Stevia rebaudiana

Hernandez, Karel Vives, Moreno-Romero, Jordi, Hern, Martha, Torre, andez de la, P, Claudia, Manríquez, erez, Leal, Darcy Ríos & Martínez-Garcia, Jaime F., 2022, Effect of light intensity on steviol glycosides production in leaves of Stevia rebaudiana plants, Phytochemistry (113027) 194, pp. 1-10 : 6

3.2. Effect of light intensity on SGs production in S. rebaudiana

Not only genetic but also environmental and growing conditions can affect SGs composition. Among the different abiotic factors, the effect of light on S. rebaudiana SGs production has been addressed by different authors ( Ceunen et al., 2012; Ceunen and Geuns, 2013a, 2013b; Jarma-Orozco et al., 2020; Shulgina et al., 2021; Yoneda et al., 2017a, 2017b). In contrast to what was reported in these works that address different aspects of light quantity and quality, for deciphering the effect of light intensity and no other variables in this study we have taken into consideration: (1) the possible negative effects caused by an excess light, avoiding this by selecting light intensities below the light saturation point; (2) that the development stage can influence the SG content ( Ceunen and Geuns, 2013b), and for this reason the different light treatments were applied only for 15 days to cultivar Morita II, being the analysis done on plants with similar physiological and developmental stage; and (3) how different light intensities affect the gene expression of the most prominent SGs biosynthetic enzymes (SrHA13H, SrUGT74G1, SrUTG76G1) and its correlation with SGs composition. In the following paragraphs, these three aspects will be developed in a greater detail.

We have selected three different light intensities (from 80 to 900 μmol m 2 s 1) that presented a positive correlation with an increased photosynthetic efficiency (based on the electron transport rate and the apparent rate of photosynthesis, Fig. 4C and D respectively). We were under the 1200 μmol m 2 ⋅ s 1 of light saturation point established in S. rebaudiana cultivar Morita II grown under field conditions ( Jarma-Orozco et al., 2020). Above this value, negative effects on photosynthetic apparatus and biomass production occur. Our conditions are clearly under this point, as even the highest light intensity treatment presents the maximum fluorescence ratio ( Fig. 4B ), in consequence photosystems are not damaged. Thus, higher photosynthesis is occurring when we increase light intensity, hence also its products, such ATP and NADPH, molecules that are used in the synthesis of specialized metabolites such as SGs. The KO and KA13H activity requires the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert ent -kaurene to ent -kaurenoic acid and finally to steviol ( Fig. 1 ) ( Zhou et al., 2021; Ko and Woo, 2020). By reconstructing the SGs pathway in E. coli , has been proved that ent -kaurenoic acid accumulates by increasing NADPH levels ( Moon et al., 2020). Therefore, we can speculate that activity of these NADPH-dependent enzymes will be dependent on photosynthesis. With higher photosynthesis rates, it is expected that the promoted activity of the KO-CPR-KA13H module could lead to the accumulation of steviol, that in turn can stimulate the following glycosylation reactions to produce SGs. This links with our results where SGs content increased at the same time that light was intensified ( Fig. 5F ). If the plant benefits from the accumulation of SGs at high light conditions is unknown.

We must take into consideration that although the total SGs at high light recorded the major levels, the proportion of stevioside was also the highest: even though rebaudioside A levels were significantly higher at maximum light, the rebaudioside A:stevioside ratio was the lowest. This is an important aspect to consider when using SGs as natural sweeteners, due to the stevioside bitterness. The type of SGs synthesized can be related with the expression of the biosynthetic enzymes. For that reason, besides the positive effect that increase photosynthetic product can have on the SGs production, we have studied if 15 days of different light intensities affect the expression of the biosynthetic genes. Several works have observed that SrKA13H, SrUGT74G1 and SrUGT76G1 expression changes upon different environmental conditions such as salinity ( Lucho et al., 2019), or under the effect of different elicitors such as salicylic acid, chitosan and hydrogen peroxide (Vazquez et al., 2019). The work of Yoneda et al. (2017b) studied the interaction of different light treatment and expression of SGs biosynthetic enzymes in S. rebaudiana . Although this work also performed different light intensities, the most important changes on gene expression and SGs accumulation were observed when red, far-red and end-of-day far-red light treatments were applied. The increase of SGs content by supplementing red or far-red light has been also reported by others ( Ceunen et al., 2012; Shulgina et al., 2021). When Yoneda et al. (2017b) studied different light intensities (from 50 to 400 μmol m 2 ⋅s 1), they did not detect changes in the expression of SrUGT74G1 (nor in the other SGs biosynthesis-related genes KO and UGT85C2) but SrUGT76G1 was overexpressed at 100 μmol m 2 ⋅s 1. Unfortunately, this work does not measure the different SGs content under different light intensities. Moreover, S. rebaudiana biomass was very different under the different treatments, as also occurs in other works where SGs content has been reported higher in long-day photoperiod ( Ceunen and Geuns, 2013a, 2013b). In our study, the expression of genes related to the three SGs synthesis enzymes (KA13H, UGT74G1 and UGT76G1) increased at higher light intensities. However, it cannot be stated that the amounts of stevioside and rebaudioside A produced at different light intensities evaluated depends exclusively on the greater expression of one gene with respect to the other. For example, at medium and high light intensity levels, no significant differences were observed in the expression of SrKA13H and SrUGT76G1, however a greater production of rebaudioside A was observed. This can be explained by the higher expression of the SrUGT74G1 enzyme along the increasing light intensities that corresponds with an accumulation of stevioside, also a precursor of rebaudioside A. Therefore, to increase the levels of rebaudioside A and obtain extracts with more sweetness, it is not only necessary to increase its direct biosynthetic enzyme, but also others involved in the previous steps of the pathway.

