Abstract
Leaves of Begonia semperflorens accumulate anthocyanins and turn red under low temperature (LT). In the present work, LT increased H2O2 content and superoxide anions production rate, causing significant increases in the activities of enzymes and contents of reduced components involved in the ascorbate-glutathione cycle (AsA-GSH cycle). As a result, LT-exposed seedlings increased the expression of genes involved in anthocyanin biosynthesis, and accumulated anthocyanin. Based on LT condition, application of N,N'-dimethylthiourea (DMTU) decreased reactive oxygen species (ROS) content, and unbalanced the AsA-GSH-controlled redox homeostasis. As a result, seedlings in the LT + DMTU group did not accumulate anthocyanin. Our results suggest that ROS may act as an important inducer in LT-induced anthocyanin biosynthesis.
As a major class of pigments in plant tissues, anthocyanins are considered stress indicators because their biosynthesis can be induced by many environmental factors, in which low temperature (LT) is a nonignorable inducer (Lo Piero, 2015; Zhang et al., 2015a).
Low temperature generally increases the expression of genes involved in anthocyanin synthesis. Anthocyanin biosynthesis involves the coordinated expression of transcription factors (TFs) and structural genes (Dixon and Steele, 1999; Takos et al., 2006) and the up-regulation of these genes results in anthocyanin accumulation. Both early (CHS, CHI, F3H, and F3′H) and late (DFR, UFGT, and ANS) anthocyanin biosynthesis genes are up-regulated in response to LT in many plants (Lo Piero, 2015; Zhang et al., 2011). LT-responsive regulatory elements have been found in the promoter regions of the Vitis vinifera genes VvCHS2, VvPAL, and VvF3H (Zhang et al., 2015b). Some TFs have also been suggested to participate in LT-induced anthocyanin accumulation. For example, HY5 and HYH were found to be necessary regulators for LT-induced anthocyanin accumulation in Arabidopsis thaliana (Gu et al., 2015). The transcript abundance of MYBA1-1 and MYB5-1 was up-regulated by LT to induce specific anthocyanin biosynthesis genes such as ANS1, ANS2, DFG1, DFR2, and UFGT2 in Actinidia chinensis (Li et al., 2017). High ambient temperature induces degradation of the HY5 protein in a COP1-activity-dependent manner to repress anthocyanin biosynthesis in A. thaliana (Kim et al., 2017).
The metabolism of carbohydrates, proteins, and lipids in plants is altered by LT to facilitate cold acclimation. LT impairs photosynthesis and causes a limitation in carbon sink for growth reduction (Wingler, 2015). The consequent excess carbon serves as a substrate for anthocyanin biosynthesis (Hughes et al., 2005; Steyn et al., 2002; Zhang et al., 2013). The imbalance between carbon assimilation and light energy produces relative excess excitation energy, which is thought to be the main driving force for anthocyanin induction under LT conditions (Das et al., 2014; Steyn et al., 2002; Zhu et al., 2016).
Both LT and relative excess excitation energy result in generation of reactive oxygen species (ROS), such as hydroxyl radicals (·OH), superoxide anions (O2·-), and hydrogen peroxide. Although well known for the damage they cause to cells, ROS are considered signalling molecules in plant stress perception (Zhou et al., 2012, 2014). Owing to its longevity, stability, and ability to cross biological membranes, H2O2 is the most ubiquitous intracellular messenger (Rhee et al., 2017). H2O2 oxidizes cysteine thiol groups of phosphatases, which are abundant in plant cells, to transmit its signal to targets (Reczek and Chandel, 2015).
Plants have developed sophisticated enzymatic and nonenzymatic mechanisms to scavenge ROS under LT conditions. Enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) play major roles in scavenging O2 and H2O2. SOD catalyzes the dismutation of O2·- to H2O2, which is subsequently converted by APX and CAT into H2O. Nonenzymatic components such as ascorbate, glutathione, carotenoids, tocopherols, and phenolic compounds (including flavonoids and anthocyanins) also serve as antioxidants and contribute to protection against ROS. The ascorbate-glutathione cycle involves APX, dehydroascorbate reductase (DHAR), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), reduced ascorbic acid (AsA), reduced glutathione (GSH), and nicotinamide adenine dinucleotide phosphate (NADPH) in a series of cyclic reactions to detoxify H2O2 and regenerate AsA and GSH (Li et al., 2010).
