrum and Rubrivivax gelatinosus. The presence of crtC and crtD genes in Rx. gelati-nosus has already been demonstrated12. This photosynthetic bacterium accumulates carot-enoids of both the spheroidene and the spirillo-xanthin series25and CrtC and CrtD are involved in both pathways12.
These results clearly indicate that when one or two key enzymes in this pathway are ex-changed, the new precursors are then generally further metabolized by the subsequent en-zymes to produce a new array of carotenoids. This modular behaviour of the key enzymes may well explain how the wide range of natu-rally occurring carotenoids can be produced with relatively minor changes to the basic bio-synthetic pathway. It is clearly more efficient for an organism to alter just one or two key en-zymes, rather than the whole set, to achieve this diversity.
Genetically engineering new carotenoids into a light-harvesting complex
The photochemical properties of carotenoids, such as the energy levels of their excited singlet states, change in a graded way as the number of conjugated double bonds increases. This property has been used extensively to investi-gate the detailed mechanism of the accessory light-harvesting role of carotenoids in the an-tenna complexes. This approach has often re-lied on extraction-reconstitution techniques3. An alternative elegant way of achieving this in vivo is to reprogramme the carotenoid biosyn-thetic machinery to produce different caroten-oids. The ability to express foreign carotenoid biosynthesis genes in R. sphaeroides created an opportunity to replace the native spher-oidenone with lycopene, a carotenoid already known to function in the light-harvesting LH2 complex of Rhodospirillum molischianum. The fact that the structure of this complex has also been determined to atomic resolution8 was further justification for choosing lycopene as a replacement carotenoid. To do so it was necessary to stop the reaction at the level of hydroxylation (crtC), and to replace the three-desaturation CrtI with a four-three-desaturation en-zyme that would be expected to favour the accumulation of lycopene. A double mutant of Rb. sphaeroides was created, in which both crtI and crtC were inactivated, providing a decisive block in the native pathway, and a foundation on which to build a new pathway. In the mutant, the native crtI and crtC genes were then replaced with the crtI gene from E. herbicola. The block in the subsequent bio-synthetic step (CrtC) in Rb. sphaeroides pre-vented modification of lycopene, and as a result, lycopene was produced from phytoene, instead of neurosporene. This carotenoid, syn-thesized in Rhodobacter for the first time, was assembled into the photosynthetic apparatus and fluorescence excitation spectra showed that it was capable of transferring absorbed light energy to the bacteriochlorophylls of the light-harvesting apparatus (G. Garcia-Asua et al., unpublished).
Prospects for the future
Photosynthetic bacteria can synthesize a wide variety of carotenoids, and genetic techniques have provided some insight into how this diversity has occurred. We anticipate that fur-ther work will enable us to direct the synthe-sis of the carotenoid biosynthetic machinery towards other carotenoids, and provide some understanding of the interactions between carotenoid biosynthetic enzymes. The func-tions of individual carotenoids can also be ex-amined using this genetic system. In this way, a structurally related series of carotenoids, whose photophysical properties change in a defined way, can be used to test how different photochemical parameters affect their function.
Acknowledgements
We thank John Olsen for Fig. 4. Our thanks also to the BBSRC for funding this research.
References
1 Van Poppel, G. (1993) Carotenoids and cancer: an update with emphasis on human intervention studies, Eur. J. Cancer 29A, 1335–1344 2 Lang, H.P. and Hunter, C.N. (1995) The
relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides, Biochem. J. 298, 197–205
3 Zurdo, J., Fernandez-Cabrera, C. and Ramirez, J.M. (1993) A structural role of the carotenoid in the light-harvesting II protein of
Rhodobacter sphaeroides, Biochem. J. 290,
531–537
4 Foote, C.S. (1976) Photosensitized oxidation and singlet oxygen: consequences in biological systems, in Free Radicals and Biological Systems (Pryer, W.A., ed.), pp. 85–133, Academic Press 5 Cogdell, R.J. (1988) The functions of pigments in
chloroplasts, in Plant Pigments (Goodwin, T.W., ed.), pp. 222–224, Academic Press
6 Hunter, C.N. et al. (1988) Oligomerization states and associations of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analysed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis, Biochemistry 27, 3459–3467 7 McDermott, G. et al. (1995) Crystal structure of an
integral membrane light-harvesting complex from photosynthetic bacteria, Nature 374, 517–521 8 Koepke, J. et al. (1996) The crystal structure of
the light harvesting complex II (B800-850) from
Rhodospirillum molischianum, Structure 4,
581–597
9 Britton, G. (1993) Biosynthesis of carotenoids, in
Carotenoids in Photosynthesis (Young, A. and
Britton, G., eds), pp. 96–126, Chapman & Hall 10 Albrecht, M., Ruther, A. and Sandmann, G.
