The effect of osmotic stress, lighting spectrum and temperature on growth and gene expression related to anthocyanin biosynthetic pathway in wild strawberry (Fragaria vesca L.) in vitro

Microshoots of wild strawberry (Fragaria vesca subsp. vesca L.) in vitro culture was derived from seedlings of the everbearing alpine variety ‘Rügen’. Ripe achenes of F. vesca were germinated in vitro as described by Miller et al. (1992). The achenes were dried and kept in the refrigerator for 3 months. They were then sterilised by rinsing with 96% ethyl alcohol and then with sterile water, incubated for 10 min in 6% sodium hypochlorite solution (Carl Roth, Karlsruhe, Germany) and again rinsed (4×) with sterile distilled water. Sterilised achenes were cut transversely by using a scalpel. The cut achenes (embryo axis part) were then placed in 200-mL glass flasks containing 30 mL of agarised Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 3% sucrose and without phytohormones. Germinated microshoots were cultivated at 22 ± 3°C under 150 μmol ∙ m^-2 ∙ s^-1 fluorescent lamp illumination and a 16-hr photoperiod in flasks with MS medium supplemented with 1 mg ∙ L^-1 benzylaminopurine, 3% sucrose and 0.8% plant agar. The medium was adjusted to pH 5.8 and autoclaved at 120°C for 30 min. The microshoots were subjected to three different treatments. For the determination of the light spectrum effect, the microshoots were transferred to a growth chamber where they were illuminated with a custom-made light-emitting diode (LED) lighting system containing four separate modules for parallel growth runs: blue, red, red + blue and red + blue + UV. The main photosynthetic photon flux (PPF) was provided by blue 452 nm (B; LedEngin LZ1-00B200, Osram Sylvania, Wilmington, MA, USA) and deep red 662 nm (R; Luxeon Rebel LXM3-PD01-0300, Philips Lumileds Lighting Co. San Jose, CA, USA) light supplemented with high-power UV-A 386 nm (LedEngin LZ440UB00-U4, Osram Sylvania, Wilmington, MA, USA) light. Photosynthetic photon flux density (PPFD) – 50 μmol ∙ m^-2 ∙ s^-1 in total was measured and regulated at the plant level using the light spectrometer FLAME-S-UV-VIS-ES (Ocean optics, Dunedin, FL, USA). Standard fluorescent lamps (broad-spectrum 400–800 nm, Philips Lighting, Eindhoven, Netherlands) were used as a control.

For the determination of the temperature effect, microshoots in flasks were grown in a climatic chamber at 15°C, 22°C (standard), 30°C and a 16-hr photoperiod. For the evaluation of the osmotic compounds (sucrose and PEG 6,000 MW) effect, the plants were transferred to MS medium supplemented with 1.5%, 3%, 6% and 9% sucrose and to MS supplemented with 3% sucrose and 5%, 10% and 12% PEG. Five explants per flask were grown in three repeats for every experiment separately. Microshoot weight in grams and colour changes were evaluated after 30 days, and the mean and the standard error of the mean were calculated. Samples of each treatment’s microshoots (0.1–0.2 g) were frozen in liquid nitrogen and kept at -70°C until RNA extraction.

Frozen samples were homogenised using a Retsch Mixer Mill 400 (Retsch GmbH, Haan, Germany). RNA was isolated using the GeneJET Plant RNA Purification Mini Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) in accordance with the manufacturer’s recommendations, from three biological replicates. The quantity and quality of RNA were measured spectrophotometrically with an Implen P330 nanophotometer (Implen GmBH, München, Germany). Samples were treated with DNase I (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) at 37°C for 30 min.

cDNA was synthesised using the RevertAid First Strand cDNA Synthesis Kit and oligo (dT)₁₈ primer (Thermo Fisher Scientific Baltics) following the manufacturer’s recommendations. The absence of genomic DNA was confirmed by PCR, and primers for elongation factor-1α subunit intron were used (Bonasera et al., 2006). PCRs were carried out using Taq DNA polymerase (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) with the following cycling parameters: 2 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 56°C and 30 s at 72°C; 10 min at 72°C.

Quantitative real-time PCR (qPCR) amplification was carried out using an Eppendorf EP Gradient S Thermocycler. Specific primers were designed for anthocyanin biosynthesis pathway genes in wild strawberry (Supplementary Table S1 in Supplementary Materials).

