Tree Physiology 36, 325–334 doi:10.1093/treephys/tpv140
Research paper
Characterization of light-dependent regulation of state transitions in gymnosperms Amy S. Verhoeven1,2, Albert Kertho1 and Mary Nguyen1 Department (OWS352), University of St Thomas, 2115 Summit Ave., St Paul, MN 55105, USA; 2Corresponding author ([email protected])
Received September 22, 2015; accepted December 3, 2015; published online January 22, 2016; handling Editor Jörg-Peter Schnitzler
The goal of this study was to characterize the light-dependent regulation of state transitions in gymnosperms. Two species of conifer were examined: eastern white pine (Pinus strobus L.) and white spruce [Picea glauca (Moench) Voss], as well as the angiosperm pumpkin (Cucurbita pepo L. subsp. pepo). Both diurnal time courses in the field and manipulated light experiments in growth chambers were conducted. Results from chlorophyll fluorescence analysis indicated that pumpkin was able to use a larger fraction of absorbed light to drive photochemistry and retain a lower reduction state at a given light intensity relative to the conifers. Results from western blots using anti-phosphothreonine demonstrate that in field conditions, conifers maintained higher light-harvesting complex II (LHCII) phosphorylation than pumpkin; however, this was likely due to a more variable light environment. Manipulated light experiments showed that general patterns of light-dependent LHCII phosphorylation were similar in conifers and pumpkin, with low levels of LHCII phosphorylation occurring in darkness and maximal levels occurring in low light conditions. However, high light-dependent dephosphorylation of LHCIII appears to be regulated differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin. Keywords: conifer, light regulation, thylakoid protein phosphorylation.
Introduction Photosynthetic organisms rely on light absorption to drive the process of photosynthesis, but are faced with dramatic fluctuations in light intensity on both a daily and seasonal scale, as well as less predictable fluctuations in light caused by factors such as wind and changes in cloud cover. In order to cope with fluctuating light environments, plants use a variety of regulatory mechanisms that allow for rapid acclimation of the photosynthetic apparatus to changing light, including thermal energy dissipation, the photosystem II (PSII) repair cycle and state transitions (Dietzel et al. 2008, Demmig-Adams et al. 2012, Tikkanen and Aro 2012, Derks et al. 2015). In high light conditions, plants engage mechanisms of thermal energy dissipation in order to safely dissipate excessive light (Demmig-Adams et al. 2012). Additionally, the highly oxidizing conditions of the PSII reaction
center necessitate rapid turnover of the D1 protein in high light conditions, and this process involves reversible phosphorylation of PSII core proteins (Tikkanen et al. 2008, Tikkanen and Aro 2012). In lower light conditions, as well as in conditions of fluctuating light, the processes of light harvesting and electron transport are optimized, at least in part, by the process of state transitions (Tikkanen and Aro 2012). State transitions involve the light-dependent reversible phosphorylation of the major light-harvesting antenna (light-harvesting complex II, LHCII), resulting in a redistribution of LHCII between PSII and photosystem I (PSI). The phosphorylated form of the mobile LCHII increases its association with PSI and this conformation has classically been described as ‘state 2’, while the dephosphorylated form of LHCII associates more strongly with PSII and is known as ‘state 1’ (Bennett 1991, Allen 1992,
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1Biology
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angiosperm species, although in moss and liverwort, there was no evidence of D1 phosphorylation (Pursiheimo et al. 1998). In a study examining conifers, relatively high phosphorylation of light-harvesting proteins was observed in very high light conditions (midday in full sunlight), suggesting that conifers may have a different light-dependent response compared with angiosperms (Verhoeven et al. 2009). The goal of this study was to investigate whether state transitions in gymnosperms have a unique light-dependent response by monitoring thylakoid protein phosphorylation in conifers, compared with pumpkin, collected under varying light environments in the field, as well as upon exposure to varying light environments in controlled laboratory conditions.
Materials and methods Plant material and sampling Sampling was performed on mature trees of white spruce [Picea glauca (Moench) Voss] and eastern white pine (Pinus strobus L.) growing in full sunlight on campus at the University of St Thomas, Minnesota (44°59′40″N, 93°05′35″W). Additionally, pumpkin plants (Cucurbita pepo L. subsp. pepo) were grown in pots and maintained outdoors in full sunlight. Diurnal time courses were conducted using a single tree (for the conifers), and pooling samples from three pumpkin plants. For the conifers, sampling was conducted on branches that had significant full sun exposure over the course of the day. On 13 July 2010, sampling was conducted predawn at 4:45 h, and again at 6:00, 7:00, 9:00, 11:00 and 13:00 h. On this date, significant cloud cover occurred late in the morning and so sampling was discontinued. Sampling was continued on 15 July 2010, which was a clear day. Samples were collected at 13:20, 16:00 and 19:00 h. At each time point, leaf temperature, light exposure and chlorophyll fluorescence were monitored. Additionally, needles/ leaves were cut and transferred to liquid nitrogen, taking care to maintain light exposure throughout that process. Needles were stored in liquid nitrogen until later isolation of thylakoids.
Manipulated light experiments For manipulated light experiments, leaf discs or needles were floated on room temperature water at controlled light intensities. Needles were collected from the trees used for diurnal measurements in the morning prior to exposure to direct sunlight, again using branches with full sun exposure for a significant portion of the day. Pumpkin leaves were collected either from plants grown in pots outdoors or from plants grown in the greenhouse in full sun conditions. Manipulated light experiments were conducted initially in June 2011, and then repeated in August 2011 and in July 2012. For dark acclimation, needles/leaves were cut and placed in plastic bags with moist paper towels and maintained in full darkness for 1 h. For light exposures, leaf discs (pumpkin) or needles were floated on room temperature water in Petri
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ietzel et al. 2008, K argul and Barber 2008, Murata 2009). D State transitions are thought to balance the excitation pressure between the photosystems, thus maintaining optimal photosynthetic efficiency in varying light environments (Dietzel et al. 2008, K argul and Barber 2008, Tikkanen and Aro 2012, Goldschmidt-Clermont and Bassi 2015). In addition to LHCII, three of the PSII core proteins are reversibly phosphorylated in a light-dependent manner, although the pattern of their light regulation is opposite to that of LHCII, with LHCII being maximally phosphorylated at low light intensities and the core proteins showing maximal phosphorylation at high light (Rintamäki et al. 1997, Tikkanen et al. 2008). Phosphorylation of PSII core proteins is thought to function in signaling protein damage and facilitating the PSII repair cycle (Tikkanen et al. 2008, Tikkanen and A ro 2012). The light-dependent phosphorylation of thylakoid proteins is regulated both by the redox state of the electron transport chain and that of the chloroplast stroma (Rintamäki et al. 2000, Aro and Ohad 2003, Puthiyaveetil et al. 2012, Rochaix et al. 2012). Protein phosphorylation is controlled by the STN7 and STN8 kinases, and dephosphorylation is regulated by the TAP38/ PPH1 and PBCP phosphatases (Bellafiore et al. 2005, Vainonen et al. 2005, Shapiguzov et al. 2010, Samol et al. 2012). In particular, LHCII is phosphorylated by a STN7 kinase, which is activated by the redox state of the plastoquinone pool in relatively low light (Bellafiore et al. 2005, Rochaix et al. 2012). There is also evidence that the kinase is deactivated by increases in thiol reducing activity of the stroma, leading to decreases in LHCII phosphorylation in high light (Rintamäki et al. 2000, Aro and Ohad 2003, Pursiheimo et al. 2003). Light-harvesting complex II is dephosphorylated by the TAP38/PPH1 phosphatase (Shapiguzov et al. 2010), although whether this enzyme is redox dependent or is constitutively active is currently unknown (Rochaix et al. 2012). A recent study by Mekala et al. (2015) demonstrates that the TAP38/PPH1 phosphatase is responsible for dephosphorylation of LHCII in high light; thus, it is possibly also redox regulated. The PSII core proteins are phosphorylated by the STN8 kinase in high light conditions (Vainonen et al. 2005) and dephosphorylated by the PBCP phosphatases (Samol et al. 2012). Several studies have examined the irradiance-dependent nature of state transitions and have found that LHCII is not phosphorylated in darkness, becomes phosphorylated in low light and is subsequently dephosphorylated in high light conditions (Rintamäki et al. 1997, 2000). In contrast, the PSII core proteins show increasing phosphorylation at high light intensities, although CP43 has been reported to remain phosphorylated in darkness in some cases ( Rintamäki et al. 1997, 2000, Pursiheimo et al. 2003). These light-dependent patterns of thylakoid protein phosphorylation have been shown to be conserved across divergent evolutionary species, with similar responses reported in a moss, liverwort and fern to that of
State transitions in gymnosperms 327
Chlorophyll fluorescence Chlorophyll fluorescence was performed at the time of sample collection using a field portable fluorescence monitoring system (FMS2, Hansatech, Kings Lynn, UK). The modulating beam was set at default values (setting of 2, gain 50) and the saturating beam was >5000 μmol m−2 s−1 (0.7 s duration). For the manipulated light experiments, needle and leaf temperature were maintained at 22 °C. Values for Fv/Fm were determined either predawn or after 1 h of dark acclimation and ΦPSII [(Fm′ − F )/Fm′ ] was determined for samples exposed to light (Genty et al. 1989). The parameter 1 − qL was used to estimate the fraction of closed QA centers [qL = (Fm′ − Fs )/(Fm′ − Fo′ ) × (Fo′ /Fs )] according to Kramer et al. (2004).
