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First published online July 7, 2006; 10.1104/pp.106.081489 Plant Physiology 142:75-87 (2006) © 2006 American Society of Plant Biologists Differential Expression and Localization of Early Light-Induced Proteins in Arabidopsis1Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE–10691 Stockholm, Sweden (M.H., H.N., M.D., B.A., I.A.); Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland (M.H.); Bio-Rad Laboratories AB, SE–17222 Sundbyberg, Sweden (H.N.); Department of Physiology and Plant Biochemistry, University of Konstanz, DE–78457 Konstanz, Germany (V.R., I.A.); Department of Cardiology, University of Maastricht-Cardiovascular Research Institute of Maastricht, 6229 ER Maastricht, The Netherlands (M.D.); Division of Cell Biology, Linköping University, S–58185 Linkoeping, Sweden (B.A.); and European Science Foundation, F–67080 Strasbourg cedex, France (B.A.)
The early light-induced proteins (Elips) in higher plants are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting chlorophyll (LHC) a/b-binding proteins. A photoprotective function was proposed for Elips. Here we showed that after 2 h exposure of Arabidopsis (Arabidopsis thaliana) leaves to light stress Elip1 and Elip2 coisolate equally with monomeric (mLhcb) and trimeric (tLhcb) populations of the major LHC from photosystem II (PSII) as based on the Elip:Lhcb protein ratio. A longer exposure to light stress resulted in increased amounts of Elips in tLhcb as compared to mLhcb, due to a reduction of tLhcb amounts. We demonstrated further that the expression of Elip1 and Elip2 transcripts was differentially regulated in green leaves exposed to light stress. The accumulation of Elip1 transcripts and proteins increased almost linearly with increasing light intensities and correlated with the degree of photoinactivation and photodamage of PSII reaction centers. A stepwise accumulation of Elip2 was induced when 40% of PSII reaction centers became photodamaged. The differential expression of Elip1 and Elip2 occurred also in light stress-preadapted or senescent leaves exposed to light stress but there was a lack of correlation between transcript and protein accumulation. Also in this system the accumulation of Elip1 but not Elip2 correlated with the degree of PSII photodamage. Based on pigment analysis, measurements of PSII activity, and assays of the oxidation status of proteins we propose that the discrepancy between amounts of Elip transcripts and proteins in light stress-preadapted or senescent leaves is related to a presence of photoprotective anthocyanins or to lower chlorophyll availability, respectively.
The chlorophyll (chl)-binding proteins in photosynthetic organisms are located either in core complexes of PSI and PSII or form antenna systems (Green and Durnford, 1996  ). The antenna systems of higher plants consist of nuclear-encoded chl a/b-binding (Cab) proteins whose primary function is the absorption of light through chl excitation and transfer of absorbed energy to photochemical reaction centers.
The Cab superfamily in Arabidopsis (Arabidopsis thaliana) consists of 20 different proteins (Jansson, 1999
In the past years several distant relatives of Cab protein superfamily with conserved chl-binding residues and a transient expression pattern were found in PSI and PSII of pro- and eukaryota. These distant relatives belong to the early light-induced protein (Elip) family of light stress proteins (Montané and Kloppstech, 2000
The Elip family members in higher plants and green algae are nuclear-encoded proteins that accumulate in thylakoid membranes in response to various abiotic stresses, when the expression of Cab proteins is down-regulated (Montané and Kloppstech, 2000
Previous studies showed that Elip from pea (Pisum sativum) is located in the nonappressed regions of the thylakoid membrane in the vicinity of PSII (Adamska and Kloppstech, 1991 Here, we investigated the localization of Elips in green light-stressed leaves of Arabidopsis and compared their expression in light stress-preadapted or senescing leaves exposed to low or high intensity light. Two closely related Elip1 and Elip2 (81.05% identity at the amino acid level) are present in Arabidopsis, thus raising the question about their redundant physiological function. Alternatively, Elip1 and Elip2 may perform their function at different intramembrane locations or during different stages of light stress. We demonstrated that although both proteins were found in isolated mLhcb and tLhcb under light stress conditions they coisolated with different LHCII subpopulations. Interestingly, during the first 2 h of light stress the amount of Elips in relation to the amount of tLhcb and mLhcb was almost equal, but a longer exposure to light stress resulted in higher Elip amounts in tLhcb as compared to mLhcb. We showed further that both proteins were differentially expressed in response to light stress in green, light stress-preadapted and senescing leaves and their expression was controlled at transcriptional and/or transcriptional/posttranslational levels. However, it is not known whether the steady-state level of Elip transcripts is controlled by their transcription and/or degradation rates. While the accumulation of Elip1 occurred in parallel to the inactivation and damage of PSII, a massive accumulation of Elip2 was assayed when approximately 40% of PSII was photodamaged and lost their D1 protein. This suggests that Elip1 and Elip2 function is related to different phases of light stress and occurs in different LHCII subpopulations.