The balance between ROS and antioxidants also functions as a signal in stress-induced anthocyanin biosynthesis. Ascorbate has been found to be an important endogenous regulator, inducing anthocyanin biosynthesis through the regulation of anthocyanin precursor biosynthesis gene expression (Page et al., 2012). Anthocyanins confer cellular protection by increasing the biosynthesis of the endogenous antioxidant GSH (Norris et al., 2016). However, how glutathione is involved in the regulation of anthocyanin remains uncertain. NADPH functions as a reducing agent and H+ donor required for dihydroflavonol 4-reductase (DFR) biosynthesis (Wu et al., 2017). The production of ROS from plasma-membrane-localized NADPH oxidase plays an important role in ultraviolet ray (ultraviolet)-induced anthocyanin biosynthesis. If ultraviolet light is attenuated, the O2·− concentration is reduced by the expression of anthocyanin biosynthesis-associated genes in Malus ×domestica peel (Zhang et al., 2014). Chloroplastic H2O2 may also activate FAH1 expression to induce anthocyanin accumulation (Maruta et al., 2014). Abscisic acid (ABA)-induced H2O2 is required for anthocyanin accumulation in the leaves of Oryza sativa seedlings (Hung et al., 2008).
To investigate the function and mechanism of H2O2 in LT-induced anthocyanin biosynthesis, we employed Begonia semperflorens ‘Super Olympia’, a perennial evergreen plant, as the plant material for our study because its leaves turn red in LT conditions (Zhang et al., 2013). We analyzed the differences in anthocyanin biosynthesis, gene expression, H2O2 generation, and antioxidant levels in the presence or absence of H2O2 under LT conditions.
Materials and Methods
Plant growth conditions and treatment
Seeds of B. semperflorens ‘Super Olympia’ were sowed in 25-cm-wide pots containing a mixture of peat and vermiculite (7:3, v:v) in a growth chamber [10 h daylength, 100 µmol·m−2·s−1 photosynthetic photon flux density (PPFD), 25/15 °C day/night temperature and 100% relative humidity]. After germination, seedlings were transplanted and supplied daily with full strength Hoagland nutrient solution in water. Seedlings with four to five leaves were employed in experiments.
Expt. 1.
Plants were treated with different concentrations of methylviologen (MV, 0.005, 0.05, 0.1, and 0.5 mm) or N,N'-dimethylthiourea (1, 2, 5, and 10 mm) by foliar spraying on both the abaxial and adaxial surfaces of the leaves every day; distilled water was used as a control. Leaf samples were harvested at 0900 HR after treatment for 0, 1, 3, 6, 9, and 15 d.
Expt. 2.
Forty seedlings were divided into four groups. Seedlings in the first group were sprayed with distilled water and grown at 25/15 °C day/night under a 10-h photoperiod with a PPFD of 300 µmol·m−2·s−1 (designated “CK”). Seedlings in the second group were sprayed with distilled water and grown at 15/5 °C day/night under a 10-h photoperiod with a PPFD of 300 µmol·m−2·s−1 (designated “LT”). Seedlings in the third group were sprayed with DMTU (every other day, 5 mm) and grown at 25/15 °C day/night under a 10-h photoperiod and with a PPFD of 300 µmol·m−2·s−1 (designated “DMTU”). Seedlings in the fourth group were treated with DMTU (every other day, 5 mm) and grown at 15/5 °C day/night under a 10-h photoperiod and with a PPFD of 300 µmol·m−2·s−1 (designated “LT + DMTU”). Leaf samples were harvested on day 12.
