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Effects of light intensity and photoperiod on improving steviol glycosides content in Stevia rebaudiana (Bertoni) Bertoni while conserving light energy consumption
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
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Effects of light intensity and photoperiod on improving steviol glycosides content in Stevia rebaudiana (Bertoni) Bertoni while conserving light energy consumption ⁎
Yuki Yoneda, Hiroshi Shimizu , Hiroshi Nakashima, Juro Miyasaka, Katsuaki Ohdoi Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Stevia rebaudiana (Asteraceae) Steviol glycosides Light intensity Photoperiod Night interruption End-of-day far-red
Stevia rebaudiana (Bertoni) Bertoni accumulates steviol glycosides (SGs) which are natural sweeteners. Leaf extracts have been widely used as a food additive because the strong sweetness intensity of SGs that is 300 times sweeter than sucrose. S. rebaudiana is a short-day (SD) perennial plant, and some studies claim that long-day (LD) treatment affects SGs accumulation. However, the optimal and detailed light regime required to increase total SGs content remains controversial. We analyzed the effect of various light intensities and day lengths to determine optimal light treatment for SGs accumulation with minimal energy consumption in growth chambers. Night-interruption (NI) treatments in conjunction with 8 h photoperiod increased leaf biomass to amounts observed under LD conditions, and resulted in higher SGs content than that observed under 12 h photoperiod while minimizing energy consumption. In addition, end-of-day far-red (EOD-FR) treatment for 15 min at 50 μmol m−2 s−1 was associated with transcription upregulation of the SGs-related gene UGT85C2, higher SGs content, and similar leaf biomass compared to that observed under 16 h photoperiod. These light treatments offer great potential for energy-efficient increases in S. rebaudiana total SGs content when plants are grown in a greenhouse or in the field with supplemental lights.
1. Introduction Stevia rebaudiana (Bertoni) Bertoni is a perennial plant that belongs to the plant family Asteraceae native to South America (Kinghorn, 2002). S. rebaudiana leaves contain sweet compounds known as steviol glycosides (SGs) which possess sweetness intensity approximately 50–450 times stronger than sucrose (Crammer and Ikan, 1986). The most abundant SG is stevioside (stev) (5–10% of the total dry matter) followed by rebaudioside A (reb-A) (2–4%) (Crammer and Ikan, 1986; Chatsudthipong and Muanprasat, 2009). Some studies have claimed that consumption of artificial sweeteners is associated with a risk of long-term weight gain, which could induce metabolic syndrome, and it has reported that saccharin induces glucose intolerance and dysbiosis (Fowler et al., 2008; Lutsey et al., 2008; Nettleton et al., 2009; Suez et al., 2014). Conversely, S. rebaudiana is used as a natural sweetener in Asia and South America with no toxicity for humans (Koyama et al., 2003; Brusick, 2008). It is possible to extract the most abundant SGs from upper large young leaves (Mohamed et al., 2011; Kumar et al., 2012); therefore, it is important to increase total leaf biomass without reducing total SGs content.
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Light is an important environmental factor for plant growth, and can be easily controlled using supplemental lights in a field or in a greenhouse. S. rebaudiana grows under a sunny climate in its native habitat (Ceunen et al., 2013a; Osman et al., 2013), but few studies have investigated optimal light intensity for SGs accumulation (Slamet and Tahardi, 1988; Kumar et al., 2013). In addition, it is difficult to isolate the effects of light intensity from other factors because these studies described experimentation in the field. S. rebaudiana is a SD plant; however, was reported that total SGs content increases under a long day (LD 16 h) compared to SD (8 h) photoperiod (Brandle and Rosa, 1992; Ceunen et al., 2013a). A natural LD environment is available during limited seasons and in limited areas of cultivatable land. However, supplemental light treatment can easily modulate photoperiod by using additional electrical energy would be required to artificially extend the photoperiod to create a LD environment (Ceunen et al., 2012). Furthermore, the percentage of SGs content which does not mean the total SGs yield per plant was higher under a SD rather than a LD photoperiod (Mohamed et al., 2011). This suggests that a SD photoperiod increases the percentage of SGs content without increasing total biomass, and total SGs yield would increase if leaf
Corresponding author. E-mail address: [email protected] (H. Shimizu).
http://dx.doi.org/10.1016/j.jarmap.2017.06.001 Received 1 December 2016; Received in revised form 31 May 2017; Accepted 3 June 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Yoneda, Y., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.06.001
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culture medium. Cuttings were composed of a stem (4 cm in length) from the shoot apex, which had leaves from the 3rd node to the apex, and the leaf pair of the 3rd node were cut in half. The cut stem was placed in water for 1 h, and then the cut end was coated with a rooting promoter (Rooton, Sumitomo Chemical Garden Products, Tokyo, Japan) and inserted into a urethane sponge. Sponges were maintained soaked in a nutrient supplement (Menedael, Menedael Co., Ltd., Osaka, Japan) diluted 100 times in water and cuttings were grown for 2 weeks under the following conditions: the 16 h photoperiod, 24 °C during light periods, 20 °C during dark periods, and 99% humidity. A fluorescent lamp (FL) (100MHF142DR, Monocoqtex, Inc., Tokyo, Japan) was used as the light source during rooting, which provided a photosynthetic photon flux (PPF, 400–700 nm) of 120 μmol m−2 s−1. PPF was measured using a light quantum meter (I-250A, LI-COR Inc., USA). For each test section, a 30 cm × 60 cm × 7 cm cultivation tank was filled with nutrient solution (OAT house A No. 1 and No. 2, OAT Agrio Co., Ltd., Tokyo, Japan) (pH 6.4 and EC 1.3 dS m−1) which was aerated using an air pump (Non-noise S100, Japan Pet Design CO., LTD., Tokyo, Japan).
biomass were increased under a SD photoperiod. The use of light emitting diodes (LEDs) has been an alternative in the artificial lighting for the increase of the photoperiod in different cultures (Goto, 2008). Using LEDs that supplied weak-intensity red light, Ceunen et al. (2012) investigated the effect of night-interruption (NI) during the dark period following a SD photoperiod (8 h) in S. rebaudiana. This NI was shown to increase total SGs content compared to plants grown under normal SD conditions. NI is a method used to delay flowering time in commercial production of chrysanthemum, which like S. rebaudiana is a SD plant (Goto, 2008). NI involves interrupting the dark period with weak-light treatment, which can affect the flowering phase (Kim et al., 2011). These results suggest that the NI is a possible alternative to growing S. rebaudiana plants under a LD photoperiod. NI effect involves the red-light photoreceptor phytochrome which has two different forms of the Pfr form (the absorption maximum: 730 nm) and the Pr form (the absorption maximum: 660 nm) (Smith and Whitelam, 1990). NI with red light of 620–640 nm and 660 nm delays the onset of flowering in cocklebur (Xanthium) and morning glory (Pharbitis nil), respectively, which thus also prolongs vegetative growth (Hendricks and Siegelman 1967; Saji et al., 1983). Phytochrome can signal different stages of photomorphogenesis when it perceives different ratios of red/far-red light (Sullivan and Deng, 2003). On the other hand, end-of-day (EOD) treatment, where a pulse of low-intensity light of a different quality is administered during the transition from the photoperiod to the dark period, also involves phytochrome. EOD-far-red (EOD-FR) treatments resulted in enhanced petiole and stem elongation and increased gibberellin (GA) content in chrysanthemum, spinach, and cowpea (Hisamatsu et al., 2005; Martínez-García et al., 1995). These studies did not show an increase in the total leaf area, but it should be noted that GA treatment affected SGs content (Hajihashemi et al., 2013). As the SGs biosynthetic pathway converges with the GA biosynthetic pathway through the formation of ent-kaurenoic acid, a change in GA content is presumed to regulate SGs content; however, the effect of EOD-FR on SGs content has not been clarified. The objective of this study was to investigate an optimal light environment for SGs accumulation and enhanced leaf biomass in S. rebaudiana with minimal electrical energy consumption. Therefore, we analyzed plant morphology and SGs content under light treatments that varied light intensity and photoperiod as well as NI and EOD light pulses. Especially, NI treatment, which has an effect to enhance vegetative growth, and EOD treatment, which may have a potential to impact on SGs content, can be expected to improve the total of SGs with saving daily light integral (DLI). In this study, SGs content was evaluated using two different approaches: a quantitative genetic analysis and high performance thin layer chromatography (HPTLC) and a transcriptional analysis of Kaurene oxidase (KO) gene and two UDP-17 glycosyltransferases (UGT) genes which are named UGT85C2 and UGT76G1. According to Mohamed et al. (2011), a glycosylation of steviol, which is a basic skeleton of SGs, to steviolmonoside, which is precursor of stevioside, is a rate limiting step, and UGT85C2 catalyzes this step. Normally, high performance liquid chromatography (HPLC) is a practical method to evaluate SGs content; however, it is difficult to evaluate SGs content in a small-scale experiment using HPLC because a certain amount dry leaf tissue is required. HPTLC method demonstrated the accuracy of the amount of SGs content (Jaitak et al., 2008). Therefore, HPTLC is a suitable alternative that can save cost and analysis time.
2.2. Experimental light treatment After rooting for 2 weeks, S. rebaudiana plants were transplanted to 18 differing light conditions (Fig. 1). Fig. 1A shows how fluorescent lamp light intensity in the 16 h photoperiod was varied between 50 and 400 μmol m−2 s−1. Fig. 1 B shows how fluorescent lamp photoperiods were varied between 8 and the 24 h. Fig. 1C illustrates NI treatments using light from a fluorescent lamp, as well as far-red (730 nm) and red (660 nm) LEDs. NI was administered by a four hours weak-light (20–50 μmol m−2 s−1) treatment during the dark period that followed the 8 h photoperiod (Fig. 1C). For these, the 8 h photoperiod with light supplied by a fluorescent lamp was followed by a 6 h dark period, which was then interrupted with 4 h of low-intensity light (20–50 μmol m−2 s−1) originating from a fluorescent lamp, or far-red or red LEDs, then followed by a further 6 h dark period. Fig. 1D shows
2. Material and methods 2.1. Plant material
Fig. 1. List of light treatments. Treatments included various light intensities(A), photoperiods (B), night-interruptions (NI) (C), and end-of-day (EOD) pulses (D). For the NI, diagonal lines indicate use of a fluorescent lamp (FL), or far-red (FR) or red (R) LEDs. For EOD, diagonal lines indicate a FR or R pulse for 5–15 min. Gray bars: photoperiods. Black bars: dark periods. DLI: daily light integral. PPF: photosynthetic photon flux.
In order to limit genetic variation, S. rebaudiana plants were propagated from multiple cuttings from a parent plant. Plants were grown hydroponically in growth chambers, using urethane sponges as the 2
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2.5. HPTLC
EOD treatments using light from far-red and red LEDs. For these, the 16 h photoperiod with light supplied by a fluorescent lamp was ended with a 5–15 min pulse of 50 μmol m−2 s−1 light from far-red or red LEDs, just prior to the dark period. All other environmental conditions were kept constant between experimental treatments, as follows: 25 °C, 41% humidity, and 535 ppm CO2 during light periods, 21 °C, 67% humidity, and 585 ppm CO2 during dark periods. Other growth conditions such as temperature, CO2 concentration, and humidity were recorded using a data logger (TR72Ii, T & D, Nagano, Japan). Light sources were a fluorescent lamp (FDA21093A, Panasonic Corporation, Kadoma, Japan), and far-red (PR2N-3LEE-SD, ProLight Opto Technology Corporation., Taoyuan, Taiwan) and red (LXM3-PD01, Lumileds Holding B.V. CA, USA) LEDs. Aside from treatments where light intensity was varied (Fig. 1A), the light intensity during main photoperiods was 120 μmol m−2 s−1. The shoot apex was constantly exposed to the same light intensity by adjusting plant position using lab jacks. Far-red irradiance of the shoot apex was adjusted using a spectroradiometer (LI-1800, LI-COR Inc., USA).
