Bachelor Thesis Chemistry
Arabidopsis thaliana under far-red light
Changes in the photosynthetic machinery
by
Dana Rademaker
11
thof July 2017
Student number
10746765
Research Institute
Faculty of Sciences
Research group
Biophysics of Photosynthesis
Responsible professor
Prof. Dr. R. Croce
Supervisor
Dr. R. Fristedt
2
Abstract
The distribution of light over a canopy follows a vertical gradient, leaving only far-red light (FR) for the bottom canopy leaves. To shed more light on the mechanisms behind plant adaptation to FR, the influence of constant FR on Arabidopsis thaliana was investigated. This was carried out by comparing three-to-four week old plants acclimated to FR with ones acclimated to white light (W). It was found that 24 hours of FR does not induce a significant reduction in the maximum PSII efficiency, whereas one week of FR acclimation has a detrimental effect on the plants. Investigating the non-photochemical quenching (NPQ) of both acclimation times shows an altered quenching mechanism in the 1wk FR acclimated plants and an inability to relax back to F0 in the dark. This
indicates that the plants are severely photodamaged upon prolonged exposure to actinic light and saturating pulses during the measurement. In these plants a strong decrease in the Chl a/Chl b ratio was observed, which was shown to be caused by degradation of the photosystems. After one week of FR acclimation the plants start to degrade their cellular content to induce a senescence-like process. After 24h FR acclimation the plants also show photoinhibition and an altered quenching mechanism. Furthermore, the 24h FR acclimated plant showed a strong decrease in the Chl a/Chl b ratio, which is not caused by changes in the amount of photosystems, since the levels of both PSI and PSII do not change. In contrast, an increase of Lhcb2 with a factor of 2,16 (±0,34) was observed. From the change in Chl a/Chl b these Lhcb2 proteins are thought to assemble into an additional LHCII trimer per PSII core for the FR acclimated plants. A complete dephosphorylation of the LHCII complexes was observed after 24h FR, indicating that the plant is in an absolute state 1, with all LHCII coordinated to PSII. State transitions of this additional LHCII trimer during the quenching experiment might explain the altered quenching mechanism for the 24h FR acclimated plant. Additionally, CP43 of the PSII core is completely dephosphorylated in the plant acclimated to 24h FR, which might indicate a structural change in the photosynthetic membranes of these plants as a further adaptation mechanism to FR. Strikingly, this complete dephosphorylation of specifically CP43 has not been reported before.
3 Figuur 1. Het elektron transport tijdens de lichtreacties van fotosynthese. Het oxygen evolving complex (OEC) kan water oxideren tot O2, waarbij fotosysteem II gereduceerd wordt en het fotosynthese proces nog eens kan doorlopen.
Populair wetenschappelijke samenvatting
Fotosynthese is het proces waarbij planten water en koolstofdioxide omzetten in zuurstof en koolwaterstoffen. De energie die dit proces nodig heeft, wordt geleverd door de zon. In de chloroplast van een plant bevinden zich eiwitcomplexen, de ‘light-harvesting complexen’ (LHCs), die licht kunnen absorberen en door kunnen geven aan andere LHCs of aan fotosystemen. Licht wordt bijvoorbeeld door LHCII geabsorbeerd en doorgegeven aan fotosysteem II (PSII). Door deze energie komt er een elektron vrij in de kern van PSII dat doorgegeven wordt aan het cytochroom b6f complex.
Dit complex geeft het elektron door aan fotosysteem I (PSI). Met de energie van een LHCI en het elektron van PSII, kan PSI NADP+ reduceren tot NADPH. Tijdens dit proces wordt lichtenergie dus omgezet in chemische energie. Deze hoog energetische cofactor wordt vervolgens in de Calvin-Bensoncyclus gebruikt om koolwaterstoffen te maken. Figuur 1 laat een schematisch overzicht zien van de lichtreacties van fotosynthese.
Een groot probleem bij gewassen in de landbouw is dat de lichtdistributie niet homogeen is, onderaan het gewas is er namelijk niet veel licht meer over voor de bladeren om te absorberen. Er is relatief veel ver-rood licht over op deze hoogte, aangezien dat in kleine mate wordt opgenomen door de hogere bladeren. De energie van dit ver-rood licht is echter niet in staat om het fotosynthese proces volledig te laten verlopen, maar het kan wel een grote invloed hebben op de plant. In dit onderzoek is onderzocht wat de invloed is van ver-rood licht op de plant Arabidopsis thaliana door een plant van drie tot vier weken bloot te stellen aan constant ver-rood licht voor 24 uur of een week. Als controle experiment zijn planten blootgesteld aan constant wit licht dat zonlicht imiteert.
