Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT-silenced lines.

Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucrose induced accumulation of CGA correlated with the increased expression of phenylalanine ammonia-lyase (PAL) rather than HQT. Transient expression of the potato MYB transcription factor StAN1 (anthocyanin 1) in tobacco increased CGA. RNAi suppression of HQT resulted in over a 90% reduction in CGA and resulted in early flowering. The reduction in total phenolics and antioxidant capacity was less than the reduction in CGA, suggesting flux was rerouted into other phenylpropanoids. Network analysis showed distinct patterns in different organs, with anthocyanins and phenolic acids showing negative correlations in leaves and flowers and positive in tubers. Some flavonols increased in flowers, but not in leaves or tubers. Anthocyanins increased in flowers and showed a trend to increase in leaves, but not tubers. HQT suppression increased biosynthesis of caffeoyl polyamines, some of which are not previously reported in potato. Decreased PAL expression and enzyme activity was observed in HQT suppressed lines, suggesting the existence of a regulatory loop between CGA and PAL. Electrophysiology detected no effect of CGA suppression on potato psyllid feeding. Collectively, this research showed that CGA in potatoes is synthesized through HQT and HQT suppression altered phenotype and redirected phenylpropanoid flux.

Changes in potato phenylpropanoid metabolism during tuber development.

Phenylpropanoid metabolite and transcript expression during different developmental stages were examined in field grown potatoes. Carbohydrate and shikimic acid metabolism was assessed to determine how tuber primary metabolism influences phenylpropanoid metabolism. Phenylpropanoid concentrations were highest in immature tubers, as were some transcript levels and enzyme activities including phenylalanine ammonia lyase (PAL). Phenylpropanoid concentration differences between mature and immature tubers varied by genotype, but in some cases were approximately three-fold. The most abundant phenylpropanoid was chlorogenic acid (5CGA), which decreased during tuber maturation. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) transcripts were highly expressed relative to other phenylpropanoid genes, but were not well correlated with 5CGA concentrations (r = -0.16), whereas HQT enzyme activity was. In contrast to 5CGA, less abundant chlorogenic isomers increased during development. Concentrations of hydroxycinnamic acid amides were higher in immature tubers, as was expression of arginine- and ornithine decarboxylases. Expression of several genes involved in carbohydrate or shikimate metabolism, including sucrose synthase and DAHP, showed similar developmental patterns to phenylpropanoid pools, as did shikimate dehydrogenase enzyme activity. Sucrose, glucose and fructose concentrations were highest in immature tubers. Exogenous treatment of potatoes with sugars stimulated phenylpropanoid biosynthesis, suggesting sugars contribute to the higher phenylpropanoid concentrations in immature tubers. These changes in phenylpropanoid expression suggest the nutritional value of potatoes varies during development.

Altered leaf colour is associated with increased superoxide-scavenging activity in aureusidin-producing transgenic plants.

The health-promoting property of diets rich in fruits and vegetables is based, in part, on the additive and synergistic effects of multiple antioxidants. In an attempt to further enhance food quality, we introduced into crops the capability to synthesize a yellow antioxidant, aureusidin, that is normally produced only by some ornamental plants. For this purpose, the snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) and aureusidin synthase (AmAs1) genes, which catalyse the synthesis of aureusidin from chalcone, were expressed in tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa) plants that displayed a functionally active chalcone/flavanone biosynthetic pathway. Leaves of the resulting transgenic plants developed a yellow hue and displayed higher superoxide dismutase (SOD) inhibiting and oxygen radical absorbance capacity (ORAC) activities than control leaves. Our results suggest that the nutritional qualities of leafy vegetables can be enhanced through the introduction of aurone biosynthetic pathways.

Tuber-specific silencing of asparagine synthetase-1 reduces the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape and yield.

Simultaneous silencing of asparagine synthetase (Ast)-1 and -2 limits asparagine (ASN) formation and, consequently, reduces the acrylamide-forming potential of tubers. The phenotype of silenced lines appears normal in the greenhouse, but field-grown tubers are small and cracked. Assessing the effects of silencing StAst1 and StAst2 individually, we found that yield drag was mainly linked to down-regulation of StAst2. Interestingly, tubers from untransformed scions grafted onto intragenic StAst1/2-silenced rootstock contained almost the same low ASN levels as those in the original silenced lines, indicating that ASN is mainly formed in tubers rather than being transported from leaves. This conclusion was further supported by the finding that overexpression of StAst2 caused ASN to accumulate in leaves but not tubers. Thus, ASN does not appear to be the main form of organic nitrogen transported from leaves to tubers. Because reduced ASN levels coincided with increased levels of glutamine, it appears likely that this alternative amide amino acid is mobilized to tubers, where it is converted into ASN by StAst1. Indeed, tuber-specific silencing of StAst1, but not of StAst2, was sufficient to substantially lower ASN formation in tubers. Extensive field studies demonstrated that the reduced acrylamide-forming potential achieved by tuber-specific StAst1 silencing did not affect the yield or quality of field-harvested tubers.

Tuber-specific silencing of the acid invertase gene substantially lowers the acrylamide-forming potential of potato.