Isolation of thylakoids To extract thylakoids, tissue was ground in liquid nitrogen with a mortar and pestle, and then homogenized with a polytron (Brinkman, Model 10/35, Fisher Scientific, Pittsburgh, PA, USA) in grinding buffer [50 mM Tricine, pH 7.6, 0.4 M sorbitol, 10 mM NaF, 10 mM MgCl2, 20% polyethylene glycol (average molecular weight 10,000, Sigma-Aldrich, St. Louis, MO, USA), 1 mM benzamidine-HCl and 5 mM 6-amino-n-hexanoic acid]. Samples were filtered through four layers of cheesecloth, centrifuged at 10,000g for 10 min at 4 °C and the pellet was resuspended in wash buffer (50 mM Tricine, pH 7.6, 10 mM NaCl, 10 mM NaF and 5 mM MgCl2) and re-centrifuged. Thylakoids were resuspended in 50 mM Tricine, pH 7.6, containing 50% glycerol, 5 mM MgCl2 and 10 mM NaCl, and stored at −80 °C. Protein concentration of thylakoids was determined by the Bio-Rad Protein assay, and chlorophyll concentration was determined in buffered acetone as described by Porra et al. (1989).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting Samples of isolated thylakoids were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. To d etermine
phosphorylation status, 10 µg of thylakoid protein was solubilized in the presence of 6 M urea and run on 15% gels containing 6 M urea (Rintamäki et al. 1997, Bergo et al. 2002). To ensure equal loading of protein, gels were stained with Coomassie Brilliant Blue and visually inspected for equivalent loading. For immunodetection, proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Fisher Scientific, Pittsburgh, PA, USA) and probed with rabbit anti-phosphothreonine (Cell Signaling Technology, Danvers, MA, USA) using the recommended dilutions (1−2 µg ml−1 antibody). Secondary antibody was goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich). Antibodies were detected by chemiluminescence (RPN2109, GE Healthcare, Pittsburgh, PA, USA) followed by exposure of the membrane to film (Kodak BioMax light film, Sigma Aldrich, St. Louis, MO, USA). Band intensity was quantified using ImageJ (NIH, Bethesda, MD, USA). To identify the phosphorylated bands, control thylakoids were prepared from pumpkin leaves exposed to low and high light following the protocol described by Rintamäki et al. (1997) and bands were compared with the published data. Additionally, direct comparisons were performed of LHC (both Lhcb1 and Lhcb4), D1, D2 and CP43 using purchased antibodies (AgriSera, Vannas, Sweden). It is noteworthy that the results of our western blots varied considerably depending on the particular lot number of antiphosphothreonine antibody that we received from Cell Signaling Technology. Most of the observed variation was in the relative phosphorylation of CP43 and the D1 protein. With one lot of the antibody, CP43 appears to be phosphorylated at most light intensities, while the other showed very low levels of CP43 phosphorylation. Additionally, the sensitivity of the antibody to the phosphorylated form of the D1 protein varied dramatically between lots. We have noted this considerable variation between purchased aliquots of anti-phosphothreonine in the recognition of CP43 and D1, although the results for LHCII phosphorylation have been consistent. Therefore, we have chosen not to attempt to draw any conclusions about light-dependent core protein phosphorylation from these data and have instead focused on changes in LHCII phosphorylation.
NADP-malate dehydrogenase assay Chloroplast redox status was estimated by measuring the activation state of the chloroplast NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.82) according to Scheibe and Stitt (1988). Leaf tissue (0.1–0.4 g) was ground in liquid nitrogen in ∼1 ml extraction buffer (50 mM sodium acetate, 0.1% w/v Triton X-100, 0.1% bovine serum albumin, 4% poly-vinyl-pyrollidone-40, 4 mM dithiothreitol (DTT), pH 6.0) that had been bubbled with N2 gas. Extracted samples were centrifuged for 10 min at ~20,000g at 4 °C, and the supernatant was collected and bubbled with N2 gas. Enzyme activity was assessed in the presence and absence of DTT according to Scheibe and Stitt (1988),
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dishes at a variety of light intensities and maintained for 1 h. The lowest light intensity was 30 µmol m−2 s−1, which used the fluorescent lights in the laboratory. Light exposures of 120, 350 and 700 µmol m−2 s−1 were maintained using a growth chamber (Model MB-60B, Percival Scientific Instruments, Perry, IA, USA) with varying distances from the light source. The high light exposure (1300 µmol m−2 s−1) was set up using halogen lamps placed above a Pyrex dish filled with water (as a heat sink) with leaf discs/needles maintained in dishes below this. Care was taken to maintain temperature and evenness of light exposure for all samples. After 1 h of light exposure, leaf discs and needles were sampled for chlorophyll fluorescence, and then samples were immediately transferred to liquid nitrogen, taking care to maintain the light environment during the transfer. Samples were stored in liquid nitrogen.
328 Verhoeven et al. and the NADP-MDH activation state was calculated from the ratio of these values. For the spruce samples, the leaf extracts were run over 5 ml Sephadex G25 (Sigma-Aldrich) c olumns that had previously been equilibrated with the reaction buffer (100 mM Tris–Cl, 1 mM ethylenediaminetetraacetic acid, 2.5 mM DTT, pH 8.0). In order to ensure that the column treatment did not alter the NADP-MDH activation state, spinach samples were tested for activity both before and after the column treatment. These tests provided similar results.
Statistical analysis
Results Diurnal time courses of chlorophyll fluorescence and phosphorylation status of photosynthetic proteins Diurnal time courses were conducted in mid-July on white pine, white spruce and pumpkin in order to establish the phosphorylation status of the conifer photosynthetic proteins in a variety of natural light environments in comparison with similar results for pumpkin, which has previously been well characterized (Bergo et al. 2002). Our goal was to conduct a diurnal time course on a clear sunny day in order to monitor the highest light exposures. The first date of sampling (13 July 2010) began sunny but clouds moved in by midday creating a variable light environment. We continued sampling 2 days later when conditions were sunny all afternoon (Figure 1a). Values for Fv/Fm measured predawn (5:00 h, Figure 1b) were quite high for all species, with values above 0.8. Reductions in ΦPSII mirrored increases in light exposure in a predictable manner for all species (Figure 1b). Figure 2 depicts example western blots probed with antiphosphothreonine antibody for all species and time points from the diurnal time courses. In pumpkin, four bands are visible, corresponding to the four photosynthetic proteins that are known to show changes in phosphorylation with light environment (LHCII, D1, D2 and CP43). In both conifer species, only three bands are visible, with the phosphorylated form of the D1 protein not showing up. The absence of the phosphorylated form of the D1 protein in conifers is likely a problem with antibody recognition, as opposed to a lack of phosphorylated D1, as mentioned in the Materials and methods section. Because the light environment varied for the individual species at the different time points, the data for relative phosphorylation of LHCII, D2 and CP43 are compiled in Figure 3 as a
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Figure 1. Light intensity (photosynthetic photon flux density, PPFD) at leaf level at the time of sampling (a) and ΦPSII or Fv/Fm (b) obtained over the course of a portion of 2 days for pumpkin (open circles), pine (filled squares) and spruce (open triangles). Time points for samples taken predawn until 11:00 h were collected in 13 July 2010, and the time points from 13:30 h until the end of the day were collected on 15 July 2010. Data represent averages ± SD (n = 3).
Figure 2. Example blots showing the phosphorylation status of thylakoid proteins of PSII (CP43, D2 and D1) as well as phospho-LHCII in samples collected from pumpkin, pine and spruce over the course of the day.
function of categories of light intensity. For pumpkin, the data show light-dependent changes in these proteins that are consistent with previously published studies (Pursiheimo et al. 2003). The LCHII proteins show low phosphorylation in the dark, high
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To test for significant differences between species in the manipulated light experiments, for fluorescence parameters, the relative phosphorylation of LHCII and the NADP-MDH activation state, repeated-measures analyses of variance were performed (JMP Statistical Software, SAS Institute, Cary, NC, USA). To test for a linear relationship between the relative phosphorylation of LHCII and 1 − qL, linear regressions were conducted using Prism (GraphPad Software, Inc., La Jolla, CA USA).
State transitions in gymnosperms 329
Manipulated light experiments
Figure 3. The relative phosphorylation of individual thylakoid proteins from samples collected under different light intensities for pumpkin (white bars), pine (gray bars) and spruce (black bars). Samples were grouped into categories of light intensity [dark, low light, medium light and high light (0, 20–100, 150–700 and >1000 µmol photons m−2 s−1)] in order to compare general light intensity effects. For these data, the time point showing the highest phosphorylation of LHC was normalized to 1, and all other time periods were expressed as a fraction of that value. The number of time points pooled and the respective light intensities during collection were as follows: 20–100 µmol photons m−2 s−1 range: pumpkin (3) 27, 63, 85, pine (2) 20, 60, spruce (3) 30, 50, 75; 150–700 µmol photons m−2 s−1 range: pumpkin (1) 660, pine (2) 250, 450, spruce (2) 175, 400; >1000 µmol photons m−2 s−1 range: pumpkin (3) 1100, 1380, 1465, pine (3) 1000, 1150, 1300, spruce (2) 1225, 1812. Where two or more samples were pooled, data represent averages ± SD.
phosphorylation in low light and decreasing phosphorylation in high light conditions (Figure 3a). The core PSII proteins D1 (not shown, but see Figure 2) and D2 (Figure 3b) show little phosphorylation in the dark, with phosphorylation increasing in high light. CP43 shows relatively high levels of phosphorylation regardless of the light environment, although it increases at higher light exposures (Figure 3c).