Measurement of ROS
The O2•- production rate was measured by analyzing the formation of nitrite from hydroxylamine in the presence of O2•- according to the method of Elstner and Heupel (1976). Frozen leaf samples (0.3 g) were homogenized with 2 mL of potassium phosphate buffer (65 mm, pH 7.8) and then centrifuged at 5000 gn for 10 min. The reaction mixture contained 0.9 mL of phosphate buffer, 0.1 mL of 10 mm hydroxylamine hydrochloride and 1 mL of supernatant. After incubation at 25 °C for 20 min, 17 mm sulfanilamide and 7 mm α-naphthylamine were added to the reaction mixture. An equal volume of ethylether was added, and the mixture was centrifuged at 1500 gn for 5 min. The absorbance of the aqueous solution was read at 530 nm.
Leaf H2O2 content was measured by monitoring absorbance of the titanium–peroxide complex at 415 nm according to the method of Brennan and Frenkel (1977). Frozen leaf samples (0.3 g) were homogenized with 2 mL of cold acetone. After centrifugation at 12,000 gn for 5 min, the supernatant was collected to determine H2O2 content. The assay mixture contained 0.5 mL of the extract, 50 µL titanium reagent (20% titanium tetrachloride in concentrated HCl, v/v), and 0.1 mL of NH3·H2O (25%). After centrifugation at 10,000 gn for 5 min, the pellets were washed with 1 mL of acetone for five times, and then dissolved in 3 mL of 2M H2SO4. Absorbance of the solution was read at 415 nm.
Determination of activities of enzymes
Frozen leaf samples (0.3 g) were homogenized in 3 mL of potassium phosphate buffer (50 mm, pH 7.8) containing 0.2 mm EDTA, 2 mm ascorbate, and 2% (w/v) PVP. The homogenate was centrifuged for 20 min at 12,000 gn, and the supernatant was used for SOD, CAT enzyme analysis. All operations were performed at 0 to 4 °C. Antioxidant enzyme activities were determined as described by Zhou et al. (2004).
Glutathione reductase [GR (EC 1.6.4.2)] activity was analyzed on the basis of the rate of decrease in absorbance at 340 nm caused by NADPH oxidation Foyer and Halliwell (1976). MDHAR (EC 1.6.5.4) activity was measured by monitoring the decrease in absorbance at 340 nm because of the NADH oxidation (Arrigoni et al., 1981). DHAR (EC1.8.5.1) activity was measured by following the formation of AsA from dehydroascorbate (DHA) at 265 nm according to Dalton et al. (1986). APX (EC 1.11.1.11) activity was determined according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm.
Determination of nonenzymatic antioxidants
Frozen leaf sample (0.3 g) was homogenized in 2 mL of 2% metaphosphoric acid containing 2 mm EDTA. After centrifugation for 10 min at 14,000 gn, the supernatant was used for measuring contents of AsA, DHA, GSH, and oxidized glutathione (GSSG). Protein content was determined according to the method of Bradford (1976) using bovine serum albumin as the standard.
AsA and DHA were measured following Law et al. (1983). To determine contents of AsA and DHA, half of the sample extract was incubated for 50 min with 200 mm phosphate buffer solution (pH 7.4) and 1.5 mm DTT. After incubation, 200 μL of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT. The other half of sample extract was used to determine AsA content in a similar manner except that 400 μL deionized H2O was substituted for DTT and NEM. Color was developed in both series of reaction mixtures (total and reduced ascorbate) with the addition of 400 μL 10% (w/v) trichloroacetic acid, 400 μL 44% H3PO4, 400 μL 4% α’-dipyridyl in 70% ethanol and 200 μL 3% (w/v) FeCl3. After incubating at 37 °C for 60 min in a water bath, the reaction mixtures were measured spectrophotometrically at 525 nm. DHA concentration was obtained by subtracting AsA concentration from the total concentration.
