Ascorbate inactivates the oxygen-evolving complex in prolonged darkness
Anna Podmaniczki1,2 | Valéria Nagy1 | André Vidal-Meireles1 | Dávid Tóth1,2 | Roland Patai3 | László Kovács1 | Szilvia Z. Tóth1
1Institute of Plant Biology, Biological Research Centre, Szeged, Hungary
2Doctoral School of Biology, University of Szeged, Szeged, Hungary
3Institute of Biophysics, Biological Research Centre, Szeged, Hungary
Correspondence
Szilvia Z. Tóth, Institute of Plant Biology, Biological Research Centre, Szeged, Temesvári krt 62, H-6726 Szeged, Hungary.
Email: toth.szilviazita@brc.hu
Funding information
Scientia Amabilis Foundation; Bolyai János Fellowship Program, Grant/Award Number:
BO/00958/19; National Research, Development and Innovation Office, Grant/
Award Numbers: ÚNKP-20-5, PD121139, GINOP-2.3.3-15-2016-00001, K132600, GINOP-2.3.2-15-2016-00026; Lendület/
Momentum Programme of the Hungarian Academy of Sciences, Grant/Award Number:
LP2014/19
Edited by:J. Ortega
Abstract
Ascorbate (Asc, vitamin C) is an essential metabolite participating in multiple physiological processes of plants, including environmental stress management and development. In this study, we acquired knowledge on the role of Asc in dark-induced leaf senescence usingArabidopsis thalianaas a model organism. One of the earliest effects of prolonged darkness is the inactivation of oxygen-evolving com-plexes (OEC) as demonstrated here by fast chlorophyllafluorescence and thermolu-minescence measurements. We found that inactivation of OEC due to prolonged darkness was attenuated in the Asc-deficientvtc2-4mutant. On the other hand, the severe photosynthetic phenotype of a psbo1 knockout mutant, lacking the major extrinsic OEC subunit PSBO1, was further aggravated upon a 24-h dark treatment.
Thepsbrmutant, devoid of the PSBR subunit of OEC, performed only slightly dis-turbed photosynthetic activity under normal growth conditions, whereas it showed a strongly diminished B thermoluminescence band upon dark treatment. We have also generated a doublepsbo1 vtc2mutant, and it showed a slightly milder photosynthetic phenotype than the single psbo1mutant. Our results, therefore, suggest that Asc leads to the inactivation of OEC in prolonged darkness by over-reducing the Mn-complex that is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits. Our study is an example that Asc may negatively affect certain cellular pro-cesses and thus its concentration and localization need to be highly controlled.
1 | I N T R O D U C T I O N
Ascorbate (Asc, also called vitamin C) is a multifunctional metabolite in eukaryotic cells. Because of economic interest in increasing the Asc
contents of plants, the metabolism and physiological roles of Asc are under intensive research, but surprisingly, there are still fundamental processes that are very poorly understood or perfectly unknown.
Asc is one of the most abundant metabolites in plants: it typically occurs in the order of 5–140μmol g−1 fresh weight (e.g., Conklin et al., 2000; Gest et al., 2013) with large variations among the specific cellular compartments (Zechmann, 2018). In plants, the best-known function of Asc is in reactive oxygen species (ROS) management (reviewed by Krieger-Liszkay et al., 2008, Foyer, 2018, Waszczak et al., 2018, Foyer et al., 2020). In non-enzymatic reactions, Asc
1 − −
Abbreviations:Asc, ascorbate; FM, maximum Chlafluorescence intensity; FV, variable fluorescence; FV/FM, indicator of photosynthetic efficiency, derived from the minimum and maximum Chlafluorescence parameters; Mn-cluster, manganese cluster of OEC; NPQ, non-photochemical quenching; OEC, oxygen-evolving complex; OJIP, fast Chlafluorescence kinetics; pmf, proton motive force; PSBO, 33 kDa extrinsic subunits of OEC; PSBR, 10 kDa extrinsic subunits of OEC; qP, photochemical quenching; RD, repeat distance of stacked thylakoid membranes; TEM, transmission electron microscopy; TFM, the time to reach FM;
superoxide dismutase, and Asc peroxidase reduces H2O2 to water (Asada, 2006; Curien et al., 2016; Foyer et al., 2020). By controlling the level of H2O2, Asc also participates in retrograde signaling (Exposito-Rodriguez et al., 2017).
