Metabolism of steviol glycosides in Stevia species

Until now, only a general outline of the in vivo dynamics of steviol glycoside (SVgly) accumulation in Stevia rebaudiana was known, with literature surveys turning up relatively few studies. The goal of the current research was to deepen our knowledge of SVgly accumulation, while seeking easy and cheap ways to stimulate it. From the results it is clear that SVgly accumulation was strongly influenced by plant development, which itself was affected by photoperiod. Both effects occurred in a cultivar-dependent manner, making extrapolation to the species level quite difficult. In general though, S. rebaudiana grown under long-day (LD) conditions accumulated greater amounts of biomass and SVglys by remaining vegetative for a prolonged amount of time. It was important to correctly denote SVgly levels, namely as amount per leaf or branch, or as amount per unit biomass. Both have different connotations, which may explain some of the contradictions found in literature. If SVgly levels were expressed per leaf, the greatest levels were commonly found in mid-stem leaves. Expressed per unit biomass, SVgly levels were greatest near the apex (under LDs) or the stem basis (under short-days, SDs), with patterns changing according to ontogenetic phase. The composition of the SVgly mixture differed widely in separated organs and dynamically changed throughout ontogeny, albeit to a lesser extent. In leaves, SVglys were strongly correlated, not only to each other but also to dry matter and free glucose levels, especially under SDs. This has possibly to to with a more controlled metabolic flux due to the more limiting role of photosynthesis under these conditions. Strong inverse correlations were found between the rebaudioside A to stevioside ratio and leaf dry mass or total SVglys per leaf. A greater ratio was found in smaller leaves near the stem apex and basis, whereas mid-stem leaves had the smallest ratio. However, the difference between both extremes proved to be cultivar-dependent.In leaves, total SVgly levels declined after flower opening, especially under SDs. This occurred largely independently of leaf dry mass. However, in specific conditions, when multi-annual cultivars exhibiting a strong lateral outgrowth after the onset of reproductive development are grown under LDs of 16 h, a harvest during flowering could still give great biomass and SVgly yields. Similar to SVglys, the levels of free SV declined at the onset of flowering, although this was clearer under LDs. Nothing is known so far about the cause of these declines during reproductive development. Despite great efforts, no clear indication was found for an endogenous SVgly-specific glycosidase activity. In addition, SVgly levels remained constant under dark stress and declined only during severe oxidative stress. Exploratory results indicated a kind of transport mechanism to the roots, although this requires further investigation.For the first time in almost 30 years, a detailed phytochemical analysis of S. phlebophylla leaves was conducted. Despite the initial report mentioning "traces of stevioside", no SVglys could be detected. Instead, a compound was extracted and characterised as a xylosylated derivative of acetylated suavioside E. Therefore, it seems likely that S. rebaudiana remains the only SVgly-producing Stevia species discovered so far. Besides the mapping of accumulation patterns, a well-known method using phytochrome activation was applied to S. rebaudiana to stimulate its production of biomass and SVglys. Illuminating plants each night with only 5 min red LED light was enough to mimic LD conditions (which prolong vegetative growth) under SDs. Transcriptional analysis of the genes involved in SVgly biosynthesis revealed fairly constant transcription during vegetative growth, both in plants under LDs and those under SDs receiving LED treatment. When plants grown under SDs moved to the reproductive stage, gene transcription decreased. While significant correlations were found between individual transcript levels, no such relationship existed between transcription and SVgly accumulation. Although field experiments are necessary for further validation of this method, it may prove to be a great asset for the optimisation of growth, field production and extraction efficiency of S. rebaudiana.status: publishe