GSH and GSSG were measured following Rao and Ormrod (1995) with minor modifications. To determine contents of GSH and GSSG, 0.1 mL of sample extract was first neutralized with 0.5 M phosphate buffer (pH 7.5). The reaction mixture contained 100 mm phosphate buffer (pH 7.5), 5 mm EDTA, 0.6 mm 5,5′-dithio-bis (2-nitrobenzoicacid) and 0.2 mm NADPH. After adding 3 U of yeast GR, the changes of absorbance in 412 nm for 1 min were recorded. For determine GSSG content, 0.5 mL of neutralized sample extract was mixed with 0.01 mL of 2-vinylpyridine. GSH concentration was obtained by subtracting GSSG concentration from the total concentration.
NADP+ and NADPH were measured following Gibon and Larher (1997) with minor modifications. Leaf sample (0.1 g) was homogenized with 0.9 mL of 0.1 N NaOH for NADPH analysis, 0.9 mL of 0.1 N HCl for NADP+ analysis. The homogenates were heated in a boiling water bath for 5 min. After cooled immediately, the homogenates were centrifuged at 10,000 gn for 10 min. The reaction mixture contained 400 mm NaCl, 100 mm Tris–HCl pH 8.0, 4 mm EDTA, 0.42 mm 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), 1.66 mm phenzine ethosulfate (PES), 14 U of glucose 6-phosphate dehydrogenase (G6PDH) at 37 °C for 40 min. The reaction was stopped by addition of 500 μL of 5 M NaCl, then centrifuged at 20,000 gn at 4 °C for 5 min. The pellets were lysed in 1 mL of 98% ethanol and the absorbance at 570 nm was measured on a spectrophotometer.
Anthocyanin quantification
Anthocyanin contents were determined according to the methods described by Mita et al. (1997), with minor modifications. Leaf samples (0.3 g) were ground with 3 mL of 1% (v/v) HCl:MeOH and then maintained under dark conditions at 4 °C for 1 d. After centrifugation at 3500 gn for 15 min, the supernatant was measured spectrophotometrically at 530 and 657 nm. One unit of anthocyanin equals one absorbance unit (A530 − 0.25 × A657) per milliliter of extraction solution.
Gene expression analysis
Gene expression of phenylalanine ammonialyase [BsPAL (Genbank accession no. KJ930039)], chalcone synthase [BsCHS (Genbank accession no. KJ930037)], flavanone 3-hydroxylas [BsF3H (Genbank accession no. KJ930038)], and anthocyanidin synthase [BsANS (Genbank accession no. KJ930039)] was examined by real-time quantitative RT-qPCR analysis. Total RNA was extracted from mixed leaf samples from three seedlings at each time point from each treatment using the CTAB method according to Liao et al. (2004) First-strand cDNA was synthetized using the Prime ScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa Bio, Dalian, China). The gene-specific primers used for real-time RT-qPCR analysis were designed using Genescript software (GenScript Biotech Corp., Nanjing, China) (Supplemental Table 1). The 18S rRNA gene (Genbank accession no. KJ959633) from B. semperflorens was used as the internal reference gene to normalize any differences in the amount of each cDNA template. The RT-qPCR cycling conditions were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 45 s. We employed the 2-△△CT method (Livak and Schmittgen, 2001) to analyze relative gene expression based on three biological replicates. The data and figures were generated using SPSS (version 16.0; IBM Corp., Armonk, NY) and Origin (version 8; OriginLab, Northampton, MA) software.
Measurement of Chlorophyll fluorescence
Chlorophyll fluorescence was measured with a pulse modulated fluorometer (FMS22; Hansatech Instruments, Norfolk, UK). Seedlings were dark adapted for at least 30 min before measurement of the photochemical quenching coefficient (qP). Minimal fluorescence (Fo) was measured under a weak modulating light, and maximal fluorescence (Fm) was induced by a saturating pulse of light (4000 µmol·m−2·s−1) applied over 0.8 s. Fv is the difference between Fo and Fm. An actinic light source was then applied to achieve a steady state of photosynthesis and to obtain steady state fluorescence yield (Fs), after which a second saturation pulse was applied for 0.7 s to obtain light adapted maximum fluorescence (Fm’). qP was calculated as (Fm’ − Fs)/(Fm’ − Fo’) according to Zhou et al. (2004). All the measurements were measured at 1000 HR.