Uit het onderzoek blijkt dat planten die 24 uur zijn blootgesteld aan ver-rood licht nog even efficiënt fotosynthese uitvoeren als planten die zijn geacclimatiseerd aan wit licht. Planten die een week aan ver-rood licht zijn gewend, vertonen een duidelijke afname in hun efficiëntie. Verder is van planten bekend dat ze mechanismes hebben om een overmaat aan energie om te zetten in warmte, dit wordt ‘quenching’ genoemd. Bij de planten geacclimatiseerd aan één week ver-rood licht werd een extra quenching mechanisme geobserveerd. Het kan zijn dat enkele van de LHCs de energie die ze absorberen niet doorgeven aan de fotosystemen, maar omzetten in warmte. Door te bepalen hoeveel eiwitten er in de bladeren van de planten zitten, kon er gekeken worden of er iets in de eiwitconcentraties in de planten veranderd. Uit de resultaten blijkt dat planten die een week aan ver-rood licht gewend zijn, bijna al hun fotosystemen hebben afgebroken en ook een deel van de LHCs. Dit komt overeen met de theorie dat er LHCs zijn die hun energie niet aan de fotosystemen doorgeven, omdat er zo weinig fotosystemen zijn. Bij de planten die 24 uur aan ver-rood licht zijn geacclimatiseerd is er ook een verandering te zien in het quenching mechanisme. In deze planten is geen afname aan de fotosystemen te zien, maar is er een verdubbeling van een component van LHCII, namelijk van Lhcb2. Deze verdubbeling zou kunnen betekenen dat er drie extra LHCs (een trimeer) aan PSII zitten gebonden die invloed kunnen hebben op het quenching mechanisme. Het kan dus geconcludeerd worden dat ver-rood licht een grote invloed heeft of de LHCs en de quenching die zij veroorzaken.
4
Contents
Abstract ... 2
Populair wetenschappelijke samenvatting ... 3
Introduction ... 5
Materials and methods ... 7
Results and discussion ... 9
Conclusion ... 17
References ... 18
5
Introduction
Plants use light as energy source to drive photosynthesis, during which light energy is used to fix carbon dioxide into sugars.1 This light energy is captured by pigments in the photosynthetic apparatus and transferred to the reaction centers of photosystems that use this energy to release an electron from a core chlorophyll (Chl) pigment to ultimately reduce NADP+ to NADPH, a reducing agent that is used during the carbon dioxide fixation. The light capturing part of photosynthesis occurs in the chloroplast thylakoid membranes (Figure 1).2,3 During photosynthesis H2O is oxidized to O2, NADP
+
is reduced to NADPH, and ATP is synthesized. H2O oxidation is catalyzed by the oxygen evolving
complex (OEC), and the further electron reactions are catalyzed by photosystem II (PSII), the cytochrome b6f complex and photosystem I (PSI). During the photosynthetic reactions a proton
gradient is formed over the thylakoid membrane, resulting in a proton motive force that chloroplast ATP synthases use to generate ATP. Figure 2 shows an overview of the electron transfer processes during photosynthesis.4
The pigments that are used to capture the light energy are organized by light-harvesting complex (LHC) proteins. These antenna complexes are associated with the core proteins of the photosystems and can transfer the captured light energy to the photosystems to drive photosynthesis. The photosystems are large protein assemblies containing hundreds of pigments that are excited upon excitation by light or transferred energy from the antennas.5 In the reaction centers of both photosystems these excitations leads to charge separation, where an electron is released from the core pigment. The spectra of both photosystems are shown in Figure 3. For PSII the core excitation needs light of 680 nm, whereas PSI needs light of 700 nm. Several antenna complexes are tightly bound to the photosystems, whereas others are loosely attached and can be released when light quality or quantity changes. This is mainly observed when the light conditions favor either the excitation of PSII or PSI. LHCII is the antenna that can be coordinated to both photosystems, depending on the available light.6 When light favors PSII, which is at shorter wavelengths, LHCII can be released from PSII and
Figure 1. The structure of a tobacco chloroplast in A) a schematic overview based on B) an electron microscopy measurement and C) a schematic overview of the organization of the thylakoid membrane based on D) an electron microscopy measurement, modified from reference 2. The stacked part of the thylakoid membranes are the grana part, the other unstacked part is called the stroma lamellae.
A
B
C
6 Figure 3. The absorption spectra of PSI and PSII, from reference 5. Dashed lines indicate the
absorption wavelengths of the primary donors of PSII (680 nm, orange) and PSI (700 nm, blue).
coordinate to PSI, thereby altering the absorption spectra for PSI. This shift of LHCII from PSII to PSI is influenced by the phosphorylation of LHCII proteins by the kinase STN7.7 This shift of the antenna is a short-term response to a change in the light availability and is called state transition.If LHCII is associated to PSII, the system is in state 1, and if it is associated to PSI, it is in state 2. On the longer term, the plant will alter its PSII:PSI ratio to balance the photosynthetic processes when the light availability changes.8 In addition to the absorption of the light, antennas can dissipate excess energy by the NPQ process.1 In this process, the absorbed energy is dissipated as heat. NPQ prevents the production of toxic reactive oxygen species that can damage PSII.9
Light availability forms a vertical gradient from the top to the bottom of the canopy, with the top leaves exposed to the full sunlight spectrum and the bottom ones to the left-over light (Figure 4A).10 Comparing the light available for the bottom leaves and their absorption spectra in Figure 4B, indicates that the leaves will not be able to absorb the light and drive their photosynthesis. The absence of absorption in the far-red part of the light spectrum is known as the red-drop.11 It is a prospect to engineer a ‘smart canopy’ in which the bottom leaves are engineered in such a way that they can use this far-red light (FR, 700-760 nm) to drive photosynthesis.12 To fulfill this prospect it is important to understand the reaction of a plant to this FR. This was the goal of this project, to investigate the influence of far-red light on the photosynthetic machinery of the model plant
Arabidopsis thaliana (Arabidopsis).