Manipulated light experiments were conducted in which leaves and needles were collected from the field and brought into the laboratory where they were maintained over a range of light intensities for 1 h, prior to sampling for chlorophyll fluorescence and thylakoid protein phosphorylation status. Values for Fv/Fm, measured after 1-h dark acclimation, were ∼0.8 for all species (Figure 4a), which was consistent with values measured in the field (Figure 1b). ΦPSII values decreased as light increased, but at a given light intensity, these values remained significantly higher in pumpkin relative to pine and spruce, suggesting that pumpkin uses a larger fraction of absorbed light to drive photosynthesis relative to the conifer species. Similarly, values for 1 − qL (an estimate of the PSII reduction status) were significantly higher for pine and spruce than for pumpkin at each light intensity, suggesting that the conifers are experiencing relatively higher excitation pressure for a given light intensity compared with pumpkin (Figure 4b). The pattern of relative phosphorylation of LHCII was similar for the three species, showing little to no phosphorylation in dark acclimated samples and maximal phosphorylation in low light, with phosphorylation decreasing as light intensity increased. Values for pumpkin and pine were very similar to each other; however, spruce maintained significantly higher phosphorylation of LHCII in high light conditions relative to the other two species.
Estimating chloroplast redox status using NADP-MDH assay The decreased phosphorylation of LHCII in high light conditions is thought to be caused by increases in the chloroplast stromal redox status that occur during excess light conditions and that
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For pine and spruce, CP43 and D2 were relatively highly phosphorylated regardless of light environment (Figure 3b and c). The relative phosphorylation of LHCII was very low in the dark and increased to high levels in low light (Figure 3a). In the higher light conditions, there were decreases in LHCII p hosphorylation, although the relative phosphorylation of LHCII remained higher in the conifers than in pumpkin. The relatively high levels of variation in the phosphorylation status of the photosynthetic proteins in our field-collected data, particularly for the two conifer species, led to questions about the consistency of the light environment during sampling. Pumpkin leaves are large and present an even surface area to a particular light environment and were therefore easy to sample in a consistent manner. The spruce needles are arranged around the branch in a variety of angles; thus, individual needles were exposed to higher variation in incident light. Similarly, the white pine needles are arranged in clusters that dangle from the plant, thus causing some self-shading within each cluster. Because of this variation, we decided to conduct a manipulated light experiment in the laboratory in order to better compare actual light environments between the species.
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Therefore, in order to estimate the redox status of the chloroplast stroma, we repeated the manipulated light exposures and measured the NADP-MDH a ctivation state at each light intensity (Figure 5). Both pumpkin and pine showed marked increases in the activation state of NADP-MDH as a function of light intensity, with a rapid increase in the activation state from 0 to 120 μmol photons m−2 s−1 that saturated at higher light intensities at an activation state of ∼75%. In contrast, spruce showed only small increases in NADP-MDH activity as light levels increased above zero, with these low values being maintained at all light intensities.
Discussion State transitions in field conditions
Figure 4. The chlorophyll fluorescence parameters ΦPSII or Fv/Fm (a), 1 − qL (b) and the relative phosphorylation of LHCII (c) for samples from pumpkin (open circles), pine (filled squares) and spruce (open triangles) as a function of light intensity. Data are averages ± SD of three experiments.
inhibit the LHCII kinase (Rintamäki et al. 2000, Aro and Ohad 2003). Given the relatively high phosphorylation of spruce LHCII in high light conditions, we were interested in assessing the chloroplast redox status, as a function of light intensity, for each species. NADP-MDH (E.C. 1.1.1.82) is a chloroplast-specific enzyme that is regulated by light through a thiol–disulfide interchange, which is triggered by photosynthetic electron transfer (Miginiac-Maslow et al. 1997). The activation state of this enzyme has been demonstrated to reflect the redox status in the chloroplast stroma (Scheibe and Stitt 1988, Foyer et al. 1992).
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Our data suggest that in field conditions, under high light environments, the sampled conifers maintained higher LHCII phosphorylation relative to pumpkin (Figures 2 and 3), although this may reflect differences in light exposure rather than differences in the regulation of state transitions. The conifers experienced more variable and dynamic light environments compared with pumpkin, due to the arrangement of needles on the branches. It is likely that for most plants growing in field conditions, the changes in leaf angle due to wind, as well as self-shading, result in more variable and dynamic light conditions than what we documented for the pumpkin leaves, as we took care to sample leaves experiencing uniform light environments. Most of the studies examining state transitions in response to light exposure are based on manipulated light treatments in laboratory settings (Rintamäki et al. 1997, Pursiheimo et al. 2003), and our results from pumpkin are quite consistent with these studies. A study examining Arabidopsis mutants lacking the LHCII kinase (stn7 mutants) found severe physiological effects under strongly fluctuating light conditions
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Figure 5. The activation state of the enzyme NADP-MDH, which is an indirect measure of the redox status of the chloroplast stroma, as a function of light intensity. Values closer to 1 indicate a higher stromal reduction state. Data are averages ± SD of three experiments for pumpkin (open circles), pine (filled squares) and spruce (open triangles).
State transitions in gymnosperms 331 (Tikkanen et al. 2010), suggesting that state transitions play a key role in balancing excitation energy distribution between photosystems particularly in dynamic light environments. The relatively high levels of LHCII phosphorylation we observed in the conifers in high light conditions in the field likely reflect this important role for state transitions in fluctuating light conditions.
Regulation of state transitions in conifers
Relationships between PSII reduction state, stromal redox status and LHCII phosphorylation As the dephosphorylation of LHCII in high light differed in spruce compared with pine and pumpkin, we decided to look more
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The manipulated light treatments allowed for more controlled light environments, which are not as reflective of natural conditions, but which provide some insight in comparing species responses. The fluorescence data reflect innate differences in maximal photosynthetic capacity of pumpkin compared with the conifers, showing that pumpkin maintains relatively high photosynthetic efficiencies and a low reduction state at higher light intensities when compared with the conifers (Figure 4a and b). This is expected, as pumpkin is an annual crop plant with a high photosynthetic capacity allowing for rapid growth, while the conifers are perennial trees, which maintain slower growth rates in general. The level of LHCII phosphorylation was very low in darkness in all species, which is consistent with previous studies. Upon transition from darkness to low light, all species showed very similar responses with dramatic increases in LHCII phosphorylation (Figure 4c). The maximal LHCII phosphorylation occurred at the lowest light intensity tested (30 μmol photons m−2 s−1) in all three species. These data are consistent with previous studies which have indicated that the LHCII kinase (STN7 in Arabidopsis) is activated by increases in the reduction state of the plastoquinone pool and its interaction with the cytochrome b6f complex (Rochaix et al. 2012), and that maximal phosphorylation occurs at light intensities well below the growth light intensity (Rintamäki et al. 1997). The data suggest that LHCII kinase activation occurs in conifers in a similar manner to the angiosperms that have been studied more extensively. Further increases in light intensity resulted in decreases in LHCII phosphorylation in all species, although for spruce, the relative phosphorylation of LHCII was maintained at significantly higher levels than for the pumpkin and pine (Figure 4c). The dephosphorylation of LHCII that occurs at higher light intensities is thought to be regulated by the thiol redox status of the chloroplast stroma, which has been suggested to inactivate the LHCII kinase by reducing disulfide bonds in the enzyme (Rintamäki et al. 2000, Aro and O had 2003, Rochaix et al. 2012), although it is also possible that an increase in the activity of the TAP38/ PPH1 phosphatase enzyme is required (Mekala et al. 2015). In order to determine whether the maintenance of relatively higher LHCII phosphorylation in spruce was due to differences in the chloroplast redox status, the activation state of the enzyme NADP-MDH was determined. Interestingly, changes in the NADP-MDH activation state, as a function of light intensity, were very similar for pumpkin and pine, with light-dependent increases consistent with the hypothesis that increases in stromal redox
status of the chloroplast cause decreases in LHCII phosphorylation in high light conditions (Rintamäki et al. 2000). The results for the NADP-MDH activation of spruce were quite different, and showed only a small increase in the activation state of this enzyme with increasing light conditions up to ∼20% activation at 300 μmol photons m−2 s−1, with no further increase upon increasing light (Figure 5). These results may suggest that spruce maintains a low chloroplast redox status in high light conditions, and that as a result, the LCHII kinase is not inhibited, leading to the maintenance of higher levels of phosphorylated LHCII in high light conditions. The large difference between spruce compared with pine and pumpkin in the light-dependent response of the NADP-MDH activation state is surprising, and it is possible that there is an alternative explanation for the observed differences. The relationship between the activation state of the NADP-MDH and the redox status of the chloroplast stroma was demonstrated in pea and spinach (Scheibe and S titt 1988, Foyer et al. 1992), but it is possible that the enzyme NADP-MDH is regulated differently in spruce and that the activation state of the enzyme does not reflect the chloroplast stromal redox status in spruce. Another possibility is that the additional purification step required to clean up the spruce leaf extracts prior to enzyme analysis may have made the spruce data less reliable than that of pine and pumpkin. In order to assess NADP-MDH activity in spruce needle extracts, it was necessary to first run the samples over Sephadex columns, presumably to remove secondary metabolites that interfered with the assay. This step was not required for either pumpkin or pine. Such techniques have been used in spruce to measure enzyme activity in previous studies (e.g., Weimar and Rothe 1986). As a control, to ensure that the technique did not disrupt the enzyme activation state, we assayed pumpkin samples both before and after extracts were run over the columns, and were able to get identical results with these controls. Therefore, we feel that our data accurately reflect the NADP-MDH activation state for spruce, but it is important to note the difference in methodology. A relatively higher fraction of phosphorylated LHCII and a relatively lower reduction status of the chloroplast stroma are both consistent with increased levels of cyclic electron transport at higher light intensity in spruce needles. Because of the difficulty of measuring rates of cyclic electron transport, little is known about the prevalence of this pathway under physiologically relevant conditions (Johnson 2011). A study examining Scots pine found evidence supporting increased use of the cyclic pathway under overwintering conditions (Ivanov et al. 2001). It would be interesting to investigate the relative flux of electrons through the cyclic pathway in white spruce compared with other species.
