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The biosynthesis of steviol glycosides is affected by both genetic and environmental factors. To evaluate the influence of total daily solar radiation or daily light integral (DLI) under long-day conditions on steviol glycoside concentration, we grew Stevia rebaudiana under ambient irradiance or varying levels of shading at different times of the year in both greenhouse and field environments, resulting in DLIs ranging from 3.55 to 20.31 mol·m·d in the greenhouse and 10.32 to 39.7 mol·m·d in the field. Total steviol glycoside concentration of selected leaves from greenhouse-grown plants increased as DLI increased up to ca. 10 mol·m·d, remaining constant with further increases in DLI, and was similar across the range of DLIs evaluated in the field. DLI influenced both the concentration and the relative proportions of specific steviol glycosides. Rebaudioside A concentration increased as DLI increased from 3.55 to 8.53 mol·m·d, remaining similar with further increases in DLI. Rebaudioside D and stevioside concentration of selected leaves from field-grown plants decreased by 22% and 13%, respectively, as DLI increased from 10.32 to 39.7 mol·m·d, while rebaudioside A and M concentrations remained similar across this DLI range. Collectively, these results indicate that the greatest influence of DLI on steviol glycoside concentration occurs under relatively low DLIs (<10 mol·m·d). However, higher DLIs can significantly affect the synthesis of minor glycosides of increasing commercial importance including rebaudioside D. The production of steviol glycosides in plant species is rare, a characteristic that Stevia rebaudiana shares with only three other known species, Stevia phlebophylla (Kinghorn et al., 1984), Rubus suavissimuss (Ohtani et al., 1992) and Angelica keiskei (Zhou et al., 2012). Steviol glycosides are tetracyclic diterpenes, which share precursors with gibberellins (Brandle and Telmer, 2007). Discovery of new diterpene glycosides has been swift in recent years, and at least 34 steviol glycosides have been identified in S. rebaudiana (Ceunen and Geuns, 2013a). The diversity of steviol glycosides results from differential glycosylation of the aglycone steviol by several cytosolic Uridine diphosphate (UDP) glycosyltransferases (Brandle and Telmer, 2007). These UDPglycosyltransferases (UGTs) transfer activated sugars, usually UDP-glucose, to acceptor molecules that produce the mixture of glycosides found in the vacuoles of leaf cells of S. rebaudiana (Richman et al., 2005). The enzymes catalyzing steps in the steviol glycoside biosynthetic pathway are beginning to be elucidated, including the glycosylation of steviol by UGT85C2, the first step in steviol glycoside formation, to form steviolmonoside (Mohamed et al., 2011). Stevioside, a triglycoside, and rebaudiosideA, a tetra-glycoside, are typically produced in the highest concentrations while glycosides, including rebaudioside B (triglycoside), rebaudiosideC (tetra-glycoside), rebaudioside D (penta-glycoside), and rebaudiosideM (hexa-glycoside; also known as rebaudioside X) are typically produced in much lower concentrations (Kennelly, 2002). UGTs are not specific to S. rebaudiana and play a large role in the diversity of secondary metabolites produced by plant species (Richman et al., 2005). At least 12 UGTs have been identified in S. rebaudiana, but only three have been directly linked to steviol glycoside synthesis (Ceunen and Geuns, 2013a; Richman et al., 2005). Several studies have explored the effects of photoperiod (Ceunen and Geuns, 2013b: Metivier and Viana, 1979; Valio and Rocha, 1977; Zaidan et al., 1980) and the effects of light quality, specifically, red light (Ceunen and Geuns, 2013b; Ceunen et al., 2012) on steviol glycoside accumulation in S. rebaudiana. However, the effects total daily radiation or the DLI have yet to be elucidated. Previous studies have quantified biomass yield potential under a range of DLIs under long photoperiods, however, no glycoside data were reported (Ermakov and Kochetov, 1996). A more recent study found that a reduction in light did not significantly affect steviol glycoside concentrations except at the crop development period described as the flower initiation stage, where rebaudioside A levels were significantly higher with 25% light reduction (Kumar et al., 2013). However, only stevioside and rebaudioside A levels were reported. The effect of DLI on synthesis of rebaudiosides B, C, D, andM has not previously been reported. The objective of this study was to evaluate the influence of DLI during long days on the concentrations and relative proportions of several steviol glycosides. Experiments were conducted in both greenhouse and field environments to create a wide range of DLIs. Materials and Methods Experiment 1 Plant material and propagation. On 14 Feb. 2012 (run 1) and again on 24 July 2012 (run 2), two Stevia rebaudiana selections from an open-pollinated population, designated 10-43-41 and 11-464, were vegetatively propagated into plug