Coisolation of Elip1 and Elip2 with PSII Antenna
Cross-linking studies indicated that Elip in pea is located in the vicinity of PSII reaction center (Adamska and Kloppstech, 1991
To investigate the distribution of Elips in collected gradient fractions anti-Elip1 or anti-Elip2 antibodies were raised and used for such analysis. The results revealed that both proteins were located mainly in fractions 11 and 12 (83.5% and 90% of the total amount of Elip1 and Elip2, respectively) with 16.5% of the total amount of Elip1 detected in the fraction 10 and 10% of the total amount of Elip2 in the fraction 13. Using discontinuous 0.5% to 10% (w/v) Suc gradients (1 mL of 0.7% steps of increasing Suc concentration) more drastic differences in the distribution of Elip1 and Elip2 were observed (data not shown). These data indicate that Elip1 and Elip2 colocalize with LHCII but their different distribution in the gradient suggests their association with different LHCII subpopulations.
When isolated from thylakoid membranes Lhcb1 to 3 proteins were mainly organized into homo- or hetero-tLhcb or mLhcb (Caffarri et al., 2004
It was reported that the tLhcb pool dissociates into the mLhcb population upon light stress (Garab et al., 2002 ). To investigate whether the distribution of Elip1 and Elip2 in mLhcb and tLhcb populations changes during the duration of light stress we isolated pigment-protein complexes at various time points of light stress exposure. The results revealed (Fig. 2C) that no significant changes in amounts of PSI and mLhcb were observed during the exposure of leaves to light stress for 3 h. In contrast, the amounts of tLhcb or PSII core complexes decreased almost linearly with the time of exposure to light stress and reached after 3 h of illumination 50% and 55% of the initial values, respectively. Immunoblot analysis showed that Elip1 and Elip2 were not detected in control leaves or leaves exposed to light stress for 1 h but their amounts increased drastically during a longer illumination period (Fig. 2D). After 2 h of illumination approximately 30% and 17% of the maximal amount of Elip1 present in 3 h illuminated leaves and 10% and 14% of Elip2 were detected in mLhcb and tLhcb, respectively. Similar to data shown in Figure 2B, the amounts of Elips in relation to amounts of tLhcb or mLhcb were comparable after 2 h of illumination (Fig. 2D). However, after 3 h of the high light exposure approximately 40% of the total Elips amount was detected in tLhcb as compared to 60% in mLhcb but due to a strong decrease of the tLhcb amount (50% of the initial value) the ratio of Elip:tLhcb was much higher (40%:50%) than Elip:mLhcb (60%:100%).