HPTLC was used for analyzing SGs content following the method of Jaitak et al. (2008). Samples from 6-week-old cuttings were spotted onto a HPTLC plate (HPTLC 60 F254, E. Merck, Darmstadt, Germany). Tissue samples, two excised pieces 0.8 cm in diameter, were taken from the sixth leaf from the bottom of plants. In a sequential manner, the samples were homogenized following the addition of 20 μl distilled water. Mobile phase consisted of ethyl acetate, ethanol, and distilled water (80: 20: 12), and each solution was measured separately. After mixing all of the solution, an expansion tank was filled with it and waited until saturated (20 min). Spots were created on HPTLC plates using 2 μl of homogenized sample from the bottom by 1 cm and each spot was separated by more than 1 cm. The plate was sprayed with a mixed solution of ethanol, acetic anhydride, and sulfuric acid (10: 0.1: 0.1) after expansion and drying. HPTLC plates were semiquantified by ImageJ (http://rsbweb.nih.gov/ij/). 2.6. Daily light integral Daily light integral (DLI) is the total amount of PPF (400–700 nm) per square meter per day. Total light energy consumption was considered in this study. Therefore, we calculated DLI including energy from far-red light (peak wavelength: 730 nm). DLI for each experimental light treatment is indicated in Fig. 1.
2.3. Morphological analysis S. rebaudiana plants of the respective conditions were sampled 4 and 6 weeks after cutting. The average measurements with standard deviation were calculated from the samples. Morphological analysis included total leaf area and fresh weight without a subset of leaves from the first leaf pair to the forth leaf pair from the bottom. These leaves had already developed prior to commencing experimental light treatments. Further morphological analysis included measurements of stem length, stem diameter, and leaf number per plant. Stem length was measured from the top of the supporting urethane sponge to the shoot apex, and stem diameter was measured around the center of a stem. Leaf area was determined by scanning leaves and measuring area by ImageJ (http://rsbweb.nih.gov/ij/).
2.7. Statistical analysis Tukey-Kramer (p < 0.05) was adopted for statistical analysis. Plants were sampled after 4 and 6 weeks cutting growth, and four plants were used in each case of morphological analysis. The following experimental treatments, which have analyses based on only three plants: 4-week-old cuttings treated with varying light intensity the PPF 50, the PPF 100 and the PPF 200, varying photoperiod the 12 h and the 24 h, varying night-interruption the NI-FL 20, the NI-FL 50, the NI-FR 20, and the NI-R 20, and varying end-of-day pulse the EOD-FR 5 min, the EOD-FR 15 min, and the EOD-R 15 min. In addition, 6-week-old cuttings treated with varying photoperiod 20 h, and varying night-interruption NI-FL 50 and NI-FR 20 were also three plants. 4-week-old cuttings treated with varying photoperiod 8 h and 10 h were not analyzed.
2.4. Quantitative RT-PCR analysis As a gene transcription analysis, a seventh leaf from the bottom of the plant was selected because this represented tissue that had developed post transplantation and experimental light treatment. After 6 weeks of cutting growth, leaf tissue was frozen in liquid nitrogen and ground using a mortar and a pestle. Leaves were sampled in the middle of photoperiod, meaning following 4 h of light during the 8 h photoperiod, and following the 8 h of light during the 16 h photoperiod. Total RNA was extracted using a RNA extraction kit (RNA suisui-P, Rizo Inc., Tsukuba, Japan) and RNA extraction column (FARBC-C50, Favorgen, Taiwan). The cDNA was synthesized from 0.5 μg of total RNA using a reverse transcription-polymerase chain reaction (PCR) kit (Revere Tra Ace, TOYOBO CO., LTD. Life Science Department, Osaka, Japan). This cDNA was then used for quantitative real-time (RT)-PCR (qPCR) in 20 μl reactions using a qPCR kit (KAPA SYBR FAST qPCR kit, Kapa Biosystems, Inc., Woburn, MA, USA). A 2-step program was used as follows: 95 °C 30 s × 1 cycle, 95 °C 3 s, and 60 °C 30 s × 40 cycles. 18S rRNA and βActin were used as internal standards. The calculation method was adopted the Vandesompele method for evaluating relative transcription levels (Vandesompele et al., 2002). Calculations were performed using Gene Transcription Macro™ (Bio-Rad, Hercules, CA, USA). Primers for used for β-Actin, 18S rRNA, UGT85C2, UGT74G1, and UGT76G1 were derived from Mohamed et al. (2011). Primers for KO were derived from Hajihashemi et al. (2013). UGT74G1 is also one of the UGT genes. UGT74G1 catalyzes the step to stev from steviolbioside. It is not reported that there is a correlation between SGs content and transcription level of UGT74G1. Three biological replicates were analyzed per experimental light treatment.
3. Results 3.1. Morphological and genetic analyses 3.1.1. Effect of light intensity on S. rebaudiana morphology and SGs-related gene transcription All of the analyzed morphological traits were seen to increase following experimental treatments with increasing light intensities. In particular, plants subject to the PPF 400 treatment possessed the significantly morphology (2A and 3 Treatment A). Total leaf area following the PPF 50, the PPF 100, and the PPF 200 treatments increased in accordance with increasing light intensity, but these differences were not significant. Despite a doubling of light intensity in the PPF 100 compared to the PPF 50 treatment, plant morphologies were similar. DLI of the PPF 400 treatment was 8 times greater the PPF 50 treatment (Fig. 1A), which resulted in a 4.6-fold increase in the total leaf area in 6-week-old cuttings. Morphological differences following experimental treatments were initially apparent in 4-week-old cuttings and more obvious in 6-week-old cuttings. Following the PPF 400 treatment, a number of leaves displayed damage, in particular those leaves in the bottom half of the cutting (Fig. 2A). Several lower leaves appeared completely withered, and others displayed middle-leaf and spot damage. Variation in light intensity did not affect KO, UGT85C2, and 3
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Fig. 2. Morphology of 6-week-old stevia cuttings following various light treatments. Variation of light intensity between 50 and 400 μmol m−2 s−1 photosynthetic photon flux (PPF) (A). Variation of photoperiod between 8 and 24 h (B). Application of night-interruption (NI) using light from a fluorescent lamp (FL), or far-red (FR) or red (R) LEDs at intensities of 20 μmol m−2 s−1 (20) or 50 μmol m−2 s−1 (50) (C). Plants grown with the 8 h and the 12 h photoperiod and no the NI are included for comparison. Application of end-of-day (EOD) FR or R light pulses of 5–15 min (D). A plant grown with the same the 16 h photoperiod and no EOD light pulse is included for comparison. Scale bar indicates 10 mm. All samples were taken from 6-week-old cuttings.
transcription levels following any varied photoperiod treatment; however, the tendency of UGT76G1 transcription levels were similar to UGT85C2.
UGT74G1 transcription levels (Fig. 4A), as no significant difference was observed in their transcription levels following each PPF treatment. In contrast, transcription level of UGT76G1 following the PPF 100 treatment significantly increased compared to other PPF treatments, with transcription levels following the PPF 100 treatment approximately 4.9 fold higher that those following the PPF 50 treatment.
3.1.3. Effect of night-interruption on S. rebaudiana morphology and SGsrelated gene transcription Between the 8 h (control), the NI-FL 20, the NI-FR 20, and the NI-R 20 treatments, stem length following the NI-FR 20 treatment was the longest among following the control and other NI treatments (Figs. 2C and 3 Treatment C). However, there were no significant changes in the total leaf area and weight, leaf number, and stem diameter. The total leaf area and weight of following the NI-FL 50 treatment were significantly increased compared to the 8 h photoperiod control (approximately 2 times larger), despite a similar DLI. DLI of the NI-FL 50 treatment (4.18 mol m−2 d−1) was less than for the control the 8 h photoperiod (3.46 mol m−2 d−1). NI treatments in general did not affect KO and UGT76G1 transcription levels and only some treatments affected UGT85C2 transcription levels. Compared to control plants grown under either the 8 h or the 16 h photoperiod, KO and UGT76G1 transcription levels did not significantly change following any of NI treatments (Fig. 4C). In addition, there was no significant difference in UGT85C2 transcription levels between the 8 h photoperiod control, the NI-FL 20, the NI-FL 50, and the NI-FR 20 treatments. However, UGT85C2 transcription levels were lower in the 12 h photoperiod and following the NI-R 20 treatment compared to the 8 h photoperiod control plants.
3.1.2. Effect of photoperiod on S. rebaudiana morphology and SGs-related gene transcription Experimental light treatments that extended the photoperiod resulted in an increase all analyzed morphological traits apart from total leaf number (Figs. 2B and 3 Treatment B). No change in the total leaf number was observed following any of the experimental light treatments. Large standard deviations were observed for average total leaf area and average total leaf weight following the 18 h and the 24 h photoperiod treatments. In addition, lateral buds were observed on cuttings grown under long photoperiods. DLI for the 24 h photoperiod treatment was three fold higher the 8 h treatment (Fig. 1B), which resulted in an approximate 2.2-fold increase in the total leaf area. In contrast, the total leaf area did not show a significant difference in the 24 h and the PPF 400 treatments (P = 0.16, data not shown). Even though there were damages to leaves under the PPF 400 condition, there were no spot damages in leaves under the 24 h condition (Fig. 2). Variation in photoperiod was seen to affect transcription levels of KO and UGT85C2, but not UGT74G1 and UGT76G1 transcription levels. KO transcription levels were highest following the 24 h photoperiod treatment (Fig. 4B), which were approximately 3.3 fold higher than following the 8 h photoperiod treatment. UGT85C2 transcription levels were highest following the 8 h and the 24 h photoperiod treatments. There was no significant difference in UGT74G1 and UGT76G1
3.1.4. Effect of end-of-day light pulse on S. rebaudiana morphology and SGs-related gene transcription The morphology of S. rebaudiana cuttings following EOD treatments were compared with control plants grown under the 16 h photoperiod alone, as 5–15 min EOD light pulses with light from a fluorescent lamp, 4
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Fig. 3. Quantitation of morphological traits of stevia cuttings following various light treatments. Experimental light treatments A–D are as described in Figs. 1 and 2. Total leaf area and fresh weight excluded the four leaves at the bottom of cuttings. Bars represent average measurements of 4-week-old (gray bars) and 6-week-old (black bars) cuttings. Error bars represent the standard deviation of the mean (n = 4 or 3, see Material and methods). Lowerand upper-case letters indicate significant difference in 4- and 6-week-old cuttings, respectively (TukeyKramer method, p < 0.05).