A reference gene β-actin was used as an internal reaction control. All reactions were performed in a 20 μL volume with DreamTaq polymerase (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) and EvaGreen dye (Biotum Ltd., Vladimir, Russia) using the following cycling parameters: 2 min at 95°C; 40 cycles of 30 s at 95°C, 45 s at 60°C and 45 s at 72°C. Later, a melting curve detection gradient from 60°C to 95°C was used. No template control reactions (NTCs) were included in all experiments. An MCE-202MultiNa system (Shimadzu Ltd, Tokyo, Japan) and a DNA-1000 Kit (Shimadzu Ltd, Tokyo, Japan) were used to measure the size and amount of PCR products.

Relative gene expression was estimated using the ΔΔC_T method (Livak and Schmittgen, 2001). The expression data were analysed using Microsoft Excel 2010.

Statistically significant differences between treatments were evaluated using one-way analysis of variance (ANOVA). Variation within treatments was determined by calculating the values of the standard error of the mean (SEM). Significant differences were assessed by using Fisher’s test at 5% and 1% probability levels.

Sucrose is important in the regulation of plant metabolism and enhances anthocyanin synthesis and accumulation in plants (Hijaz et al., 2018; Solfanelli et al., 2006). In the current study, wild strawberry microshoots grown on MS growth medium with 3% sucrose reached the average weight of 0.3 g (Table 1). Similar results were established in cultivated strawberry (Abdullah et al., 2013). However, differences in microshoot weight were not significant among strawberry plants grown on MS medium with 1.5% and 3% sucrose. Microshoots were healthy and did not show stress signs, and the colour of petioles was very rarely reddish. The weight of the microshoot significantly decreased up to one-third, reaching 0.13 g and 0.10 g in plants grown on MS medium with 6% and 9% sucrose, respectively. Microshoots with reddish petioles were more common, especially when the concentration of sucrose in the medium was the highest. We propose that the reduction in microshoot weight and petiole colouring was influenced by osmotic stress, thus reducing or blocking plant metabolism, except in the case of adaptational pathways, which have been activated. Since increased amounts of sucrose (4%–8%) in the medium caused enzyme-like superoxide dismutase (SOD) activation and protein and polyamine accumulation in potato microshoots in vitro, Sajid and Aftab (2022) suggest that this indicates a response to the oxidative stress induced by osmotic stress.

The expression of the phenylpropanoid pathway, specifically anthocyanin biosynthesis genes in wild strawberry, was evaluated. Our study showed that the expression of regulatory Myb10, WD40 genes was significantly lower in microshoots grown on MS medium with 1.5% sucrose than in those grown on standard MS medium with 3% sucrose (Figure 1). The decrease in the expression of regulatory genes was possibly related to the decrease in growth due to an insufficient amount of sucrose.

Figure 1.

Compared to the control, the expression of the CHI (chalcone isomerase) and CHS (chalcone synthase) genes was significantly lower in the plants grown on MS medium with 6% sucrose but higher in the microshoots grown on medium with 9% sucrose. These results suggest that sucrose levels of 6% and lower in the growth medium are possibly too low to induce osmotic stress in microshoots. The reduction in gene expression under 6% sucrose was more likely to be related to the regulatory properties of sucrose than to osmotic stress. Sucrose is known to regulate the expression of many gene systems, including ABA synthesis. In turn, an increase in the ABA level during stress increases the expression of WD40 and other anthocyanin synthesis genes and anthocyanin accumulation (Li et al., 2014; Liu et al., 2018; Mattioli et al., 2020).

UFGT (UDP glucose: flavonol 3-O-glucosyl-transferase) could be distinguished from other studied genes because its expression decreased at the peak of osmotic stress induced by 9% sucrose. The expression of other genes (Myb10, WD40) under the same conditions increased or tended to increase. Increased expression of most genes in the microshoots grown on MS medium with 9% sucrose remained intense due to osmotic stress and might be related to anthocyanin synthesis.