To test whether Elip1 and Elip2 are differentially expressed under light stress conditions we analyzed the accumulation of both proteins and their transcripts in leaves exposed to increasing light intensities from 100 µmol m–2 s–1 (low light conditions) to 2,000 µmol m–2 s–1 (severe light stress conditions). Northern-blot analysis revealed (Fig. 3A
) that although the level of transcript accumulation for these two genes increased in a light intensity-dependent manner, different accumulation profiles were observed for each of these mRNAs. While Elip1 transcripts accumulated almost linearly with an increasing light intensity starting from 500 µmol m–2 s–1 and reaching the maximal level at 2,000 µmol m–2 s–1, a comparable amount of Elip2 transcripts was induced at 500 µmol m–2 s–1 and 1,000 µmol m–2 s–1 (Fig. 3A). Exposure of leaves to 1,500 µmol m–2 s–1 resulted in a 6-fold increase of Elip2 transcripts but higher light intensities did not significantly influence this accumulation level. In agreement with reports published for barley (Hordeum vulgare; Pötter and Kloppstech, 1993
Immunoblotting analysis revealed that Elips accumulated in thylakoid membranes with kinetics resembling the accumulation pattern of their transcripts (Fig. 3B). Similarly, the amount of Lhcb2 protein followed the level of transcript amount and decreased with an increasing light intensity (Fig. 3B). The amounts of  / -subunits of the CF1-ATP-synthase complex (CF1-/) assayed as a control did not change significantly during the light stress treatment (Fig. 3B). To assess how the increasing irradiance influences the translational activity of Elip1 and Elip2 transcripts we analyzed the distribution of these mRNAs between polysome-bound and -free fractions. During the first step of protein synthesis in the cytoplasm, mRNAs are integrated into the ribosomal complex and polysomes are formed. Analysis of the mRNA content of polysomes is assumed to be indicative of active protein synthesis. Our results revealed that over 99% of Elip1 and Elip2 mRNAs were detected in the polysomal fraction, indicating their simultaneous translation into corresponding proteins. The distribution of Lhcb2 transcripts, assayed for comparison, demonstrated (Fig. 3C) that also these mRNAs were actively translated. Less than 1% of the total amount of Elip1, Elip2, and Lhcb2 transcripts were present in polysome-free fractions under all light intensities tested. Interestingly, exposure of leaves to light stress resulted in a drastic decrease of polysomal complexes followed by the enrichment in monosomes (Fig. 3D). While under low light conditions 71% of ribosomes formed polysomes and only 29% were present as monosomes, under light stress conditions the polysome amount was reduced to 55% and monosomes increased to 45%. This suggests a selective translation of induced Elip1 and Elip2 mRNAs under light stress conditions.
It was reported that the accumulation of the carotenoid biosynthesis-related (Cbr) protein, a homolog of Elip in the green alga Dunaliella salina, occurs in parallel with the accumulation of photodamaged PSII reaction centers (Jin et al., 2001
As reported before (Andersson and Aro, 2001 ), the exposure of leaves to light stress led to reduced amounts of D1 protein, probably when the degradation rate exceeded the rate of its de novo synthesis (Fig. 4C). The loss of D1 protein occurred in parallel to the inactivation of PSII, and the steady-state level of D1 in leaves exposed to 2,000 µmol m–2 s–1 was reduced to approximately 50% as compared to the amount present in control leaves. Quantifications of Elip signals in immunoblots revealed (Fig. 4D) that Elip1 accumulated almost linearly with increasing light intensities and its level of accumulation correlated with increasing photoinactivation (Fig. 4, A and B) and photodamage (Fig. 4C) of PSII reaction centers. In contrast, the level of Elip2 accumulation was low (7%–13% of the maximal value) in leaves exposed to 100 to 1,000 µmol m–2 s–1 and a massive accumulation (92%–100%) of these proteins occurred only at 1,500 to 2,000 µmol m–2 s–1, when at least 40% of PSII reaction centers were photodamaged (Fig. 4C).