photoperiod control plants. For the 8 h photoperiod control plants, the product of average total leaf area and average UGT85C2 transcription level was 216.3. However, for plants subject to the NI-FL 50 treatment, the same product was 439.6. Plants following the NI-FL 50 treatment were 2 fold larger the 8 h photoperiod control plants. In addition, DLI of the NI-FL 50 treatment was slightly higher (approximately 1.2 fold) than for the 8 h photoperiod alone (Fig. 1C). Even though DLI of the NIFL 50 treatment was less (approximately 0.8 fold) than for the 12 h photoperiod, the 439.6 figure for total leaf area × UGT85C2 transcription following the NI-FL 50 treatment was larger than 124.6 for total leaf area × UGT85C2 transcription for the 12 h photoperiod control plants. When considering leaf biomass following EOD light pulse treatments, there was no significant difference in the total leaf area (Fig. 3 Treatment D). Average total leaf area in the 16 h photoperiod control plants was 27.9 cm2/plant and 37.2 cm2/plant following the EOD-FR 15 min treatment. Plants following the EOD-FR 15 min treatment were 1.3 fold larger than those subjected to a 16 h photoperiod alone. The product of average total leaf area and UGT85C2 transcription level in the 16 h photoperiod plants was 53. In contrast, the same product following the EOD-FR 15 min treatment was 524.1. This figure for total leaf area × UGT85C2 transcription following the EOD-FR 15 min
or red or far-red LEDs were administered following the 16 h photoperiod (Fig. 1D). Compared to control plants, EOD treatments resulted in a significant difference in the total leaf weight and leaf number only (Figs. 2D and 3 Treatment D). No significant change in stem length was observed by ANOVA, whereas only the EOD-FR 15 min treatment significantly elongated than control by t-test (P < 0.05, data not shown). Despite an increase in leaf number following all EOD treatments, no increase in leaf biomass was observed following the EOD-FR 15 min and the EOD-R 15 min treatments. KO and UGT74G1 transcription levels were unaffected by EOD light pulse treatments (Fig. 4D). However, UGT85C2 and UGT76G1 transcription levels following the EOD-FR 15 min treatment were higher than in the 16 h photoperiod control plants (approximately 7.5 fold and 4.6-fold transcription, respectively). Similar transcription levels were observed for UGT85C2 and UGT76G1 between the 16 h photoperiod control, the EOD-FR 5 min, and the EOD-R 15 min treatments. 3.2. Relationship between leaf biomass and SGs-related gene transcription For the NI experimental light treatments, average total leaf area following the NI-FL 50 treatment was 44.0 cm2/plant, which was near double the 23.5 cm2/plant average leaf area observed in the 8 h 5
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Fig. 4. Relative expression of SGs-related genes. Experimental light treatments A–D are as described in Figs. 1 and 2. Each bar represents the mean. Error bars represent the standard deviation of the mean (n = 3). Letters indicate significant difference (Tukey-Kramer method, p < 0.05). All samples were taken from 6-week-old cuttings.
than all of NI treatment plants, stev spot was detected 0.32-fold relative to the 8 h photoperiod control. Reb-A spot of the NI-FL20, the NI-FL50, and the NI-FR20 treatments were 0.53, 0.79, and 1.00-fold, respectively relative to the 8 h photoperiod control. It was same as stev spot that reb-A spot from the 12 photoperiod treatment sample was lower than the 8 h control (0.61-fold relative to the 8 h treatment). Stev and reb-A spots from the NI-R 20 treatment were the smallest among all of treatments (0.01 and 0.48-fold, respectively). Total leaf area × SGs (sum of stev and reb-A) of the NI-FL 50 treatment was 1.26 and 1.44 times higher than in the 8 h and the 12 h photoperiod treatments, respectively (Fig. 5A line graphs).
treatment was approximately 9.9 fold larger than in the 16 h photoperiod control plants even though DLI of both treatments was approximately the same (Fig. 1D).
3.3. Stevioside and rebaudioside A content HPTLC analysis of samples extracted and total leaf area × SGs (sum of stev and reb-A) following the NI treatment are shown in Fig. 5A. The NI-FL20, the NI-FL50, and the NI-FR20 treatments produced detectable stev spot were 0.67, 0.55, and 0.43-fold relative to the 8 h photoperiod control. Even though the DLI of the 12 h photoperiod plant was larger
Fig. 5. HPTLC analysis. Experimental light treatments of samples are as described in Figs. 1 and 2. Black bars: stevioside (stev). White bars: rebaudioside-A (reb-A). Line graphs: Total leaf area × SGs (sum of stev and reb-A) (TLS). Left axis indicated fold changes relative to the 8 h treatment and right axis indicated TLS of each condition (A). Left axis indicated fold changes relative to the 16 h treatment and right axis indicated TLS of each condition (B). All samples were taken from 6-week-old cuttings.
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steviolmonoside. UGT85C2 transcription levels have been shown to correlate with SGs content (Mohamed et al., 2011; Guleria et al., 2011). Mohamed et al. (2011) suggests that the glycosylation of steviol to steviolmonoside is the rate-limiting step for SGs content. UGT76G1 converts stev to reb-A. However, although a correlation between UGT76G1 transcription level and SGs content has been suggested, the relationship between UGT76G1 transcription levels and reb-A content are still incompletely understood. We analyzed UGT74G1, which converts steviolbioside to stev. Many studies have investigated this enzyme, but its correlation with SGs content is not clear. Our results showed there were no significant changes in KO, UGT85C2, and UGT74G1 transcription levels. These results suggest that light intensity has no effect on transcription of SGs-related genes. In field experiments, Kumar et al. (2013) revealed that diminished light environments did not result in significant changes in SGs such as stev and reb-A. However, leaf biomass was increased by strong light. Our results support this study, although it is difficult to make a simple comparison as our experiments were conducted in growth chambers, and this genetic analysis was an indirect investigation for SGs. In addition, UGT76G1 transcription level following the PPF 100 treatment was significantly increased. There was no significant difference in UGT85C2 transcription level among all PPF treatments. Analysis of leaf biomass with varying light intensity showed that leaf biomass tended to increase with additional light intensity. Several studies have demonstrated that the correlation between SGs-related genes and SGs content (Kumar et al., 2012; Mohamed et al., 2011). These results suggest that normal SGs content can be obtained under strong light intensity because of increasing leaf biomass.
HPTLC analysis of samples extracted following EOD treatment showed stev and reb-A spots strongly detectable in all EOD treatment samples compared to the 16 h photoperiod control except stev spot in the EOD-FR 5 min treatment (Fig. 5B). Most strongly detection of reb-A was a sample from the EOD-FR 15 min treatment (8.22-fold relative to the 16 h). Total leaf area × SGs (sum of stev and reb-A) of all three EOD treatments were more than 4.21 times higher than the 16 h photoperiod treatment (Fig. 5B line graphs). The highest total leaf area × SGs (sum of stev and reb-A) was the EOD-FR 15 min treatment (8.62-fold relative to the 16 h). 4. Discussion 4.1. Effect of light intensity on S. rebaudiana Our study showed that the more light that S. rebaudiana was subjected to, the greater the leaf biomass that could accumulate. Because S. rebaudiana received more light, photosynthetic activity was optimized. As a result, S. rebaudiana subject to high PPF in this study were seen to increase all morphological traits analyzed. It was reported that dry leaf biomass under 100 W/m2 (approximately 400 μmol m−2 s−1 under natural daylight) was approximately 2 fold greater than under 50 W/m2 (approximately 200 μmol m−2 s−1 under natural daylight) (Ermakov and Kochetov, 1996). This indicates that increasing light intensity increases leaf biomass. There was no significantly different morphology between plants subject to the PPF 50 and the PPF 100 treatments. The light saturation point of S. rebaudiana under various nitrogen supply levels in a growth chamber was approximately 1000–1600 μmol m−2 s−1 (Barbet-Massin et al., 2015). Therefore, there is a possibility that effects of the PPF 50 and the PPF 100 treatments were small scale from the point of view of morphological change. The tendency of morphological changes in 4week-old cuttings was not obvious than 6-week-old cuttings. This result means that 4-week-old cuttings were still early vegetative growth phase and there was a time delay from receiving a light stimulation to morphological changes. Ceunen and Geuns (2013b) measured the average of SGs level under LD and SD condition in several growth phase, and the difference between LD and SD condition in the late vegetative growth phase was obvious than the early vegetative growth phase. Therefore, 4-week-old cuttings were difficult to evaluate morphological differences in each treatment. The leaves of plants subject to the PPF 400 treatment were damaged and exhibited brown coloring, and these symptoms resembled a photodamage. Under excess light condition, plants produce various reactive oxygen species (ROS) such as hydrogen peroxide or singlet oxygen, and ROS cause damage to D1 reaction center protein of photosystem-II (PS II) and photosystem-I (PSI) in the thylakoid membrane (Melis, 1999; Li et al., 2009). Therefore, it is thought that the photodamage under the PPF400 condition was caused by the damage to photosynthesis apparatus. However, light intensity for the PPF 400 treatment was set 400 μmol m−2 s−1, and such a light environment is not considered as strong light in an outer natural environment. Prior to commencing experimental light treatments, S. rebaudiana was grown under 120 μmol m−2 s−1 for 2 weeks. Thus, as there was no acclimation period prior to the PPF 400 treatment, the damage observed following the PPF 400 treatment might be due to a lack of acclimatization. Recent investigations have demonstrated that KO, UGT85C2, and UGT76G1, which are SGs-related genes, are likely to be involved in SGs accumulation (Kumar et al., 2012; Mohamed et al., 2011). SGs share their biosynthetic pathway with GA, and KO, which works on the substrate ent-kaurenoic acid, is a common enzyme for SGs and GA biosynthesis. In this study, we examined KO transcription levels as an index for the relationship between SGs and GA content. UGT85C2 and UGT76G1 are involved in SGs biosynthesis, but are not involved in GA biosynthesis as the enzyme products act downstream of SGs biosynthesis. UGT85C2 converts steviol, which is a basic SGs structure, to
4.2. Effects of photoperiod on S. rebaudiana A LD photoperiod means that DLI is high, that is, S. rebaudiana was able to maintain a high photosynthetic output. Therefore, each morphological trait analyzed significantly increased except leaf number. Total leaf area following the 24 h photoperiod treatment was larger than for the 8 h photoperiod, yet standard deviation of the 24 h photoperiod average was also large. In addition, there were several lateral buds emerging from each node for this treatment. It seems that S. rebaudiana subject to the 24 h photoperiod suffered morphological defects as a result of the absence of a dark period. Photosynthesis follows a circadian rhythm, so a dark period is important in some species. For example, tomato undergoes necrosis under continuous light conditions (Arthur et al., 1930). UGT85C2 transcription level was highest in the 8 h and the 24 h photoperiod treatments. This result shows that it is possible to affect SGs-related gene transcription with two different photoperiods, namely 8 and 24 h. In contrast, the KO transcription level in the 8 h photoperiod was significantly lower than in the 24 h photoperiod treatment. It has been suggested that KO transcription level is related to SGs content, but the KO enzyme is involved in both SGs and GA biosynthesis. Therefore, it is possible that there are two different signaling pathways for upregulating UGT85C2, which would allow for specificity of this enzyme to be involved in both SGs and GA content. LD photoperiods increase GA content in rosette and woody plants (García-Martinez and Gil, 2001). Therefore, S. rebaudiana subject to the 24 h photoperiod might have enhanced GA content, which, because SGs and GA share the same biosynthetic pathway, may explain why UGT85C2 transcription levels were affected. A number of studies have shown that total SGs content would increase under a LD photoperiod (Metivier and Viana, 1979; Brandle and Rosa, 1992; Ceunen and Geuns, 2013a, 2013b). Even though growth conditions in these experiments were different compared to previous studies, our investigation also observed the effect of a LD photoperiod on SGs-related gene transcription. The growth period in this study was set to 6 weeks post-cutting 7
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because S. rebaudiana grown under the 8 h photoperiod had flower buds emerge approximately 7 weeks post-cutting. This result means that S. rebaudiana grown under the 8 h photoperiod was in a late vegetative growth stage at 6 weeks post-cutting. In contrast, S. rebaudiana grown under the 24 h photoperiod was still growing with no bud emergence approximately 7 weeks post-cutting. S. rebaudiana grown under the 24 h photoperiod might be in an early or middle vegetative growth stage at 6 weeks post-cutting. These results mean that the growth stages at which samples were taken were different for the variation in photoperiod experiment. Ceunen and Geuns (2013b) investigated the levels of free steviol in plants at several growth stages grown under LD and SD photoperiods, and showed that different steviol accumulation patterns were associated with each growth stage for each photoperiod. The upregulation of UGT85C2 in the 8 h photoperiod treatment was thus potentially the result of a different growth stage. In addition, a previous study also demonstrated that the percentage of SGs content in total of upper leaves under SD photoperiods was higher than under LD photoperiods (Mohamed et al., 2011). Previous researches were described that LD photoperiod treatment has been affected the total SGs content because of increasing leaf biomass even though the percentage of SGs content was high in SD treatment (Brandle and Rosa, 1992; Ceunen et al., 2013a). Our results may support previous studies in the genetic level. Evans et al. (2015) investigated effects of DLI on SGs content, and total of stev, reb-A, rebaudioside B, rebaudioside C, and rebaudioside D increased as the DLI increased up to 8–10 mol m−2 s−1. In their study, greenhouse-grown S. rebaudiana were subjected to two different light quality conditions, and this difference caused opposite results on stev concentration. The concentration of stev under a higher R/FR condition increased with a corresponding increase in DLI, but the other experiment decreased like our results. Therefore, they suggest that the phytochrome photostationary state may mediate SGs content. Our experiments were used fluorescent lamps (R/FR ratio = 11.4), so there is a possibility to change the SGs content if a higher R/FR ratio lamp were changed.