Figure 2. An overview of the photosynthetic reactions occurring in the thylakoid membrane (left) and the subsequent dark reactions of the Calvin-Benson cycle (right), picture modified from reference 4. Black arrows represent the electron flow from water to the NADPH, which is used in the Calvin-Benson cycle. (1) Light energy is absorbed by the antennas of PSII, (2) which causes the release of an electron of its internal pigment. (3) This electron is used to reduce plastoquinone (PQ), which travels to the cytochrome b6f complex. (4) The electron is used to reduce plastocyanin (PC), that transfers the energy to PSI. (5) The electrons are used to reduce NADP+ to NADPH that is ultimately used in the Calvin-Benson cycle. During the processes in PSII and Cyt b6f protons are pumped into the lumen, creating a proton gradient over the thylakoid membrane. (6)This gradient generates a proton motive force that is used by the ATP synthase complex to generate ATP.
7 Previous research into the influence of FR on Arabidopsis focused on the early development after germination.13 It was found that plants contain FR-sensing complexes called phytochromes that are involved in the early stage of plant development, the greening of plants (de-etiolation), the elongation of the stem and the shade avoidance response.14,15 The phytochromes are activated by the absorption of red light (660-670 nm) and inactivated by absorbing FR (725-735 nm).16 In the active form these proteins locate to the cell nucleus where they are involved in gene expression. Since these complexes will be in their inactive form it is hypothesized that the plants will show less greening and longer stems during exposure to FR. Research that focused on FR-enriched illumination of plants has shown that this condition protects PSI from photoinhibition in fluctuating light17, inhibits shoot branching18, induces flowering19, promotes root growth, increases leaf length20 and bends leaves upward by modifying the petiole angle.
The influence of pure FR was investigated in this project by comparing plants acclimated to FR for 24 hours (24h) or for one week (1wk) with a white light (W) acclimated control plant. The maximum PSII efficiency (Fv/Fm) was determined to check the health of the plants. A Fv/Fm value of 0,83 is
conserved for several plant species and serves as a probe for healthy photosynthesis.10, 21This was followed by measuring the absorption spectra of the leaves to indicate if the absorption of plants acclimated to FR changes with respect to plants acclimated to W. Then the photosynthetic protein content of the leaves and the phosphorylation state of several of these proteins was investigated. At last, the rough PSII/PSI ratio of the plant acclimated to either FR or W was compared to indicate the long term acclimation response to FR.
Materials and methods
Growth conditions
Wild type Arabidopsis plants were grown for 3-4 weeks in a 16h light/day period (22°C day/18°C night) in a controlled climate chamber at a photon flux of 120 mol m-2 s-1 of white light (W16h) and
relative humidity of 70%. The plants were transferred to either constant white light (W) or far-red light (FR) of 743 nm (the spectra of the chambers are given in Supplementary Figure 1). The W chamber was set to a photon flux of 100 mol m-2 s-1, humidity of 60% and temperature of 22,5°C. The FR chamber is homemade and was set to either 58 mol m-2 s-1 for 1wk acclimation or 156 mol m-2 s-1 for24h acclimation. The chamber was further covered with black sheets in order to keep light other than FR out. The humidity was set to 75% and the temperature to 22°C.
Chlorophyll Fluorescence Analyses21
Imaging of
F
v/F
m and NPQ was performed on detached leaves with the DualPam-100 (Fluorescence & P700 photosynthesis analyzer). A quenching protocol was used with Actinic light 1028 mol m-2 s-1 and saturating pulses of 4000 mol m-2 s-1, 800 ms. After 42 seconds the actinic light was turned on and after 522 seconds it was turned off. During the light period a saturating pulse was used every 20Available light in the canopy
A
0 0,2 0,4 0,6 0,8 1 1,2 1,4 350 550 750 Ab so rp tio n (a .u .) Wavelength (nm)B
Absorption of ArabidopsisFigure 4. A) The available light in a canopy, from reference 10. These spectra show the low light availability in the bottom of the canopy and the relatively high abundance of FR there. B) The absorption of a four weeks old Arabidopsis thaliana leaf, measured by an integrating sphere. This spectrum indicates the low absorption of far-red light.
8 seconds and during the dark period every 50 seconds. Plants were dark adapted for at least 30 min prior to measurements. In addition to the DualPam measurement, the NPQ values of whole plants was determined with a FluorCam 700 MF System (Photon Systems Instruments) using the quenching analysis 1 effect settings (Act1 70% (170 mol m-2 s-1); Act2 40% (600 mol m-2 s-1); Super 40% (1150 mol m-2 s-1), 800 ms). FluorCam version 1.6 software was used for the imaging system and to process images. To compute the
F
v/F
m and NPQ of the plant, the values of individual leaves were averaged. An overview of the different stages during these quenching experiments is shown in Supplementary Figure 2.Chlorophyll composition
The chlorophyll concentration and ratio between Chl a/Chl b was determined by fitting spectra of 80% acetone (-20°C) samples of the isolated thylakoids (dilution 200x) with the spectra of the individual pigments.22 The spectra were recorded with a Cary 4000 UV-VIS spectrophotometer (Agilent technologies). The chlorophyll content per area for the 24h acclimated plants was determined by grinding a certain area of the leaf in liquid nitrogen. This grinded leaf was mixed vigorously in 80% acetone (-20°C) and spun down (13300 rpm, 5 min). The supernatant was used for measuring the absorption (dilution 20x). Furthermore, the absorption of the whole leaf was measured, using leaves of four-week-old plants acclimated to W (24h), FR (24h) and FR (1wk). For this purpose the extension of the Cary 4000 with an integrated sphere (Agilent technologies) was used.