We investigated the expression pattern of Elip1 and Elip2 transcripts and proteins in Arabidopsis leaves preadapted to moderate light stress conditions and in senescent leaves exposed to low or high light conditions. Exposure of low light-acclimated leaves to moderate light stress conditions was accompanied by a massive accumulation of anthocyanins (Merzlyak and Chivkunova, 2000
The expression of senescence-associated gene 12 (SAG12) was assayed as a molecular marker for naturally induced senescence. This gene encodes a Cys protease (Lohman et al., 1994 ) and is expressed as a consequence of natural aging of tissues (Noh and Amasino, 1999). As expected, a high level of SAG12 transcript accumulation was detected in yellow leaves under low light conditions but their amounts were strongly reduced after the exposure of such leaves to light stress (Fig. 5A). The SAG12 transcripts were neither detected in red nor in green leaves, both under low light or light stress conditions (Fig. 5A). Immunoblot analysis revealed that the accumulation of Elip1 and Elip2 in thylakoid membranes followed the induction of corresponding transcripts only in green leaves exposed to light stress but not in red or yellow leaves (compare Fig. 5, A and B). In red and yellow leaves exposed to light stress amounts of Elip1 were reduced to 70% and 90%, respectively, as compared to green leaves, although its transcripts were induced to a higher extent. Only 5% and 13% of Elip2 amounts present in green leaves were detected in thylakoid membranes of red and yellow leaves exposed to light stress, respectively, and these amounts did not correlate with the high level of Elip2 transcripts induced in yellow leaves (compare Fig. 5, A and B). As expected, no Elip1 and Elip2 were detected in green, red, or yellow leaves exposed to low light intensity.
For comparison, the levels of Lhcb2 and D1 protein from PSII reaction center were assayed in green, red, and yellow leaves under the same experimental conditions. The results revealed (Fig. 5B) that the amount of Lhcb2 decreased to a different extent in red and yellow leaves as compared with green leaves. While red leaves still contained 80% of the Lhcb2 amount present in green leaves, only 15% this protein was assayed in yellow leaves. No significant differences in the amount of Lhcb2 were detected in low or high light-treated green, red, and yellow leaves (Fig. 5B). Comparable amounts of D1 protein were detected in green and red leaves but its level was reduced to 18% in yellow leaves under low light conditions as compared to amounts present in green leaves (Fig. 5B). In yellow leaves exposed to light stress the amount of D1 protein was reduced to 7% (Fig. 5B) probably due to a disturbed balance between the rates of protein synthesis and degradation (Andersson and Aro, 2001 The discrepancy between transcript and protein levels for Elip1 and Elip2 in red and yellow leaves suggests the existence of a posttranscriptional control in the expression of both genes. This posttranscriptional regulation can occur at translational or posttranslational levels. To investigate whether Elip1 and Elip2 transcripts induced in red and yellow leaves by light stress are actively translated we analyzed the distribution of mRNAs between polysome-bound and -free fractions (Fig. 5C). For comparison, polysomal fractions from green leaves exposed to light stress were also analyzed. Our results revealed (Fig. 5C) that 75% and 97% of total Elip1 and 80% and 70% of total Elip2 mRNAs induced by light stress in green and red leaves, respectively, were found to be bound to polysomes and to be actively translated. The remaining amounts of these transcripts were detected in the polysome-free fractions. However, in yellow leaves 40% of the amount of Elip1 mRNAs were found as free RNAs.
It was shown (Adamska et al., 2001
To test whether red leaves showed a better protection of photosynthetic apparatus against light stress due to the accumulation of anthocyanins we measured the photosynthetic activity before and after the exposure to light stress in correlation to amounts of D1 protein and Elips. The similar measurements were performed for green and yellow leaves for comparison. The results revealed (Table I ) that the photosynthetic activity assayed as changes in chl fluorescence induction kinetics and expressed as Fv/Fm was reduced to 58% and 72% after exposure of green and yellow leaves to light stress, respectively. This photoinactivation correlated with decreased amounts of D1 protein whose level was reduced to 55% and 80% in green and yellow leaves. Interestingly, only a minor reduction of Fv/Fm (93%) and the D1 amount (93%) was assayed in red leaves. Similar to results shown in Figure 4 also in this experimental system the amount of Elip1 correlated with the degree of photoinactivation and photodamage of PSII. The highest amount of Elip1 accumulated in green leaves (this value was set as 100%) followed by yellow (90%) and red (70%) leaves. In contrast, Elip2 was strongly induced only in green leaves (this value was set as 100%) but not in red (7%) or yellow (15%) leaves, which were less photodamaged.