experiments, it is difficult to discuss whether red or far-red treatment worked or not because leaf biomass under NI treatments at 20 μmol m−2 s−1 was not increased. The effect of vegetative growth could not be detected by NI treatment with single red or far-red light treatment during dark period, but transcription levels of UGT85C2 and SGs content under the NI-FR 20 condition were higher than the NI-R 20 condition. These results suggest that red and far-red light act on genetic and sweet components levels without concern for NI treatment. This means that it may be a participation of the photostationary state of phytochrome and following the change of endogenous GA contents. In our results, even though leaf biomass did not change, stem length under the NI-FR 20 condition was highest among NI experiments. Stem elongation was involved in GA contents, and GA contents can be increased by far-red in bean, Phaseolus vulgaris (Beall et al., 1996). Far-red enrich environment induce the change of the photostationary state of phytochrome which means the reduction of far-red light absorbing form of Pfr is occurred, and stem elongation is related to the photostationary state (Morgan and Smith 1976; Holmes and Smith, 1977; Smith and Whitelam, 1990). GA and SGs biosynthetic pathways partially share; therefore, the change of endogenous GA contents by containing far-red during dark period may not negatively affect UGT85C2 transcription levels and SGs content than single red light during dark period. Taken together, NI treatment can affect vegetative growth (Kim et al., 2011; Ceunen et al., 2012), and the light intensity more than 20 μmol m−2 s−1 during dark period may be important in order to obtain the effect of NI treatment in S. rebaudiana according to our results. The leaf biomass of the NI-FL50 treatment was near double larger than control because the light intensity may be fulfilled vegetative growth function as NI treatment. In addition, the total leaf area × UGT85C2 and the total leaf area × SGs in the NI-FL50 treatment was higher than the 8 h control. These results suggest that the NIFL50 treatment has a potential to increase the total yield of SGs with similar light environment like SD. 4.4. Effects of end-of-day light pulses on S. rebaudiana
4.3. Effects of night-interruption on S. rebaudiana Stem length in the EOD-FR 15 min treatment was greater than control in our results; however, little study examined the effect of EOD treatment in S. rebaudiana. Hisamatsu et al. (2008) demonstrated the effect of EOD-FR treatment on stem elongation in same Asteraceae family, chrysanthemum (Chrysanthemum morifolium Ramat.). EOD-FR treatment leads to stem or petiole elongation, and this response is caused by GA, auxin, and brassinosteroid in cowpea (Vigna sinensis L.) or Arabidopsis (Arabidopsis thaliana). (Martínez-García and GarcíaMartínez, 1995; Kozuka et al., 2010). The difference between EOD-FR and EOD-R is recognized by phytochrome, and GA 2β-hydroxylation, which is induced with stem elongation, may be controlled by its (Smith, 1982; Kamiya and García-Martínez, 1999). Therefore, stem elongation in the EOD-FR 15 min treatment was also observed in S. rebaudiana. In contrast, the EOD-FR 5 min treatment had no effect on stem elongation compared to control. Franklin et al. (2003) suggested that the involvement of phytochromes B, D and E in stem elongation by EOD-FR treatment. Responses of phytochrome can be classified to the very-lowfluence response, the low-fluence response, and the high-irradiance response, and phytochrome B is mediated by the low-fluence response (Kircher et al., 1999). The low-fluence response can be occurred when the amount of light energy is over a certain amount (integral of light intensities and exposure times) (Bunsen and Roscoe, 1863; Briggs, 1960). These results suggest that the amount of light energy by EOD treatment with 50 μmol m−2 s−1 for 5 min was not satisfied the condition of the low-fluence response in S. rebaudiana. The primary objective of the EOD light pulse experiments was to evaluate whether EOD treatments would upregulate SGs-related gene transcription. However, the EOD treatments were conducted before results were obtained indicating the optimal photoperiod for SGs-related gene transcription. Therefore, we included control plants grown
Although DLI of the NI-FL 50 treatment was only slightly higher the 8 h photoperiod treatment, leaf biomass was significantly increased. In addition, leaf biomass in plants grown under the 12 h photoperiod and those that were administered the NI treatment were similar despite higher DLI of the 12 h photoperiod. the NI treatment is used to delay flowering in SD plants such as Asteraceae when grown commercially. SD plants have a critical day length and flowering is induced when the photoperiod falls below it. In other words, S. rebaudiana subject to the NI-FL 50 treatment might be regulated like under a LD photoperiod because of the NI, which maintains their vegetative growth. In Cymbidium, NI induced vegetative growth (Kim et al., 2011); therefore, NI treatment with more than 50 μmol m−2 s−1 light for 4 h in the middle of the dark period night is suitable to affect vegetative grown in S. rebaudiana. The NI-FL 20, the NI-F 20, and the NI-R treatments did not increase leaf biomass compared to the 8 h and the 12 h photoperiod control treatments. This result means that NI treatment using the light intensity less than 20 μmol m−2 s−1 was not sufficient to increase leaf biomass in S. rebaudiana. In our study we did not observe significant change in UGT85C2 transcription level between the 8 h photoperiod control and the NI-FL 20, the NI-FL50, and the NI-FR 20 treatments. Compared to other NI treatments, only S. rebaudiana grown under the NI-R 20 treatment was not subject to far-red light during the dark period. NI treatment with red light in many short plants can contribute to the inhibition of flowering, and phytochrome, red/far-red reversible photoreceptor, regulates flowering (Downs, 1956; Smith and Whitelam, 1990). These results suggest that NI treatment with red light has an effect to vegetative growth in SD plants. Ceunen et al. (2012) showed that NI by red LEDs promotes the vegetative growth and SGs content. However, in our 8
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Science 72, 1263–1266. Briggs, W.R., 1960. Light dosage and phototropic responses of corn and oat coleoptiles. Plant Physiology 35 (6), 951. Brusick, D.J., 2008. A critical review of the genetic toxicity of steviol and steviol glycosides. Food and Chemical Toxicology 46 (7), S83–S91. Bunsen, R., Roscoe, H., 1863. Photochemische untersuchungen. Annalen der Physik 193 (12), 529–562. Ceunen, S., Geuns, J.M.C., 2013a. Steviol glycosides: chemical diversity, metabolism, and function. Journal of Natural Products 76 (6), 1201–1228. Ceunen, S., Geuns, J.M.C., 2013b. Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods. Phytochemistry 89, 32–38. Ceunen, S., Werbrouck, S., Geuns, J.M.C., 2012. Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. Journal of Plant Physiology 169, 749–752. Chatsudthipong, V., Muanprasat, C., 2009. Stevioside and related compounds: therapeutic benefits beyond sweetness. 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under the 16 h photoperiod because a number of studies claimed that the 16 h photoperiod affected total SGs content. As a result, the EOD-FR 15 min treatment upregulated UGT85C2 transcription. the EOD-FR treatment is well known to cause stem elongation by changing GA1 content (Martínez-García and García-Martínez, 1995; Kamiya and García-Martínez, 1999; Hisamatsu et al., 2005). Increased UGT85C2 transcription was possibly the indirect result of the EOD-FR treatment via upregulation of GA content. No less than 15 min of 50 μmol m−2 s−1 far-red irradiation is required to increase UGT85C2 transcription, as no effect was seen with the EOD-FR 5 min treatment. DLIs of the 16 h photoperiod control and EOD treatments were almost same; however, total leaf area × UGT85C2 transcription level and total leaf area × SGs (sum of stev and reb-A) in the EOD-FR 15 min treatment were larger than the 16 h control. In other words, the percentage of SGs was increased by the EOD-FR 15 min treatment even though leaf area did not change compared to the 16 h control. Since SGs and GA biosynthesis pathway from ent-kaurenoic acid goes through same pathway (Kinghorn, 2002), the increase of SGs may be linked the increase of GA1 by EOD-FR treatment (Martínez-García and GarcíaMartínez, 1995; Kamiya and García-Martínez, 1999; Hisamatsu et al., 2005). These results suggest that there is a possibility of improvement of SGs accumulation by EOD-FR 15 min treatment. In addition, in this treatment were used LEDs which can save light energy consumption compared to florescent lamp. 5. Conclusion We analyzed the transcription levels of SGs-related genes quantitatively in order to evaluate the SGs content indirectly. As a result, transcription induction of UGT85C2 was a similar tendency with stev and reb-A even though a correlation between other SGs-related genes and SGs content was not as obvious in this study. The change in UGT85C2 transcription did not depend on light intensity (50–400 μmol m−2 s−1), while a SD photoperiod upregulated UGT85C2. In addition, in spite of a light environment with lower DLI, leaf biomass following the NI-FL 50 treatment was comparable to plants grown under a LD photoperiod with no decrease in SGs content. However, sample size was small for morphological analysis (n = 3–4), which means that a rough trend can only obtain from our results. S. rebaudiana subject to the EOD-FR 15 min treatment had increased UGT85C2 transcription levels and stev and reb-A contents. These results suggest that, even if low DLI is present in the field, it is possible to improve the SGs yield from S. rebaudiana by using supplemental lights to add slight amounts of light energy, including far-red light, in a directed manner. Acknowledgements The authors wish to thank Michiho Ito, Associate Professor, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, for S. rebaudiana was provided. This study has been partly supported by the programs of the Grantin-Aid for Scientific Research (B, 25292150) from the Japan Society for the Promotion of Science. References Arthur, J.M., Guthrie, J.D., Newell, J.M., 1930. Some effects of artificial climates on the growth and chemical composition of plants. American Journal of Botany 17 (5), 416–482. Barbet-Massin, C., Giuliano, S., Alletto, L., Daydé, J., Berger, M., 2015. Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Sevia rebaudiana Bertoni. PLoS One 10 (7), e0133067. Beall, F.D., Yeung, E.C., Pharis, R.P., 1996. Far-red light stimulates internode elongation, cell division, cell elongation, and gibberellin levels in bean. Canadian Journal of Botany 74 (5), 743–752. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Canadian Journal of Plant
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Melis, A., 1999. Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage in vivo? Trends in Plant Science 4 (4), 130–135. Metivier, J., Viana, A.M., 1979. Determination of microgram quantities of stevioside from leaves of Stevia rebaudiana Bert. by two-dimensional thin layer chromatography. Journal of Experimental Botany 30 (4), 805–810. Mohamed, A.A.A., Ceunen, S., Geuns, J.M.C., Van den Ende, W., De Ley, M., 2011. UDPdependent glycosyltransferases involved in the biosynthesis of steviol glycosides. Journal of Plant Physiology 168 (10), 1136–1141. Morgan, D.C., Smith, H., 1976. Linear relationship between phytochrome photoequilibrium and growth in plants under simulated natural radiation. Nature 262 (5565), 210–212. Nettleton, J.A., Lutsey, P.L., Wang, Y., Lima, J.A., Michos, E.D., Jacobs, D.R., 2009. Diet soda intake and risk of incident metabolic syndrome and type 2 diabetes in the MultiEthnic Study of Atherosclerosis (MESA). Diabetes Care 32 (4), 688–694. Osman, M., Samsudin, N.S., Faruq, G., Nezhadahmadi, A., 2013. Factors affecting microcuttings of Stevia using a mist-chamber propagation box. The Scientific World Journal 2013 (Article ID 940201, 10 pages). Saji, H., Daphne, V.P., Furuya, M., 1983. Studies on the photoreceptors for the promotion
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Effects of light intensity and photoperiod on improving steviol glycosides content in Stevia rebaudiana (Bertoni) Bertoni while conserving light energy consumption ⁎
Yuki Yoneda, Hiroshi Shimizu , Hiroshi Nakashima, Juro Miyasaka, Katsuaki Ohdoi Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Stevia rebaudiana (Asteraceae) Steviol glycosides Light intensity Photoperiod Night interruption End-of-day far-red
Stevia rebaudiana (Bertoni) Bertoni accumulates steviol glycosides (SGs) which are natural sweeteners. Leaf extracts have been widely used as a food additive because the strong sweetness intensity of SGs that is 300 times sweeter than sucrose. S. rebaudiana is a short-day (SD) perennial plant, and some studies claim that long-day (LD) treatment affects SGs accumulation. However, the optimal and detailed light regime required to increase total SGs content remains controversial. We analyzed the effect of various light intensities and day lengths to determine optimal light treatment for SGs accumulation with minimal energy consumption in growth chambers. Night-interruption (NI) treatments in conjunction with 8 h photoperiod increased leaf biomass to amounts observed under LD conditions, and resulted in higher SGs content than that observed under 12 h photoperiod while minimizing energy consumption. In addition, end-of-day far-red (EOD-FR) treatment for 15 min at 50 μmol m−2 s−1 was associated with transcription upregulation of the SGs-related gene UGT85C2, higher SGs content, and similar leaf biomass compared to that observed under 16 h photoperiod. These light treatments offer great potential for energy-efficient increases in S. rebaudiana total SGs content when plants are grown in a greenhouse or in the field with supplemental lights.