The increase in the expression of most studied genes (except UFGT) as well as the reddish colour of petioles in the medium with 9% sucrose indicated that anthocyanin biosynthesis starts when osmotic stress reaches a certain threshold. The decrease in UFGT expression during sucrose stress shows that catechin and proanthocyanidin synthesis was not induced at the same time. Because flavonoid biosynthesis in Fragaria plants is tissue-specific, anthocyanins are mostly synthesised in generative organs, but not in leaves or stems. Several genes that regulate anthocyanin biosynthesis have been identified in Arabidopsis (Jeong et al., 2018). Therefore, we do not yet know the whole picture of anthocyanin biosynthesis regulation in response to sucrose.

PEG is widely used in modelling drought-induced stress in an in vitro system (Sakthivelu et al., 2008; Verma et al., 2013). In our study, the lowest concentration of PEG (5%) in the MS medium caused a significant reduction in the weight of wild strawberry microshoots to 0.12 g, which was 2.5 times lower than in control microshoots grown on the MS medium with 3% sucrose (Table 1). According to Yosefi et al. (2022), PEG-induced water stress negatively affected shoot fresh and dry weight and physiological and biochemical traits. However, an increase in the PEG amount in the growth medium to 12% resulted in larger microshoots (0.44 g). Therefore, the variation was also larger. Microshoots were pale and watery, but no reddish petioles were observed. In this case, osmotic stress was different from stress caused by sucrose. The microshoots were not growing, they were soaked with PEG and their weight increased due to PEG concentration in the tissues.

The addition of 5%–12% of PEG to the MS medium resulted in reduced expression of all studied genes (Figure 2). PEG concentrations of 5% and 10% in the growth medium were the most inhibiting for the expression of WD40 and CHS genes, while the expression reduction of DFR (dihydroflavonol reductase), EGL (enhancer of glabra) and UFGT genes was significant when 12% of PEG was applied. It is possible that a lower concentration of PEG blocks single metabolic pathways and induces some adaptation reactions, while higher concentrations inhibit the whole metabolism. In Arabidopsis, it was demonstrated that MYB12 or PAP1 overexpression and flavonoid over-accumulation were key to enhanced tolerance to drought stress (Nakabayashi et al., 2014). Cui et al. (2017) noticed that drought stress has a synergistic effect with other stresses that induce gene (UFGT and MYBA1) expression and anthocyanin accumulation in grapevine, even though drought stress alone does not have an effect.

Figure 2.

The expression of the anthocyanin pathway genes in microshoots varied under different osmotic conditions, possibly due to peculiarities of the plant metabolism and the nature of osmotic compounds. Plants cannot use PEG in the same way as sucrose – although PEG enters the plant, it is not metabolised.

Our experiments with wild strawberry microshoots showed that their weight depends on the light spectrum during growth in vitro. The highest average weight (0.34 g) of wild strawberry microshoots was observed under blue + red lights (452 + 662 nm) (Table 1), and it was significantly higher than that of plants grown under red (662 nm) (0.27 g) and blue + red + UV (452 + 662 + 386 nm) (0.24 g) lights, but did not differ significantly from the plants grown under blue (452 nm) (0.31 g) and fluorescent lights (0.28 g). The average weight of microshoots grown under red light (0.27 g) was close to that of the control (0.28 g). This could imply that red light predominates in fluorescent light. Microshoots grown under fluorescent, red and blue + red + UV lights had partially reddish petioles that show increased anthocyanin synthesis (Figure 3). Lower plant weight and accumulation of anthocyanins under blue + red + UV lights in our experiment suggest that microshoots experience stress, which may be related to UV damage. It is known that UV light causes stress and induces the accumulation of anthocyanins in plants (Deroles, 2009; Guo et al., 2009).

Figure 3.

Although no reliable differences in the expression of flavonoid biosynthetic pathway genes were found, most genes’ expression tended to increase when shoots were exposed to blue and blue + red lights for 3 days and to blue + red + UV lights for 9 days (Supplementary Figure S1 in Supplementary Materials). The increase in CHS and UFGT genes’ expression correlated with the increase in the anthocyanin content in lettuce under UV light (Goto et al., 2016). UV light damages cells and increases the concentration of free radicals. Therefore, it intensifies antioxidant flavonoid synthesis.