Protein oxidation results mainly in introduction of carbonyl groups, which can be easily detected. We measured the oxidation status of proteins in green, red, or yellow leaves that were exposed at low or high light conditions (Fig. 6, C and D). The results revealed that several oxidized proteins were detected in green leaves and their content increased dramatically in yellow leaves. Several additional oxidized proteins were detected in green leaves under high light conditions (Fig. 6C). Interestingly, only few oxidized proteins were detected in red leaves and their content did not change significantly after the light stress treatment. A Coomassie blue-stained immunoblot membrane, shown as a control, confirmed that the equal amount of proteins was loaded in each line (Fig. 6D). The diffused protein pattern in samples isolated from yellow leaves results from their modification by photooxidation.
Differential Expression of Elip1 and Elip2 in Green Arabidopsis Leaves Exposed to Light Stress Is Regulated at the Transcript Level and Related to the Degree of PSII Photodamage
We showed that the accumulation of Elip1 and Elip2 transcripts and proteins was induced in a light intensity-dependent manner but the induction kinetics differed for both genes. While Elip1 transcripts and proteins accumulated almost linearly with increasing light intensities, the induction of Elip2 transcripts and proteins occurred stepwise. Furthermore, much higher photon fluency rates were required for elip2 than elip1 genes for a comparable expression level. While significant amounts of Elip1 transcripts and proteins were induced already at 500 µmol m–2 s–1, a comparable amount of Elip2 transcripts and proteins were detected only at 1,500 µmol m–2 s–1 (Fig. 3). The induced Elip1 and Elip2 transcripts were simultaneously translated into correspondent proteins that accumulated in thylakoid membranes. Interestingly, the amount of accumulated Elip1 but not Elip2 correlated with the degree of photoinactivation and photodamage of PSII reaction centers. While Elip1 accumulation and the degree of photoinactivation and photodamage of PSII reaction centers increased almost in parallel in green (Fig. 4), red, and yellow leaves (Table I), the comparable amounts of Elip2 were induced only when approximately 40% of PSII reaction centers were damaged. Thus, the expression pattern of Elip1 resembles that reported for its Cbr homolog in Dunaliella, where the induction of Cbr followed the accumulation of photodamaged D1 protein in 160 kD complex (Jin et al., 2001
The differential expression of four high light-induced A to D (Hli) proteins, related to higher plants Ohps, was reported for the cyanobacterium Synechocystis PCC 6803 (He et al., 2001
A differential expression of elip1 and elip2 genes might be connected with the differences in their promoter regions. It was reported that the transcription factor HY5 promotes the light induction of elip1 but not elip2 gene during greening of etiolated Arabidopsis seedlings (Harari-Steinberg et al., 2001
We demonstrated that the level of transcript accumulation for Elip1 in red and yellow leaves exposed to light stress increased 2- or 6-fold, respectively, as compared with green leaves, while the protein amount was down-regulated. Also the enhanced level of Elip2 transcript accumulation present in yellow leaves exposed to light stress was not accompanied by the accumulation of the correspondent protein. Since the induced Elip RNAs were actively translated as shown by their association with polysomes the reduced amounts of proteins could be a result of lower rates of translation, import into plastids, insertion into plastid membranes, or an increased rate of the degradation.