Statistical analysis
Data from four to six replicates per treatment were analyzed. Means were analyzed using a one-way analysis of variance using Data Processing System software [DPS 3.01 (Tang and Feng, 2002)]. Multiple comparisons between means were conducted using Tukey’s test at P < 0.05.
Results
Effect of MV and DMTU on ROS and anthocyanin accumulation
Treatment of DMTU had negative effect on H2O2 content and O2•- production rate in B. semperflorens (Fig. 1A and C). Over the first 6 d, DMTU treatment decreased the H2O2 content and O2•- production rate in a dose-dependent manner. From days 9 to 15, the H2O2 content and O2•- production rate in DMTU-treated seedlings (except for the seedlings treated with 10 mm DMTU) returned to the levels observed in seedlings in the CK group. With the exception of the lowest concentration (0.005 mm) of MV, MV treatment increased the H2O2 content and O2•- production rate in B. semperflorens (Fig. 1B and D). ROS accumulation increased over time in seedlings treated with 0.5 mm MV, whereas in plants treated with 0.05 and 0.1 mm MV, ROS accumulation significantly increased starting at day 1 and peaked at day 6.
The dose-dependent response of hydrogen peroxide (H2O2) contents (A and B), superoxide anions (O2•-) production rate (C and D), and anthocyanin contents (E and F) to exogenous N,N'-dimethylthiourea [DMTU (1, 2, 5, and 10 mm)] and methylviologen [MV (0.005, 0.05, 0.1, and 0.5 mm)] in leaves of Begonia semperflorens. Leaf samples were harvested at 0900 HR after different treatments for 0, 1, 3, 6, 9, and 15 d. Values and error bars indicate means ±sd (n = 3–6). Means denoted by the same letter do not significantly differ at P < 0.05 in a single group according to Tukey’s test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04488-18
DMTU had no significant effect on anthocyanin content at any treatment concentration (Fig. 1E). Treatment with 0.005 and 0.5 mm MV had no significant effect on anthocyanin content, whereas treatment with 0.05 and 0.1 mm MV significantly increased anthocyanin content by day 3, and the level then plateaued at day 9 in B. semperflorens (Fig. 1F).
These results suggested that anthocyanin accumulation may be related to ROS accumulation; thus, ROS accumulation may be an inducer of anthocyanin biosynthesis in B. semperflorens.
Effects of LT and DMTU on anthocyanin and ROS accumulation and the expression of genes involved in anthocyanin biosynthesis
To further explore the relationship between anthocyanin content and ROS accumulation, we verified the effect of DMTU (5 mm) on LT-induced anthocyanin biosynthesis, a phenomenon reported in our previous studies (Zhang et al., 2013, 2016).
As shown in Fig. 2E–H, LT significantly increased ROS and anthocyanin accumulation, while DMTU had no significant effect on either. Compared with the LT condition, the combination of LT and DMTU treatment (LT + DMTU) significantly decreased the H2O2 content and O2•- production rate. Although seedlings in the LT + DMTU group had a higher O2•- production rate than seedlings in the CK group, no significant difference in H2O2 content was observed between seedlings in the CK and LT + DMTU groups.