Ascorbate also plays a role in photoprotection by being a reduc-tant for violaxanthin de-epoxidase (VDE), the enzyme responsible for the conversion of violaxanthin to zeaxanthin in the thylakoid mem-branes of vascular plants (Hallin et al., 2016). Zeaxanthin accumulation results in increased excitation energy dissipation in the form of non-photochemical quenching (NPQ) (Ballottari et al., 2014; Holt et al., 2005; Nicol et al., 2019; Sacharz et al., 2017) and it also acts as a scavenger of ROS (Dall'Osto et al., 2012). The role of Asc in NPQ is thoroughly demonstrated in vascular plants (Forti et al., 1999; Hager &
Holocher, 1994; Müller-Moulé et al., 2002; Saga et al., 2010); on the other hand, Asc is not required as a reductant for the activity of Chlo-rophycean-type VDE (Vidal-Meireles et al., 2020).
Ascorbate can reduce amino acid radicals (e.g. tyrosine, trypto-phan) effectively (Gebicki et al., 2010), which enables electron dona-tion to TyrZ
+ in photosystem II (PSII) units with inactive oxygen-evolving complexes (OEC), typically found in heat-stressed plants and green algae (Tóth et al., 2009). This PSII alternative electron donor function of Asc is physiologically relevant, as it slows down donor-side induced photoinhibition of PSII and enables recovery from heat stress (Tóth et al., 2011). Ascorbate also provides electrons to PSII and PSI in bundle sheath cells of NADP-malic enzyme-type C4 plants, which are deficient in OEC activity. The probable physiological role of this process is to support PSI cyclic electron transport, responsible for the generation of ATP in these cells (Ivanov et al., 2007).
On the other hand, we demonstrated previously that Asc is capa-ble of inactivating the Mn4CaO5(Mn)-cluster inChlamydomonas rein-hardtiiwhen accumulated to the mM range upon sulfur deprivation (Nagy et al., 2016, 2018). It has also been shown that in isolated PSII membranes lacking the extrinsic OEC proteins, bulky reductants, such as Asc, may directly reduce the Mn-cluster (Tamura et al., 1990).
Remarkably, the potentially negative effect of Asc on OEC activity has not been investigated in vascular plants in vivo, and in this respect, the protective roles of the extrinsic OEC proteins have not been clearly demonstrated either.
In the present study, the effect of Asc on OEC was investigated in plants subjected to prolonged darkness, which is a widely used model system to study senescence in plants (Buchanan-Wollaston et al., 2005). Leaf senescence is a highly controlled and active process requiring transcriptional and metabolic reprogramming to attain the organized breakdown and remobilization of valuable resources to seeds and other parts of the plant (Liebsch & Keech, 2016; Maillard et al., 2015). It is age-dependent though it is also influenced by a range of environmental factors, such as light, drought, and pathogen attack (Jing et al., 2009; Sade et al., 2018). In prolonged darkness,
levels, sugar content, redox homeostasis, and ROS-induced stress effects (Jing et al., 2008; Rosenwasser et al., 2011; Zentgraf &
Hemleben, 2008).
One of the early events occurring upon prolonged darkness is the decrease in photosynthetic activity, as assessed by the FV/FM and photochemical quenching (qP) parameters, which is at least partially due to the inactivation of OEC (e.g., Sobieszczuk-Nowicka et al., 2018). Later, photosynthetic complexes, including PSII, PSI, cytb6f, and ATP synthase, are degraded (Barros et al., 2017; Kunz et al., 2009; Yamazaki et al., 2000). Regarding the chloroplast ultra-structure, it was observed that thylakoids become swollen after a few days of darkness, and at a later stage, the number of plastoglobules increases, and finally chloroplasts are disintegrated (Niu & Guo, 2012;
Sobieszczuk-Nowicka et al., 2018). At later stages, degradation of chlorophyll (Chl) is observed. In the process of dark-induced senes-cence, autophagy (Avin-Wittenberg et al., 2018; Barros et al., 2017;
Elander et al., 2018; Fan et al., 2019) and vesicle formation play important roles (Wang & Blumwald, 2014).