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Figure 6. 1 − qL (a) or NADPH-MDH activation state (b) as a function of relative LHCII phosphorylation levels in pumpkin (open circles), pine (filled squares) and spruce (open triangles). The lines depict estimates of best fit for a linear regression in (a) and the curves depict estimates of best fit for a second-order polynomial in (b) using Graphpad Prism.
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activation state increased from very low values up to 0.5 or so, there were corresponding decreases in LHCII phosphorylation from maximal levels down to 60–70% of maximal LCHII phosphorylation. Subsequent decreases in LHCII phosphorylation were accompanied by much smaller increases in NADP-MDH activation. There was no relationship between these parameters in spruce. Based on previous studies, it is not surprising that there is no correlation between the PSII reduction status and the LHCII phosphorylation state of pumpkin, as it has been demonstrated that the dephosphorylation of LHCII in high light is regulated by increased stromal redox status, which is consistent with our results. For pine, and more so for spruce, the correlation between PSII reduction status and LHCII phosphorylation may indicate that some regulation of LHCII dephosphorylation occurs via redox sensing of electron transport. For pine, but not for spruce, the data also support a role for the stromal redox status in regulating LHCII dephosphorylation in high light conditions. The higher PSII excitation pressure experienced by conifers in high light conditions likely involves different strategies for coping with excess energy relative to annual plants, where a larger fraction of light can be used to drive photosynthesis. Both proton gradient-dependent thermal energy dissipation and redox- regulated phosphorylation of thylakoid proteins contribute to reducing the excitation energy transfer to PSII. A recent study by Demmig-Adams et al. (2015) suggested that in conifers, thylakoid unstacking is correlated with higher levels of non-photochemical quenching (NPQ), which has apparently not been observed in herbaceous annual plants. Thylakoid unstacking in high light might be expected to be associated with higher LHCII phosphorylation (observed here), given the biophysical constraints of the phosphorylated form of LHCII fitting within the granal regions of thylakoids. Although the physiological role of high light-induced dephosphorylation of LHCII is not understood, Mekala et al. (2015) speculate that the dephosphorylation of LHCII in high light may function to protect against the excitation imbalance and energy losses in PSII upon subsequent decreases in light intensity. They suggest that upon shifting from high to low light, if PSII had a smaller antenna, it would not have enough excitation energy to satisfy the electron needs of PSI (with a large antenna), which would lead to energy losses in NPQ processes within PSI. It is possible that in conifers, and other plants in sink-limited conditions, this could be a useful strategy to dissipate excess energy. This would be consistent with a recent suggestion by Demmig-Adams et al. (2015) that spillover quenching in PSI may be an important component of NPQ in evergreen species. The process of dephosphorylation of LHCII is controlled by both a kinase and a phosphatase, both of which can be activated and deactivated, allowing for complex regulation of LHCII phosphorylation (Rochaix et al. 2012, Mekala et al. 2015). It is not surprising that there are species variations in this process of optimization, as the myriad possible regulatory pathways for electron flow and
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closely at the relationship between LHCII phosphorylation and an estimate of the PSII reduction state (1 − qL, Figure 6a) as well as an estimate of the stromal redox status (NADP-MDH activation, Figure 6b). Dark values were excluded for this analysis. In general, both conifers retained higher phosphorylation of LHCII at a given perceived PSII excitation pressure (estimated as 1 − qL, Figure 6a). For example, when the value for 1 − qL is ∼0.3, pumpkin LHCII shows very little phosphorylation while, for both conifers, LHCII is highly phosphorylated at this excitation pressure. Linear regressions indicated the highest correlation between these parameters for spruce, followed by pine and then pumpkin (goodness of fit = 0.62, 0.71 and 0.92 for pumpkin, pine and spruce, respectively). The slope was significantly nonzero for spruce, although not for pine or pumpkin (P values of 0.12, 0.07 and 0.01 for pumpkin, pine and spruce, respectively). The relationship between the chloroplast stromal redox status (estimated as the activation state of the enzyme NADP-MDH) and the level of LHCII phosphorylation for both pumpkin and pine showed more of a curvilinear relationship. As the NADP-MDH
State transitions in gymnosperms 333 photoprotection work in concert to optimize the balance of photosynthesis and photoprotection in variable environmental conditions. It is possible that improved understanding of species variation will help to better elucidate the physiological role of high light-dependent dephosphorylation of LHCII in state transitions.
Conclusions
Conflict of interest None declared.
Funding Funding was provided by an American Society of Plant Biology SURF award to A.K., a University of St. Thomas Young Scholars award to A.K., and a University of St. Thomas Collaborative Inquiry Award to M.N.
References Allen JF (1992) Protein phosphorylation in regulation of photosynthesis. Biochim Biophys Acta 1098:275–335. Aro E-M, Ohad I (2003) Redox regulation of thylakoid protein phosphorylation. Antioxid Redox Signal 5:55–67. Bellafiore S, Barneche F, Peltier G, Rochaix J-D (2005) State transitions and light adaptation require chloroplast thylakoid protein kinase STN7. Nature 433:892–895. Bennett J (1991) Protein phosphorylation in green plant chloroplasts. Annu Rev Plant Physiol Plant Mol Biol 42:281–311. Bergo E, Pursiheimo S, Paakkarinen V, Giacometti GM, Donella-Deana A, Andreucci F, Barbato R, Aro E-M (2002) Rapid and highly specific
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We demonstrated that conifers growing in high light conditions in the field retain relatively high LHCII phosphorylation, which is likely due in part to the field conditions resulting in more variable light exposure relative to laboratory manipulations of light. The general patterns of light-dependent phosphorylation of LHCII proteins occur in conifers in a manner that is similar to that of angiosperms and other monitored species, with low levels of LHCII phosphorylation occurring in darkness and the maximal levels of LHCII phosphorylation occurring in low light conditions. However, the dephosphorylation that occurs in high light conditions appears to be regulated somewhat differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions when the PSII reduction state was quite high. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin. Differential regulation of state transitions in different species may reflect species variation in strategies for regulation of excitation energy transfer in order to balance photosynthesis and photoprotection.
monitoring of reversible thylakoid protein phosphorylation by polyclonal antibody to phosphothreonine-containing proteins. J Plant Physiol 159:371–377. Demmig-Adams B, Cohu CM, Muller O, Adams WW III (2012) Modulation of photosynthetic energy conversion efficiency in nature: from seconds to seasons. Photosynth Res 113:75–88. Demmig-Adams B, Muller O, Stewart JJ, Cohu CM, Adams WW III (2015) Chloroplast thylakoid structure in evergreen leaves employing strong thermal energy dissipation. J Photochem Photobiol B 152:357–366. Derks A, Schaven K, Bruce D (2015) Diverse mechanisms for photoprotection in photosynthesis. Dynamic regulation of photosystem II excitation in response to rapid environmental change. Biochim Biophys Acta 1847:468–485. Dietzel L, Brautigam K, Pfannschmidt T (2008) Photosynthetic acclimation: state transitions and adjustment of photosystem stoichiometry— functional relationships between short-term and long-term light quality acclimation in plants. FEBS J 275:1080–1088. Foyer CH, Lelandais M, Harbinson J (1992) Control of the quantum efficiencies of photosystems I and II, electron flow, and enzyme activation following dark-to-light transitions in pea leaves. Plant Physiol 99:979–986. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92. Goldschmidt-Clermont M, Bassi R (2015) Sharing light between two photosystems: mechanism of state transitions. Curr Opin Plant Biol 25:71–78. Ivanov AG, Sane PV, Zeinalov Y, Malmberg G, Gardeström P, Huner NPA, Öquist G (2001) Photosynthetic electron transport adjustments in overwintering Scots pine (Pinus sylvestris L.). Planta 213:575–585. Johnson GN (2011) Physiology of PSI cyclic electron transport in higher plants. Biochim Biophys Acta 1807:384–389. Kargul J, Barber J (2008) Photosynthetic acclimation: structural reorganisation of light harvesting antenna—role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins. FEBS J 275:1056–1068. Kramer DM, Johnson G, Kiirats O, Edwards GE (2004) New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth Res 79:209–218. Mekala NR, Surosa M, Rantala M, Aro E-M, Tikkanen M (2015) Plants actively avoid state transitions upon changes in light intensity: role of light-harvesting complex II protein dephosphorylation in high light. Plant Physiol 168:721–734. Miginiac-Maslow M, Issakidis E, Lemaire M, Ruelland E, Jacquot J-P, Decottignies P (1997) Light-dependent activation of NADP-malate dehydrogenase: a complex process. Aust J Plant Physiol 24:529–542. Murata N (2009) The discovery of state transitions in photosynthesis 40 years ago. Photosynth Res 99:155–160. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394. Pursiheimo S, Rintamäki E, Baena-Gonzalez E, Aro E-M (1998) Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis. FEBS Lett 42:178–182. Pursiheimo S, Martinsuo P, Rintamäki E, Aro E-M (2003) Photosystem II protein phosphorylation follows four distinctly different regulatory patterns induced by environmental cues. Plant Cell Environ 26: 1995–2003. Puthiyaveetil S, Ibrahim IM, Allen JF (2012) Oxidation-reduction signalling components in regulatory pathways of state transitions and photosystem stoichiometry adjustment in chloroplasts. Plant Cell Environ 35:347–359.