Effects of low temperature [LT (15/5 °C day/night)] and N,N'-dimethylthiourea [DMTU (5 mm)] on relative transcript levels for four key genes in anthocyanin biosynthesis [BsPAL (A), BsCHS (B), BsF3H (C), and BsANS (D)], hydrogen peroxide (H2O2) contents (E), superoxide anions (O2•-) production rate (F), and anthocyanin contents (G) in leaves of Begonia semperflorens. Leaf samples were harvested on day 12. Seedlings were sprayed with distilled water and grown at 25/15 °C day/night (CK); seedlings were sprayed with distilled water and grown at 15/5 °C day/night (LT); seedlings were sprayed with DMTU (every other day, 5 mm) and grown at 25/15 °C day/night (DMTU); seedlings were sprayed with DMTU (every other day, 5 mm) and grown at 15/5 °C day/night (LT + DMTU). Values and error bars indicate means ±sd (n = 3–6). Means denoted by the same letter do not significantly differ at P < 0.05 in a single group according to Tukey’s test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04488-18
Changes in anthocyanin contents were in line with ROS accumulation: LT increased the anthocyanin content in B. semperflorens, while treatment with 5 mm DMTU prevented this increase (Fig. 2E–H). These results suggested that ROS accumulation corresponded to anthocyanin content in B. semperflorens.
To explore the function of ROS in promoting anthocyanin accumulation, we analyzed the expression of genes involved in anthocyanin biosynthesis. As shown in Fig. 2A–D, LT significantly up-regulated the expression of BsPAL, BsCHS, BsF3H, and BsANS. DMTU down-regulated BsPAL and BsCHS expression, but had no effect on the expression of BsF3H and BsANS. Compared with seedlings in the LT group, the expression levels of all four genes were down-regulated in seedlings exposed to LT and DMTU treatment. We therefore concluded that the combination of LT and DMTU regulated anthocyanin content at the transcriptional level by modulating the expression of late biosynthesis-related genes.
Effects of LT and DMTU on chlorophyll fluorescence, enzyme activities, and AsA-GSH-controlled redox homeostasis
In B. semperflorens, the activities of antioxidant enzymes, including SOD, CAT, APX, MDHAR, DHAR, and GR, were unaffected by DMTU treatment, while they were increased under LT conditions (Fig. 3). The combination of LT and DMTU treatment decreased the activities of these four enzymes in B. semperflorens compared with LT alone. The activities of SOD and MDHAR were higher in seedlings in the LT + DMTU group than in seedlings in the CK group, while no significant differences in the activities of four other enzymes (CAT, DHAR, APX, and GR) were observed between the two groups (Fig. 3).
Effects of low temperature [LT (15/5 °C day/night)] and N,N'-dimethylthiourea [DMTU (5 mm)] on activities of superoxide dismutase [SOD (A)], catalase [CAT (B)], monodehydroascorbate reductase [MDHAR (C)], dehydroascorbate reductase [DHAR (D)], ascorbate peroxidase [APX (E)], and glutathione reductase [GR (F)] in leaves of Begonia semperflorens. Leaf samples were harvested on day 12. Seedlings were sprayed with distilled water and grown at 25/15 °C day/night (CK); seedlings were sprayed with distilled water and grown at 15/5 °C day/night (LT); seedlings were sprayed with DMTU (every other day, 5 mm) and grown at 25/15 °C day/night (DMTU); seedlings were sprayed with DMTU (every other day, 5 mm) and grown at 15/5 °C day/night (LT + DMTU). Values and error bars indicate means ±sd (n = 3–6). Means denoted by the same letter do not significantly differ at P < 0.05 in a single group according to Tukey’s test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04488-18
As shown in Table 1, LT increased AsA, GSH, and NADPH contents and decreased DHA, GSSG, and NADP+ contents. Compared with LT alone, the combination of DMTU treatment and LT decreased the contents of AsA, GSH, and NADPH in B. semperflorens to the same levels as those observed in seedlings in the CK group.
Effects of low temperature [LT (15/5 °C day/night)] and N,N'-dimethylthiourea [DMTU (5 mm)] on the contents of components involved in the ascorbate-glutathione cycle [ascorbate peroxidase (APX), reduced ascorbic acid (AsA), dehydroascorbate (DHA), reduced glutathione (GSH), oxidized glutathione (GSSG), nicotinamide adenine dinucleotide phosphate (NAPPH), and nicotinamide adenine dinucleotide phosphate (NADP+)] in leaves of Begonia semperflorens.z
As shown in Table 2, DMTU treatment had no effects on Fm, Fo, qP, and Fv/Fm. LT decreased the level of Fm and qP, and had no effect on the level of Fo. As a result, the level of Fv/Fm significantly decreased in seedlings in LT group. The combination of DMTU treatment and LT showed the same effects on Fm, qP, and Fo.
