Klaus Hahlbrock

Biosynthesis of Flavonoids

Interest in the biosynthesis of flavonoids was first stimulated by studies on genetic aspects of flower colour (cf. Harborne, 1967; Hess, 1968) and by chemical speculations on the mode of formation of the carbon skeleton of this class of compounds (Birch and Donovan, 1953; Robinson, 1955); tracer studies were first applied to the problem around 1957. Investigations with intact plants or plant tissues led to a basic knowledge of the precursors required and to an understanding of some details of the biosynthesis of flavonoids. From this, a general picture of the interrelationships between various classes of these compounds has emerged. In the course of these tracer experiments, however, it became apparent that a more detailed knowledge of the nature and sequence of the individual biosynthetic steps and their regulation could only be gained by investigations of the enzymes involved.

Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension culture of parsley (Petroselinum hortense)

Abstract 1. 1. In cell suspension cultures of parsley ( Petroselinum hortense ) the degree of increase of activity of phenylalanine ammonia-lyase caused by white light depends on the growth stage of the cultures. Maximum enzyme stimulation is observed at approximately the tenth day of growth. 2. 2. Changes in the activities of eight enzymes directly related to the biosynthesis of apiin ( 7-O-[β- D -apiofuranosyl -(1 → 2)-β- D -glucosyl ]-5,7,4′- trihydroxyflavone and graveobiosid B (3′-methoxyyapiin) were measured over a period of 24 h after illumination of cell suspension cultures of parsley. 3. 3. The enzymes investigated were phenylalanine ammonia-lyase, trans -cinnamic acid 4-hydroxylase, p -coumarete:CoA ligase, chalcone-flavanone isomerase, UDP-glucose:apigenin 7- O -glucosyltransferase, UDP-apiose:apigenin 7- O -glucoside-[1 → 2]-apiosyltransferase, UDP-apiose synthetasem and S -adenosylmethionine:luteolin3′- O -methyltransferase. 4. 4. Two groups of enzymes could be distinguished on the basis of their responses to light. The first group comprises the three enzymes acting on substrates of the phenylpropanoid type (phenylalanine, cinnamic acid and p -coumaric acid); the second group includes all those enzymes involved exclusively in the formation of flavonoid compounds and their glycosides. 5. 5. A hypothesis is discussed which would correlate three other enzymes assumed to take part in the same biosynthetic pathway with one of these groups of enzymes.

A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations

SummarySeveral genomic clones from Petroselinum hortense, Zea mays and Antirrhinum majus all homologous to cloned Petroselinum chalcone synthase cDNA were isolated using the λgt WES cloning system.Clones containing the chalcone synthase structural gene were identified by hybridization to cDNA from Petroselinum hortense, genomic wildtype, mutant and revertant DNA.Among the 5 different clones from Petroselinum hortense, PH3 is the most likely candidate to contain at least a portion of the chalcone synthase gene.None of the 4 Zea mays clones appeared to contain part of the chalcone synthase gene.Among the 2 different clones from Antirrhinum majus, AM3 contains the portion of the chalcone synthase structural gene which is altered in the mutant nivea recurrens (nivrec). This mutant is considered to be due to the integration of a transposable element. In revertants of nivrec to niv+ the wildtype locus is restored molecularly.

Further studies on the relationship between the rates of nitrate uptake, growth and conductivity changes in the medium of plant cell suspension cultures

SummaryThe changes in packed cell volume and in nitrate content and conductivity of the medium during the growth cycle of cell suspension cultures from Petroselinum hortense Hoffm., Glycine max Merr., and Haplopappus gracilis A. Grey in a chemically defined medium were compared. In all three cases sigmoidal curves obtained for large decreases in the conductivity were paralleled by similar curves for the rates of nitrate depletion from the medium until this nutrient was completely exhausted. Further decreases in the conductivity subsequent to nitrogen starvation proceeded at relatively slow rates and ceased when the cultures entered into the stationary phase of the growth cycle. Thus the previously reported method of deriving growth curves indirectly from conductivity measurements (Hahlbrock and Kuhlen, Planta 108: 271–278, 1972; Hahlbrock et al., Planta 118: 75–84, 1974) might be generally applicable for this particular medium. The method seems to be based on a continuous uptake by the cells of ionic constituents throughout all stages of actual growth, even beyond the stage of nitrate exhaustion.Cell suspension cultures from Cicer arietinum L. and Acer pseudoplatanus L. in two different, more complex media were used for similar experiments, in which the changes in packed cell volume and in the conductivity of the medium were recorded. As with the results obtained with the fully synthetic medium, the mirror-images of the curves obtained for the decline in conductivity initially paralleled the growth curves. However, the two curves became incongruous after a certain growth stage was reached. These results are discussed with respect to the composition of the media used and to the apparent limitations of the method of determining specific growth stages by monitoring conductivity changes in the medium.