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Shapiguzov A, Ingelsson B, Samol I, Andres C, Kessler F, Rochaix J-D, Vener AV, Goldschmidt-Clermont M (2010) The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis. Proc Natl Acad Sci USA 107:4782–4787. Tikkanen M, Aro EM (2012) Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants. Biochim Biophys Acta 1817:232–238. Tikkanen M, Nurmi M, Kangasjarvi S, Aro EM (2008) Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light. Biochim Biophys Acta 1777:1432–1437. Tikkanen M, Grieco M, Kangasjarvi S, Aro EM (2010) Thylakoid protein phosphorylation in higher plant chloroplasts optimizes electron transfer under fluctuating light. Plant Physiol 152:723–735. Vainonen JP, Hansson M, Vener AV (2005) STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins. J Biol Chem 280:33679–33686. Verhoeven AS, Osmolak A, Morales P, Crow J (2009) Seasonal changes in abundance and phosphorylation status of photosynthetic proteins in eastern white pine and balsam fir. Tree Physiol 29:361–374. Weimar M, Rothe GM (1986) Preparation of extracts from mature spruce needles for enzymatic analyses. Physiol Plant 69:692–698.
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Rintamäki E, Salonen M, Suoranta U, Carlberg I, Andersson B, Aro EM (1997) Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins. J Biol Chem 272:30476– 30482. Rintamäki E, Martinsuo P, Pursiheimo S, Aro EM (2000) Cooperative regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts. Proc Natl Acad Sci USA 97:11644–11649. Rochaix JD, Lemeille S, Shapiguzov A, Samol I, Fucile G, Willig A, Goldshmidt-Clermont M (2012) Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment. Philos Trans R Soc B Biol Sci 367:3466–3474. Samol I, Shapiguzov A, Ingelsson B, Fucile G, Crevecoeur M, Vener AV, Rochaix JD, Goldshmidt-Clermont M (2012) Identification of a photosystem II phosphatase involved in light acclimation in Arabidopsis. Plant Cell 24:2596–2609. Scheibe R, Stitt M (1988) Comparison of NADP-malate dehydrogenase activation, QA reduction and O2 evolution in spinach leaves. Plant Physiol Biochem 26:473–481.
Research paper
Characterization of light-dependent regulation of state transitions in gymnosperms Amy S. Verhoeven1,2, Albert Kertho1 and Mary Nguyen1 Department (OWS352), University of St Thomas, 2115 Summit Ave., St Paul, MN 55105, USA; 2Corresponding author ([email protected])
Received September 22, 2015; accepted December 3, 2015; published online January 22, 2016; handling Editor Jörg-Peter Schnitzler
The goal of this study was to characterize the light-dependent regulation of state transitions in gymnosperms. Two species of conifer were examined: eastern white pine (Pinus strobus L.) and white spruce [Picea glauca (Moench) Voss], as well as the angiosperm pumpkin (Cucurbita pepo L. subsp. pepo). Both diurnal time courses in the field and manipulated light experiments in growth chambers were conducted. Results from chlorophyll fluorescence analysis indicated that pumpkin was able to use a larger fraction of absorbed light to drive photochemistry and retain a lower reduction state at a given light intensity relative to the conifers. Results from western blots using anti-phosphothreonine demonstrate that in field conditions, conifers maintained higher light-harvesting complex II (LHCII) phosphorylation than pumpkin; however, this was likely due to a more variable light environment. Manipulated light experiments showed that general patterns of light-dependent LHCII phosphorylation were similar in conifers and pumpkin, with low levels of LHCII phosphorylation occurring in darkness and maximal levels occurring in low light conditions. However, high light-dependent dephosphorylation of LHCIII appears to be regulated differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin. Keywords: conifer, light regulation, thylakoid protein phosphorylation.
Introduction Photosynthetic organisms rely on light absorption to drive the process of photosynthesis, but are faced with dramatic fluctuations in light intensity on both a daily and seasonal scale, as well as less predictable fluctuations in light caused by factors such as wind and changes in cloud cover. In order to cope with fluctuating light environments, plants use a variety of regulatory mechanisms that allow for rapid acclimation of the photosynthetic apparatus to changing light, including thermal energy dissipation, the photosystem II (PSII) repair cycle and state transitions (Dietzel et al. 2008, Demmig-Adams et al. 2012, Tikkanen and Aro 2012, Derks et al. 2015). In high light conditions, plants engage mechanisms of thermal energy dissipation in order to safely dissipate excessive light (Demmig-Adams et al. 2012). Additionally, the highly oxidizing conditions of the PSII reaction
center necessitate rapid turnover of the D1 protein in high light conditions, and this process involves reversible phosphorylation of PSII core proteins (Tikkanen et al. 2008, Tikkanen and Aro 2012). In lower light conditions, as well as in conditions of fluctuating light, the processes of light harvesting and electron transport are optimized, at least in part, by the process of state transitions (Tikkanen and Aro 2012). State transitions involve the light-dependent reversible phosphorylation of the major light-harvesting antenna (light-harvesting complex II, LHCII), resulting in a redistribution of LHCII between PSII and photosystem I (PSI). The phosphorylated form of the mobile LCHII increases its association with PSI and this conformation has classically been described as ‘state 2’, while the dephosphorylated form of LHCII associates more strongly with PSII and is known as ‘state 1’ (Bennett 1991, Allen 1992,
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1Biology
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angiosperm species, although in moss and liverwort, there was no evidence of D1 phosphorylation (Pursiheimo et al. 1998). In a study examining conifers, relatively high phosphorylation of light-harvesting proteins was observed in very high light conditions (midday in full sunlight), suggesting that conifers may have a different light-dependent response compared with angiosperms (Verhoeven et al. 2009). The goal of this study was to investigate whether state transitions in gymnosperms have a unique light-dependent response by monitoring thylakoid protein phosphorylation in conifers, compared with pumpkin, collected under varying light environments in the field, as well as upon exposure to varying light environments in controlled laboratory conditions.
Materials and methods Plant material and sampling Sampling was performed on mature trees of white spruce [Picea glauca (Moench) Voss] and eastern white pine (Pinus strobus L.) growing in full sunlight on campus at the University of St Thomas, Minnesota (44°59′40″N, 93°05′35″W). Additionally, pumpkin plants (Cucurbita pepo L. subsp. pepo) were grown in pots and maintained outdoors in full sunlight. Diurnal time courses were conducted using a single tree (for the conifers), and pooling samples from three pumpkin plants. For the conifers, sampling was conducted on branches that had significant full sun exposure over the course of the day. On 13 July 2010, sampling was conducted predawn at 4:45 h, and again at 6:00, 7:00, 9:00, 11:00 and 13:00 h. On this date, significant cloud cover occurred late in the morning and so sampling was discontinued. Sampling was continued on 15 July 2010, which was a clear day. Samples were collected at 13:20, 16:00 and 19:00 h. At each time point, leaf temperature, light exposure and chlorophyll fluorescence were monitored. Additionally, needles/ leaves were cut and transferred to liquid nitrogen, taking care to maintain light exposure throughout that process. Needles were stored in liquid nitrogen until later isolation of thylakoids.