Effects of low temperature [LT (15/5 °C day/night)] and N,N'-dimethylthiourea [DMTU (5 mm)] on chlorophyll fluorescence in leaves of Begonia semperflorens.z
Discussion
ROS may induce anthocyanin accumulation in B. semperflorens
ROS, especially H2O2 and O2•-, function as signaling molecules in many physiological processes. DMTU treatment decreased H2O2 and O2•- levels in B. semperflorens, an effect that diminished gradually over 6 d (Fig. 1A and C). Although different concentrations showed different trends, the application of MV promoted H2O2 and O2•- accumulation in B. semperflorens (Fig. 1B and D). These results suggested that DMTU and MV treatments were effective in our experiments.
While DMTU had no effect on anthocyanin content (Fig. 1E), concentrations of MV (0.05 and 0.1 mm) that significantly promoted H2O2 and O2•- accumulation (Fig. 1B and D) also significantly induced anthocyanin accumulation (Fig. 1F).
By up-regulating late biosynthetic and corresponding regulatory genes, ROS may induce anthocyanin accumulation in A. thaliana (Xu et al., 2017). Chloroplastic H2O2 may activate FAH1 expression to induce anthocyanin accumulation (Maruta et al., 2014). If ultraviolet light is attenuated, the O2•- concentration and the expression of anthocyanin biosynthesis-associated genes is reduced in apple peel (Zhang et al., 2014). ABA-induced H2O2 is required for anthocyanin accumulation in the leaves of rice seedlings (Hung et al., 2008). We therefore hypothesized that the application of MV induced anthocyanin accumulation by increasing H2O2 and O2•- levels.
ROS was involved in LT-induced anthocyanin biosynthesis in B. semperflorens
Our previous studies showed that the leaves of B. semperflorens turned red and produced anthocyanin under LT conditions (Zhang et al., 2013, 2016). To explore the mechanism of ROS-mediated induction of anthocyanin biosynthesis, we analyzed the changes in anthocyanin content and ROS production in B. semperflorens under LT conditions.
LT conditions of 15/5 °C led to a decrease in Fv/Fm (Table 2), implying photoinhibition occurred in LT-exposed seedlings. A decrease in Fv/Fm was the result of a decline in Fm with no increase in Fo; photoinhibition (a decrease in Fv/Fm level) was ascribed to the conformational changes in photosystem II (PSII) related to thermal energy dissipation (Pagliano et al., 2006). Additional application of DMTU alleviated the photoinhibition caused by LT treatment through scavenging ROS (Fig. 2).
As shown in Fig. 3 and Table 1, the levels of both enzymatic antioxidants (SOD, CAT, and APX) and nonenzymatic antioxidants (AsA and GSH) were significantly increased in response to the oxidative stress caused by LT. LT-exposed seedlings still exhibited a lower O2•- production rate and H2O2 content than seedlings in the CK group (Fig. 1).
By increasing the contents of AsA, GSH, and NADPH while decreasing the contents of DHA, GSSG, and NADP+, LT increased the ratios of AsA/DHA, GSH/GSSG, and NADPH/NADP+ in LT-exposed seedlings (Table 1). Higher ratios of AsA/DHA, GSH/GSSG, and NADPH/NADP+ may trigger the reduction of cysteine residues, which then act as retrograde signals from chloroplasts to nucleus (Noctor and Foyer, 2016). Ascorbate has been found to be an important endogenous regulator, inducing anthocyanin biosynthesis via the regulation of anthocyanin precursor biosynthesis gene expression (Page et al., 2012). Anthocyanins protect cells by increasing the biosynthesis of the endogenous antioxidant GSH (Norris et al., 2016). However, how glutathione is involved in the regulation of anthocyanin metabolism remains uncertain. NADPH functions as a reducing agent and H+ donor required for DFR biosynthesis (Wu et al., 2017).