1. Introduction Stevia rebaudiana (Bertoni) Bertoni is a perennial plant that belongs to the plant family Asteraceae native to South America (Kinghorn, 2002). S. rebaudiana leaves contain sweet compounds known as steviol glycosides (SGs) which possess sweetness intensity approximately 50–450 times stronger than sucrose (Crammer and Ikan, 1986). The most abundant SG is stevioside (stev) (5–10% of the total dry matter) followed by rebaudioside A (reb-A) (2–4%) (Crammer and Ikan, 1986; Chatsudthipong and Muanprasat, 2009). Some studies have claimed that consumption of artificial sweeteners is associated with a risk of long-term weight gain, which could induce metabolic syndrome, and it has reported that saccharin induces glucose intolerance and dysbiosis (Fowler et al., 2008; Lutsey et al., 2008; Nettleton et al., 2009; Suez et al., 2014). Conversely, S. rebaudiana is used as a natural sweetener in Asia and South America with no toxicity for humans (Koyama et al., 2003; Brusick, 2008). It is possible to extract the most abundant SGs from upper large young leaves (Mohamed et al., 2011; Kumar et al., 2012); therefore, it is important to increase total leaf biomass without reducing total SGs content.
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Light is an important environmental factor for plant growth, and can be easily controlled using supplemental lights in a field or in a greenhouse. S. rebaudiana grows under a sunny climate in its native habitat (Ceunen et al., 2013a; Osman et al., 2013), but few studies have investigated optimal light intensity for SGs accumulation (Slamet and Tahardi, 1988; Kumar et al., 2013). In addition, it is difficult to isolate the effects of light intensity from other factors because these studies described experimentation in the field. S. rebaudiana is a SD plant; however, was reported that total SGs content increases under a long day (LD 16 h) compared to SD (8 h) photoperiod (Brandle and Rosa, 1992; Ceunen et al., 2013a). A natural LD environment is available during limited seasons and in limited areas of cultivatable land. However, supplemental light treatment can easily modulate photoperiod by using additional electrical energy would be required to artificially extend the photoperiod to create a LD environment (Ceunen et al., 2012). Furthermore, the percentage of SGs content which does not mean the total SGs yield per plant was higher under a SD rather than a LD photoperiod (Mohamed et al., 2011). This suggests that a SD photoperiod increases the percentage of SGs content without increasing total biomass, and total SGs yield would increase if leaf
Corresponding author. E-mail address: [email protected] (H. Shimizu).
http://dx.doi.org/10.1016/j.jarmap.2017.06.001 Received 1 December 2016; Received in revised form 31 May 2017; Accepted 3 June 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Yoneda, Y., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.06.001
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culture medium. Cuttings were composed of a stem (4 cm in length) from the shoot apex, which had leaves from the 3rd node to the apex, and the leaf pair of the 3rd node were cut in half. The cut stem was placed in water for 1 h, and then the cut end was coated with a rooting promoter (Rooton, Sumitomo Chemical Garden Products, Tokyo, Japan) and inserted into a urethane sponge. Sponges were maintained soaked in a nutrient supplement (Menedael, Menedael Co., Ltd., Osaka, Japan) diluted 100 times in water and cuttings were grown for 2 weeks under the following conditions: the 16 h photoperiod, 24 °C during light periods, 20 °C during dark periods, and 99% humidity. A fluorescent lamp (FL) (100MHF142DR, Monocoqtex, Inc., Tokyo, Japan) was used as the light source during rooting, which provided a photosynthetic photon flux (PPF, 400–700 nm) of 120 μmol m−2 s−1. PPF was measured using a light quantum meter (I-250A, LI-COR Inc., USA). For each test section, a 30 cm × 60 cm × 7 cm cultivation tank was filled with nutrient solution (OAT house A No. 1 and No. 2, OAT Agrio Co., Ltd., Tokyo, Japan) (pH 6.4 and EC 1.3 dS m−1) which was aerated using an air pump (Non-noise S100, Japan Pet Design CO., LTD., Tokyo, Japan).
biomass were increased under a SD photoperiod. The use of light emitting diodes (LEDs) has been an alternative in the artificial lighting for the increase of the photoperiod in different cultures (Goto, 2008). Using LEDs that supplied weak-intensity red light, Ceunen et al. (2012) investigated the effect of night-interruption (NI) during the dark period following a SD photoperiod (8 h) in S. rebaudiana. This NI was shown to increase total SGs content compared to plants grown under normal SD conditions. NI is a method used to delay flowering time in commercial production of chrysanthemum, which like S. rebaudiana is a SD plant (Goto, 2008). NI involves interrupting the dark period with weak-light treatment, which can affect the flowering phase (Kim et al., 2011). These results suggest that the NI is a possible alternative to growing S. rebaudiana plants under a LD photoperiod. NI effect involves the red-light photoreceptor phytochrome which has two different forms of the Pfr form (the absorption maximum: 730 nm) and the Pr form (the absorption maximum: 660 nm) (Smith and Whitelam, 1990). NI with red light of 620–640 nm and 660 nm delays the onset of flowering in cocklebur (Xanthium) and morning glory (Pharbitis nil), respectively, which thus also prolongs vegetative growth (Hendricks and Siegelman 1967; Saji et al., 1983). Phytochrome can signal different stages of photomorphogenesis when it perceives different ratios of red/far-red light (Sullivan and Deng, 2003). On the other hand, end-of-day (EOD) treatment, where a pulse of low-intensity light of a different quality is administered during the transition from the photoperiod to the dark period, also involves phytochrome. EOD-far-red (EOD-FR) treatments resulted in enhanced petiole and stem elongation and increased gibberellin (GA) content in chrysanthemum, spinach, and cowpea (Hisamatsu et al., 2005; Martínez-García et al., 1995). These studies did not show an increase in the total leaf area, but it should be noted that GA treatment affected SGs content (Hajihashemi et al., 2013). As the SGs biosynthetic pathway converges with the GA biosynthetic pathway through the formation of ent-kaurenoic acid, a change in GA content is presumed to regulate SGs content; however, the effect of EOD-FR on SGs content has not been clarified. The objective of this study was to investigate an optimal light environment for SGs accumulation and enhanced leaf biomass in S. rebaudiana with minimal electrical energy consumption. Therefore, we analyzed plant morphology and SGs content under light treatments that varied light intensity and photoperiod as well as NI and EOD light pulses. Especially, NI treatment, which has an effect to enhance vegetative growth, and EOD treatment, which may have a potential to impact on SGs content, can be expected to improve the total of SGs with saving daily light integral (DLI). In this study, SGs content was evaluated using two different approaches: a quantitative genetic analysis and high performance thin layer chromatography (HPTLC) and a transcriptional analysis of Kaurene oxidase (KO) gene and two UDP-17 glycosyltransferases (UGT) genes which are named UGT85C2 and UGT76G1. According to Mohamed et al. (2011), a glycosylation of steviol, which is a basic skeleton of SGs, to steviolmonoside, which is precursor of stevioside, is a rate limiting step, and UGT85C2 catalyzes this step. Normally, high performance liquid chromatography (HPLC) is a practical method to evaluate SGs content; however, it is difficult to evaluate SGs content in a small-scale experiment using HPLC because a certain amount dry leaf tissue is required. HPTLC method demonstrated the accuracy of the amount of SGs content (Jaitak et al., 2008). Therefore, HPTLC is a suitable alternative that can save cost and analysis time.
2.2. Experimental light treatment After rooting for 2 weeks, S. rebaudiana plants were transplanted to 18 differing light conditions (Fig. 1). Fig. 1A shows how fluorescent lamp light intensity in the 16 h photoperiod was varied between 50 and 400 μmol m−2 s−1. Fig. 1 B shows how fluorescent lamp photoperiods were varied between 8 and the 24 h. Fig. 1C illustrates NI treatments using light from a fluorescent lamp, as well as far-red (730 nm) and red (660 nm) LEDs. NI was administered by a four hours weak-light (20–50 μmol m−2 s−1) treatment during the dark period that followed the 8 h photoperiod (Fig. 1C). For these, the 8 h photoperiod with light supplied by a fluorescent lamp was followed by a 6 h dark period, which was then interrupted with 4 h of low-intensity light (20–50 μmol m−2 s−1) originating from a fluorescent lamp, or far-red or red LEDs, then followed by a further 6 h dark period. Fig. 1D shows
2. Material and methods 2.1. Plant material
Fig. 1. List of light treatments. Treatments included various light intensities(A), photoperiods (B), night-interruptions (NI) (C), and end-of-day (EOD) pulses (D). For the NI, diagonal lines indicate use of a fluorescent lamp (FL), or far-red (FR) or red (R) LEDs. For EOD, diagonal lines indicate a FR or R pulse for 5–15 min. Gray bars: photoperiods. Black bars: dark periods. DLI: daily light integral. PPF: photosynthetic photon flux.
In order to limit genetic variation, S. rebaudiana plants were propagated from multiple cuttings from a parent plant. Plants were grown hydroponically in growth chambers, using urethane sponges as the 2
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2.5. HPTLC
EOD treatments using light from far-red and red LEDs. For these, the 16 h photoperiod with light supplied by a fluorescent lamp was ended with a 5–15 min pulse of 50 μmol m−2 s−1 light from far-red or red LEDs, just prior to the dark period. All other environmental conditions were kept constant between experimental treatments, as follows: 25 °C, 41% humidity, and 535 ppm CO2 during light periods, 21 °C, 67% humidity, and 585 ppm CO2 during dark periods. Other growth conditions such as temperature, CO2 concentration, and humidity were recorded using a data logger (TR72Ii, T & D, Nagano, Japan). Light sources were a fluorescent lamp (FDA21093A, Panasonic Corporation, Kadoma, Japan), and far-red (PR2N-3LEE-SD, ProLight Opto Technology Corporation., Taoyuan, Taiwan) and red (LXM3-PD01, Lumileds Holding B.V. CA, USA) LEDs. Aside from treatments where light intensity was varied (Fig. 1A), the light intensity during main photoperiods was 120 μmol m−2 s−1. The shoot apex was constantly exposed to the same light intensity by adjusting plant position using lab jacks. Far-red irradiance of the shoot apex was adjusted using a spectroradiometer (LI-1800, LI-COR Inc., USA).