Thylakoid isolation6
Everything was performed under weak light at 4°C or on ice. Three-week-old Arabidopsis plants that were acclimated for 1wk or 24h to constant W or FR were harvested and homogenized in ~80 mL of 20 mM Tricine-NaOH, pH 7.8, 0,4 M sorbitol, 5 mM MgCl2, 5 mM EDTA and GmbH protease
inhibitor* with a metal blender for four periods of 8 seconds at high speed. The homogenate was filtered through 4 layers of nylon mesh, after which the filtrate was centrifuged (10 min, 1400g). The pellet was suspended in 20 mM Tricine-NaOH, pH 7.8, 0,15M sorbitol, 5 mM MgCl2, 2,5 mM
EDTA, and GmbH protease inhibitor* and centrifuged (10 min, 4000g). The pellet was suspended in 20 mM Hepes, pH 7.5, 15 mM NaCl, and 5 mM MgCl2 and centrifuged (10 min, 5500g). The pellet
was suspended in 2 ml 10 mM Hepes, pH 7.5, 0,4M Sorbitol, 15 mM NaCl, 5 mM MgCl2 and stored
at -80°C. *For the 24h thylakoid isolation 1 mM acido e amino caproic acid and 0,2 mM benzamidina were used as protease inhibitors.
First dimension polyacrylamide gel electrophoreses and immunoblotting6
Thylakoid membrane proteins diluted in loading buffer (2% SDS, 8% sucrose, 0,2 mM EDTA, 10 mM Tris (pH 6.8), and 4% -Mercaptoethanol, stained with bromophenol blue) with a final concentration of 0,1 mg Chl/ml were separated by sodium dodecyl sulfate polyacrylamide gel electrophoreses (SDS-PAGE, 0,75 mm, 6% acrylamide stacking gel and 12 % separation gel), and the proteins were transferred to a nitrocellulose membrane (Amersham) through wet-transfer. The proteins were then visualized using a ponceau stain. The membranes were blocked with skimmed fat free dry milk (10% in TTBS (50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0,001% (v/v) Tween20), stirred for 1h at RT) and incubated with primary antibodies for D1DE-loop, PsaB, Lhcb1, Lhcb2, Lhca1, CP43 or phosphorylated threonine (purchased from Agrisera). In the case of the primary antibody for phosphorylated threonine the membrane was blocked with 5% BSA instead of milk. After overnight incubation at 4°C the membranes were washed with TTBS (4*10 min) at RT, after which the membranes were incubated with secondary anti-rabbit antibody (1:10.000 dilution) conjugated with horseradish peroxidase (Agrisera) in 20 ml TTBS for 1h at RT. The membranes were washed with TTBS (5*5 min and 2*15 min). The bands were visualized by using a peroxide
9 incubation (5 min) and chemiluminescence detection on a LAS-3000 imager (Fuji). The blots were quantified using ImageJ software.
Blue-Native gels23
During all steps the samples were kept on ice and in low light. Isolated thylakoids were divided into aliquots of 8 g Chl / aliquot and spun down (13300 rpm, 10 min). The pellet was washed with 15 l 5mM EDTA. The suspension was spun down (13300 rpm, 4 min) and the pellet was suspended in 11,5 l 25BTH2OG buffer (0,25 M BisTris/HCl (pH 7,0), 20% (w/v) glycerol) and 2 l n-dodecyl -D-maltopyranoside (-DDM, 5% in 25BTH2OG buffer). The sample was incubated on ice for 13 min with tapping every 2 min. The suspension was spun down (1200g, 9 min) and the pellet was discarded and 1,5 l loading buffer (100 mM BisTris (pH 7,0), 0,5M ACA, 30% (w/v) sucrose, 50 mg/ml Serva Blue G) was added to the supernatant to a final Chl concentration of 0,5 mg/ml. The samples were loaded on BN gels (0,75 mm, 4% stacking and 4-12,5% acrylamide separation gel) based on 7,5 g Chl/well. The gels were run using a 50 mM BisTris/HCl (pH 7,0) anode buffer and a 15mM BisTris/HCl (pH 7,0) and 50 mM Tricine cathode buffer with and without 0,01% Serva Blue G. The Serva Blue G cathode buffer was used until the sample reached halfway the gel, then the cathode buffer was replaced for the one without Serva Blue G. Second dimension separation of the native protein complexes were performed on a SDS-PAGE gel (1,0 mm, 6% stacking and 12% acrylamide separation gel). The bands were visualized using a Serva Blue G Coomassie stain (0,025% in 10% acetic acid).