It was shown in the past that the chl a availability is crucial for the stable insertion of Elips into etioplast membranes of barley (Adamska et al., 2001
Investigation of the global gene expression pattern in naturally senescing leaves of poplar (Populus tremula) trees revealed that transcripts for Elips, together with those for metallothioneins and Cys proteases, were the most abundant (Bhalerao et al., 2003
Our data revealed that red leaves contained very high amounts of anthocyanins as compared with green or yellow leaves. Although the role of anthocyanins is not very well understood, it was postulated that they could be involved in several protective mechanisms, including the modification of the quantity and quality of light (Barker et al., 1997
Anthocyanins absorb mainly light between 270 and 290 nm (UV-B), 310 and 320 nm (UV-A), and 500 and 550 nm (green light; Barker et al., 1997
There is only very limited information available on the intrathylakoid location of Elip family members. It was demonstrated that Elip from pea is located in PSII (Adamska and Kloppstech, 1991
It is still unclear whether Elips are attached to the preexisting homo- or hetero-tLhcb and/or form heterotrimers with Lhcb1, Lhcb2, or Lhcb3. Mutation analysis has shown that Trp-16, Tyr-17, and Arg-21 residues within the N-terminal domain of the mature Lhcb1 are needed for trimerization of LHCII complexes (Hobe et al., 1995
Different distribution of Elip1 and Elip2 in the gradient fractions suggests their coisolation with different LHCII subpopulations. Native LHCII trimers are not homogeneous but consist of homo- and heterotrimers in different combinations of the various Lhcb1 to 3 isoforms (Larsson et al., 1987
Based on past expression and localization studies it was proposed that Elips might be involved in binding of chls released during proteolytic degradation of photodamaged D1 protein from PSII (Adamska and Kloppstech, 1991
It was shown that high intensity light induces the monomerization of tLhcb (Garab et al., 2002 Based on our data we can assume that the function of Elip1 and Elip2 in Arabidopsis is not redundant but connected to various locations within LHCII at different stages of the light stress treatment.
Growth of Plants and Stress Conditions
Arabidopsis plants (Arabidopsis thaliana L. cv Columbia and cv Wassilewskaja) were grown in a growth chamber at 20°C at a photon flux density of 100 µmol m–2 s–1 under the light regime of 8-h dark/16-h light. Plants were cultivated either hydroponically (Norén et al., 2004 For preadaptation to higher light intensities, 50- to 60-d-old, low light-grown plants were transferred to a moderate light intensity of 300 µmol m–2 s–1 for 4 d. A massive accumulation of anthocyanins occurred during this period (Fig. 6B). For natural senescence, plants were grown for 80 to 110 d under conditions described above prior to collection of senescing leaves. During this period a massive loss of chls occurred (Fig. 6A). Detached mature green leaves, anthocyanins containing red leaves, and naturally senescing yellow leaves floating on water were exposed to light stress for 3 h at a photon flux density of 1,800 µmol m–2 s–1 provided by white fluorescent lamps (Osram Power star HQI-E 250W/D). The spectrum of the lamp covered a visible light region from 380 to 720 nm. The temperature of the water was kept constant between 22°C and 25°C. Photon fluency rates were measured with a photometer (Skye, Techtum Laboratory AB). Control leaves were incubated at 20 µmol m–2 s–1 for the same time. After treatment leaves were frozen in liquid nitrogen and stored at –80°C for further analysis. For light intensity-dependent induction of Elips (transcripts and proteins) and the association of mRNAs with polysomes leaves were exposed to increasing light intensities from 100 to 2,000 µmol m–2 s–1 for 3 h at 25°C. For isolation of polysomes and monosomes leaves were exposed to low light (20 µmol m–2 s–1) or high light (1,800 µmol m–2 s–1) for 3 h at 25°C.