HPTLC was used for analyzing SGs content following the method of Jaitak et al. (2008). Samples from 6-week-old cuttings were spotted onto a HPTLC plate (HPTLC 60 F254, E. Merck, Darmstadt, Germany). Tissue samples, two excised pieces 0.8 cm in diameter, were taken from the sixth leaf from the bottom of plants. In a sequential manner, the samples were homogenized following the addition of 20 μl distilled water. Mobile phase consisted of ethyl acetate, ethanol, and distilled water (80: 20: 12), and each solution was measured separately. After mixing all of the solution, an expansion tank was filled with it and waited until saturated (20 min). Spots were created on HPTLC plates using 2 μl of homogenized sample from the bottom by 1 cm and each spot was separated by more than 1 cm. The plate was sprayed with a mixed solution of ethanol, acetic anhydride, and sulfuric acid (10: 0.1: 0.1) after expansion and drying. HPTLC plates were semiquantified by ImageJ (http://rsbweb.nih.gov/ij/). 2.6. Daily light integral Daily light integral (DLI) is the total amount of PPF (400–700 nm) per square meter per day. Total light energy consumption was considered in this study. Therefore, we calculated DLI including energy from far-red light (peak wavelength: 730 nm). DLI for each experimental light treatment is indicated in Fig. 1.
2.3. Morphological analysis S. rebaudiana plants of the respective conditions were sampled 4 and 6 weeks after cutting. The average measurements with standard deviation were calculated from the samples. Morphological analysis included total leaf area and fresh weight without a subset of leaves from the first leaf pair to the forth leaf pair from the bottom. These leaves had already developed prior to commencing experimental light treatments. Further morphological analysis included measurements of stem length, stem diameter, and leaf number per plant. Stem length was measured from the top of the supporting urethane sponge to the shoot apex, and stem diameter was measured around the center of a stem. Leaf area was determined by scanning leaves and measuring area by ImageJ (http://rsbweb.nih.gov/ij/).
2.7. Statistical analysis Tukey-Kramer (p < 0.05) was adopted for statistical analysis. Plants were sampled after 4 and 6 weeks cutting growth, and four plants were used in each case of morphological analysis. The following experimental treatments, which have analyses based on only three plants: 4-week-old cuttings treated with varying light intensity the PPF 50, the PPF 100 and the PPF 200, varying photoperiod the 12 h and the 24 h, varying night-interruption the NI-FL 20, the NI-FL 50, the NI-FR 20, and the NI-R 20, and varying end-of-day pulse the EOD-FR 5 min, the EOD-FR 15 min, and the EOD-R 15 min. In addition, 6-week-old cuttings treated with varying photoperiod 20 h, and varying night-interruption NI-FL 50 and NI-FR 20 were also three plants. 4-week-old cuttings treated with varying photoperiod 8 h and 10 h were not analyzed.
2.4. Quantitative RT-PCR analysis As a gene transcription analysis, a seventh leaf from the bottom of the plant was selected because this represented tissue that had developed post transplantation and experimental light treatment. After 6 weeks of cutting growth, leaf tissue was frozen in liquid nitrogen and ground using a mortar and a pestle. Leaves were sampled in the middle of photoperiod, meaning following 4 h of light during the 8 h photoperiod, and following the 8 h of light during the 16 h photoperiod. Total RNA was extracted using a RNA extraction kit (RNA suisui-P, Rizo Inc., Tsukuba, Japan) and RNA extraction column (FARBC-C50, Favorgen, Taiwan). The cDNA was synthesized from 0.5 μg of total RNA using a reverse transcription-polymerase chain reaction (PCR) kit (Revere Tra Ace, TOYOBO CO., LTD. Life Science Department, Osaka, Japan). This cDNA was then used for quantitative real-time (RT)-PCR (qPCR) in 20 μl reactions using a qPCR kit (KAPA SYBR FAST qPCR kit, Kapa Biosystems, Inc., Woburn, MA, USA). A 2-step program was used as follows: 95 °C 30 s × 1 cycle, 95 °C 3 s, and 60 °C 30 s × 40 cycles. 18S rRNA and βActin were used as internal standards. The calculation method was adopted the Vandesompele method for evaluating relative transcription levels (Vandesompele et al., 2002). Calculations were performed using Gene Transcription Macro™ (Bio-Rad, Hercules, CA, USA). Primers for used for β-Actin, 18S rRNA, UGT85C2, UGT74G1, and UGT76G1 were derived from Mohamed et al. (2011). Primers for KO were derived from Hajihashemi et al. (2013). UGT74G1 is also one of the UGT genes. UGT74G1 catalyzes the step to stev from steviolbioside. It is not reported that there is a correlation between SGs content and transcription level of UGT74G1. Three biological replicates were analyzed per experimental light treatment.
3. Results 3.1. Morphological and genetic analyses 3.1.1. Effect of light intensity on S. rebaudiana morphology and SGs-related gene transcription All of the analyzed morphological traits were seen to increase following experimental treatments with increasing light intensities. In particular, plants subject to the PPF 400 treatment possessed the significantly morphology (2A and 3 Treatment A). Total leaf area following the PPF 50, the PPF 100, and the PPF 200 treatments increased in accordance with increasing light intensity, but these differences were not significant. Despite a doubling of light intensity in the PPF 100 compared to the PPF 50 treatment, plant morphologies were similar. DLI of the PPF 400 treatment was 8 times greater the PPF 50 treatment (Fig. 1A), which resulted in a 4.6-fold increase in the total leaf area in 6-week-old cuttings. Morphological differences following experimental treatments were initially apparent in 4-week-old cuttings and more obvious in 6-week-old cuttings. Following the PPF 400 treatment, a number of leaves displayed damage, in particular those leaves in the bottom half of the cutting (Fig. 2A). Several lower leaves appeared completely withered, and others displayed middle-leaf and spot damage. Variation in light intensity did not affect KO, UGT85C2, and 3
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Fig. 2. Morphology of 6-week-old stevia cuttings following various light treatments. Variation of light intensity between 50 and 400 μmol m−2 s−1 photosynthetic photon flux (PPF) (A). Variation of photoperiod between 8 and 24 h (B). Application of night-interruption (NI) using light from a fluorescent lamp (FL), or far-red (FR) or red (R) LEDs at intensities of 20 μmol m−2 s−1 (20) or 50 μmol m−2 s−1 (50) (C). Plants grown with the 8 h and the 12 h photoperiod and no the NI are included for comparison. Application of end-of-day (EOD) FR or R light pulses of 5–15 min (D). A plant grown with the same the 16 h photoperiod and no EOD light pulse is included for comparison. Scale bar indicates 10 mm. All samples were taken from 6-week-old cuttings.
transcription levels following any varied photoperiod treatment; however, the tendency of UGT76G1 transcription levels were similar to UGT85C2.
UGT74G1 transcription levels (Fig. 4A), as no significant difference was observed in their transcription levels following each PPF treatment. In contrast, transcription level of UGT76G1 following the PPF 100 treatment significantly increased compared to other PPF treatments, with transcription levels following the PPF 100 treatment approximately 4.9 fold higher that those following the PPF 50 treatment.
3.1.3. Effect of night-interruption on S. rebaudiana morphology and SGsrelated gene transcription Between the 8 h (control), the NI-FL 20, the NI-FR 20, and the NI-R 20 treatments, stem length following the NI-FR 20 treatment was the longest among following the control and other NI treatments (Figs. 2C and 3 Treatment C). However, there were no significant changes in the total leaf area and weight, leaf number, and stem diameter. The total leaf area and weight of following the NI-FL 50 treatment were significantly increased compared to the 8 h photoperiod control (approximately 2 times larger), despite a similar DLI. DLI of the NI-FL 50 treatment (4.18 mol m−2 d−1) was less than for the control the 8 h photoperiod (3.46 mol m−2 d−1). NI treatments in general did not affect KO and UGT76G1 transcription levels and only some treatments affected UGT85C2 transcription levels. Compared to control plants grown under either the 8 h or the 16 h photoperiod, KO and UGT76G1 transcription levels did not significantly change following any of NI treatments (Fig. 4C). In addition, there was no significant difference in UGT85C2 transcription levels between the 8 h photoperiod control, the NI-FL 20, the NI-FL 50, and the NI-FR 20 treatments. However, UGT85C2 transcription levels were lower in the 12 h photoperiod and following the NI-R 20 treatment compared to the 8 h photoperiod control plants.
3.1.2. Effect of photoperiod on S. rebaudiana morphology and SGs-related gene transcription Experimental light treatments that extended the photoperiod resulted in an increase all analyzed morphological traits apart from total leaf number (Figs. 2B and 3 Treatment B). No change in the total leaf number was observed following any of the experimental light treatments. Large standard deviations were observed for average total leaf area and average total leaf weight following the 18 h and the 24 h photoperiod treatments. In addition, lateral buds were observed on cuttings grown under long photoperiods. DLI for the 24 h photoperiod treatment was three fold higher the 8 h treatment (Fig. 1B), which resulted in an approximate 2.2-fold increase in the total leaf area. In contrast, the total leaf area did not show a significant difference in the 24 h and the PPF 400 treatments (P = 0.16, data not shown). Even though there were damages to leaves under the PPF 400 condition, there were no spot damages in leaves under the 24 h condition (Fig. 2). Variation in photoperiod was seen to affect transcription levels of KO and UGT85C2, but not UGT74G1 and UGT76G1 transcription levels. KO transcription levels were highest following the 24 h photoperiod treatment (Fig. 4B), which were approximately 3.3 fold higher than following the 8 h photoperiod treatment. UGT85C2 transcription levels were highest following the 8 h and the 24 h photoperiod treatments. There was no significant difference in UGT74G1 and UGT76G1
3.1.4. Effect of end-of-day light pulse on S. rebaudiana morphology and SGs-related gene transcription The morphology of S. rebaudiana cuttings following EOD treatments were compared with control plants grown under the 16 h photoperiod alone, as 5–15 min EOD light pulses with light from a fluorescent lamp, 4
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Fig. 3. Quantitation of morphological traits of stevia cuttings following various light treatments. Experimental light treatments A–D are as described in Figs. 1 and 2. Total leaf area and fresh weight excluded the four leaves at the bottom of cuttings. Bars represent average measurements of 4-week-old (gray bars) and 6-week-old (black bars) cuttings. Error bars represent the standard deviation of the mean (n = 4 or 3, see Material and methods). Lowerand upper-case letters indicate significant difference in 4- and 6-week-old cuttings, respectively (TukeyKramer method, p < 0.05).