Based on the results above, we therefore suggested that the expression levels of genes involved in anthocyanin biosynthesis (BsPAL, BsCHS, BsF3H, and BsANS) were up-regulated (Fig. 2A–D) by both the over-accumulation of ROS (Fig. 2E–F) and the changes in the levels of AsA-GSH cycle components (Table 1); anthocyanins were eventually biosynthesized in B. semperflorens under LT conditions (Fig. 2G–H).
To further verify the function of LT-mediated ROS accumulation in anthocyanin biosynthesis, we analyzed the anthocyanin biosynthesis under LT conditions in the absence or presence of excess ROS in B. semperflorens.
As shown in Table 1, although seedlings in the LT + DMTU group showed higher MDHAR activity than those in the CK group, DHAR activity was the same in both groups (Fig. 3). As a result, the reduction of DHA to AsA, catalyzed by MDHAR and DHAR, was inefficient in seedlings in the LT + DMTU group. The content of AsA in seedlings in the LT + DMTU group therefore did not show a significant increase compared with CK-seedlings (Table 1). Compared with LT seedlings, the activity of APX in LT + DMTU seedlings was decreased to the same level as that in CK seedlings (Fig. 3) because of no decrease in AsA content (Table 1). Along with the chemical reduction of DHA to AsA, GSH is oxidized to GSSG, which receives H+ from NADPH and is then converted to GSH by GR (Choudhury et al., 2014). Compared with seedlings in the LT group, oxidation of GSSG to GSH in the LT + DMTU group was inefficient because of the decreased NADPH content and GR activity (Fig. 3), causing an increase in GSSG content (Table 1). The result of these alterations is an imbalance in the AsA-GSH cycle, albeit with a small increase in MDHAR activity (compared with CK seedlings); the AsA-GSH cycle in seedlings of LT + DMTU was no longer able to scavenge H2O2.
Compared with LT alone, the combination of DMTU treatment and LT significantly reduced the O2•- production rate and H2O2 content in seedlings. However, the O2•- production rate was still higher than that in CK seedlings, while H2O2 content was not (Fig. 2E-F). Seedlings in the LT + DMTU group exhibited higher activity of SOD (compared with CK seedlings), which converts O2•- to H2O2 (de Haan et al., 1995). The enzymes APX and CAT can then detoxify H2O2 to H2O and O2 (Choudhury et al., 2014). The AsA-GSH cycle plays an important role in detoxifying excess H2O2 generated by SOD in plant cells (Choudhury et al., 2014). However, neither the activities of APX and CAT nor the contents of AsA and GSH were increased in LT + DMTU seedlings compared with CK seedlings (Fig. 3; Table 1). We therefore concluded that the detoxification of H2O2 under the higher O2•- production rate observed in seedlings in the LT + DMTU group was mainly due to the addition of DMTU and the inability of the AsA-GSH cycle to function efficiently.
ROS function as signals in stress-induced anthocyanin biosynthesis (Xu et al., 2017), while anthocyanins possess significant antioxidant activity to decrease ROS contents (Silva et al., 2017). Although seedlings in the LT + DMTU group exhibited a higher O2•- production rate than seedlings in the CK group (Fig. 2F), they did not display a significant up-regulation of four anthocyanin biosynthetic genes (BsPAL, BsCHS, BsF3H, and BsANS) or significantly increased anthocyanin contents (Fig. 2A–D and G–H). H2O2 oxidizes cysteine residues in effector proteins and is one of the most ubiquitous intracellular messengers, owing to its longevity, stability, and ability to cross biological membranes (Rhee et al., 2017; Zhou et al., 2015). Together, the results suggested that ROS was involved in LT-induced anthocyanin biosynthesis in B. semperflorens.
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Gene graph and primer sequences used for RT-qPCR.