Manipulated light experiments For manipulated light experiments, leaf discs or needles were floated on room temperature water at controlled light intensities. Needles were collected from the trees used for diurnal measurements in the morning prior to exposure to direct sunlight, again using branches with full sun exposure for a significant portion of the day. Pumpkin leaves were collected either from plants grown in pots outdoors or from plants grown in the greenhouse in full sun conditions. Manipulated light experiments were conducted initially in June 2011, and then repeated in August 2011 and in July 2012. For dark acclimation, needles/leaves were cut and placed in plastic bags with moist paper towels and maintained in full darkness for 1 h. For light exposures, leaf discs (pumpkin) or needles were floated on room temperature water in Petri
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ietzel et al. 2008, K argul and Barber 2008, Murata 2009). D State transitions are thought to balance the excitation pressure between the photosystems, thus maintaining optimal photosynthetic efficiency in varying light environments (Dietzel et al. 2008, K argul and Barber 2008, Tikkanen and Aro 2012, Goldschmidt-Clermont and Bassi 2015). In addition to LHCII, three of the PSII core proteins are reversibly phosphorylated in a light-dependent manner, although the pattern of their light regulation is opposite to that of LHCII, with LHCII being maximally phosphorylated at low light intensities and the core proteins showing maximal phosphorylation at high light (Rintamäki et al. 1997, Tikkanen et al. 2008). Phosphorylation of PSII core proteins is thought to function in signaling protein damage and facilitating the PSII repair cycle (Tikkanen et al. 2008, Tikkanen and A ro 2012). The light-dependent phosphorylation of thylakoid proteins is regulated both by the redox state of the electron transport chain and that of the chloroplast stroma (Rintamäki et al. 2000, Aro and Ohad 2003, Puthiyaveetil et al. 2012, Rochaix et al. 2012). Protein phosphorylation is controlled by the STN7 and STN8 kinases, and dephosphorylation is regulated by the TAP38/ PPH1 and PBCP phosphatases (Bellafiore et al. 2005, Vainonen et al. 2005, Shapiguzov et al. 2010, Samol et al. 2012). In particular, LHCII is phosphorylated by a STN7 kinase, which is activated by the redox state of the plastoquinone pool in relatively low light (Bellafiore et al. 2005, Rochaix et al. 2012). There is also evidence that the kinase is deactivated by increases in thiol reducing activity of the stroma, leading to decreases in LHCII phosphorylation in high light (Rintamäki et al. 2000, Aro and Ohad 2003, Pursiheimo et al. 2003). Light-harvesting complex II is dephosphorylated by the TAP38/PPH1 phosphatase (Shapiguzov et al. 2010), although whether this enzyme is redox dependent or is constitutively active is currently unknown (Rochaix et al. 2012). A recent study by Mekala et al. (2015) demonstrates that the TAP38/PPH1 phosphatase is responsible for dephosphorylation of LHCII in high light; thus, it is possibly also redox regulated. The PSII core proteins are phosphorylated by the STN8 kinase in high light conditions (Vainonen et al. 2005) and dephosphorylated by the PBCP phosphatases (Samol et al. 2012). Several studies have examined the irradiance-dependent nature of state transitions and have found that LHCII is not phosphorylated in darkness, becomes phosphorylated in low light and is subsequently dephosphorylated in high light conditions (Rintamäki et al. 1997, 2000). In contrast, the PSII core proteins show increasing phosphorylation at high light intensities, although CP43 has been reported to remain phosphorylated in darkness in some cases ( Rintamäki et al. 1997, 2000, Pursiheimo et al. 2003). These light-dependent patterns of thylakoid protein phosphorylation have been shown to be conserved across divergent evolutionary species, with similar responses reported in a moss, liverwort and fern to that of
State transitions in gymnosperms 327
Chlorophyll fluorescence Chlorophyll fluorescence was performed at the time of sample collection using a field portable fluorescence monitoring system (FMS2, Hansatech, Kings Lynn, UK). The modulating beam was set at default values (setting of 2, gain 50) and the saturating beam was >5000 μmol m−2 s−1 (0.7 s duration). For the manipulated light experiments, needle and leaf temperature were maintained at 22 °C. Values for Fv/Fm were determined either predawn or after 1 h of dark acclimation and ΦPSII [(Fm′ − F )/Fm′ ] was determined for samples exposed to light (Genty et al. 1989). The parameter 1 − qL was used to estimate the fraction of closed QA centers [qL = (Fm′ − Fs )/(Fm′ − Fo′ ) × (Fo′ /Fs )] according to Kramer et al. (2004).
Isolation of thylakoids To extract thylakoids, tissue was ground in liquid nitrogen with a mortar and pestle, and then homogenized with a polytron (Brinkman, Model 10/35, Fisher Scientific, Pittsburgh, PA, USA) in grinding buffer [50 mM Tricine, pH 7.6, 0.4 M sorbitol, 10 mM NaF, 10 mM MgCl2, 20% polyethylene glycol (average molecular weight 10,000, Sigma-Aldrich, St. Louis, MO, USA), 1 mM benzamidine-HCl and 5 mM 6-amino-n-hexanoic acid]. Samples were filtered through four layers of cheesecloth, centrifuged at 10,000g for 10 min at 4 °C and the pellet was resuspended in wash buffer (50 mM Tricine, pH 7.6, 10 mM NaCl, 10 mM NaF and 5 mM MgCl2) and re-centrifuged. Thylakoids were resuspended in 50 mM Tricine, pH 7.6, containing 50% glycerol, 5 mM MgCl2 and 10 mM NaCl, and stored at −80 °C. Protein concentration of thylakoids was determined by the Bio-Rad Protein assay, and chlorophyll concentration was determined in buffered acetone as described by Porra et al. (1989).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting Samples of isolated thylakoids were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. To d etermine
phosphorylation status, 10 µg of thylakoid protein was solubilized in the presence of 6 M urea and run on 15% gels containing 6 M urea (Rintamäki et al. 1997, Bergo et al. 2002). To ensure equal loading of protein, gels were stained with Coomassie Brilliant Blue and visually inspected for equivalent loading. For immunodetection, proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Fisher Scientific, Pittsburgh, PA, USA) and probed with rabbit anti-phosphothreonine (Cell Signaling Technology, Danvers, MA, USA) using the recommended dilutions (1−2 µg ml−1 antibody). Secondary antibody was goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich). Antibodies were detected by chemiluminescence (RPN2109, GE Healthcare, Pittsburgh, PA, USA) followed by exposure of the membrane to film (Kodak BioMax light film, Sigma Aldrich, St. Louis, MO, USA). Band intensity was quantified using ImageJ (NIH, Bethesda, MD, USA). To identify the phosphorylated bands, control thylakoids were prepared from pumpkin leaves exposed to low and high light following the protocol described by Rintamäki et al. (1997) and bands were compared with the published data. Additionally, direct comparisons were performed of LHC (both Lhcb1 and Lhcb4), D1, D2 and CP43 using purchased antibodies (AgriSera, Vannas, Sweden). It is noteworthy that the results of our western blots varied considerably depending on the particular lot number of antiphosphothreonine antibody that we received from Cell Signaling Technology. Most of the observed variation was in the relative phosphorylation of CP43 and the D1 protein. With one lot of the antibody, CP43 appears to be phosphorylated at most light intensities, while the other showed very low levels of CP43 phosphorylation. Additionally, the sensitivity of the antibody to the phosphorylated form of the D1 protein varied dramatically between lots. We have noted this considerable variation between purchased aliquots of anti-phosphothreonine in the recognition of CP43 and D1, although the results for LHCII phosphorylation have been consistent. Therefore, we have chosen not to attempt to draw any conclusions about light-dependent core protein phosphorylation from these data and have instead focused on changes in LHCII phosphorylation.
NADP-malate dehydrogenase assay Chloroplast redox status was estimated by measuring the activation state of the chloroplast NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.82) according to Scheibe and Stitt (1988). Leaf tissue (0.1–0.4 g) was ground in liquid nitrogen in ∼1 ml extraction buffer (50 mM sodium acetate, 0.1% w/v Triton X-100, 0.1% bovine serum albumin, 4% poly-vinyl-pyrollidone-40, 4 mM dithiothreitol (DTT), pH 6.0) that had been bubbled with N2 gas. Extracted samples were centrifuged for 10 min at ~20,000g at 4 °C, and the supernatant was collected and bubbled with N2 gas. Enzyme activity was assessed in the presence and absence of DTT according to Scheibe and Stitt (1988),
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dishes at a variety of light intensities and maintained for 1 h. The lowest light intensity was 30 µmol m−2 s−1, which used the fluorescent lights in the laboratory. Light exposures of 120, 350 and 700 µmol m−2 s−1 were maintained using a growth chamber (Model MB-60B, Percival Scientific Instruments, Perry, IA, USA) with varying distances from the light source. The high light exposure (1300 µmol m−2 s−1) was set up using halogen lamps placed above a Pyrex dish filled with water (as a heat sink) with leaf discs/needles maintained in dishes below this. Care was taken to maintain temperature and evenness of light exposure for all samples. After 1 h of light exposure, leaf discs and needles were sampled for chlorophyll fluorescence, and then samples were immediately transferred to liquid nitrogen, taking care to maintain the light environment during the transfer. Samples were stored in liquid nitrogen.
328 Verhoeven et al. and the NADP-MDH activation state was calculated from the ratio of these values. For the spruce samples, the leaf extracts were run over 5 ml Sephadex G25 (Sigma-Aldrich) c olumns that had previously been equilibrated with the reaction buffer (100 mM Tris–Cl, 1 mM ethylenediaminetetraacetic acid, 2.5 mM DTT, pH 8.0). In order to ensure that the column treatment did not alter the NADP-MDH activation state, spinach samples were tested for activity both before and after the column treatment. These tests provided similar results.