Purification and properties of an o-dihydricphenol meta-O-methyltransferase from cell suspension cultures of parsley and its relation to flavonoid biosynthesis

Abstract 1. 1. An O -methyltransferase directly related to flavone glycoside biosynthesis has been isolated from cell suspension cultures of parsley. The enzyme has been purified 82-fold by MnCl 2 and (NH 4 ) 2 SO 4 precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The enzyme catalyses the transfer of the methyl group of S -adenosyl- l -methionine to the meta position of o -dihydric phenols. 2. 2. Highest enzyme activities were obtained after illumination of the cells for 24 h with high intensities of white light from fluorescent lamps. 3. 3. The enzyme has a pH optimum around pH 9.7. It requires Mg 2+ and is not inhibited by p -chloromercuribenzoate or iodoacetamide. A molecular weight of about 48 000 was estimated from the elution volume after chromatography on a Sephadex G-100 column. 4. 4. Only o -dihydric phenols can function as substrates for the enzyme and only the meta -hydroxyl group is methylated. Luteolin (5,7,3′,4′-tetrahydroxyflavone) and its 7- O -glucoside with apparent K m values of 4.6·10 −5 and 3.1·10 −5 M, respectively, are the best substrates tested, whereas eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) ( K m = 1.2·10 −3 M) and caffeic acid ( K m = 1.6·10 −3 M) had much lower affinity for the enzyme. With luteolin as substrate the apparent K m value for S -adenosyl- l -methionine is 1.5·10 −4 M. 5. 5. Cell cultures also contain enzymatic activity for the synthesis of S -adenosyl- l -methionine from l -methionine and ATP. In contrast to the O -methyltransferase the activity of this enzyme is not influenced by illumination of the cultures.

Light-induced flavone biosynthesis and activity of phenylalanine ammonia-lyase and UDP-apiose synthetase in cell suspension cultures of Petroselinum hortense

SummaryThe formation of flavone glycosides in cell suspension cultures from parsley leaf petiols after illumination with high intensities of white light was demonstrated. Changes in the activities of two of the enzymes involved in flavone glycoside biosynthesis (phenylalanine ammonia-lyase and UDP-apiose synthetase) were measured over a period of 14 days. The two enzymes reached maximum specific activities after significantly different periods of time.

Relationship between organ development and activity of enzymes involved in flavone glycoside biosynthesis in young parsley plants

Abstract The activities of five enzymes directly related to the biosynthesis of apiin, (7- O [β- d -apiofuranosyl(1→2)β- d -glucosyl]-5,7,4′-trihydroxylfavone, and to the corresponding glycoside of chrysoeriol (3′-methoxyapigenin) as well as the accumulation of these flavone glycosides were studied during the growth of young parsley plants ( Petroselinum hortense ). The enzymes examined were phenylalanine ammonia-lyase (E.C. 4.3.1.5), chalcone-flavanone isomerase, UDP-apiose synthetase, and a glucosyl- and an apiosyltransferase. On a fresh weight basis, the glycoside content as well as the activities of all of the five enzymes were found to be relatively high in very young cotyledons or leaves and decreased to a rather low level within a few days. The striking similarity among the five curves obtained when enzyme activities were plotted versus growth time suggests that the isolation of other enzymes involved in this biochemical pathway is likely to be most successful in the initial stages of cotyledon or leaf development.

Determination of Specific Growth Stages of Plant Cell Suspension Cultures by Monitoring Conductivity Changes in the Medium

SummaryConductivity changes in the medium of cultured soybean (Glycine max L.) cells were shown to be strictly correlated with nitrate uptake and growth of the cultures. A continuous record of the conductivity was used as a simple and reliable method of determining specific growth stages and concomitant peaks in the activities of nitrate reductase and phenylalanine ammonia-lyase.

Flavanone synthase: simple and rapid assay for the key enzyme of flavonoid biosynthesis.

Abstract It is suggested that flavanone synthase activity should be measured when the key reaction of flavonoid biosynthesis is to be tested. A simple and rapid procedure for the determination of flavanone synthase activity, based on extraction of the 14C-labelled product(s) into ethylacetate, is described. The enzyme can be stored under appropriate conditions for several weeks without significant loss of activity. Results obtained with cell suspension cultures of parsley indicate that the activity of flavanone synthase is regulated differently from the activity of phenylalanine ammonia-lyase, an enzyme frequently referred to as a key enzyme of flavonoid biosynthesis.

Relationship between growth of parsley and soybean cells in suspension cultures and changes in the conductivity of the culture medium.

SummaryChanges in conductivity and pH during the growth cycle of cell suspensions derived from parsley (Petroselinum hortense) and soybean (Glycine max) have been investigated. Measurement of the conductivity of the medium represents a simple, rapid, and reliable method for the precise determination of the growth phase of a culture. The accuracy of this method has been tested by using phenylalanine ammonia-lyase, an enzyme that has a characteristically short, distinct period of activity during the growth cycle of soybean cell suspensions. It is suggested that an automatic regulation of the conductivity of the medium might be employed for growing plant cells in a continuous culture at a defined stage of growth.