The total RNA was isolated with a RNeasy kit (Qiagen, GmbH), spotted on the Hybond-N+ membrane (Amersham Biosciences) at four different concentrations, 5.0, 2.5, 1.25, and 0.63 µg (data shown in Fig. 3), or 1.0, 0.5, and 0.25 µg (data shown in Fig. 5) with a dot-blot apparatus (Schleicher & Schuell), and the membrane was used for hybridization as described (Heddad and Adamska, 2000
For northern blots 5 µg RNA was separated in 1.2% agarose gel and transferred to Hybond-N+ membrane prior to the hybridization as described (Heddad and Adamska, 2000
The frozen plant material (5 g) was ground in liquid nitrogen and the resulting tissue powder was resuspended at 4°C in 50 mL polysome buffer containing 400 mM KCl, 50 mM Tris-HCl, pH 8.3, 10 mM Mg acetate, 250 mM Suc, 2% (w/v) Triton X-100, and 0.005% (v/v) For investigations of ribosomal profiles 1 to 2 g of frozen leaves were ground in liquid nitrogen and resuspended in five volumes of buffer A (200 mM Tris-HCl, pH 8.9, 200 mM KCl, 35 mM MgCl2, 0.6 M sorbitol, 12.5 mM EGTA, and 15 mM dithiothreitol). After differential centrifugation at 10,000g and 30,000g for 10 min, homogenate was filtrated through Miracloth (Calbiochem). After adding Triton X-100 to a final concentration of 2% (w/v), ribosomes were pelleted by centrifugation at 160,000g for 3.5 h at 4°C over a 1.5 M Suc cushion in buffer B (40 mM Tris-HCl, pH 8.9, 20 mM KCl, 10 mM MgCl2, 5 mM EGTA, and 5 mM dithiothreitol). The pellets were gently resuspended in buffer C (10 mM Tris-HCl, pH 7.6, 25 mM KCl, and 5 mM MgCl2) and centrifuged at 8,000g in the Eppendorf centrifuge. The polysomal suspension was loaded onto a linear 15% to 55% (w/v) Suc density gradient prepared in buffer C and centrifuged at 260,000g for 70 min. Gradient fractions containing polysomes and monosomes were collected by monitoring the absorption at 254 nm.
Leaves frozen in liquid nitrogen were homogenized in extraction medium containing 300 mM sorbitol, 20 mM HEPES-NaOH pH 7.4, 5 mM MgCl2, 2.5 mM EDTA, and 10 mM KCl. The homogenate was filtrated through Miracloth (Calbiochem), mixed (1:1, v/v) with 3x concentrated sample buffer (Laemmli, 1970
Polyclonal antibodies were raised against synthetic peptides of Elip1 and Elip2 selected shortly after the predicted N-terminal cleavage sites. These peptide sequences were unique for Arabidopsis Elip1 and Elip2 and consisted of amino acids 48 to 60 for mature Elip1 (EGGPTNEDSSPAP) and amino acids 45 to 57 for mature Elip2 (QGDPIKEDPSVPST; AgriSera AB). The peptides were conjugated to keyhole limpet hemocyanin carrier protein prior to immunization of rabbits. The IgG fraction was purified by affinity chromatography on the G-protein-sepharose column (AgriSera AB). These antibodies were highly specific for Elip1 or Elip2 and did not show cross-reactivity with a second Elip form or with any other thylakoid membrane protein. The polyclonal D1 and Lhcb2 antibodies were purchased from Agrisera AB and the antibody against
Immunoblotting was carried out according to Towbin et al. (1979)
For isolation and assay of oxidized proteins, leaves frozen in liquid nitrogen were homogenized in extraction medium as described above, supplemented with 2% (v/v)
Thylakoid membranes were prepared from fresh or frozen leaf material as described (Tidholm et al., 2002
To establish optimal conditions for the isolation of pigment-protein complexes from light-stressed leaves on green native gels (0.5 mg chl/mL) were solubilized either with Triton X-100:LDS mixture, 15 mM DM, or 15 mM OGP. For the solubilization with OGP or DM (data shown in Fig. 2, A and B) leaves were homogenized in medium containing 300 mM sorbitol, 20 mM HEPES, pH 7.4, 5 mM MgCl2, 2.5 mM EDTA, and 10 mM KCl and an aliquot of homogenate corresponding to 20 µg chl was mixed (10:1, v/v) with 1 M Tris, pH 6.6, and 30% glycerol, and supplemented with DM or OGP. Solubilization was carried out at room temperature for 5 min in the dark, with brief vortexing and samples corresponding to 2 to 4 µg chl separated on native green gels as described below. For the solubilization with Triton X-100:LDS mixture (10% stock solutions were mixed at the ratio 12:1, v/v) samples were treated as previously described (Peter and Thornber, 1991