Statistical analysis
Results Diurnal time courses of chlorophyll fluorescence and phosphorylation status of photosynthetic proteins Diurnal time courses were conducted in mid-July on white pine, white spruce and pumpkin in order to establish the phosphorylation status of the conifer photosynthetic proteins in a variety of natural light environments in comparison with similar results for pumpkin, which has previously been well characterized (Bergo et al. 2002). Our goal was to conduct a diurnal time course on a clear sunny day in order to monitor the highest light exposures. The first date of sampling (13 July 2010) began sunny but clouds moved in by midday creating a variable light environment. We continued sampling 2 days later when conditions were sunny all afternoon (Figure 1a). Values for Fv/Fm measured predawn (5:00 h, Figure 1b) were quite high for all species, with values above 0.8. Reductions in ΦPSII mirrored increases in light exposure in a predictable manner for all species (Figure 1b). Figure 2 depicts example western blots probed with antiphosphothreonine antibody for all species and time points from the diurnal time courses. In pumpkin, four bands are visible, corresponding to the four photosynthetic proteins that are known to show changes in phosphorylation with light environment (LHCII, D1, D2 and CP43). In both conifer species, only three bands are visible, with the phosphorylated form of the D1 protein not showing up. The absence of the phosphorylated form of the D1 protein in conifers is likely a problem with antibody recognition, as opposed to a lack of phosphorylated D1, as mentioned in the Materials and methods section. Because the light environment varied for the individual species at the different time points, the data for relative phosphorylation of LHCII, D2 and CP43 are compiled in Figure 3 as a
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Figure 1. Light intensity (photosynthetic photon flux density, PPFD) at leaf level at the time of sampling (a) and ΦPSII or Fv/Fm (b) obtained over the course of a portion of 2 days for pumpkin (open circles), pine (filled squares) and spruce (open triangles). Time points for samples taken predawn until 11:00 h were collected in 13 July 2010, and the time points from 13:30 h until the end of the day were collected on 15 July 2010. Data represent averages ± SD (n = 3).
Figure 2. Example blots showing the phosphorylation status of thylakoid proteins of PSII (CP43, D2 and D1) as well as phospho-LHCII in samples collected from pumpkin, pine and spruce over the course of the day.
function of categories of light intensity. For pumpkin, the data show light-dependent changes in these proteins that are consistent with previously published studies (Pursiheimo et al. 2003). The LCHII proteins show low phosphorylation in the dark, high
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To test for significant differences between species in the manipulated light experiments, for fluorescence parameters, the relative phosphorylation of LHCII and the NADP-MDH activation state, repeated-measures analyses of variance were performed (JMP Statistical Software, SAS Institute, Cary, NC, USA). To test for a linear relationship between the relative phosphorylation of LHCII and 1 − qL, linear regressions were conducted using Prism (GraphPad Software, Inc., La Jolla, CA USA).
State transitions in gymnosperms 329
Manipulated light experiments
Figure 3. The relative phosphorylation of individual thylakoid proteins from samples collected under different light intensities for pumpkin (white bars), pine (gray bars) and spruce (black bars). Samples were grouped into categories of light intensity [dark, low light, medium light and high light (0, 20–100, 150–700 and >1000 µmol photons m−2 s−1)] in order to compare general light intensity effects. For these data, the time point showing the highest phosphorylation of LHC was normalized to 1, and all other time periods were expressed as a fraction of that value. The number of time points pooled and the respective light intensities during collection were as follows: 20–100 µmol photons m−2 s−1 range: pumpkin (3) 27, 63, 85, pine (2) 20, 60, spruce (3) 30, 50, 75; 150–700 µmol photons m−2 s−1 range: pumpkin (1) 660, pine (2) 250, 450, spruce (2) 175, 400; >1000 µmol photons m−2 s−1 range: pumpkin (3) 1100, 1380, 1465, pine (3) 1000, 1150, 1300, spruce (2) 1225, 1812. Where two or more samples were pooled, data represent averages ± SD.
phosphorylation in low light and decreasing phosphorylation in high light conditions (Figure 3a). The core PSII proteins D1 (not shown, but see Figure 2) and D2 (Figure 3b) show little phosphorylation in the dark, with phosphorylation increasing in high light. CP43 shows relatively high levels of phosphorylation regardless of the light environment, although it increases at higher light exposures (Figure 3c).
Manipulated light experiments were conducted in which leaves and needles were collected from the field and brought into the laboratory where they were maintained over a range of light intensities for 1 h, prior to sampling for chlorophyll fluorescence and thylakoid protein phosphorylation status. Values for Fv/Fm, measured after 1-h dark acclimation, were ∼0.8 for all species (Figure 4a), which was consistent with values measured in the field (Figure 1b). ΦPSII values decreased as light increased, but at a given light intensity, these values remained significantly higher in pumpkin relative to pine and spruce, suggesting that pumpkin uses a larger fraction of absorbed light to drive photosynthesis relative to the conifer species. Similarly, values for 1 − qL (an estimate of the PSII reduction status) were significantly higher for pine and spruce than for pumpkin at each light intensity, suggesting that the conifers are experiencing relatively higher excitation pressure for a given light intensity compared with pumpkin (Figure 4b). The pattern of relative phosphorylation of LHCII was similar for the three species, showing little to no phosphorylation in dark acclimated samples and maximal phosphorylation in low light, with phosphorylation decreasing as light intensity increased. Values for pumpkin and pine were very similar to each other; however, spruce maintained significantly higher phosphorylation of LHCII in high light conditions relative to the other two species.
Estimating chloroplast redox status using NADP-MDH assay The decreased phosphorylation of LHCII in high light conditions is thought to be caused by increases in the chloroplast stromal redox status that occur during excess light conditions and that
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For pine and spruce, CP43 and D2 were relatively highly phosphorylated regardless of light environment (Figure 3b and c). The relative phosphorylation of LHCII was very low in the dark and increased to high levels in low light (Figure 3a). In the higher light conditions, there were decreases in LHCII p hosphorylation, although the relative phosphorylation of LHCII remained higher in the conifers than in pumpkin. The relatively high levels of variation in the phosphorylation status of the photosynthetic proteins in our field-collected data, particularly for the two conifer species, led to questions about the consistency of the light environment during sampling. Pumpkin leaves are large and present an even surface area to a particular light environment and were therefore easy to sample in a consistent manner. The spruce needles are arranged around the branch in a variety of angles; thus, individual needles were exposed to higher variation in incident light. Similarly, the white pine needles are arranged in clusters that dangle from the plant, thus causing some self-shading within each cluster. Because of this variation, we decided to conduct a manipulated light experiment in the laboratory in order to better compare actual light environments between the species.
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Therefore, in order to estimate the redox status of the chloroplast stroma, we repeated the manipulated light exposures and measured the NADP-MDH a ctivation state at each light intensity (Figure 5). Both pumpkin and pine showed marked increases in the activation state of NADP-MDH as a function of light intensity, with a rapid increase in the activation state from 0 to 120 μmol photons m−2 s−1 that saturated at higher light intensities at an activation state of ∼75%. In contrast, spruce showed only small increases in NADP-MDH activity as light levels increased above zero, with these low values being maintained at all light intensities.
Discussion State transitions in field conditions
Figure 4. The chlorophyll fluorescence parameters ΦPSII or Fv/Fm (a), 1 − qL (b) and the relative phosphorylation of LHCII (c) for samples from pumpkin (open circles), pine (filled squares) and spruce (open triangles) as a function of light intensity. Data are averages ± SD of three experiments.
inhibit the LHCII kinase (Rintamäki et al. 2000, Aro and Ohad 2003). Given the relatively high phosphorylation of spruce LHCII in high light conditions, we were interested in assessing the chloroplast redox status, as a function of light intensity, for each species. NADP-MDH (E.C. 1.1.1.82) is a chloroplast-specific enzyme that is regulated by light through a thiol–disulfide interchange, which is triggered by photosynthetic electron transfer (Miginiac-Maslow et al. 1997). The activation state of this enzyme has been demonstrated to reflect the redox status in the chloroplast stroma (Scheibe and Stitt 1988, Foyer et al. 1992).
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Our data suggest that in field conditions, under high light environments, the sampled conifers maintained higher LHCII phosphorylation relative to pumpkin (Figures 2 and 3), although this may reflect differences in light exposure rather than differences in the regulation of state transitions. The conifers experienced more variable and dynamic light environments compared with pumpkin, due to the arrangement of needles on the branches. It is likely that for most plants growing in field conditions, the changes in leaf angle due to wind, as well as self-shading, result in more variable and dynamic light conditions than what we documented for the pumpkin leaves, as we took care to sample leaves experiencing uniform light environments. Most of the studies examining state transitions in response to light exposure are based on manipulated light treatments in laboratory settings (Rintamäki et al. 1997, Pursiheimo et al. 2003), and our results from pumpkin are quite consistent with these studies. A study examining Arabidopsis mutants lacking the LHCII kinase (stn7 mutants) found severe physiological effects under strongly fluctuating light conditions
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Figure 5. The activation state of the enzyme NADP-MDH, which is an indirect measure of the redox status of the chloroplast stroma, as a function of light intensity. Values closer to 1 indicate a higher stromal reduction state. Data are averages ± SD of three experiments for pumpkin (open circles), pine (filled squares) and spruce (open triangles).
State transitions in gymnosperms 331 (Tikkanen et al. 2010), suggesting that state transitions play a key role in balancing excitation energy distribution between photosystems particularly in dynamic light environments. The relatively high levels of LHCII phosphorylation we observed in the conifers in high light conditions in the field likely reflect this important role for state transitions in fluctuating light conditions.