(1997) Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter
sphaeroides, J. Bacteriol. 179, 7462–7467
11 Komori, M. et al. (1998) A null lesion in the rhodopin 3,4-desaturase of Rhodospirillum
rubrum unmasks a cryptic branch of the
carotenoid biosynthetic pathway, Biochemistry 37, 8987–8994
12 Ouchane, S. et al. (1997) Pleiotropic effects of
puf interposon mutagenesis on carotenoid
biosynthesis in Rubrivivax gelatinosus, J. Biol.
Chem. 272, 1670–1676
13 Giuliano, G., Pollock, D. and Scolnik, P.A. (1986) The gene crtI mediates the conversion of phytoene into colored carotenoids in
Rhodopseudomonas capsulata, J. Biol. Chem.
261, 12925–12929
14 Fraser, P.D. et al. (1992) Expression in E. coli, purification and reactivation of the recombinant
Erwinia uredovora phytoene desaturase, J. Biol. Chem. 270, 19891–19895
448
perspectives
15 Armstrong, G.A., Alberti, M. and Hearst, J.E. (1990) Conserved enzymes mediate the early reactions of carotenoid biosynthesis in non-photosynthetic and non-photosynthetic prokaryotes,
Proc. Natl. Acad. Sci. U. S. A. 87, 9975–9979
16 Bartley, G.E. et al. (1990) Carotenoid desaturases from Rhodobacter capsulatus and Neurospora
crassa are structurally and functionally conserved
and contain domains homologous to flavoprotein disulfide oxidoreductases, J. Biol. Chem. 265, 16020–16024
17 Pecker, I. et al. (1992) A single polypeptide catalysing the conversion of phytoene to
z-carotene is transcriptionally regulated during tomato fruit ripening, Proc. Natl. Acad.
Sci. U. S. A. 89, 4962–4966
18 Linden, H., Vioque, A. and Sandmann, G. (1992) Isolation of a carotenoid biosynthesis gene coding for z-carotene desaturase from Anabaena PCC 7120 by heterologous complementation,
FEMS Microbiol. Lett. 106, 99–104
19 Windhoevel, U., Sandmann, G. and Boeger, P. (1997) Genetic engineering of resistance to bleaching herbicides affecting phytoene desaturase and lycopene cyclase in cyanobacterial caroteno-genesis, Pestic. Biochem. Phys. 57, 68–78 20 Lang, H.P. et al. (1995) Complete DNA
sequence, specific Tn5 insertion map and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides,
J. Bacteriol. 177, 2064–2073
21 Coomber, S.A. and Hunter, C.N. (1989) Construction of a physical map of the 45 kb photosynthetic gene cluster of Rhodobacter
sphaeroides, Arch. Microbiol. 4, 454–458
22 Zurdo, J. et al. (1991) Dimeric carotenoid inter-action in the light-harvesting antenna of purple phototrophic bacteria, Biochem. J. 274, 881–884 23 Hundle, B. et al. (1994) Functional assignment of
Erwinia herbicola Eho10 carotenoid genes
expressed in Escherichia coli, Mol. Gen. Genet. 245, 406–416
24 Hunter, C.N. et al. (1994) Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and
Rhodobacter sphaeroides, J. Bacteriol. 176,
3692–3697
25 Schmidt, K. (1978) Biosynthesis of carotenoids, in The Photosynthetic Bacteria (Clayton, R.K. and Sistrom, W.R., eds), pp.729–750, Plenum Press
Guillermo Garcia-Asua, Helen P. Lang and C. Neil Hunter*are at the Dept of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK S10 2TN;
Richard J. Cogdell is at the Dept of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, UK G12 8QQ.