trays [128-cell size (12 mL volume)] in a soilless medium consisting of 50% fine perlite (Perlite Vermiculite Packaging Industries Inc., North Bloomfield, OH) and 50% potting mix composed of peat, perlite, lime, and starter fertilizer (Suremix Perlite; Michigan Grower Products Inc., Galesburg, MI). The cuttings were rooted in a glass-covered greenhouse under a 16-h photoperiod [ambient daylight plus day-extension lighting provided by highpressure sodium (HPS) lamps from 0600 to 2200 HR] at an air temperature of 24 C and bench temperature of 26 C. Bench temperature was regulated by running hot water through tubes inlayed in a Styrofoam sheet on the benchtop. The watering regime consisted of intermittent overhead mist supplemented with a water-soluble fertilizer providing (mg·L) 50 N, 8 P, 42 K, 22 Ca, 12 Mg, 1.0 Fe and Cu, 0.5 Mn and Zn, 0.3 B, and 0.1 Mo (MSU Prop Special; GreenCare Fertilizers Inc., Kankakee, IL).When plant roots reached all corners of the plug cell, rooted cuttings were transplanted into 10-cm round plastic containers (480 mL volume) filled with 100% Suremix Perlite potting mix and moved to the greenhouse environment described below. Greenhouse environment and plant culture. Plants were grown under a photoperiod of 16 h (0600 to 2200 HR) achieved by supplementing the natural photoperiod (42 N lat.) with day-extension lighting provided by 400WHPS lamps (LU400; GE,Mississaugua, Canada) supplying a supplemental photosynthetic photon flux of 90 mmol·m·s at plant Received for publication 4 Dec. 2014. Accepted for publication 17 Mar. 2015. This project was supported by PureCircle SDN BHD, the USDA National Institute of Food and Agriculture and AgBioResearch at Michigan State University. J.M.E. and R.M.W. designed the experiments and wrote the manuscript. J.M.E. conducted the experiments and analyzed data. J.M.E., V.A.V., and R.M.B. conducted the steviol glycoside quantification. Corresponding author. E-mail: warnerry@msu. edu. HORTSCIENCE VOL. 50(10) OCTOBER 2015 1479 height as measured by a custom-made line quantum sensor with 10 photodiodes (Apogee Instruments Inc., Logan, UT). The HPS lamps were operated by an environmental control computer (Priva Integro 724; Priva, Vineland Station, Ontario, Canada). When irradiance was less than 580 mmol·m·s for at least 10 min supplemental lighting was activated. Lighting would shut off only when irradiance was greater than 580 mmol·m·s for a minimum of 20 min. Average temperatures and standard deviations are reported in Table 1. Plants were irrigated as needed with reverse osmosis water supplemented with a water-soluble fertilizer supplying (mg·L) 125 N, 12 P, 100 K, 65 Ca, 12 Mg, 1.0 Fe and Cu, 0.5 Mn and Zn, 0.3 B, and 0.1 Mo (MSU RO Water Special; GreenCare Fertilizers Inc.). On 17 Apr. 2012 (run 1) and 1 Oct. 2012 (run 2) plants were pinched back one node and sprayed with 200 mg·L ethephon (Florel; SouthernAgricultural Insecticides Inc., Hendersonville, NC) diluted with reverse osmosis water to abort any flower buds that may have been present. At the onset of the experiment, the number of lateral branches exceeding 3 cm in length and the number of nodes per branch were recorded and 36 representative plants per genotype were selected for the experiment. Each DLI environment described below was replicated three times, and each replicate consisted of four plants per genotype. Plants were placed into their respective treatments on 3 May 2012 (run 1) and 5 Oct. 2012 (run 2), and spaced 23 cm from midpot to midpot. After 4 weeks in treatment, all plants were repotted into 15-cm square plastic containers (2506 mL) filled with the same medium and spaced 36 cm from midpot to midpot. Plants were grown in treatment for 9 weeks following the ethephon application. DLI treatments. Six different DLIs were created using two different woven shadecloths (OLS 50, OLS 30; Ludvig Svensson Inc., Charlotte, NC) or no shadecloth, over two time periods (May–July and October– Dec. 2012, runs 1 and 2, respectively), resulting in average DLIs ranging from 3.55 to 20.31 mol·m·d (Table 1). Tissue sampling and steviol glycoside analysis. Following 9 weeks in treatments, leaf tissue samples were collected for quantification of steviol glycosides. Ten fully expanded leaves selected from about 66% of the height of the plant (from the base) were collected (representing young, but fully expanded leaves) and dried at 65 C for at least 72 h, then transferred to 15-mL conical tubes and held at –20 C. Ten samples per genotype from each treatment and replicate were selected for analysis. Samples were allowed to warm to room temperature before seven stainless steel ball bearings (3.97 mm diameter, 440C-G16, VXB Ball Bearings; NationSkander California Corp., Anaheim, CA) were added to each tube. Samples were ground via shaking with an adapted paint shaker for 15 min until the sample was a fine powder. Ten to twelve milligram samples were transferred to 1.5-mL

Daily Light Integral Influences Steviol Glycoside Biosynthesis and Relative Abundance of Specific Glycosides in Stevia