Using wild type (WT, Col-0), Asc-deficient (vtc2-4),psbo1, double psbo1 vtc2, andpsbr1knockoutArabidopsis thalianamutants, we dem-onstrate here that Asc contributes to the early inactivation of OEC in prolonged darkness, probably by over-reducing the Mn-complex. We also show that the PSBO1 and PSBR subunits of OEC protect the oxygen-evolving machinery by hindering the access of Asc to the Mn-cluster.
2 | M A T E R I A L S A N D M E T H O D S 2.1 | Plant material and growth conditions
The Asc-deficient vtc2-4 mutant with Col-0 background (ABRC CS69540;At4g26850; see Lim et al., 2016) originates from the labora-tory of Dr. John F. Golz (University of Melbourne). Seeds ofpsbo1 (SALK 093396; At5g66570) and psbr (SALK 114496; At1g79040) mutant lines with Col-0 background were provided by Prof.
Dr. Cornelia Spetea (University of Gothenburg) and Dr. Yagut Allahverdiyeva (University of Turku).
AllArabidopsis thalianagenotypes were initially grown in a growth chamber under short-day conditions (8 h light, 24C; 16 h dark, 20C), at approximately 80μmol photons m−2s−1in the light period. When the plants were 7 weeks old, they were transferred to complete dark-ness in the middle of the day (for 24 or 96 h) or left in the growth light conditions. In separate experiments, 10-week old plants were also transferred to darkness.
2.2 | PCR reactions
CTCTGCACAA-30, and 50-GCCTTTTCAGAAATGGATAAATAGCCTTG CTTCC-30 were used as the gene-specific forward and reverse, and T-DNA-specific primers, respectively. For PSBO1, 50-AAAAATAAC AGCAAAGATGCCAAGTTCA-30, 50-GGAGACAAAAACAAACAAACA ACGGCTA-30, and 50-GCGTGGACCGCTTGCAACT-30 were used as the gene-specific forward and reverse, and T-DNA-specific primers respectively.
2.3 | Ascorbate and Chl content determination
For Asc content determination, approximately 20–40 mg of Ara-bidopsis leaves were frozen in liquid nitrogen, ground into a fine pow-der using MM400 laboratory mill (Retsch) at freezing temperature.
Ascorbate content of leaves was determined by HPLC, based on Kovács et al. (2016). The Chl content was measured according to Porra et al. (1989).
2.4 | Fast Chl a fluorescence (OJIP) measurements
Fluorescence measurements were carried out at room temperature with a Handy-PEA instrument (Hansatech Instruments). Leaf samples, dark-adapted for approximately 30 min or for 24–96 h, were illumi-nated with continuous red light (3500μmol photons m−2s−1, 650 nm peak wavelength; the spectral half-width 22 nm; the light emitted by the LEDs is cut off at 700 nm by a near-infrared short-pass filter). The light was provided by an array of three light-emitting diodes focused on a circle of 5 mm diameter on the sample surface. The first reliably measured point of the fluorescence transient was at 20μs, which was taken as F0. The length of the measurements was 5 s.
2.5 | Heat treatment
Whole leaves were submerged in a water bath in darkness. Inactiva-tion of OECs was achieved by a heat treatment at 38C or 40C for 15 min in a water bath, according to Tóth et al. (2011); the effect of heat treatment on OEC activity was tested with the aid of thermolu-minescence (TL) measurements (not shown). Special attention was paid at keeping the leaves in the dark during and after the heat treat-ment in order to avoid donor-side-induced photoinhibition (Tóth et al., 2011).