77K measurements for the PSI/PSII ratio
Isolated thylakoids were diluted in a 10 mM Hepes, pH 7.5, 0,4M Sorbitol, 15 mM NaCl, 5 mM MgCl2 solution (200x dilution) and were frozen in liquid nitrogen. The fluorescence of this sample in
the range of 600-800 nm was measured upon excitation of Chl a at 440 nm with a FluoroLog Spectrophotometer (Horiba). The average of three measurements was used.
Pph mutant experiment
Arabidopsis thaliana pph mutants were grown at W16h for four weeks, after which they were
acclimated to W (100 mol m-2 s-1) or FR (156 mol m-2 s-1)for four days. Then the phenotype was documented and compared to wild type plants.24
Results and discussion
Phenotypes
The first observation was that WT Arabidopsis seeds do not germinate under FR, indicating the need for other light qualities to initiate the greening process (data not shown). The phenotype of Arabidopsis plants after acclimation to FR changes with respect to the phenotype of plants acclimated to W, which is shown in Figure 5. It can be seen that after 24h of FR, the leaves reach for light and curl slightly inside, which might indicate that the plant does not feel enough light. After 1wk FR the leaves are curled inside completely and they become smaller and thinner, compared to the W acclimated leaves. Furthermore, it can be seen that the W plant grows and develops new leaves and the FR acclimated plant does not. This indicates that FR is not sufficient for the plant to grow new leaves. Additionally, the plant is more pale green after acclimation to FR than to W, which indicates the degradation of chlorophyll. Increase of the stems and the curling of the leaves might be assigned to the phytochromes, since these changes have been observed before.17,18
10
Photosynthetic efficiency
To investigate the efficiency of the photosynthesis of plants acclimated to 24h or 1wk of W or FR, DualPam measurements were carried out. These measurements are shown in Figure 6. On the top the original fluorescence is shown and on the bottom the corresponding NPQ values. The NPQ traces of both the 1wk and the 24h FR acclimated leaves show two parts when the actinic light is on. In the first 180s the NPQ value seems to flatten out at a lower NPQ than the W sample, roughly at half the NPQ value. This is also observed in FluorCam measurements for the 24h FR acclimated plant (Supplementary Figure 3). However, after these 180 seconds the NPQ increases linearly until the actinic light is turned off. In the 1wk FR acclimated plants, this is a massive increase and the plant
Figure 5. Phenotypes of Arabidopsis after acclimation to 24h or 1wk of constant white (W) or far-red (FR) light.
Figure 6. The fluorescence and NPQ spectra of Arabidopsis plants acclimated for 1wk or 24h to white or far-red light. The white bar means that the actinic light was on during the measurement and the black bar means it was off.
11 cannot relax back to its initial F0 value after the light is off. This indicates that the PSII core is
photodamaged and is no longer able to do photochemistry. In the 24h FR acclimated plants the shape of the trace differs from the W acclimated plants. The maximum NPQ value for the FR and W acclimated plants is similar, only the shape of the trace changes. The inability to fully relax back to F0
again indicates that there is photoinhibition of the PSII core.
Acclimation 24h 1wk Fv/Fm W 0,834 (± 0,010) 0,827 (± 0,011) FR 0,824 (± 0,010) 0,742 (± 0,008) Chl a/Chl b W 3,45 (± 0,02) 3,79 (± 0,03) FR 2,94 (± 0,03) 2,69 (± 0,19) Chl per area (mg/cm2) W 0,0212 (±0,0006) - FR 0,0187 (±0,0004) -
With the DualPam the maximum quantum yield of PSII (
F
v/F
m)
could also be determined. The results for these measurements are listed in Table 1. These values indicate that acclimation to FR does not influence the PSII efficiency after 24h of acclimation, since theF
v/F
m value is still within the error range of the value measured for the W acclimated plant. However, after 1wk the plant starts to suffer, which leads to a decrease of theF
v/F
m to 0,742 (±0,008). This value indicates that the plant is severely stressed. It was also tested if the photosensitive reaction seen during the DualPam measurement was reversible by acclimating plants first to 24h FR, followed by a recovery period of 24h W. The NPQ traces of these measurements are shown in Figure 7. From these data it can be concluded that the effect of 24h FR can be reversed by 24h W. However, this does come with a price for the PSII efficiency, since theF
v/F
m value after recovery decreased to 0,794 (±0,019). This decrease might be caused by the sudden transition from FR to W and the inability for the FR plant to cope with this change. As shown by the DualPam measurements the plant is photosensitive after 24h FR, which may cause some inhibition in the recovering plant. More tests should be carried out to investigate if the plant can recover from 24h of FR completely and how long this would take. The results should also be compared to the NPQ trace of a plant acclimated to 48h of constant W to determine if the lower maximum NPQ value is also observed after longer exposure to W.Figure 7. The NPQ values from a DualPam measurement of Arabidopsis plants acclimated for 24h to white light (W 24h), far-red light (FR 24h) or to 24h far-red light followed by a 24h recovery period in white light (Recovery).