Anthocyanin accumulation might also be associated with stress conditions in plants caused by an improper light spectrum, or it may occur as a consequence of the change in the content of endogenic carbohydrates. According to Miranda and Williams (2007), blue and yellow light increased the level of sucrose in plants developed in vitro compared to white light. The photochemical efficiency of photosystem II (PSII) is usually low under blue light, but the rate of photochemical reaction is high. The morphogenetic effect, fresh and dry weight, and chlorophyll and nitrogen content of microshoots depend on the proportion of those two parameters (Miranda and Williams, 2007). Blue and red lights are necessary for photosynthesis, but they also have regulatory properties and influence plant metabolism and morphogenesis (Samuolienė et al., 2013). Tripathy and Brown (1995) noticed the root-perceived photomorphogenic inhibition of the shoot and decreased chlorophyll content in wheat seedlings induced by red light. In our experiment, microshoots were almost without roots, but under red and fluorescent lights, we noticed signs of stress that appeared in the red petioles of some of the microshoots and slightly less intensive growth and proliferation compared with microshoots grown under blue and blue + red lights. According to Zhang et al. (2018), blue light causes an increase in CHI, DFR, ANS (anthocyanidin synthase) and FLS (flavonol synthase) expression and a decrease in CHS and F3H (flavanone 3-hydroxylase) expression. Red light not only increases the amount of anthocyanins but also potentially contributes to the synthesis of proanthocyanidins by inducing leucocyanidin reductase and anthocyanin reductase.

Microshoots grown at 22°C were found to have the highest average fresh weight (0.34 g) (Table 1). This is the standard temperature for cultivating wild strawberry plants in vitro. The mean fresh weight of microshoots (0.3 g) grown at 30°C did not differ significantly from the mean fresh weight of microshoots grown under standard conditions. Vitrification and necrosis were observed in some microshoots grown at 30°C, indicating the stress condition of the plants. The mean fresh weight of wild strawberry microshoots grown at 15°C was significantly lower than the weight of microshoots grown under standard conditions and reached only 0.13 g. Despite their reduced size, these microshoots looked quite healthy. However, the colour of the petioles was more reddish than the colour of the microshoots grown at 22°C. This shows anthocyanin accumulation in the microshoots.

The anthocyanin biosynthesis is strongly affected by temperature as some genes regulating cold resistance are involved in this pathway (Christie et al., 1994). During winter, anthocyanins protect the leaves of evergreen plant species by reducing damage caused by low-temperature stress. Anthocyanin accumulation at low temperatures and degradation at high temperatures have been noticed in plants’ fruits and tissue cultures (Deroles, 2009; Gaiotti et al., 2018). In this study, the influence of lower (15°C) and higher (30°C) temperatures on the gene expression of the anthocyanin biosynthesis pathway was evaluated (Figures 4 and 5). At 15°C, the expression of CHI and CHS genes was higher than that of the control (in the case of CHS, for at least 2 weeks); only WD40 expression was lower (significantly lower after 3 weeks). Okutsu et al. (2018) indicated that the level of gene expression of the anthocyanin biosynthesis pathway is affected by the timing of plant exposure to high-temperature stress. Here, the duration of exposure at 15°C had no discernible effect on the expression of the genes tested (Figure 4). The expression of the regulatory WD40 gene, as well as CHI and DFR, was almost the same as that in the control, when microshoots were grown at 30°C for 1 week. On the other hand, the expression of the CHS gene increased and may be explained by the primary reaction to stress (Figure 5). After 4 weeks at the same temperature, the expression of WD40, CHI, CHS and DFR genes decreased substantially. It is in line with the findings of most researchers, and it could be a sign that gene expression decreases under long-term high-temperature stress. It was shown that high ambient temperatures repress anthocyanin biosynthesis through a COP1-HY5 signalling module (Kim et al., 2017). However, transcript levels and activity of PsANS and PsUFGT remained stable or increased when a high temperature (35°C) was applied to plums (Niu et al., 2017). Exposure to light and high temperature (32°C) induced the expression of MYB16 in apple callus, resulting in inhibition of anthocyanin biosynthesis (Wang et al., 2016). The authors suggest that the light-induced change in anthocyanin biosynthesis is primarily caused by altered MYB10 transcript levels, while temperature affects the expression of bHLH3/33, MYB16, MYB17, MYB111 and other repressors (Lightbourn et al., 2007).

Figure 4.

Figure 5.