2.6 | Immunoblot analysis and protein content determination
–
Protease Inhibitor Cocktail [Roche]), and incubated in the dark at 4C for 30 min with vigorous shaking, before centrifugation at 20000gfor 12 min at 4C. The supernatant was collected into a new Eppendorf tube and the Chl(a + b) content of the extract was determined. An amount equivalent to 2 μg Chl (a+b) was then mixed with 6× Laemmli buffer (375 mM Tris/HCl [pH 6.8], 60% [v/v] glycerin, 12.6%
[w/v] sodium dodecyl sulfate, 600 mM dithiothreitol, 0.09% [w/v]
bromophenol blue) and incubated at 43C for 30 min before loading on the gel. Proteins separated by SDS-PAGE (Perfect Blue Twin Gel System, Peqlab) were transferred to a polyvinylidene difluoride mem-brane (Bio-Rad Immun-Blot LF-PVDF) using a wet transfer tank blot-ting system (Cleaver Scientific Ltd). Blots were blocked with 5% (w/v) milk (in 1% TBS-T) for 1 h at room temperature with agitation. Poly-clonal PSBO (recognizing both PSBO1 and PSBO2), PSBO1, PSBP, PSBQ, PSBR, PsbA, CP43, and CP47 antibodies (produced in rabbit) were purchased from Agrisera AB. The incubation was done overnight at 4C, in dilutions recommended by the manufacturer. Blots were incubated in secondary antibody (Bio-rad goat anti-rabbit IgG horse-radish peroxidase conjugate) diluted to 1:10000 in 1% TBS-T for 1 h at room temperature with agitation. Immunochemical detection was carried out with the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific), according to the instructions of the manufacturer. Exposure time varied between 10 and 30 s.
2.7 | Thermoluminescence measurements
Thermoluminescence measurements were carried out using a custom-made TL apparatus, similar to the one described in Ducruet and Vass (2009). Intact leaves fixed with a copper ring placed on a copper sample holder, connected to a cold finger immersed in liquid nitrogen.
A heater coil (SEI 10/50, Thermocoax) placed under the sample holder, ensured the desired temperature of the sample during the measurement. Dark-adapted samples were illuminated at 4C by two single-turnover saturating Xe flashes (Excelitas LS-1130-3 Flashpac with FX-1163 Flashtube with reflector) of 1.5μs duration at half-peak intensity (Sipka et al., 2019), and the sample was heated to 70C in darkness with a heating rate of 20C min−1. The emitted TL was mea-sured with an end-window photomultiplier (H10721-20, Hamamatsu) simultaneously with recording the temperature.
2.8 | Electron microscopy
Approximately 2 mm3leaf samples from all groups (n= 3/group) were cut and immersed into a modified Karnovsky fixative solution (pH 7.4), which contained 2% paraformaldehyde (Sigma-Aldrich) and 2.5% glutaraldehyde (Polysciences) in phosphate buffer. Samples were
in each concentration. Afterwards, leaf tissues were proceeded through propylene oxide (Molar Chemicals Kft), then embedded in an epoxy-based resin, Durcupan ACM (Sigma-Aldrich). After polymeriza-tion for 48 h at 56C, resin blocks were etched and 50 nm thick ultrathin sections were cut using an Ultracut UCT ultramicrotome (Leica). Sections were mounted on a single-hole, formvar-coated cop-per grid (Electron Microscopy Sciences), and contrast of the samples was enhanced by staining with 2% uranyl acetate in 50% ethanol (Molar Chemicals Kft, Electron Microscopy Sciences) and 2% lead cit-rate in distilled water (Electron Microscopy Sciences).
Ultrathin sections from the leaves were screened at 3000× mag-nification on a JEM-1400 Flash transmission electron microscope (JEOL) until 25–30 granum cross-sections were identified from each sample (n= 3/group). For quantitative measurements of the repeat distance, images of thylakoid membrane structures were recorded at
×50000 magnification using a 2 k×2 k Matataki (JEOL) scientific complementary metal-oxide-semiconductor camera. Finally, images were evaluated using the built-in measurement module of the microscope.
