1. Accumulation pattern of steviol glycosides in Stevia rebaudiana Bertoni and its stimulation by red light Stijn Ceunen Laboratory of Functional Biology K.U.Leuven Belgium

2. Contents Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana  General outline: what is known so far?  Fate of steviol glycosides  Photoperiodism  Experimental set-up  Results Stimulation of steviol glycoside accumulation by red light  Phytochrome response: principles  Experimental set-up  Results  Practical application: simulating long-days on the field  Acknowledgments

3. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana General outline: what is known so far? Source: Bondarev et al. (2003)

4. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana General outline: what is known so far?  Leaves >> Flowers > Stems > Seeds >> Roots  All organs have the three major steviol glycosides (SVglys), yet proportions can differ in each organ  Leaves: SVgly proportions may vary greatly depending on plant age and phase of plant development

5. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana General outline: what is known so far?  Steviol glycoside content within leaves Large natural variability in SVgly content, yet general observations can be made Vegetative state: gradual increase Maximal content: between flower bud formation and initial flowering (max. 10% flowers); afterwards: decline Upper young, actively growing leaves: higher SVgly content compared to more mature leaves which finished their growth

6. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana Fate of steviol glycosides  Transport to roots Cfr. monoterpene catabolism in Mentha piperita But: no significant amounts of SVglys were ever reported in roots  Transport to generative organs Cfr. ecdysteroids in e.g. Ajuga reptans But: only small amounts are found within flowers  In situ degradation by β-glycosidases Cfr. various secondary metabolites, e.g. cyanogenic glucosides,... But: preliminary incubation experiments didn’t yield significant amounts of steviol or glucose

7. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana Photoperiodism  Stevia rebaudiana is an obligate short-day (SD) plant with a critical daylength of 12-13 h  Long-days (LDs) delay flowering and increase SVgly accumulation  Compared to SD cultivation, plants under LD have: Larger, more ovate leaves Greater plant height, due to longer internodes and greater internode number Greater leaf area Greater water content More dry matter Greater protein and total soluble carbohydrate content Greater SVgly content due to proportionally more SV biosynthesis compared to total soluble carbohydrate content

8. Spatial and temporal accumulation of steviol glycosides within Stevia rebaudiana Experimental set-up

11. Stimulation of steviol glycoside accumulation by red light Is there an inexpensive way to easily simulate LD conditions during SD regime (e.g. during autumn)?  Yes

12. Stimulation of steviol glycoside accumulation by red light Phytochrome response: principles  Light-sensitive pigment found within leaves  Sensitive to red (660 nm) and far-red (730 nm) light  Regulates the time of flowering, seed germination, elongation of seedlings, etc…  Pr / Pfr ratio determines if certain processes will take place  As daylength is sensed by phytochrome, activation by red light might simulate LDs by prolonging vegetative growth and avoiding flower bud formation

13. Stimulation of steviol glycoside accumulation by red light Experimental set-up  First experiment Control SD group: 12 h photoperiod LED-treated SD group: 12 h photoperiod + 1 h red LED light (λ max 660 nm) during the night Samples were taken every week for SVgly analysis  Second experiment Control SD group: 8 h photoperiod LED-treated SD group: 8 h photoperiod + 5’, 15’, 30’ or 1 h red LED light (λ max 631 nm) during the night Samples were taken at week 0, 4 and 9 for SVgly analysis

14. Stimulation of steviol glycoside accumulation by red light Results  First experiment

15. Stimulation of steviol glycoside accumulation by red light

16. Results  First experiment

17. Stimulation of steviol glycoside accumulation by red light Results  Second experiment

21. 9.8x more in 16 weeks

23. 26.5x more in 16 weeks

24. Stimulation of steviol glycoside accumulation by red light Practical application: simulating long-days on the field  Scaling up to a field: belts of red LED lights  Only 5 min. illumination is sufficient  In our experiment: SD conditions (e.g. < 12 h photoperiod, cfr. Paraguay): 87 mg SVglys / plant after 10 weeks SD+LED conditions: 4032 mg SVglys / plant after 25 weeks (46x more!)  Ideally, 5 crops / year: 435 mg / plant  LED plants: 2 crops / year: 8064 mg / plant  18x more yield per plant

25. Stimulation of steviol glycoside accumulation by red light Practical application: simulating long-days on the field  Advantages Less labour Low amounts of electricity needed (solar power!) LEDs have long lifespans (> 20,000 hours; at 5 min / night, this theoretically means 657 years) Small investment per hectare (± 10,000 euro) Easier extraction due to greater percentage yield Temperate climate: at least two-month prolongation of growth season, thus more production of plant biomass and sweetener Continuous vegetative propagation of interesting cultivars

26. Acknowledgments Prof. Dr. Jan Geuns for supervising the research Prof. Dr. Stefaan Werbrouck for providing the phytotron and for setting up the first experiment Uria, my wife, for making the pencil drawings of Stevia plants in different developmental stages Mr. Christophe Pépin for providing the young Stevia seedlings Tom, Hilde and Amal for excellent technical support