*Author for correspondence
(tel 144 114 222 4191; fax 144 114 272 8697; e-mail [email protected]).
Plants produce an extraordinarily diverse range of compounds, which are increasingly providing leads in drug development. Advances in high throughput screening techniques have allowed pharmaceutical companies to explore this chemodiversity and have raised awareness for the potential use of secondary metabolites as therapeutic agents: over one hundred thousand secondary metabolites have been identified to date. The challenge for phytochemists is to exploit these techniques to develop new means of furthering our understanding of secondary metabolism. One obvious challenge is the need to avoid the repetitive isolation of common struc-tures with known biological activity. Further-more, in many biological systems, compounds may act at very low concentrations (e.g. peptides that play a role in signaling), and it is thus necessary to develop techniques to improve the detection threshold.
In this third edition of Phytochemical Methods, Jefferey B. Harborne describes some of the latest advances in phytochemical research. The first chapter introduces the reader to the different methods used to extract, isolate and identify plant compounds and this is followed by a dis-cussion on how to interpret the data obtained.
The final part of the introduction focuses on phytochemical procedures in different research fields and emphasizes the importance of phyto-chemistry in solving problems in plant system-atics, physiology, ecology and genetics.
Subsequent chapters describe methods for identifying different plant secondary metab-olites. The compounds are classified according to their biosynthetic origin and are illustrated by representative formulae, together with common chemical variations. For comparative purposes, the most commonly occurring structures in each class of compounds are presented in tables show-ing their Rf values, colour reactions and spectral properties; other details include their taxonomic distribution and localization in the plant. The methods used most widely in the identification of these compounds are outlined and each chap-ter concludes with a comprehensive list of refer-ences published in the past decade.
The author uses his many years of experience in the field to guide the reader in the selection of appropriate methods, and even provides in-formation about which authentic markers are commercially available – and if they are not available, which plant source may be used to isolate the reference compound. Further infor-mation about the stability of different types of compounds, enzymatic influence and artefact for-mation is also very useful. One of the strengths of this book is that traditional methods are de-scribed with original literature references, and information is also provided about suitable alter-native techniques that can be used to replace them. The book is organized in a logical way and the data is easily accessible and not complicated by the inclusion of too much theory. This ensures that the reader can quickly access information on
the status of our knowledge of different types of compounds, and select suitable methods for a preliminary characterization.
The book aims to be a simple guide to mod-ern methods of plant analysis. Phytochemical Methods fully achieves this objective and is an excellent introduction for any student new to the field of plant chemistry. Indeed, it is a suitable entry point for students in related fields such as pharmacognosy, biochemistry and natural prod-uct organic chemistry. The book has very few limitations. My main criticisms are that the high performance liquid chromatography (HPLC) methodology could be expanded for the differ-ent classes of compounds and placed before the traditional methods in each chapter. The appli-cation of phytochemistry to research on medici-nal plants could also be somewhat expanded. Finally, I was surprised to discover that the sec-tion on methods for isolating and characterizing macromolecules fails to discuss the analysis of plant polypeptides. But, perhaps this is because protein purification has been extensively dis-cussed in other books on lab techniques.
The extensive experience and knowledge of the author and the continuous updating of infor-mation in the book ensures that this latest edition maintains a timely quality. I strongly recom-mend it as a reference manual for phytochem-istry courses but also as a handbook for every research laboratory in the field of plant analysis.
Lars Bohlin
Division of Pharmacognosy, Dept of Pharmacy, Biomedical Centre, Uppsala University, Box 579, S-751 23 Uppsala, Sweden (fax + 46 18 50 91 01;
e-mail [email protected])
The ABC of
phytochemistry
Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis by J.B. Harborne
