Activity of phenylalanine ammonia-lyase (PAL) and concentrations of anthocyanins and phenolics in developing strawberry fruit

Studies on regulation of production of phenolics in strawberry (Fragaria X ananassa Duch,) fruit were initiated by monitoring phenylalanine ammonia-lyase (PAL) activity and levels of anthocyanins, flavonoids, tannins, and other soluble phenols throughout fruit ontogeny in 'Tillikum'. PAL catalyzes the first step in the biosynthesis of phenylpropanoids, which are further modified into a wide variety of phenolic compounds. Peak in PAL activity (1 mol· s -l = 1 kat) of 90 pkat· mg -l protein was detected at 5 and 27 days after anthesis (DAA), when fruit was green and nearly ripe, respectively. PAL activity was only 10% of peak values in the white berry stage, when. fruit growth was most rapid. The second peak in PAL activity was followed by a rapid drop, to nearly zero in red-ripe fruit at 30 DAA. Total soluble phenols reached a maximum level soon after anthesis, just before the first peak in PAL activity, then declined to a low constant value well in advance of fruit ripening. Similar changes were observed in levels of tannins and flavonoids that, at anthesis, accounted for 44% and 51% of the soluble phenols, respectively. The concentration of anthocyanin was very low throughout most of fruit development, but beginning at 23 DAA it increased from 0.53 mg·g -l fresh weight in 3 days. This accumulation paralleled the second rise in PAL activity. Accordingly, strawberry fruit have a developmental- dependent expression of PAL activity and accumulation of phe- nolic substances derived from the phenylpropanoid pathway. Many distinctive developmental features of fleshy fruits, such as loss of astringency and appearance of characteristi c color at ripening, are related to changes in the synthesis and accumu- lation of phenolic compounds. For example, astringency is a tannin effect and the color of ripe fruit often results from ac- cumulated anthocyanins. Besides anthocyanins and condensed tannins (proanthocyanins), fruit also contain other flavonoids, as well as simple phenols such as phenolic acids and cinnamic acid derivatives (see review by Macheix et al., 1990). The type and amount of phenolic compounds in plant tissues depend on genotype and developmental stage (Hahlbrock and Griseback, 1-979). Tissues of fleshy fruits commonly have high levels of cinnamic acid derivatives, flavonoids, and tannins during early development, but accumulate anthocyanins only near maturity (Billet et al., 1978; Hyodo, 1971; Kataoka et al., 1983). In- formation on metabolism of phenolics is important not only because they influence the quality of fruit and related processed products, but also because of their roles in developmental phys- iology and defense mechanisms. Understanding phenolic metabolism in plant cells and tissues requires knowledge of the biosynthetic reactions and their reg- ulation. The majority of monophenolic compounds, most of which are phenolic acids and phenylpropanoids, are interme- diates and derivatives of the shikimate and phenylpropanoid pathways. Flavonoids, including anthocyanins and condensed tannins, are derived from p-coumaric acid of the phenylpropa- noid pathway. Another group of tannins, the hydrolyzable tan- nins, are ester derivatives of gallic acid that maybe synthesized via the shikimate or phenylpropanoid pathways (Ishikura et al., 1984). Conversion of L-phenylalanine to trans -cinnamic acid is the initial step of the phenylpropanoid pathway. This ammonia elimination reaction is catalyzed by L-phenylalanine ammonia- lyase (PAL) (EC 4.3.1.5), a key regulatory enzyme in the bio-