Introduction

While light energy is converted to useful chemical energy during photosynthesis, excess light is harmful to the plants. Most of the excess energy, which is not required for photosynthetic CO2 assimilation, is dissipated as heat via non-photochemical quenching (NPQ) of chlorophyll fluorescence (Horton et al. 1996; Niyogi 2000; Zulfugarov et al. 2010, 2014). Zeaxanthin (Zx), a component of the violaxanthin (Vx) cycle, participates in the non-photochemical protection mechanism in vascular plants and algae (Demmig et al. 1987; Krause and Weis 1991; Pfündel and Bilger 1994; Jahns et al. 2009). In the Vx cycle, also known as the xanthophyll cycle, Vx is reversibly converted to Zx via the intermediate antheraxanthin (Ax). In the forward de-epoxidation reaction, two epoxy groups are removed stepwise by Vx de-epoxidase (VDE) in the thylakoid lumen. The backward epoxidation reaction is catalyzed by Zx epoxidase (ZE) in the chloroplast stroma.

Given the important function of Zx as a photoprotector against photoinhibition, the majority of studies have focused on the regulation of VDE in the Vx cycle. There have been several reports regarding the regulatory factors of de-epoxidation involved in the light-driven lumen acidification and ascorbate availability (Pfündel and Dilley 1993; Hager and Holocher 1994; Neubauer and Yamamoto 1994; Bratt et al. 1995), Vx availability and temperature (Siefermann and Yamamoto 1974; Bilger and Björkman 1991; Arvidsson et al. 1997), Vx orientation (Gruszecki et al. 1999), aggregation of light-harvesting complex II (LHCII), and lipid properties of thylakoid membrane (Jahns 1995; Gruszecki et al. 2006; Jahns et al. 2009; Schaller et al. 2010). A structural study of VDE suggested that the activation of VDE at low pH involves its dimerization that permits the parallel de-epoxidation of two epoxide rings of Vx (Arnoux et al. 2009).

However, there are far fewer studies on the regulation of ZE. Zx retention has been observed in plants under severe stress such as in overwintering evergreen plants, suggesting a long-term regulation mechanism (Adams et al. 2002; Öquist and Huner 2003). Moreover, significant inhibition of Zx epoxidation has been observed under prolonged illumination of high intensity or by chilling under light (Jahns 1995; Reinhold et al. 2008). This short-term down-regulation of Zx epoxidation was found to be independent of trans-thylakoid pH gradient (Xu et al. 1999; Gilmore and Ball 2000). Reinhold et al. (2008) attributed the short-term down-regulation of ZE to its direct modification, possibly by photooxidation.

Recently, we showed that the down-regulation of Zx epoxidation is a key factor that confers chilling-tolerance to the japonica rice cultivar compared with an indica one cultivar (Kim et al. 2017). Although previous studies mostly focused on the activity of VDE as a key regulator of the non-photochemical protection mechanism (Latowski et al. 2004; Szabó et al. 2005; Chen and Gallie 2012; Murchie and Ruban 2019), no significant differences have been observed in the activity of VDE between the two cultivars. In this review, we suggest that reversible down-regulation of Zx epoxidation is important in the leaves of a vascular plant at various light intensities at room temperature, and this reversible down-regulation mechanism is a general mechanism that functions in vascular plants at room temperature under various light conditions as well as under stress conditions in the presence of light. Under stress condition, a rather irreversible down-regulation mechanism is working, as we have mentioned earlier. Still, the actual mechanism for this down-regulation of ZE activity is uncertain. We will discuss about the redox regulated activation of ZE activity in the low light, which is proposed recently (Nikkanen et al. 2019), but cannot explain its down-regulation under higher light. We have proposed that the down-regulation of ZE activity is by phosphorylation of itself or one of its regulators (Kim et al. 2017), but still without suggesting candidate(s) for the regulator(s).

NPQ Development and Zx Accumulation Kinetics in a Dicot and Two Monocot Plant Leaves Under Light of Various Intensities

Faster NPQ development and Zx accumulation kinetics shown in leaves at room temperature under light of higher intensity can be due to down-regulation of ZE activity rather than up-regulation of VDE activity. To prove this hypothesis, we compared NPQ development and Zx accumulation kinetics in rice, barley, and spinach leaves under different light intensities.

The seeds of the rice (Oryza sativa L.) cultivar Dongjin-byeo were grown in a growth chamber under a 14-h light period with a PPFD of 100 μmol m–2 s–1 under a day/light temperature regime of 28 °C/25 °C. Barley (Hordeum vulgare L. ‘Albori’) seeds were grown at a PPFD of 50 μmol m–2 s–1 under a photoperiod of 14 h at 25 °C. For all the experiments, fully expanded first leaves of 1-week-old barley were used. Fresh spinach (Spinacia oleracea L.) leaves were obtained from a local market. The leaves of spinach, rice, and barley were floated on distilled water in Petri dishes with the adaxial side up and dark-adapted at 25 °C for 3 h to remove Zx that is present in plants under non-stressed growth condition. The dark-adapted leaf segments floated on distilled water were then exposed to different PPFDs (50–2100 μmol m–2 s–1) at 25 °C. The light provided using 500-W halogen lamps was passed through a 10-cm-deep water bath.