Regulation of state transitions in conifers
Relationships between PSII reduction state, stromal redox status and LHCII phosphorylation As the dephosphorylation of LHCII in high light differed in spruce compared with pine and pumpkin, we decided to look more
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The manipulated light treatments allowed for more controlled light environments, which are not as reflective of natural conditions, but which provide some insight in comparing species responses. The fluorescence data reflect innate differences in maximal photosynthetic capacity of pumpkin compared with the conifers, showing that pumpkin maintains relatively high photosynthetic efficiencies and a low reduction state at higher light intensities when compared with the conifers (Figure 4a and b). This is expected, as pumpkin is an annual crop plant with a high photosynthetic capacity allowing for rapid growth, while the conifers are perennial trees, which maintain slower growth rates in general. The level of LHCII phosphorylation was very low in darkness in all species, which is consistent with previous studies. Upon transition from darkness to low light, all species showed very similar responses with dramatic increases in LHCII phosphorylation (Figure 4c). The maximal LHCII phosphorylation occurred at the lowest light intensity tested (30 μmol photons m−2 s−1) in all three species. These data are consistent with previous studies which have indicated that the LHCII kinase (STN7 in Arabidopsis) is activated by increases in the reduction state of the plastoquinone pool and its interaction with the cytochrome b6f complex (Rochaix et al. 2012), and that maximal phosphorylation occurs at light intensities well below the growth light intensity (Rintamäki et al. 1997). The data suggest that LHCII kinase activation occurs in conifers in a similar manner to the angiosperms that have been studied more extensively. Further increases in light intensity resulted in decreases in LHCII phosphorylation in all species, although for spruce, the relative phosphorylation of LHCII was maintained at significantly higher levels than for the pumpkin and pine (Figure 4c). The dephosphorylation of LHCII that occurs at higher light intensities is thought to be regulated by the thiol redox status of the chloroplast stroma, which has been suggested to inactivate the LHCII kinase by reducing disulfide bonds in the enzyme (Rintamäki et al. 2000, Aro and O had 2003, Rochaix et al. 2012), although it is also possible that an increase in the activity of the TAP38/ PPH1 phosphatase enzyme is required (Mekala et al. 2015). In order to determine whether the maintenance of relatively higher LHCII phosphorylation in spruce was due to differences in the chloroplast redox status, the activation state of the enzyme NADP-MDH was determined. Interestingly, changes in the NADP-MDH activation state, as a function of light intensity, were very similar for pumpkin and pine, with light-dependent increases consistent with the hypothesis that increases in stromal redox
status of the chloroplast cause decreases in LHCII phosphorylation in high light conditions (Rintamäki et al. 2000). The results for the NADP-MDH activation of spruce were quite different, and showed only a small increase in the activation state of this enzyme with increasing light conditions up to ∼20% activation at 300 μmol photons m−2 s−1, with no further increase upon increasing light (Figure 5). These results may suggest that spruce maintains a low chloroplast redox status in high light conditions, and that as a result, the LCHII kinase is not inhibited, leading to the maintenance of higher levels of phosphorylated LHCII in high light conditions. The large difference between spruce compared with pine and pumpkin in the light-dependent response of the NADP-MDH activation state is surprising, and it is possible that there is an alternative explanation for the observed differences. The relationship between the activation state of the NADP-MDH and the redox status of the chloroplast stroma was demonstrated in pea and spinach (Scheibe and S titt 1988, Foyer et al. 1992), but it is possible that the enzyme NADP-MDH is regulated differently in spruce and that the activation state of the enzyme does not reflect the chloroplast stromal redox status in spruce. Another possibility is that the additional purification step required to clean up the spruce leaf extracts prior to enzyme analysis may have made the spruce data less reliable than that of pine and pumpkin. In order to assess NADP-MDH activity in spruce needle extracts, it was necessary to first run the samples over Sephadex columns, presumably to remove secondary metabolites that interfered with the assay. This step was not required for either pumpkin or pine. Such techniques have been used in spruce to measure enzyme activity in previous studies (e.g., Weimar and Rothe 1986). As a control, to ensure that the technique did not disrupt the enzyme activation state, we assayed pumpkin samples both before and after extracts were run over the columns, and were able to get identical results with these controls. Therefore, we feel that our data accurately reflect the NADP-MDH activation state for spruce, but it is important to note the difference in methodology. A relatively higher fraction of phosphorylated LHCII and a relatively lower reduction status of the chloroplast stroma are both consistent with increased levels of cyclic electron transport at higher light intensity in spruce needles. Because of the difficulty of measuring rates of cyclic electron transport, little is known about the prevalence of this pathway under physiologically relevant conditions (Johnson 2011). A study examining Scots pine found evidence supporting increased use of the cyclic pathway under overwintering conditions (Ivanov et al. 2001). It would be interesting to investigate the relative flux of electrons through the cyclic pathway in white spruce compared with other species.
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Figure 6. 1 − qL (a) or NADPH-MDH activation state (b) as a function of relative LHCII phosphorylation levels in pumpkin (open circles), pine (filled squares) and spruce (open triangles). The lines depict estimates of best fit for a linear regression in (a) and the curves depict estimates of best fit for a second-order polynomial in (b) using Graphpad Prism.
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activation state increased from very low values up to 0.5 or so, there were corresponding decreases in LHCII phosphorylation from maximal levels down to 60–70% of maximal LCHII phosphorylation. Subsequent decreases in LHCII phosphorylation were accompanied by much smaller increases in NADP-MDH activation. There was no relationship between these parameters in spruce. Based on previous studies, it is not surprising that there is no correlation between the PSII reduction status and the LHCII phosphorylation state of pumpkin, as it has been demonstrated that the dephosphorylation of LHCII in high light is regulated by increased stromal redox status, which is consistent with our results. For pine, and more so for spruce, the correlation between PSII reduction status and LHCII phosphorylation may indicate that some regulation of LHCII dephosphorylation occurs via redox sensing of electron transport. For pine, but not for spruce, the data also support a role for the stromal redox status in regulating LHCII dephosphorylation in high light conditions. The higher PSII excitation pressure experienced by conifers in high light conditions likely involves different strategies for coping with excess energy relative to annual plants, where a larger fraction of light can be used to drive photosynthesis. Both proton gradient-dependent thermal energy dissipation and redox- regulated phosphorylation of thylakoid proteins contribute to reducing the excitation energy transfer to PSII. A recent study by Demmig-Adams et al. (2015) suggested that in conifers, thylakoid unstacking is correlated with higher levels of non-photochemical quenching (NPQ), which has apparently not been observed in herbaceous annual plants. Thylakoid unstacking in high light might be expected to be associated with higher LHCII phosphorylation (observed here), given the biophysical constraints of the phosphorylated form of LHCII fitting within the granal regions of thylakoids. Although the physiological role of high light-induced dephosphorylation of LHCII is not understood, Mekala et al. (2015) speculate that the dephosphorylation of LHCII in high light may function to protect against the excitation imbalance and energy losses in PSII upon subsequent decreases in light intensity. They suggest that upon shifting from high to low light, if PSII had a smaller antenna, it would not have enough excitation energy to satisfy the electron needs of PSI (with a large antenna), which would lead to energy losses in NPQ processes within PSI. It is possible that in conifers, and other plants in sink-limited conditions, this could be a useful strategy to dissipate excess energy. This would be consistent with a recent suggestion by Demmig-Adams et al. (2015) that spillover quenching in PSI may be an important component of NPQ in evergreen species. The process of dephosphorylation of LHCII is controlled by both a kinase and a phosphatase, both of which can be activated and deactivated, allowing for complex regulation of LHCII phosphorylation (Rochaix et al. 2012, Mekala et al. 2015). It is not surprising that there are species variations in this process of optimization, as the myriad possible regulatory pathways for electron flow and
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closely at the relationship between LHCII phosphorylation and an estimate of the PSII reduction state (1 − qL, Figure 6a) as well as an estimate of the stromal redox status (NADP-MDH activation, Figure 6b). Dark values were excluded for this analysis. In general, both conifers retained higher phosphorylation of LHCII at a given perceived PSII excitation pressure (estimated as 1 − qL, Figure 6a). For example, when the value for 1 − qL is ∼0.3, pumpkin LHCII shows very little phosphorylation while, for both conifers, LHCII is highly phosphorylated at this excitation pressure. Linear regressions indicated the highest correlation between these parameters for spruce, followed by pine and then pumpkin (goodness of fit = 0.62, 0.71 and 0.92 for pumpkin, pine and spruce, respectively). The slope was significantly nonzero for spruce, although not for pine or pumpkin (P values of 0.12, 0.07 and 0.01 for pumpkin, pine and spruce, respectively). The relationship between the chloroplast stromal redox status (estimated as the activation state of the enzyme NADP-MDH) and the level of LHCII phosphorylation for both pumpkin and pine showed more of a curvilinear relationship. As the NADP-MDH
State transitions in gymnosperms 333 photoprotection work in concert to optimize the balance of photosynthesis and photoprotection in variable environmental conditions. It is possible that improved understanding of species variation will help to better elucidate the physiological role of high light-dependent dephosphorylation of LHCII in state transitions.
Conclusions
Conflict of interest None declared.
Funding Funding was provided by an American Society of Plant Biology SURF award to A.K., a University of St. Thomas Young Scholars award to A.K., and a University of St. Thomas Collaborative Inquiry Award to M.N.
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We demonstrated that conifers growing in high light conditions in the field retain relatively high LHCII phosphorylation, which is likely due in part to the field conditions resulting in more variable light exposure relative to laboratory manipulations of light. The general patterns of light-dependent phosphorylation of LHCII proteins occur in conifers in a manner that is similar to that of angiosperms and other monitored species, with low levels of LHCII phosphorylation occurring in darkness and the maximal levels of LHCII phosphorylation occurring in low light conditions. However, the dephosphorylation that occurs in high light conditions appears to be regulated somewhat differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions when the PSII reduction state was quite high. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin. Differential regulation of state transitions in different species may reflect species variation in strategies for regulation of excitation energy transfer in order to balance photosynthesis and photoprotection.
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