12
The chlorophyll content
To investigate whether or not the absorption of the leaves changes upon acclimation to FR, this absorption was measured using an integrating sphere. The absorption spectra are shown in Figure 8. From these spectra it can be seen that Arabidopsis plants do not absorb at wavelengths longer that 734 nm (1wk FR) or 760 nm (24h W or FR). Since the FR chamber supplies light in the range of 700-790 nm, the plants should be able to absorb some light. Interestingly, a long FR irradiance (1wk) leads to less absorption of FR. This might indicate that the plant degrades FR absorbing units in the leaves, such as PSI subunits and antennas.5 This is supported by the observation that plants die after two weeks of FR acclimation (data not shown). The hypothesis could be further tested by determining the composition of the thylakoid membranes after 24h and 1wk FR acclimation.
From the Chl a/Chl b values listed in Table 1 it can be seen that FR acclimated plants show a decrease in this ratio, which indicates either or both a decrease in the photosystems (mainly Chl a) or a relative increase in the light harvesting complexes (both Chl a and Chl b). The different Chl a/Chl b values of plants acclimated to 24h of W or FR, 3,45 (±0,02) to 2,94 (±0,03), respectively, might indicate that the FR acclimated plant contains three trimers per core compared to two trimers for the W acclimated plants if the PSI/PSII is kept constant.25 After 1wk the plants acclimated to FR show an even larger decrease in Chl a/Chl b to 2,69 (±0,19) compared to W with a value of 3,79 (±0,03). This supports the hypothesis that far-red absorbing proteins are degraded, resulting in less Chl a and a lower Chl a/Chl b ratio. Higher Chl a/Chl b observed for plants acclimated to a long time of constant W has been reported before.26 Plants acclimated to 24h FR also show a slightly decreased Chl per area value, which might be caused by the extension of the leaves observed in Figure 4. This might dilute the chlorophyll content per area, which is also shown in the absorption of the leaf in Figure 8 for the 24h FR acclimated plant.
Protein content
To explain the photosensitivity and the decreased Chl a/Chl b in the FR treated plants, the thylakoids of plants acclimated to 1wk or 24h of W or FR were isolated and separated on SDS-PAGE or BN-gels. In Figure 9A the relative protein levels of plants acclimated to 1wk W or FR is visualized, using antibodies against PsaB, D1, Lhcb2 and Lhca1. It can be seen that especially the photosystems (PsaB for PSI and D1 for PSII) are reduced in the FR samples. Also the LHCs seem to be reduced in the FR Figure 8. The absorption of an Arabidopsis leaf acclimated for 24h to white light (W 24h) or far-red light (FR 24h) or for one week to far-red light (FR 1wk) measured by an integrated sphere. On the left the crude spectra are shown and on the right the spectra are normalized to 679 nm.
13 plants, but not as much as the photosystems. Since the plants also seem to show the lower PSII efficiency, the plants probably have insufficient light to grow and are degrading the protein content to survive. The lower D1 content might explain the sensitivity during the DualPam measurement, since it has not enough resources to efficiently perform photosynthesis and consequently NPQ. Also Figure 9B shows a reduction in all protein assemblies in the FR. This is in agreement with the theory that the proteins, especially the red-absorbing PSI, are degraded after 1wk of FR.
The experiments were repeated with plants acclimated to 24h W or FR to investigate the influence of FR without the plant being too stressed, as seen for the 1wk FR. These SDS-PAGE separated proteins are shown in Figure 10, using antibodies against D1, PsaB, Lhca1, Lhcb1, Lhcb2, CP43 and phosphorylated threonine. From these immunoblotting experiments it can be seen that the levels of D1, PsaB, Lhca1, Lhcb1, and CP43 do not change significantly. On the contrary, the Lhcb2 content seems to increase with a factor of 2,16 (± 0,34) in the FR sample. Since Lhcb2 is composed of both Chl a and Chl b, this increase would explain the decrease in Chl a/Chl b, since there is more Chl b.27 The unchanged levels of the photosystems indicate that the decrease in the Chl a/Chl b ratio is caused by this increase in LHCII. In order to absorb more light the plant apparently produces more antennas. Therefore, the observed Chl a/Chl b ratio for the FR plant corresponds to three LHCII trimers per PSII core and the ratio for the W acclimated plant for two trimers per core.25
In addition to this change in Lhcb2, Figure 10 shows a change in the phosphorylation state of several thylakoid proteins. It can be seen that in the FR thylakoids both CP43 and the LHCII antennas are not phosphorylated, whereas they are phosphorylated in the W sample. Phosphorylation of LHCII antennas is known to be involved in the transition from state 1 to state 2, where LHCII protein complexes, especially Lhcb2, moves from PSII to PSI.6 This phosphorylation is performed by the STN7 kinase.7 The absence of LHCII phosphorylation could indicate that the LHCII antennas are Figure 9. A) SDS-PAGE of isolated thylakoids of Arabidopsis plants (3 weeks) acclimated to constant white (W) or far-red (FR) light for one week. The gels were transferfar-red to a cellulose membrane. The proteins are visualized using a Ponceau stain (left) and antibodies against PsaB, D1, Lhcb2 and Lhca1 (from top to bottom, right picture). The loading is based on 1,0 g Chl. B) BN-PAGE of isolated thylakoids of Arabidopsis plants (3 weeks) acclimated to W of FR for 1wk. The loading is based on 7,5 g Chl. The picture was taken of the gel directly.