photoperiod control plants. For the 8 h photoperiod control plants, the product of average total leaf area and average UGT85C2 transcription level was 216.3. However, for plants subject to the NI-FL 50 treatment, the same product was 439.6. Plants following the NI-FL 50 treatment were 2 fold larger the 8 h photoperiod control plants. In addition, DLI of the NI-FL 50 treatment was slightly higher (approximately 1.2 fold) than for the 8 h photoperiod alone (Fig. 1C). Even though DLI of the NIFL 50 treatment was less (approximately 0.8 fold) than for the 12 h photoperiod, the 439.6 figure for total leaf area × UGT85C2 transcription following the NI-FL 50 treatment was larger than 124.6 for total leaf area × UGT85C2 transcription for the 12 h photoperiod control plants. When considering leaf biomass following EOD light pulse treatments, there was no significant difference in the total leaf area (Fig. 3 Treatment D). Average total leaf area in the 16 h photoperiod control plants was 27.9 cm2/plant and 37.2 cm2/plant following the EOD-FR 15 min treatment. Plants following the EOD-FR 15 min treatment were 1.3 fold larger than those subjected to a 16 h photoperiod alone. The product of average total leaf area and UGT85C2 transcription level in the 16 h photoperiod plants was 53. In contrast, the same product following the EOD-FR 15 min treatment was 524.1. This figure for total leaf area × UGT85C2 transcription following the EOD-FR 15 min
or red or far-red LEDs were administered following the 16 h photoperiod (Fig. 1D). Compared to control plants, EOD treatments resulted in a significant difference in the total leaf weight and leaf number only (Figs. 2D and 3 Treatment D). No significant change in stem length was observed by ANOVA, whereas only the EOD-FR 15 min treatment significantly elongated than control by t-test (P < 0.05, data not shown). Despite an increase in leaf number following all EOD treatments, no increase in leaf biomass was observed following the EOD-FR 15 min and the EOD-R 15 min treatments. KO and UGT74G1 transcription levels were unaffected by EOD light pulse treatments (Fig. 4D). However, UGT85C2 and UGT76G1 transcription levels following the EOD-FR 15 min treatment were higher than in the 16 h photoperiod control plants (approximately 7.5 fold and 4.6-fold transcription, respectively). Similar transcription levels were observed for UGT85C2 and UGT76G1 between the 16 h photoperiod control, the EOD-FR 5 min, and the EOD-R 15 min treatments. 3.2. Relationship between leaf biomass and SGs-related gene transcription For the NI experimental light treatments, average total leaf area following the NI-FL 50 treatment was 44.0 cm2/plant, which was near double the 23.5 cm2/plant average leaf area observed in the 8 h 5
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Fig. 4. Relative expression of SGs-related genes. Experimental light treatments A–D are as described in Figs. 1 and 2. Each bar represents the mean. Error bars represent the standard deviation of the mean (n = 3). Letters indicate significant difference (Tukey-Kramer method, p < 0.05). All samples were taken from 6-week-old cuttings.
than all of NI treatment plants, stev spot was detected 0.32-fold relative to the 8 h photoperiod control. Reb-A spot of the NI-FL20, the NI-FL50, and the NI-FR20 treatments were 0.53, 0.79, and 1.00-fold, respectively relative to the 8 h photoperiod control. It was same as stev spot that reb-A spot from the 12 photoperiod treatment sample was lower than the 8 h control (0.61-fold relative to the 8 h treatment). Stev and reb-A spots from the NI-R 20 treatment were the smallest among all of treatments (0.01 and 0.48-fold, respectively). Total leaf area × SGs (sum of stev and reb-A) of the NI-FL 50 treatment was 1.26 and 1.44 times higher than in the 8 h and the 12 h photoperiod treatments, respectively (Fig. 5A line graphs).
treatment was approximately 9.9 fold larger than in the 16 h photoperiod control plants even though DLI of both treatments was approximately the same (Fig. 1D).
3.3. Stevioside and rebaudioside A content HPTLC analysis of samples extracted and total leaf area × SGs (sum of stev and reb-A) following the NI treatment are shown in Fig. 5A. The NI-FL20, the NI-FL50, and the NI-FR20 treatments produced detectable stev spot were 0.67, 0.55, and 0.43-fold relative to the 8 h photoperiod control. Even though the DLI of the 12 h photoperiod plant was larger
Fig. 5. HPTLC analysis. Experimental light treatments of samples are as described in Figs. 1 and 2. Black bars: stevioside (stev). White bars: rebaudioside-A (reb-A). Line graphs: Total leaf area × SGs (sum of stev and reb-A) (TLS). Left axis indicated fold changes relative to the 8 h treatment and right axis indicated TLS of each condition (A). Left axis indicated fold changes relative to the 16 h treatment and right axis indicated TLS of each condition (B). All samples were taken from 6-week-old cuttings.
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steviolmonoside. UGT85C2 transcription levels have been shown to correlate with SGs content (Mohamed et al., 2011; Guleria et al., 2011). Mohamed et al. (2011) suggests that the glycosylation of steviol to steviolmonoside is the rate-limiting step for SGs content. UGT76G1 converts stev to reb-A. However, although a correlation between UGT76G1 transcription level and SGs content has been suggested, the relationship between UGT76G1 transcription levels and reb-A content are still incompletely understood. We analyzed UGT74G1, which converts steviolbioside to stev. Many studies have investigated this enzyme, but its correlation with SGs content is not clear. Our results showed there were no significant changes in KO, UGT85C2, and UGT74G1 transcription levels. These results suggest that light intensity has no effect on transcription of SGs-related genes. In field experiments, Kumar et al. (2013) revealed that diminished light environments did not result in significant changes in SGs such as stev and reb-A. However, leaf biomass was increased by strong light. Our results support this study, although it is difficult to make a simple comparison as our experiments were conducted in growth chambers, and this genetic analysis was an indirect investigation for SGs. In addition, UGT76G1 transcription level following the PPF 100 treatment was significantly increased. There was no significant difference in UGT85C2 transcription level among all PPF treatments. Analysis of leaf biomass with varying light intensity showed that leaf biomass tended to increase with additional light intensity. Several studies have demonstrated that the correlation between SGs-related genes and SGs content (Kumar et al., 2012; Mohamed et al., 2011). These results suggest that normal SGs content can be obtained under strong light intensity because of increasing leaf biomass.
HPTLC analysis of samples extracted following EOD treatment showed stev and reb-A spots strongly detectable in all EOD treatment samples compared to the 16 h photoperiod control except stev spot in the EOD-FR 5 min treatment (Fig. 5B). Most strongly detection of reb-A was a sample from the EOD-FR 15 min treatment (8.22-fold relative to the 16 h). Total leaf area × SGs (sum of stev and reb-A) of all three EOD treatments were more than 4.21 times higher than the 16 h photoperiod treatment (Fig. 5B line graphs). The highest total leaf area × SGs (sum of stev and reb-A) was the EOD-FR 15 min treatment (8.62-fold relative to the 16 h). 4. Discussion 4.1. Effect of light intensity on S. rebaudiana Our study showed that the more light that S. rebaudiana was subjected to, the greater the leaf biomass that could accumulate. Because S. rebaudiana received more light, photosynthetic activity was optimized. As a result, S. rebaudiana subject to high PPF in this study were seen to increase all morphological traits analyzed. It was reported that dry leaf biomass under 100 W/m2 (approximately 400 μmol m−2 s−1 under natural daylight) was approximately 2 fold greater than under 50 W/m2 (approximately 200 μmol m−2 s−1 under natural daylight) (Ermakov and Kochetov, 1996). This indicates that increasing light intensity increases leaf biomass. There was no significantly different morphology between plants subject to the PPF 50 and the PPF 100 treatments. The light saturation point of S. rebaudiana under various nitrogen supply levels in a growth chamber was approximately 1000–1600 μmol m−2 s−1 (Barbet-Massin et al., 2015). Therefore, there is a possibility that effects of the PPF 50 and the PPF 100 treatments were small scale from the point of view of morphological change. The tendency of morphological changes in 4week-old cuttings was not obvious than 6-week-old cuttings. This result means that 4-week-old cuttings were still early vegetative growth phase and there was a time delay from receiving a light stimulation to morphological changes. Ceunen and Geuns (2013b) measured the average of SGs level under LD and SD condition in several growth phase, and the difference between LD and SD condition in the late vegetative growth phase was obvious than the early vegetative growth phase. Therefore, 4-week-old cuttings were difficult to evaluate morphological differences in each treatment. The leaves of plants subject to the PPF 400 treatment were damaged and exhibited brown coloring, and these symptoms resembled a photodamage. Under excess light condition, plants produce various reactive oxygen species (ROS) such as hydrogen peroxide or singlet oxygen, and ROS cause damage to D1 reaction center protein of photosystem-II (PS II) and photosystem-I (PSI) in the thylakoid membrane (Melis, 1999; Li et al., 2009). Therefore, it is thought that the photodamage under the PPF400 condition was caused by the damage to photosynthesis apparatus. However, light intensity for the PPF 400 treatment was set 400 μmol m−2 s−1, and such a light environment is not considered as strong light in an outer natural environment. Prior to commencing experimental light treatments, S. rebaudiana was grown under 120 μmol m−2 s−1 for 2 weeks. Thus, as there was no acclimation period prior to the PPF 400 treatment, the damage observed following the PPF 400 treatment might be due to a lack of acclimatization. Recent investigations have demonstrated that KO, UGT85C2, and UGT76G1, which are SGs-related genes, are likely to be involved in SGs accumulation (Kumar et al., 2012; Mohamed et al., 2011). SGs share their biosynthetic pathway with GA, and KO, which works on the substrate ent-kaurenoic acid, is a common enzyme for SGs and GA biosynthesis. In this study, we examined KO transcription levels as an index for the relationship between SGs and GA content. UGT85C2 and UGT76G1 are involved in SGs biosynthesis, but are not involved in GA biosynthesis as the enzyme products act downstream of SGs biosynthesis. UGT85C2 converts steviol, which is a basic SGs structure, to
4.2. Effects of photoperiod on S. rebaudiana A LD photoperiod means that DLI is high, that is, S. rebaudiana was able to maintain a high photosynthetic output. Therefore, each morphological trait analyzed significantly increased except leaf number. Total leaf area following the 24 h photoperiod treatment was larger than for the 8 h photoperiod, yet standard deviation of the 24 h photoperiod average was also large. In addition, there were several lateral buds emerging from each node for this treatment. It seems that S. rebaudiana subject to the 24 h photoperiod suffered morphological defects as a result of the absence of a dark period. Photosynthesis follows a circadian rhythm, so a dark period is important in some species. For example, tomato undergoes necrosis under continuous light conditions (Arthur et al., 1930). UGT85C2 transcription level was highest in the 8 h and the 24 h photoperiod treatments. This result shows that it is possible to affect SGs-related gene transcription with two different photoperiods, namely 8 and 24 h. In contrast, the KO transcription level in the 8 h photoperiod was significantly lower than in the 24 h photoperiod treatment. It has been suggested that KO transcription level is related to SGs content, but the KO enzyme is involved in both SGs and GA biosynthesis. Therefore, it is possible that there are two different signaling pathways for upregulating UGT85C2, which would allow for specificity of this enzyme to be involved in both SGs and GA content. LD photoperiods increase GA content in rosette and woody plants (García-Martinez and Gil, 2001). Therefore, S. rebaudiana subject to the 24 h photoperiod might have enhanced GA content, which, because SGs and GA share the same biosynthetic pathway, may explain why UGT85C2 transcription levels were affected. A number of studies have shown that total SGs content would increase under a LD photoperiod (Metivier and Viana, 1979; Brandle and Rosa, 1992; Ceunen and Geuns, 2013a, 2013b). Even though growth conditions in these experiments were different compared to previous studies, our investigation also observed the effect of a LD photoperiod on SGs-related gene transcription. The growth period in this study was set to 6 weeks post-cutting 7
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because S. rebaudiana grown under the 8 h photoperiod had flower buds emerge approximately 7 weeks post-cutting. This result means that S. rebaudiana grown under the 8 h photoperiod was in a late vegetative growth stage at 6 weeks post-cutting. In contrast, S. rebaudiana grown under the 24 h photoperiod was still growing with no bud emergence approximately 7 weeks post-cutting. S. rebaudiana grown under the 24 h photoperiod might be in an early or middle vegetative growth stage at 6 weeks post-cutting. These results mean that the growth stages at which samples were taken were different for the variation in photoperiod experiment. Ceunen and Geuns (2013b) investigated the levels of free steviol in plants at several growth stages grown under LD and SD photoperiods, and showed that different steviol accumulation patterns were associated with each growth stage for each photoperiod. The upregulation of UGT85C2 in the 8 h photoperiod treatment was thus potentially the result of a different growth stage. In addition, a previous study also demonstrated that the percentage of SGs content in total of upper leaves under SD photoperiods was higher than under LD photoperiods (Mohamed et al., 2011). Previous researches were described that LD photoperiod treatment has been affected the total SGs content because of increasing leaf biomass even though the percentage of SGs content was high in SD treatment (Brandle and Rosa, 1992; Ceunen et al., 2013a). Our results may support previous studies in the genetic level. Evans et al. (2015) investigated effects of DLI on SGs content, and total of stev, reb-A, rebaudioside B, rebaudioside C, and rebaudioside D increased as the DLI increased up to 8–10 mol m−2 s−1. In their study, greenhouse-grown S. rebaudiana were subjected to two different light quality conditions, and this difference caused opposite results on stev concentration. The concentration of stev under a higher R/FR condition increased with a corresponding increase in DLI, but the other experiment decreased like our results. Therefore, they suggest that the phytochrome photostationary state may mediate SGs content. Our experiments were used fluorescent lamps (R/FR ratio = 11.4), so there is a possibility to change the SGs content if a higher R/FR ratio lamp were changed.
