E L S E V I E R Biochemical Education 26 (1998) 315-316

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Biochemical Education

Waxes: a forgotten topic in lipid teaching

Eva Domfnguez, Antonio Heredia* Departamento de Bioquimica y Biologia Moh'cuhm Facultad de Ciencia,~; Universidad de M~ilaga, 29071 M~ilaga, Spain

Abstract

This paper provides a brief review of the biological importance of the lipids categorized as waxes and describes some of the organic chemistry of these compounds. A short laboratory practical exercise on the extraction of plant waxes and their analysis by TLC is presented. © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

1. Introduction

The topic l ipids is a key theme in a standard course of General Biochemistry or Biomolecules. Most books of biochemistry begin the theme with a description of the physico-chemical characteristics and structures of the different classes of lipids. Some of the most commonly used textbooks include in their classification a class of lipids named waxes, and include a definition such as 'esters of long-chain aliphatic acids with long-chain aliphatic alcohols' [1]. As we shall show, this classifica- tion is no longer adequate and is too narrow considering our current knowledge on waxes and their importance in several aspects of biology, chemistry and modern biotechnology [2]. We believe that more detailed and factual information should be included in a standard course of biochemistry.

The term wax is derived from the Anglo-Saxon word w e a x which was used to describe the material in the honeycomb of bees. Similar substances isolated from plants were also called weax . This etymological relation- ship tell us that the similarities between different waxes are not concerned with their chemical and physical properties. We can classify waxes, depending on the source, into three categories: animal, vegetable and mineral but in this short communication we will concen- trate on the characteristics of plant waxes because of their importance, and because the major animal waxes, wax esters, are usually quite well described in the textbooks: e.g. honey wax and spermaceti.

The interface between plant tissue and the environ- ment is provided by protective, extracellular envelopes in which lipids provide the waterproofing layer of wax. This superficial material is synthesized by specialized cells in

*Corresponding author.

the outermost layers of the plant tissue. These waxes are generally markedly different in their chemical composi- tion from any lipids found in the internal tissues and cellular organelles of the same plant, They are commonly referred by several terms: e.g. surface lipids, surface waxes, cuticular lipids or cuticular waxes. The organiza- tion of waxes has been studied for over 150years, but only since the mid-1960s it has been possible to establish details of their ultrastructure and chemistry and, conse- quently, to carry out systematic studies on their biosynthesis and physiology [2]. At the present time our knowledge of cuticular waxes is really quite extensive.

2. Isolation and types of plant waxes

The amount of plant cuticular waxes produced is dependent on growth conditions, whilst chemical compo- sition is less influenced by environmental factors. From a general point of view, wax yield for leaves and fruits of many species is very variable, ranging between 20 and 600 i t g / cm 2. Cuticular waxes from the surfaces of fruits and leaves are generally isolated by brief (20-60 s) immersion (total surface wax) or washing (adaxial/ abaxial surface wax) fresh plant tissue with organic solvents such as hexane or chloroform at room tempera- ture [3]. From a chemical point of view, plant cuticular waxes are composed of complex mixtures of organic compounds which differ one from another in the number, relative abundance and homologue distribution of the constituent classes. Most waxes contain long-chain aliphatic components which may be further classified according to the distribution of their major homologues. It is noticeable that, except for the occasional existence of some alkenes, all the aliphatic chains are saturated. Table 1 gives a resume of the principal classes of constit-

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316 E. I)omOlguez, A. Heredia/Biochemical Education 26 (1998) 315-316

Table 1 Principal constituents occurring in plant waxes

Type of compound General structure Range Most common Source

Hydrocarbons CtL-(CIt3), ,-CH~ C>-C~, C.,,, C~, Most plants Ketoncs R , - C O - R : Ce~-C,~ C>, C,~ Bra.ssica. roses fl-Diketones R , -COCHeCO-R, - - C>. C.,,. C,, Eucalyplus, barley, carnation, grasses Monoesters R~-COO-R3 C~,,-C,,, C4a. Ca,,, C~. C,,, Most plants Polyesters - - - - ('j.-C,~. ~,~-hydroxyacids. 5-7/mol Gymnosperms Primary alcohols R - C H , O H C,.-C,,, C2,,. C., Most plants Aldehydes R - C H O C,a-C,4 C_.,,. C.~. C,,, Grapes. apple, B oleracea, pea Alkanoic acids R - C O O H C,:-C,,, (':4, C:,,. (_7~ Most plants Terpencs - - - - Ursolic acid. oleanolic acid and others Apple, grape

uents occurring in plant cuticular waxes. From this Table one can conclude that plant waxes have so far only been considered as long-chain esters. In contrast with this definition, a rich range of organic compounds are included in this type of lipids. Common features ascribed to plant waxes are a high degree of crystallinity and a strongly hydrophobic character, together with a low chemical reactivity, which confers on these organic compounds an important physiological role as molecular barriers of plant surfaces. This fact must be emphasized in the presentation of these topics in a standard biomole- cules course.

Table 2 R~ values of the most important compounds of plant waxes using benzene and a mixture of chloroform/ethanol as mobile phases.

R, benzene R, chloroform/ethanol (99:1)

Alkanoic acids 11 II Hydroxy-/~-diketones I).06 0.1)5 Primary alcohols 0.16 1). 15 Secondary alcohols I).43 0.36 Aldehydes I).65 I).42 Ketones tl.76 11.53 fl-Diketones 11.76 11.39 Esters 0.84 0.65 Alkanes I).91 0.83

3. A short laboratory class experiment

The qualitative separation of most of the major classes of aliphatic wax components can be achieved by one-dimensional silica gel-G thin layer chromatography (TLC). This might constitute the subject of a short laboratory class experiment illustrating the different wax components isolated from fresh plant fruits or leaf tissues. Typical good wax samples may easily be extracted and isolated by following the above procedure from leaves of citrus species, grapes, Brass(ca cultivars, tomato fruits and apple cultivars. Alternatively, conifer needles are a good source of specific waxes: such as those containing long-chain secondary alcohols. For further analysis, lipid samples are dissolved in any suitable organic solvent (e.g. chloroform) giving a concentration of 5-10 mg/ml, and only a small aliquot of 50-100 pg is applied to the layer using a fine glass pipette. The TLC separation can be achieved using benzene or a mixture of chloroform/ethanol as running solvents (mobile phases). Table 2 shows the corresponding R, values of thc most important constituents of plant waxes using these organic

solvents as mobile phases. Each class of wax components on TLC plates can finally be visualized by sulphuric acid charring or after spraying with a fluorescent dye solution of 0.3% 2'7'-dichlorofluoresceine in ethanol and after air drying, the chromatogram is inspected under UV light. The wax components appear as quenched areas on a fluorescent background.

In summary, these comments offer an opportunity to update topics concerning the teaching of lipid compo- nents and in addition to present a short laboratory class experiment to illustrate, from a qualitative point of view, the most important classes of plant waxes.

References

[1] H. G. Garrett, C. M. Grisham, Biochemistry, Harcourt Brace, New York, 1995.

[2] R. J. Hamilton, Waxes: Chemistry, Molecular Biology and Functions, The Oily Press, Dundee, Scotland, 1995.

[3] T. J. Walton, Waxes, cutin and suberin. Methods in Plant Biochemistry 4 (1990) 11)5-158.