14 Figure 10. On the left a ponceau stain of isolated thylakoids of Arabidopsis plants acclimated to 24h of white (W) or far-red (FR) light separated on a SDS-PAGE gel. The loading is based on Chl (0,1 g/l, given numbers in l). On the right the immunoblots are shown with antibodies against D1, PsaB, Lhca1, Lhcb1, Lhcb2, CP43 and phosphorylated threonine (from top to bottom). The phosphorylated threonine shows three bands that indicate CP43, D1&D2 and LHCII (from top to bottom).
associated with only PSII, which can be explained by the light availability. PSII antennas normally absorb at lower wavelength than PSI antennas and since there is only light of 740 nm, PSII has more trouble absorbing light than PSI. Therefore, PSII recruits all available antennas in order to start the photosynthesis process. Dephosphorylation of the LHCII proteins can indicate that the kinase, STN7, is inactivated under the FR conditions or that the phosphatase TAP38 is highly activated.28 The conditions used during the DualPam measurements might induce phosphorylation of LHCII complexes and the movement of these complexes to PSI. This might explain the different shape observed for the NPQ during the measurement in Figure 6. After several pulses STN7 might be activated and the LHCII complexes might be phosphorylated, which causes them to move to PSI through the thylakoid membrane. This movement might induce the quenching after 180 seconds. This theory could be tested by determining the phosphorylation state of the LHCII complexes before and after several saturating pulses.
In addition to LHCII, CP43 is dephosphorylated in the FR sample. Phosphorylation and dephosphorylation of CP43 is known to be involved in PSII repair after oxidative damage to D1.29 Phosphorylation of the PSII core components D1, D2 and CP43 have been shown to be necessary for the disassembly of PSII, which is followed by the separation of the dimer in monomers. The phosphorylated monomer travels to the stroma lamellae and gets dephosphorylated. Then the CP43 protein detaches from the monomer and a new D1 protein is incorporated, after which CP43 attaches again to the PSII monomer and two monomers form a dimer. The absence of phosphorylation of CP43 might indicate that the plant is no longer able to repair photodamaged PSII cores or that it was not in need of repairing them under FR. An inability to repair the PSII cores on a short term would explain the NPQ curve for FR acclimation shown in Figure 6. After several saturating pulses and a certain amount of actinic light, the PSII cores might get photodamaged and the leaf might not be able to repair the proteins. This would explain the inability to relax back to F0 during the dark period of the
15 DualPam measurements in Figure 6. This theory could be proven by measuring D1 turnover in FR acclimated plants prior and after exposure to several high light pulses. The Photosystem II Core Phosphatase (PBCP) has been implicated as phosphatase for all PSII core proteins, which cannot explain the high selectivity for CP43.30 The absence of phosphorylation might be assigned to the STN8 kinase, which is known to phosphorylate D1, D2 and CP43.31 It might be that the phosphorylation side of CP43 is shielded due to a change in the PSII organization and thus shielded from the kinase. This could be investigated by electron microscopy. The dephosphorylation of CP43 in FR acclimated plants is in contradiction with earlier studies, which indicated decreased phosphorylation of D1, D2 and LHCII, but not of CP43 after 1h of FR illumination.32 This difference might be caused by the different FR acclimation times.
To investigate if the native membrane bound protein complexes differ between the 24h samples, a second dimension SDS-PAGE of a BN-gel was performed and pictures of the gels are shown in Figure 11B. This gel shows a small difference between the W and FR sample in the band of PSI-LHCI, PSII core dimer. In this band the FR sample shows one spot that is more apparent in the FR sample than the W sample. This spot could be linked to the light harvesting complexes coordinated to PSI, such as Lhca3 or Lhca2, or subunits of PSI itself, such as PsaD, PsaF or PsaL.33 These gels should be investigated in more detail with immunoblotting.
Photosystems
Since FR is more in the absorption range of PSI, it is expected that the ratio between PSI and PSII might be changed in the plant acclimated to 24h FR. From the immunoblots in Figure 10 it was seen that the relative levels of PSI and PSII do not change, which implicates that the PSI/PSII ratio does not change. This was confirmed with a 77K fluorescence measurement, which measures the
Supercomplexes LHCII trimer
FR
W
PSII monomer LHCII-CP24-CP29 PSI-LHCI, PSII core dimer W 24h FR 24hA
B
Figure 11. A) Isolated thylakoids of plants acclimated to 24h of white (W) or far-red (FR) light separated on a BN gel. The loading was 15 l (0,5 g Chl/l). B) The BN-gels run in the second dimension on a SDS-PAGE gel.
16 fluorescence after excitation of Chl a at 440 nm. The normalized graph is shown in Figure 12. The peaks at 680 nm originate from PSII and the peak at 730 nm from PSI. From the normalized graph it can be seen that the ratio between PSI and PSII does not change after 24h of either W or FR, which is in agreement with the immunoblots. The broad peak shown at 622 for the W trace is due to a lower concentration of isolated thylakoids in this measurement. This peak can be ignored for the assessment of the PSI/PSII ratio.