The NPQ development kinetics at room temperature were compared as reported previously (Kim et al. 2017) using the Stern–Volmer equation: NPQ = (Fm − Fm′)/Fm′, where Fm′ is the maximum yield of fluorescence in the light-acclimated leaves and Fm was measured after 10 min of dark adaption at room temperature for photoinhibition at 25 °C. Chlorophyll fluorescence quenching was analyzed using a pulse-amplitude modulated fluorometer (PAM-2000, Walz, Effeltrich, Germany). The illumination of spinach, rice, and barley leaves at four to five different PPFDs varying from low (50–100, 60–150, and 180–300 μmol m–2 s–1 for barley, rice, and spinach, respectively) to moderately high (300–600 μmol m–2 s–1 for barley and rice, and 700–1400 μmol m–2 s–1 for spinach), and high (1000, 1200, and 2100 μmol m–2 s–1 for barley, rice, and spinach, respectively) led to different NPQ development kinetics (Fig. 1a–c). The pattern of kinetics was highly similar among the three plants in each light group: NPQ increased rapidly or exponentially and saturated under light at high PPFDs (high light, HL), increased exponentially and saturated but rather slowly under light at moderately high PPFDs (medium light, ML), and increased initially but then decreased under light at low PPFDs (low light, LL). The saturated level of NPQ was approximately 3.6, 2.4, and 1.5 for spinach, rice, and barley, respectively.

Fig. 1
figure 1

Development of non-photochemical quenching (NPQ) and time course of zeaxanthin formation under light of various intensities at 25 °C in the leaves of spinach (a, d), rice (b, e), and barley (c, f). Light intensity is presented as photosynthetic photon flux density (PPFD) and is expressed as μmol m−2 s−1. After the measurement of NPQ, pigments were extracted with 100% cold-acetone from three segments of leaves at each measuring point and analyzed by a high-performance liquid chromatography system. % VAZ = % (violaxanthin + antheraxanthin + zeaxanthin) (n = 3, mean value ± SD; n = 1 ~ 2 for 0 h samples, rice high light (1200 μmol m−2 s−1) treated samples and barley samples)

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As Zx accumulation is closely related to NPQ development, Zx accumulation kinetics at room temperature were also compared among the three plant species under light of various intensities (Fig. 1d–f). After the measurement of NPQ, pigments were extracted with 100% cold-acetone from three segments of leaves at each measuring point, and the Vx cycle components were determined as described by Kim et al. (2017) following the method of Thayer and Björkman (1990) with some modifications. The pattern of accumulation kinetics was highly similar to that of the NPQ development kinetics. Within 2 h of illumination, the relative amount of accumulated Zx reached a steady-state in all light groups. Remarkably, the fluctuations with respect to the relative amount of Zx were more prominent than those of the NPQ development kinetics in the plants under LL. Under HL, approximately 80%, 70%, and 50% of the Vx cycle pigments were converted from Vx to Zx in spinach, rice, and barley, respectively.

Effects of Salicylaldoxime on NPQ Development and Zx Accumulation in the Leaves of Three Vascular Plants Under Light of Various Intensities

To ensure that the differences in the rate of Zx formation at different PPFDs are due to the availability of Vx to de-epoxidase or the activity of epoxidase as reported in Jahns (1995), the leaves of three plants were pre-inhibited using 5 mM salicylaldoxime (SA), an epoxidase inhibitor (Pfündel and Bilger 1994), and then exposed to PPFDs varying from 50 to 2100 μmol m–2 s–1 (Fig. 2). Both the NPQ development kinetics and the rate of Zx formation in the leaves of three plants under low and moderately high PPFDs increased, reaching the level of leaves under high PPFDs. However, the treatment with SA did not significantly alter the availability of Vx in all the three plant species (Fig. 2d–f, Tables S1, S2, S3). This indicates that the differences in the rate of Zx formation in the three plant leaves under different PPFDs depend on the differences in the activity of ZE, and not on the availability of Vx to VDE. Although our data shown in this study are consistent with our conclusion, we cannot exclude the possible side effects of SA.

Fig. 2
figure 2

Development of non-photochemical quenching (NPQ) and time course of zeaxanthin formation under light of various intensities at 25 °C in the salicylaldoxime (SA)-treated leaves of spinach (a, d), rice (b, e), and barley (c, f). Light intensity is presented as photosynthetic photon flux density (PPFD) and expressed as μmol m−2 s−1. To inhibit epoxidation, pre-darkened leaves were infiltrated with 5 mM SA through the cut petiole at 25 °C for 3 h in dark before the light treatment. After the measurement of NPQ, pigments were extracted with 100% cold-acetone from three segments of leaves at each measuring point and analyzed by a high-performance liquid chromatography system. % VAZ = % (violaxanthin + antheraxanthin + zeaxanthin) (n = 3, mean value ± SD; n = 1 ~ 2 for 0 h samples, rice high light (1200 μmol m−2 s−1) treated samples and barley samples)