Some popular processed foods including French fries contain small amounts of toxic acrylamide. Efforts to lower the accumulation of this reactive compound by modifying the production process have a negative effect on sensory characteristics and are not broadly applicable. This study optimized a method developed more than a decade ago to lower the accumulation of the acrylamide precursors glucose and fructose in cold-stored tubers. In contrast to the original application, which lowered hexose content by one-third through constitutive expression of an antisense copy of the cold-inducible acid invertase (Inv) gene, the current approach was based on tuber-specific expression of an Inv-derived inverted repeat. Stored tubers of transgenic plants contained as little as 2% of the reducing sugars that accumulated in controls. This decline in glucose and fructose formation is counterbalanced by increased sucrose and starch levels. However, it did not trigger any phenotypic changes and also did not affect the formation of free asparagine, ascorbic acid, phenylalanine, and chlorogenic acid. Importantly, French fries from the low-invertase tubers contained up to 8-fold reduced amounts of acrylamide. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with the low-hexose potatoes would reduce the average daily intake of acrylamide by one-fourth.

Tastier and healthier alternatives to French fries.

The effect of both the origin and shape of potato cuts on fry quality was investigated in this study. Linear strips from the inner core of tubers were compared to those from outer tissues, both before and after processing, and strips from either specific tissues or whole peeled tubers were also evaluated against ring-shaped cuts. Both strips and rings had 0.7 cm sides and, in most cases, a volume of 4.9 cm(3). They were analyzed for moisture content, antioxidants, asparagine, and reducing sugars. The material was then blanched, dipped in 0.5% disodium acid pyrophosphate and 0.3% glucose, dried at 77 degrees C, par-fried in soybean oil at 191 degrees C, and finish-fried at 168 degrees C. The fried product was analyzed for sensory characteristics and oil, salt, and acrylamide content. Our results showed that strips from the inner core absorbed 28% more oil and exhibited inferior sensory characteristics compared to strips from the outer parts. The extended drying and frying times needed to match the crispness and flavor of inner strips to those of regularly fried outer strips resulted in a further increased absorption of oil and, importantly, triggered a 163% increase in levels of the toxic Maillard reaction product acrylamide. Potato rings consisted of higher dry matter material, contained more antioxidants, and had a lower surface-to-volume ratio than the conventional linear strips. Upon processing, they also absorbed 22% less oil, contained 26% less salt, and displayed superior sensory properties. Thus, ring fries may represent an attractive alternative to French fries as processed staple food.

LC-MS analysis of solanidane glycoalkaloid diversity among tubers of four wild potato species and three cultivars (Solanum tuberosum).

Secondary metabolites in potato tubers include both phytonutrients and plant defense compounds. The extent these small molecules vary among different potato genotypes is not well characterized. LC-MS analysis of tuber extracts from seven potato genotypes showed that one large source of small molecule variation is the glycoalkaloids. Glycoalkaloids are involved in the resistance of potatoes to pathogens and pests, but they also have implications for human health and nutrition. This study focused on glycoalkaloids with solanidane or solanidane-like aglycones, of which over 50 were tentatively identified, many of which appeared to be novel glycoalkaloids. Results suggested the variety of glycoalkaloids in potatoes is considerably greater than previously thought. Dissecting the role of these many glycoalkaloids in human health or pest and pathogen resistance will be a formidable undertaking.

Rapid screening of ascorbic acid, glycoalkaloids, and phenolics in potato using high-performance liquid chromatography.

Evaluation of phenolic metabolism in potato tubers (Solanum tuberosum) would be facilitated by faster analytical methods. A high-throughput HPLC method was developed for the qualitative and quantitative determination in potato of numerous phenolic compounds, the sum of the glycoalkaloids chaconine and solanine, plus ascorbic acid. Following a fast extraction, HPLC run times of 12 min were achieved with the use of a monolithic RP C18 column. UV and MS analyses were used to characterize the phenolic complement in extracts from two white-fleshed varieties. Over 30 compounds were identified, some of which are thought to possess either nutritional value or are involved in plant disease resistance. This method is expected to be useful for germplasm mining and for varietal development programs in which large numbers of lines are generated.

Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv. syringae B301 D-R.

Strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae secrete a family of structurally closely related peptide derivatives dubbed syringolins, of which syringolin A is the major variant. The function of syringolins in the interaction of P. syringae pv. syringae with their host plants presently is unknown. It is hypothesized that they may constitute virulence factors. However, syringolins are determinants recognized and reacted to by nonhost plant species, and syringolin A has been shown to induce hypersensitive death of cells colonized by powdery mildew in wheat and, thus, to reprogram a compatible interaction into an incompatible one. Syringolin A is an unusual derivative of a tripeptide that contains a 12-membered ring consisting of the amino acids 5-methyl-4-amino-2-hexenoic acid and 3,4-dehydrolysine, two nonproteinogenic amino acids. Here we report the cloning, sequencing, and analysis of genes involved in the biosynthesis of syringolin A. The genes encode proteins consisting of modules typical for nonribosomal peptide synthetases and type I polyketide synthetases, as well as proteins likely involved in the transcriptional regulation of syringolin A biosynthesis and in syringolin A export. The structure and arrangement of the modules lead to the formulation of a model explaining the synthesis of the tripeptide, including the formation of the two nonproteinogenic amino acids in the ring structure of syringolin A.