Figures

Fig. 4. Photosynthetic parameters in S. rebaudiana plants cultivar Morita II treated with different light intensities. (A) Morphological appearance of plants 15 days after being subjected to three light intensities: (left) low light (80 μmol m2⋅s 1), (center) medium light (300 μmol m2⋅s 1) and (right) high light intensity (900 μmol m2⋅s 1). (B) Typical measurement obtained from the IMAGIM-PAM chlorophyll fluorometer. (left) Representation of Quantum yield of photochemical energy conversion in PS II (Y(II)) and Pulse-Amplitude-Modulation (PAM) values during the time of the experiment. Different phases has been highlighted: I, initial pulse of actinic light; II, increased steps of actinic light; III, maximum saturation point; IV, recovery after exposure; (right) images of the plants during the different phases of the measurements. (C) Maximum quantum yield of PSII (Fv/Fm), (D) Electron transport rate (ETR) and (E) Apparent rate of photosynthesis (PS) obtained at the different light intensities. The error bars represent the standard deviation of the mean (n = 5). Different letters above each bar indicate significant differences (One Way ANOVA, Tukey, P <0.05).

Fig. 1. Schematic overview of the metabolic pathway for steviol glycosides (SGs) synthesis and its subcellular organization in S. rebaudiana. In the chloroplast, the ent-kaurene is synthesized through the MEP pathway. Afterwards, the ent-kaurene oxidase (KO) is responsible for the conversion of ent-kaurene to ent- kaurenoic acid, that is the substrate for the ent-kaurenoic acid 13 hydroxylase (KA13H) to synthesize steviol. Both KO and KA13H are located in the endoplasmic reticulum in close proximity to the cytochrome P450 reductase (CPR) that acts as a necessary NADPH donor. The regulation of KO and KA13H activities is crucial as ent-kaurenoic acid is a branching point in the pathway that leads either to the synthesis of gibberellins or SGs. If KA13H activity is favored, steviol will be produced. From steviol, by consecutive rounds of glycosylation performed by diverse uridinediphosphate glycosyltransferases (UGTs) located in the cytoplasm, multiple SGs are synthesized. The most abundant SGs, stevioside and rebaudioside A, are produced downstream by the activity of the highly promiscuous UGT74G1 and UGT76G1 enzymes. The stevioside and rebaudioside A accumulated are subsequently transported and stored in the vacuole. Representation adapted from Singh et al. (2017) and Libik-Konieczny et al. (2021).

Fig. 5. Effect of three light intensities on the production of stevioside and rebaudioside A and the expression of their biosynthetic enzymes in S. rebaudiana cultivar Morita II. Quantification of (A) stevioside and (B) rebaudioside A. Each bar represents the mean. The error bars represent the standard deviation of the mean. Different letters above each bar indicate significant differences (One Way ANOVA, Tukey, P <0.05, n = 5). Gene expression levels of (C) SrKA13H, (D) SrUGT74G1 (E) and SrUGT76G1. Transcription levels were normalized against two reference genes. The mean is represented by an x. The different letter above each box plot indicates significant differences (One Way ANOVA, Tukey, P <0.05, n = 8). (F) Sum of stevioside and rebaudioside A (data from A and B) and ratio of rebaudioside A:stevioside (on the top).