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Reversible Down-Regulation of ZE Activities is a General Mechanism Working on Vascular Plant Leaves Under Light of Various Intensities

The leaves of plants tolerant to photoinhibitory stress are expected to have relatively high VDE activity compared with that in the leaves of sensitive plants. However, as reported earlier, a light-chilling resistant rice cultivar reversibly down-regulates the rate of Zx epoxidation rather than promoting the rate of Vx de-epoxidation, serving the photosystems in thylakoid membranes with the large amount of photoprotectors (Zx) (Kim et al. 2017). Under the same light-chilling condition, in the leaves of a light-chilling-sensitive rice cultivar, large amounts of Zx were re-converted to Ax and Vx due to relatively higher ZE activity, resulting in the slower accumulation of Zx in the thylakoid membranes compared with that in the tolerant plants. Extending this idea, the results of the present study suggest that the down-regulation of Zx epoxidation is a key factor that regulates Zx accumulation in vascular plant leaves (including both monocots and dicots) at various light intensities, and this down-regulation mechanism is a general mechanism that works at room temperature as well as under other stresses in the light including light-chilling.

Irreversible Down-Regulation of ZE Activities During Photoinhibition of PSII

The down-regulation of Zx epoxidation by a different mechanism was proposed by Reinhold et al. (2008) using Arabidopsis leaves under prolonged illumination of high-intensity light or under light-chilling stress. The gradual retardation of Zx epoxidation when authors increased light stress during pre-illumination was referred to the gradual down-regulation of the Zx epoxidase activity. Increasing the light intensity or the illumination time or decreasing the temperature during pre-illumination which decreases the PSII quantum efficiency after the pre-illumination treatment, also delays the epoxidation rates. Authors show that Zx epoxidation was retarded in thylakoids isolated from pre-illuminated leaves and on the basis of this data suggest that modification of the Zx epoxidase is most probably involved in the light-induced down-regulation. They speculated that the down-regulation of Zx epoxidation was due to the modification of ZE induced by photooxidation. The down-regulation of ZE protein by degradation has also been suggested by Schwarz et al. (2015) using Arabidopsis leaves under drought stress, not under high light. In Arabidopsis, pea and tobacco plant leaves under photoinhibitory stress condition, ZE protein degradation is also reported together with D1 protein degradation as a photoprotective mechanism by Bethmann et al. (2019).

The degradation of ZE protein in this irreversible mechanism in induced by photooxidation. However, the content of ROS produced in the chilling-tolerant rice cultivar was less than that produced by chilling-sensitive rice cultivar (Kim et al. 2017). Further, the down-regulated ZE quickly re-activated in the dark, suggesting that the down-regulation is a reversible process. Therefore, this irreversible mechanism is different from the above-mentioned reversible down-regulation mechanism. However, the involvement of irreversible inactivation of ZE by ROS or its degradation cannot be excluded, and this irreversible mechanism can be partially involved under mild stress conditions and is likely involved under severe stress conditions.

ZE Inactivation Mechanism for Reversible Down-Regulation

According to the data in our published paper (Kim et al. 2017) and the data shown in Figs. 1 and 2, we hypothesized that the reversible down-regulation of Zx epoxidation in vascular plants at room temperature under various light conditions as well as under stress conditions in the presence of light might be regulated by phosphorylation of ZE. Based on the research using stn7/stn8 mutants of Arabidopsis, Reinhold et al. (2008) suggest that phosphorylation is not involved in the short-term down-regulation of Zx epoxidation. However, this result still does not exclude the participation of other chloroplast kinases, because there are at least an overall set of 15 chloroplast-localized protein kinases are present in diverse vascular plants (Reiland et al. 2009; Bayer et al. 2012). However, we still cannot answer whether ZE itself is phosphorylated for its inactivation or other reaction partners of ZE that can influence ZE activity are phosphorylated.

Recently, several papers have been published suggesting the redox regulation of the activity of ZE. At light-limiting conditions, both Arabidopsis mutants defective in NADPH thioredoxin (Trx) reductase C (NTRC) (Naranjo et al. 2016) and Arabidopsis trxm mutants with silenced Trx m proteins (Da et al. 2018) accumulate higher levels of Zx than wild-type plants. Although the redox state of ZE was not altered in ntrc mutants (Naranjo et al. 2016), Da et al. (2018) provides evidences suggesting that by either NTRC or via Trx m, the sulfhydryl group of the ZE is reduced and activated through oligomerization. Because of the degradation of ZE in trxm mutants, Da et al. (2018) suggests that the redox-dependent oligomerization of ZE stabilizes and activate ZE, as well.

In conclusion, ZE can be somewhat inactive in darkness possibly with the oxidation of Trx and is activated and stabilized in the LL by the Trx system. However, we do not know how this stabilized ZE or activated ZE is degraded or inactivated under HL. Therefore, under HL, the current redox regulation mechanism need further evidences to explain the irreversible down-regulation of ZE by concomitant degradation of D1 and ZE proteins (Bethmann et al. 2019), and the reversible down-regulation mechanism of ZE activity by phosphorylation (Kim et al. 2017) need further regulators that influence ZE activity by phosphorylation.