2.9 | Statistics
The presented data are based on at least three independent experi-ments. The exact number of biological replicates are indicated in the figure legends. When applicable, averages and standard errors (±SE) were calculated. The significance of the mean differences between the genotypes and the treatments were analyzed byANOVAwith Tukey post-hoc test atP< 0.05 level using OriginPro 9.5 software.
3 | R E S U L T S
3.1 | Photosystem II activity is heavily affected by prolonged darkness
Prolonged darkness, generating synchronized physiological changes, is a widely used method to study leaf senescence in plants (Buchanan-Wollaston et al., 2005), and it is also a common event in nature. In order to assess the effect of darkness on PSII, with particular atten-tion to OEC activity, 7-week-old WTArabidopsisplants were placed in the dark for up to 96 h under well-watered conditions. The fast Chl afluorescence (OJIP) kinetics of the WT plants showed a typical pat-tern: The O-J phase (approximately 0–2 ms), representing the reduc-tion of the acceptor side of PSII, was responsible for approximately 40% of variable fluorescence. The J-I phase, during which the cytb6f complex becomes reduced (2–30 ms), accounted for approximately 40% of the fluorescence rise. Finally, the I-P phase (30–300 ms),
rep-the fast Chlafluorescence transients altered: The FMvalue gradually decreased, the fluorescence kinetics became decelerated, and the K peak emerged with a maximum at around 300μs. During the dark treatment, the FV/FMparameter decreased in two steps. After 42 h of darkness, a value of approximately 0.6 was detected, whereas at the end of the second phase (60–96 h) the FV/FM value reached a low, approximately 0.3, value (Figure 1B). Since we aimed at studying the early events of PSII inactivation, we chose the 24 h time point for fur-ther experiments.
The K peak represents a single charge separation and its emer-gence is typical for photosynthetic samples with compromised OEC activity (Srivastava et al., 1997; Tóth et al., 2009). We treated entire leaves at 38 and 40C to obtain samples with partially and fully inhibited O2evolution, respectively (Tóth et al., 2009); by comparing the heat-treated and the dark-treated samples, clear similarities could be observed in the OJIP kinetics (cf. Figure 1A,C), suggesting that the Mn-cluster became inactivated upon prolonged darkness. We also note that when OEC is inactivated, Asc acts as an alternative electron donor to PSII and provides electron to TyrZ
+ with a halftime of approximately 22 ms in intactArabidopsisleaves; this phenomenon is reflected in the fluorescence peak at around 1 s (Tóth et al., 2009, Figure 1C).
Next, we investigated whether the effect of prolonged darkness on OEC is age-dependent. Figure 1D shows that older (10-week-old) plants were much less affected by the 24 h dark treatment than 7-week-old plants.
3.2 | Ascorbate is a key player in the inactivation of OEC upon prolonged darkness
In order to study the effects of Asc exerted on the Mn-cluster, an Asc-deficient mutant, vtc2-4, was included in the study. VTC2 encodes for GDP-L-galactose phosphorylase, catalyzing the first com-mitted step of Asc biosynthesis (Conklin et al., 2000; Dowdle et al., 2007; Müller-Moulé et al., 2003; Müller-Moulé et al., 2004).
Thevtc2-4T-DNA line contains approximately 80% less Asc than the WT, and its growth and photosynthetic characteristics are not affected under standard conditions (Lim et al., 2016).