Chlorophyll degradation
The pph mutant lacks the gene for making pheophytin pheophorbide hydrolase (pheophytinase), which means that the plant is not able to degrade chlorophyll molecules.24 The phenotype of two wild type (WT) and pph Arabidopsis plants was documented before and after four days of constant FR, which is shown in Figure 13. From these pictures it can be seen that the pph mutant dies already after four days of FR, whereas the WT plants are just slightly affected. This indicates that the plant needs to degrade chlorophyll in order to survive under FR.
Figure 12. The fluorescence spectra of isolated thylakoids after excitation at 440 nm measured at 77K. The peaks are normalized to 682 nm.
Figure 13. Phenotype of WT plants and pph mutants of Arabidopsis grown for four weeks after which they were transferred to FR.
17
Conclusion
The influence of one week and 24 hours of far-red light on Arabidopsis thaliana plants was investigated. The first observation was the changing phenotype with longer leaves in the FR acclimated plants. This has been observed before in experiments investigating the role of the phytochromes.17, 18 From DualPam measurements it became evident that there is additional quenching in the 1wk FR treated plants relative to W acclimated plants (Figure 6). The FR acclimated plant was also not able to relax back to its initial F0 value, which indicates that PSII is photodamaged. Since this
happens only after several pulses under actinic light, the prolonged light exposure is believed to induce the damage on the photosystem that leads to uncontrolled NPQ. Furthermore, a strong decrease in the Fv/Fm indicates that the plant isnot doing healthy photosynthesis. Further absorption
measurements of an intact leaf with an integrating sphere supports the theory that photosystems are degraded in the plant acclimated to 1wk FR. It can be seen that the overall absorption is reduced in this sample (Figure 8) and that the absorption in the FR after 734 nm is completely gone. This indicates the loss of PSI-LHCI complexes, which are known to absorb in this area. The decreased Chl a/Chl b ratio and immunoblots confirms that both PSI and PSII are largely degraded, making the plant extremely photosensitive. Therefore, one week of acclimation to FR induces a senescence-like process.
After 24h FR acclimation, the DualPam measurement does not show the uncontrolled NPQ increase. The trace still consists of two parts, but the maximum NPQ values for the FR and W acclimated plants are similar. The FR acclimated plant does show photoinhibition, since it is not able to fully relax back to its initial F0 value. The Fv/Fm value suggests that the plant is doing healthy
photosynthesis, although the Chl a/Chl b value is strongly decreased. Both immunoblots and 77K emission spectra show that the PSI/PSII ratio has not changes in the 24h FR acclimated plant. The decrease in the Chl a/Chl b ratio indicates that the FR acclimated plants contains roughly three LHCII trimers per PSII core, compared to two trimers per PSII core for the W acclimated plant. According to a SDS-PAGE separation of the thylakoid proteins and subsequent immunoblotting, this additional LHCII trimer consists of Lhcb2, which shows an increase of a factor 2,16 (± 0,34) in the FR acclimated plant. From the blots it is also observed that LHCII complexes are not phosphorylated in the plants acclimated to 24h FR. This indicates that the plants are in absolute state 1, where all LHCII antennas are associated with PSII. During the DualPam measurements these proteins might get phosphorylated by the kinase STN7, due to the availability of light and the activation of the kinase. A state transition might be induced, causing the trimer to dissociate from PSII to go to PSI. During this trip through the thylakoid membrane it might induce quenching. The theory could be tested by looking at the structure of PSII and its associated antennas with electron microscopy and more detailed investigation of the 2D gels protein content, for example by immunoblotting PSII and Lhcb2 and quantifying these blots. The inability to relax back to F0 during the DualPam measurements again
indicates photodamage. This might be explained by the complete dephosphorylation of CP43 observed in the 24h FR acclimated plants. The phosphorylation of CP43 is known to be involved in the PSII core repair-cycle, which might be inactive in the 24h FR acclimated plant. Importantly, such opposed behavior of the tightly controlled phosphorylation of the PSII core proteins has never been observed before. The impact of this unusual CP43 phosphorylation behavior could be tested by measuring D1 turnover in plants acclimated to FR when they are subjected to several high light pulses.
In further studies it would also be interesting to determine the relative PsbS levels, since this is one of the major proteins associated with NPQ.34 In addition to this the relative concentrations of the carotenoid pigments should be determined, since they are also involved in several quenching mechanisms. Once the influence of FR has been investigated, it might be possible to change the photosynthetic machinery to make photosynthesis in FR more efficient.
18
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20
Supplementary figures
Supplementary Figure 1. Spectra of the W (100 E), FR (58 E) and FR (156 E) chambers.
0 10 20 30 40 50 60 70 80 350 450 550 650 750 850 In te n si ty ( W /cm 2/n m ) Wavelength
Spectra of the light chambers
W 100 uE FR 58 uE FR 156 uE
Supplementary Figure 3. Fluor Cam measurement for the 24h samples acclimated to white (W) or far-red (FR) light.
0 50 100 150 Fl u o re sce n ce ( a.u .) Time (s)
Quenching experiment
F0 F0’ Fv Fm Fp Saturating pulse Actinic light onActinic light off
Fm’
Low light on
Supplementary Figure 2. An overview of the stages during the quenching experiments with the FluorCam. The DualPam measurement shows the same stages, with longer exposure times.