For routinely used isolation of pigment-protein complexes (shown in Fig. 2, C and D) 2% (v/v) DM was used and the solubilization took place on ice for 10 min as described for greening barley (Hordeum vulgare) leaves (Peter and Thornber, 1991
For extraction of pigments two leaf discs with 12 mm diameter were agitated gently in the dark for 24 h at 4°C in 1 mL of 80% (v/v) acetone (chls and carotenoids) or 3 M mixture of HCl:water:MeOH (1:3:16, v/v/v, anthocyanins) according to Gould et al. (2000)
Chl fluorescence induction kinetics were measured at room temperature on detached leaves using either an imaging/pulse-amplitude modulation fluorimeter (Walz GmbH) for data shown in Figure 4 or a FlurCam fluorimeter (PSI Instruments) for data shown in Figure 5. Leaves were preadapted in the dark for 15 min and then exposed to a saturating 1 s light flash. The minimal fluorescence (F0) in the absence of actinic light and maximal fluorescence (Fm) after a saturating light flash were measured and the variable fluorescence (Fv = Fm – F0) was calculated as described (Butler and Kitajima, 1975
The effective quantum yield of PSII photochemistry (PSII yield) was calculated as described (Maxwell and Johnson, 2000
Results shown in Figures 1 and 2 are the average of five to seven independent experiments using 10 to 15 leaves of the same age collected from three to five plants. Results shown in Figure 3 are the average of three independent experiments using 8 to 10 (sections A and B), 50 to 60 (section C), and 10 to 20 (section D) leaves per point collected from three, or eight to 10 plants, respectively. Data shown in Figure 4 are the average of three independent experiments using three to four leaves per point. The measurement of Fv/Fm and PSII yield were performed on three leaves per point on six different leaf areas. Data shown in Figures 5 and 6 are the average of three independent experiments using 20 leaves per point collected from eight to 10 plants. Sequence data from this article can be found in the GenBank under the following accession numbers: At3g22840 (Elip1) and At4g14690 (Elip2).
We thank Dr. Ralf Oelmüller (University of Jena, Germany) for providing polyclonal antibodies against /-subunits of the CF1-ATP-synthase complex and Amine Heddad (Stockholm) for the assisting with the promoter analysis. We would like also to thank Lars Hiertas Minne (Sweden) for the sponsoring peptide antibodies against Elip1 and Elip2 (to M.H.). Received April 4, 2006; accepted June 30, 2006.
1 This work was supported by research grants from the Swedish Research Council, the Swedish Strategic Foundation and the Carl Tryggers Foundation (to I.A. and B.A.), the Deutsche Forschungsgemeinschaft (grant nos. AD92/7–1 and AD92/7–2 to I.A.), and the Konstanz University grant (to I.A.). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Iwona Adamska (iwona.adamska{at}uni-konstanz.de). www.plantphysiol.org/cgi/doi/10.1104/pp.106.081489 * Corresponding author; e-mail iwona.adamska{at}uni-konstanz.de; fax 49–7531–88–3042.
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ABSTRACT
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F/Fm = [Fm' – Ft]/Fm'), where Fm' represented the maximal fluorescence in the light and Ft was the steady-state fluorescence yield in the light measured immediately before the saturating light flash. The PSII yield represents the proportion of the light absorbed by chl associated with PSII that is used in photochemistry. 