Under control conditions, the Asc-deficient mutant showed neg-ligible alterations compared to the WT with respect to the OJIP tran-sient (Figure 2A), which is in agreement with our previous results on anotherVTC2mutant, calledvtc2-1(Tóth et al., 2009). However, the effect of prolonged darkness was milder in the vtc2-4mutant as compared to the WT: (1) In contrast to the WT, the K peak did not emerge in the Asc-deficient mutant (Figure 2B), (2) Variable fluores-cence (FV) decreased less (Figure 2C), and (3) the time to reach FM
(TF ) reflecting the time needed for the reduction of the
photosyn-The Mn-cluster of vascular plants is shielded from the bulk solu-tion by four lumen-exposed extrinsic proteins (PSBO, PSBP, PSBQ, and PSBR, with apparent molecular masses of 33, 23, 17, and 10 kDa, respectively); these extrinsic proteins are required for the stabilization and retention of Mn and Cl− cofactors and Ca2+ binding (Loll et al., 2005; Popelkova et al., 2011). InArabidopsis thaliana, PSBO is encoded by two genes (PSBO1, At5g66570; PSBO2, At3g50820), just as PSBP (PSBP1, At1g06680; PSBP2, At2g30790) and PSBQ (PSBQ1, At4g21280; PSBQ2, At4g05180); PSBR is encoded by a single gene, At1g79040 (reviewed by Ifuku et al., 2010, Bricker et al., 2012).
The PSBO protein is essential for photoautotrophic growth in
growth, low PSII activity, and are susceptible to photoinactivation, whereaspsbo2knockout plants show WT levels of PSII activity and growth rate (Allahverdiyeva et al., 2009; Lundin et al., 2007). The PSBP protein is essential for photoautotrophy, whereas the PSBQ protein is dispensable in higher plants (Allahverdiyeva et al., 2013; Yi et al., 2007; Yi et al., 2009). The absence of PsbR leads to decreased oxygen evolution rate and slow QA− re-oxidation (Allahverdiyeva et al., 2007; Allahverdiyeva et al., 2013; Liu et al., 2009b; Suorsa et al., 2006), though it is to be noted that the exact location and func-tion of PSBR is yet unknown (van Bezouwen et al., 2017).
We decided to includepsbo1andpsbrknockout mutants in our F I G U R E 1 The effects of prolonged darkness and heat stress on the fast Chlafluorescence (OJIP) transients ofArabidopsis thaliana. (A) OJIP transients ofArabidopsiskept for 0–96 h in complete darkness. (B) FV/FMvalues ofArabidopsiskept in the dark for 0–96 h. (C) The effects of 38C and 40C heat treatments (intact leaves treated for 15 min in water bath) on OJIP transients. (D) The effects of 24-h darkness on OJIP transients of 7 (7-W) and 10 weeks old (10-W)Arabidopsisplants. The fluorescence transients are the average of 6–12 measurements taken on individual plants
complementation and/or by independent lines and they all are in Col-0 background (Allahverdiyeva et al., 2007; Lundin et al., 2007;
Suorsa et al., 2006).
Thepsbo1 mutant showed remarkable differences in the OJIP transient relative to the WT (Figure 2A): The F0value was very high and the FM value was lower than in the WT; the O-J phase was altered with a peak reminiscent of the K peak, with a maximum at around 300μs; the FMvalue was reached at around 1.4 s instead of 180–200 ms. These alterations are typical of plant leaves with par-tially inactivated OECs (Figure 1C, Tóth et al., 2009, Tóth et al., 2011).
The absence of PSBR led to increased J step, indicating that QA−reoxidation was slowed down (Figure 2A, Tóth et al., 2007), in accordance with the results of Liu et al. (2009b).
Upon the 24 h dark treatment, the K peak in thepsbo1mutant became well defined (Figure 2B), the FVvalue decreased, and the TFM
parameter increased slightly further (Figure 2C,D), indicating that OEC
In order to confirm the effect of prolonged darkness on OEC activity, thermoluminescence (TL) measurements were carried out on entire leaves. Thermoluminescence is light emission from a pre-illuminated photosynthetic sample during temperature increase pro-viding information about the redox components of the photosynthetic apparatus (for a review, see Ducruet, 2013). In the case of leaves, freezing before TL measurements is not recommended due to cellular
In order to confirm the effect of prolonged darkness on OEC activity, thermoluminescence (TL) measurements were carried out on entire leaves. Thermoluminescence is light emission from a pre-illuminated photosynthetic sample during temperature increase pro-viding information about the redox components of the photosynthetic apparatus (for a review, see Ducruet, 2013). In the case of leaves, freezing before TL measurements is not recommended due to cellular