Anthocyanins and related compounds in potatoes (Solanum tuberosum L.)
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Coloured potatoes may have economic value as natural food colourants and as food products such as novelty potato crisps and coloured potato salads. This thesis investigated the biochemistry and physiology of anthocyanins and related compounds in Solanum tuberosum L., and the relationship to tuber colour. These factors were discussed in terms of consumer requirements. Phenolic acids, flavonoids and anthocyanins were surveyed and quantified in the tubers (skin and flesh), flowers and leaves of twenty nine cultivars of S. tuberosum and eight other Solanum species (S. acaule, S. berthaultii, S. gourlayi, S. oplocense, S. sanctaerosae, S. spars/pilum, S. speggazzinii, S. stenotomum). The main anthocyanin found in red tubers was pelargonidin-3-(p-coumaroyl-rutinoside)-5-glucoside (200-2000μg/gFW) with lower amounts of peonidin-3-(p-coumaroyl-rutinoside)-5-glucoside (20-400μg/gFW), Light to medium pmple coloured tubers contained mostly petunidin-3- (p-coumaroyl-rutinoside)-5-glucoside (400-2000μg/gFW) pills low concentrations of malvidin-3-(p-coumaroyl- rutinoside)-5-glucoside (20-200μg/gFW), whilst dark purple black coloured tubers contained a similar concentration of petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside (1000-2000μg/gFW) to the light to medium purple tubers, but with high concentrations of malvidin-3-(p-coumaroyl-rutinoside)-5-glucoside (2000- 5000μg/gFW). Red and purple tubers also contained a number of minor anthocyanins, with the same aglycones as above, but mostly as the 3-rutinosides. Tubers contained high concentrations of phenolic acids (2000-5000μg/gFW), with chlorogenic acid making up 60-90%. Apart from the anthocyanins, there were low concentrations of other flavonoids (200-300μg/gFW). The major anthocyanin present in the flowers was petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside. Flowers and leaves contained higher concentrations of flavonoids (1000-3000μg/gFW), the major flavonoids being quercetinglycosides. The flavonoid patterns of flowers and leaves fell into two different categories with some cultivars containing high concentrations of quercetin-glycosides, whilst others contained low concentrations. Tubers of the other available Solanum species did not show the range of colours shown by S. tuberosum cultivars, and were mostly white or light purple, with petunidin-3-(pcoumaroyl-rutinoside)-5-glucoside being the major anthocyanin (when present) in the skin of tubers of the other Solanum species. The major anthocyanin in flowers of the other Solanum species was petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside, with levels similar to those found in S. tuberosum flowers. Low levels of anthocyanin were found in the leaves of the other Solanum species whereas in S. tuberosum no anthocyanins were found in the leaves. There was considerable variation among plants and species in both phenolic acid and flavonoid concentrations, but generally a similar pattern was found in the other Solanum species as in S. tuberosum cultivars, except that S. tuberosum flowers contained lower concentrations of total phenolic acids and flavonoids on average, and tubers and leaves contained high concentrations of flavonoids. The expression of different pathways appeared to depend on the species, plant tissue and environmental factors. Diseased tubers contained higher concentrations of phenolic acids, flavanones and flavonols than healthy tubers, and some flavonols which were not present in healthy tubers were produced in diseased tubers. There was differential expression of anthocyanins, flavonoids and phenolic acids in the different parts of the plant (tubers, flowers and leaves). These compounds also responded differently to light, with anthocyanins showing a large increase, flavonoids a smaller increase, and phenolic acids no change in concentration, in minitubers after indirect exposure to light. The biosynthesis of anthocyanins in tubers was investigated throughout tuber development and during storage. Newly initiated tubers contained no anthocyanin, and subsequent production of colour occurred firstly at the stem end of the developing tuber, and then proceeded to the bud end. Anthocyanin concentrations increased throughout the development of the tuber, reaching a maximum at a tuber size which was dependent on the cultivar (about 150-200g for Desiree). Concentrations were higher at the stem end of the tuber than the bud end for most of tuber development although, as the maximum anthocyanin concentration was reached, the distribution of anthocyanin over the tuber became more uniform. This suggested that the transport of some compound (carbohydrate or "trigger”) was responsible for the initiation of anthocyanin biosynthesis. Concentration of other flavonoids also increased and followed a similar pattern to that of the anthocyanins, with maximum concentrations occurring in Desiree tubers about 150-200g. Phenolic acid concentrations also increased during tuber development, although these reached a maximum concentration in slightly smaller tubers (about 70-100g). Microscopical studies of anthocyanin-containing cells showed that the difference in colour intensities of the different cultivars was because of differences in the amount of anthocyanin produced in individual cortex cells, in the proportion of cortical cells within a layer producing anthocyanin, and the number of layers of coloured cells. Totally white tubers did not contain any anthocyanin coloured cells, whilst more highly coloured tubers contained a greater number of more highly coloured cells in these layers. Additionally, the strongly coloured tubers had increased amounts of anthocyanin present in the phellem cells and intense1y coloured deposits of anthocyanin around the cell walls of these cells. Cold storage (4°C) of tubers caused an increased concentration of anthocyanins, especially at the bud end of the tuber, so that the distribution pattern of anthocyanins was reversed from that found in developing tubers. Storage at higher temperatures (10°C and above) caused a decrease in anthocyanin concentration. These changes in anthocyanin concentration were thought to be related to sprouting and also the sugar concentration within the tuber. Cooking (boiling, steaming or crisping) of tubers (until they were ready to eat) did not result in any significant loss in anthocyanin colour, although after two to three times the normal cooking time some loss of anthocyanin colour occurred. Anthocyanin colour may have been affected by the high concentration of starch or sugars found in tubers. Addition of amylose, amylopectin, α-cyclodextrin and β-cyclodextrin to anthocyanin solutions caused a decrease in anthocyanin colour, whilst the addition of sugars (glucose, sucrose and maltose) resulted in increased anthocyanin colour. Tissue-cultured minitubers were used to investigate the effects of light on anthocyanin biosynthesis. Although anthocyanins were produced in the dark in field grown tubers, the presence of light on the plant leaves was necessary for anthocyanin production in some cultivars. Anthocyanin concentration increased with increased light intensities in all cultivars studied (to a maximum of about 3.2ng/cm² of surface area in Desirée minitubers), and the activities of enzymes (phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, dihydroflavonol reductase and glycosyltransferase) showed related increases. Biosynthesis of anthocyanins was a high irradianceresponse and required at least eight hours of exposure to light for a significant increase in anthocyanin concentration to occur, after which anthocyanin concentration (to a similar maximum concentration) increased linearly with increasing time of exposure. Both phytochrome and cryptochrome light receptors were thought to be involved because light of blue, red and purple wavelengths enhanced anthocyanin production, compared with white light of similar intensity. The regulation of anthocyanin biosynthesis in potato tubers is different from most other plants and tissues because direct light exposure of the tuber is not necessary for anthocyanin production. However, for maximum anthocyanin synthesis the exposure of the plant leaves to light is required. It is proposed that this synthesis of anthocyanins intubers in the dark requires genetic capability of the tubers, a supply of carbohydrates, and is mediated by a "trigger" compound produced after the exposure of the leaves to light and transported to the tubers.