experiments, it is difficult to discuss whether red or far-red treatment worked or not because leaf biomass under NI treatments at 20 μmol m−2 s−1 was not increased. The effect of vegetative growth could not be detected by NI treatment with single red or far-red light treatment during dark period, but transcription levels of UGT85C2 and SGs content under the NI-FR 20 condition were higher than the NI-R 20 condition. These results suggest that red and far-red light act on genetic and sweet components levels without concern for NI treatment. This means that it may be a participation of the photostationary state of phytochrome and following the change of endogenous GA contents. In our results, even though leaf biomass did not change, stem length under the NI-FR 20 condition was highest among NI experiments. Stem elongation was involved in GA contents, and GA contents can be increased by far-red in bean, Phaseolus vulgaris (Beall et al., 1996). Far-red enrich environment induce the change of the photostationary state of phytochrome which means the reduction of far-red light absorbing form of Pfr is occurred, and stem elongation is related to the photostationary state (Morgan and Smith 1976; Holmes and Smith, 1977; Smith and Whitelam, 1990). GA and SGs biosynthetic pathways partially share; therefore, the change of endogenous GA contents by containing far-red during dark period may not negatively affect UGT85C2 transcription levels and SGs content than single red light during dark period. Taken together, NI treatment can affect vegetative growth (Kim et al., 2011; Ceunen et al., 2012), and the light intensity more than 20 μmol m−2 s−1 during dark period may be important in order to obtain the effect of NI treatment in S. rebaudiana according to our results. The leaf biomass of the NI-FL50 treatment was near double larger than control because the light intensity may be fulfilled vegetative growth function as NI treatment. In addition, the total leaf area × UGT85C2 and the total leaf area × SGs in the NI-FL50 treatment was higher than the 8 h control. These results suggest that the NIFL50 treatment has a potential to increase the total yield of SGs with similar light environment like SD. 4.4. Effects of end-of-day light pulses on S. rebaudiana
4.3. Effects of night-interruption on S. rebaudiana Stem length in the EOD-FR 15 min treatment was greater than control in our results; however, little study examined the effect of EOD treatment in S. rebaudiana. Hisamatsu et al. (2008) demonstrated the effect of EOD-FR treatment on stem elongation in same Asteraceae family, chrysanthemum (Chrysanthemum morifolium Ramat.). EOD-FR treatment leads to stem or petiole elongation, and this response is caused by GA, auxin, and brassinosteroid in cowpea (Vigna sinensis L.) or Arabidopsis (Arabidopsis thaliana). (Martínez-García and GarcíaMartínez, 1995; Kozuka et al., 2010). The difference between EOD-FR and EOD-R is recognized by phytochrome, and GA 2β-hydroxylation, which is induced with stem elongation, may be controlled by its (Smith, 1982; Kamiya and García-Martínez, 1999). Therefore, stem elongation in the EOD-FR 15 min treatment was also observed in S. rebaudiana. In contrast, the EOD-FR 5 min treatment had no effect on stem elongation compared to control. Franklin et al. (2003) suggested that the involvement of phytochromes B, D and E in stem elongation by EOD-FR treatment. Responses of phytochrome can be classified to the very-lowfluence response, the low-fluence response, and the high-irradiance response, and phytochrome B is mediated by the low-fluence response (Kircher et al., 1999). The low-fluence response can be occurred when the amount of light energy is over a certain amount (integral of light intensities and exposure times) (Bunsen and Roscoe, 1863; Briggs, 1960). These results suggest that the amount of light energy by EOD treatment with 50 μmol m−2 s−1 for 5 min was not satisfied the condition of the low-fluence response in S. rebaudiana. The primary objective of the EOD light pulse experiments was to evaluate whether EOD treatments would upregulate SGs-related gene transcription. However, the EOD treatments were conducted before results were obtained indicating the optimal photoperiod for SGs-related gene transcription. Therefore, we included control plants grown
Although DLI of the NI-FL 50 treatment was only slightly higher the 8 h photoperiod treatment, leaf biomass was significantly increased. In addition, leaf biomass in plants grown under the 12 h photoperiod and those that were administered the NI treatment were similar despite higher DLI of the 12 h photoperiod. the NI treatment is used to delay flowering in SD plants such as Asteraceae when grown commercially. SD plants have a critical day length and flowering is induced when the photoperiod falls below it. In other words, S. rebaudiana subject to the NI-FL 50 treatment might be regulated like under a LD photoperiod because of the NI, which maintains their vegetative growth. In Cymbidium, NI induced vegetative growth (Kim et al., 2011); therefore, NI treatment with more than 50 μmol m−2 s−1 light for 4 h in the middle of the dark period night is suitable to affect vegetative grown in S. rebaudiana. The NI-FL 20, the NI-F 20, and the NI-R treatments did not increase leaf biomass compared to the 8 h and the 12 h photoperiod control treatments. This result means that NI treatment using the light intensity less than 20 μmol m−2 s−1 was not sufficient to increase leaf biomass in S. rebaudiana. In our study we did not observe significant change in UGT85C2 transcription level between the 8 h photoperiod control and the NI-FL 20, the NI-FL50, and the NI-FR 20 treatments. Compared to other NI treatments, only S. rebaudiana grown under the NI-R 20 treatment was not subject to far-red light during the dark period. NI treatment with red light in many short plants can contribute to the inhibition of flowering, and phytochrome, red/far-red reversible photoreceptor, regulates flowering (Downs, 1956; Smith and Whitelam, 1990). These results suggest that NI treatment with red light has an effect to vegetative growth in SD plants. Ceunen et al. (2012) showed that NI by red LEDs promotes the vegetative growth and SGs content. However, in our 8
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Science 72, 1263–1266. Briggs, W.R., 1960. Light dosage and phototropic responses of corn and oat coleoptiles. Plant Physiology 35 (6), 951. Brusick, D.J., 2008. A critical review of the genetic toxicity of steviol and steviol glycosides. Food and Chemical Toxicology 46 (7), S83–S91. Bunsen, R., Roscoe, H., 1863. Photochemische untersuchungen. Annalen der Physik 193 (12), 529–562. Ceunen, S., Geuns, J.M.C., 2013a. Steviol glycosides: chemical diversity, metabolism, and function. Journal of Natural Products 76 (6), 1201–1228. Ceunen, S., Geuns, J.M.C., 2013b. Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods. Phytochemistry 89, 32–38. Ceunen, S., Werbrouck, S., Geuns, J.M.C., 2012. Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. Journal of Plant Physiology 169, 749–752. Chatsudthipong, V., Muanprasat, C., 2009. Stevioside and related compounds: therapeutic benefits beyond sweetness. 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under the 16 h photoperiod because a number of studies claimed that the 16 h photoperiod affected total SGs content. As a result, the EOD-FR 15 min treatment upregulated UGT85C2 transcription. the EOD-FR treatment is well known to cause stem elongation by changing GA1 content (Martínez-García and García-Martínez, 1995; Kamiya and García-Martínez, 1999; Hisamatsu et al., 2005). Increased UGT85C2 transcription was possibly the indirect result of the EOD-FR treatment via upregulation of GA content. No less than 15 min of 50 μmol m−2 s−1 far-red irradiation is required to increase UGT85C2 transcription, as no effect was seen with the EOD-FR 5 min treatment. DLIs of the 16 h photoperiod control and EOD treatments were almost same; however, total leaf area × UGT85C2 transcription level and total leaf area × SGs (sum of stev and reb-A) in the EOD-FR 15 min treatment were larger than the 16 h control. In other words, the percentage of SGs was increased by the EOD-FR 15 min treatment even though leaf area did not change compared to the 16 h control. Since SGs and GA biosynthesis pathway from ent-kaurenoic acid goes through same pathway (Kinghorn, 2002), the increase of SGs may be linked the increase of GA1 by EOD-FR treatment (Martínez-García and GarcíaMartínez, 1995; Kamiya and García-Martínez, 1999; Hisamatsu et al., 2005). These results suggest that there is a possibility of improvement of SGs accumulation by EOD-FR 15 min treatment. In addition, in this treatment were used LEDs which can save light energy consumption compared to florescent lamp. 5. Conclusion We analyzed the transcription levels of SGs-related genes quantitatively in order to evaluate the SGs content indirectly. As a result, transcription induction of UGT85C2 was a similar tendency with stev and reb-A even though a correlation between other SGs-related genes and SGs content was not as obvious in this study. The change in UGT85C2 transcription did not depend on light intensity (50–400 μmol m−2 s−1), while a SD photoperiod upregulated UGT85C2. In addition, in spite of a light environment with lower DLI, leaf biomass following the NI-FL 50 treatment was comparable to plants grown under a LD photoperiod with no decrease in SGs content. However, sample size was small for morphological analysis (n = 3–4), which means that a rough trend can only obtain from our results. S. rebaudiana subject to the EOD-FR 15 min treatment had increased UGT85C2 transcription levels and stev and reb-A contents. These results suggest that, even if low DLI is present in the field, it is possible to improve the SGs yield from S. rebaudiana by using supplemental lights to add slight amounts of light energy, including far-red light, in a directed manner. Acknowledgements The authors wish to thank Michiho Ito, Associate Professor, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, for S. rebaudiana was provided. This study has been partly supported by the programs of the Grantin-Aid for Scientific Research (B, 25292150) from the Japan Society for the Promotion of Science. References Arthur, J.M., Guthrie, J.D., Newell, J.M., 1930. Some effects of artificial climates on the growth and chemical composition of plants. American Journal of Botany 17 (5), 416–482. Barbet-Massin, C., Giuliano, S., Alletto, L., Daydé, J., Berger, M., 2015. Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Sevia rebaudiana Bertoni. PLoS One 10 (7), e0133067. Beall, F.D., Yeung, E.C., Pharis, R.P., 1996. Far-red light stimulates internode elongation, cell division, cell elongation, and gibberellin levels in bean. Canadian Journal of Botany 74 (5), 743–752. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Canadian Journal of Plant
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Melis, A., 1999. Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage in vivo? Trends in Plant Science 4 (4), 130–135. Metivier, J., Viana, A.M., 1979. Determination of microgram quantities of stevioside from leaves of Stevia rebaudiana Bert. by two-dimensional thin layer chromatography. Journal of Experimental Botany 30 (4), 805–810. Mohamed, A.A.A., Ceunen, S., Geuns, J.M.C., Van den Ende, W., De Ley, M., 2011. UDPdependent glycosyltransferases involved in the biosynthesis of steviol glycosides. Journal of Plant Physiology 168 (10), 1136–1141. Morgan, D.C., Smith, H., 1976. Linear relationship between phytochrome photoequilibrium and growth in plants under simulated natural radiation. Nature 262 (5565), 210–212. Nettleton, J.A., Lutsey, P.L., Wang, Y., Lima, J.A., Michos, E.D., Jacobs, D.R., 2009. Diet soda intake and risk of incident metabolic syndrome and type 2 diabetes in the MultiEthnic Study of Atherosclerosis (MESA). Diabetes Care 32 (4), 688–694. Osman, M., Samsudin, N.S., Faruq, G., Nezhadahmadi, A., 2013. Factors affecting microcuttings of Stevia using a mist-chamber propagation box. The Scientific World Journal 2013 (Article ID 940201, 10 pages). Saji, H., Daphne, V.P., Furuya, M., 1983. Studies on the photoreceptors for the promotion
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