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Plant Physiol. 2006 Aug; 141(4): 1264–1273.
PMCID: PMC1533944

Suppression of Both ELIP1 and ELIP2 in Arabidopsis Does Not Affect Tolerance to Photoinhibition and Photooxidative Stress1

Abstract

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

High light, in combination with adverse environmental factors that reduce the CO2 assimilation rate, such as too low or too high temperatures, drought, salinity, nutrient deprivation, etc., compromises the photosynthetic function.

The imbalance between the rate of excitation arrival in the photosynthetic centers and the rate of the use occurring in these environmental situations results in an overexcitation of the photosystems. This condition favors the formation of highly reactive oxygen species that may produce photooxidative damages of the constituents (pigments, proteins, and lipids) of the photosynthetic machinery. Plants have protective mechanisms against photooxidative damage, such as decreased light absorption, removal of excess excitation inside the photosystems, scavenging of reactive oxygen species, and up- and down-regulation of photosynthesis-related genes (Demmig-Adams and Adams, 1992).

ELIPs (early light-induced proteins) are thylakoid proteins, encoded by nuclear genes, expressed in plants exposed to stress (Meyer and Kloppstech, 1984; Adamska, 2001). ELIPs are synthesized in the cytoplasm, imported into the chloroplast, and inserted in thylakoids via a pathway involving cpSRP43 (Hutin et al., 2002). ELIPs have three transmembrane domains, with the I and III α-helices showing a high homology with the corresponding helices of Cab (chlorophyll a/b-binding) proteins (Grimm et al., 1989). Comparison of the amino acid sequences between ELIPs and Cabs shows that ELIPs contain four putative chlorophyll-binding residues in the helices I and III (Green and Kühlbrandt, 1995). Furthermore, spectroscopic analysis of ELIP isolated from pea (Pisum sativum) leaves indicates the presence of chlorophyll a and lutein, with a high chlorophyll to lutein ratio (Adamska et al., 1999). These data allow to make the hypothesis that ELIPs are more probably involved in energy dissipation than in light harvesting (Montané and Kloppstech, 2000).

With respect to other Cab proteins that are constitutively expressed in thylakoids, ELIPs show a transient accumulation. The transcripts and the corresponding proteins are induced during the first hours of greening of etiolated seedlings (Grimm and Kloppstech, 1987; Cronshagen and Herzfeld, 1990; Pötter and Kloppstech, 1993), when the developing photosynthetic apparatus is very susceptible to photooxidation (Caspi et al., 2000). In mature plants they are absent until the plants are exposed to photoinhibitory conditions, such as high light (Adamska et al., 1992b; Pötter and Kloppstech, 1993), high light and cold (Montané et al., 1997), high salinity (Sävenstrand et al., 2004), UV-B irradiance (Adamska et al., 1992a), or desiccation (Zeng et al., 2002). This expression pattern, together with the putative ability to bind pigments, suggested that ELIPs may have a photoprotective function, for example, by binding free chlorophylls to avoid the formation of reactive oxygen species (Hutin et al., 2003) or by binding xanthophyll pigments to dissipate the excess of absorbed light energy (Król et al., 1999). This hypothesis was further supported by the finding that chaos, an Arabidopsis (Arabidopsis thaliana) mutant unable to rapidly accumulate ELIPs, is strongly susceptible to photooxidative stress and that its phenotype can be rescued by constitutive expression of ELIPs (Hutin et al., 2003).

Recently, we investigated the phototolerance of two Arabidopsis mutants lacking one or the other of the two ELIPs present in this species. Both mutants behave as the wild type as far as sensitivity to light and cold-induced, short-term photoinhibition is concerned, and differ only slightly in chlorophyll accumulation kinetics during greening (Casazza et al., 2005). Since in this very early stage both ELIPs are expressed, it is possible that, in the single mutants, the remaining ELIP is sufficient to allow normal chloroplast development and sensitivity to stress. For this reason, we generated a double null ELIP mutant (elip1/elip2 [elip1/2]) that lacks both ELIPs and investigated its phenotype in those conditions where ELIPs are expressed in the wild type, i.e. during deetiolation and during exposure to high light and cold.

RESULTS

Isolation of the elip1/2 Mutant

Two elip1/2 mutants were obtained by crossing the elip1 and elip2 independently originated single mutants, carrying a T-DNA insertion in one or in the other Elip gene and previously characterized by Casazza et al. (2005). To identify plants homozygous for T-DNA insertion either in Elip1 and in Elip2, PCR analysis on genomic DNA was performed on plants of the F2 generation using specific primers for the Elips and for the T-DNA sequence (see “Materials and Methods” and Fig. 1 for one of the two mutants). To verify that in these plants ELIPs were not expressed, leaves of the wild type and of the mutant were exposed to high light (750 μE m−2 s−1) and cold (4°C) for 8 h to induce ELIP1 and ELIP2 accumulation; afterward, total proteins were extracted, fractionated by SDS-urea polyacrylamide gel, and analyzed by western blot. Figure 1D shows the total absence of both ELIPs in the mutant plants exposed to a treatment that induces both ELIPs in the wild type.

Figure 1.
Validation of the elip1/2 mutant. A and B, The elip1/2 mutant was obtained by crossing mutants bearing a T-DNA insertion in the Elip1 (A) or Elip2 (B) gene (introns are indicated in white, exons in black, and T-DNA insertions in gray). C, The screening ...

The phenotype of the double elip mutants, grown in our standard conditions (120 μE m−2 s−1, 22°C in long day), was completely undistinguishable from the wild type during the entire life cycle, with the exception of a slight reduction (approximately 10%) in the total chlorophyll content (Chltot), while the chlorophyll a to chlorophyll b ratio (Chl a/b) was identical to that of the wild type (Fig. 2). This latter observation indicates that the external antenna system of PSII is not significantly altered. Furthermore, since both double mutants behave similarly, results of only one of the two is reported in the following sections.

Figure 2.
Phenotype and chlorophyll content of mature wild-type (wt; left) and mutant (right) plants. Chlorophyll content of wild-type and elip1/2 plants grown for 21 d at 14 h light (120 μE m−2 s−1)/10 h dark is provided.

Chlorophyll, ELIPs, and Photoinhibition during Deetiolation

It was known that ELIPs are expressed in young developing seedlings and in Arabidopsis the absence of one or the other ELIP led to a slight reduction in the rate of chlorophyll accumulation during greening of etiolated seedlings in continuous light (Casazza et al., 2005). Figure 3 shows chlorophyll and ELIP content together with maximal PSII photochemical efficiency (Fv/Fm) during deetiolation of wild-type and elip1/2 seedlings at different light intensities. In wild type, the exposure to continuous light of 120 μE m−2 s−1 causes a progressive accumulation of chlorophyll that reaches a concentration of 838 μg g−1 fresh weight after 7 d (Fig. 3A); the double mutant shows a slower greening kinetic, reaching a chlorophyll content corresponding to 80% of that of the wild type after 7 d. At higher light intensities, the chlorophyll content of the wild type progressively decreases; that of the mutant also decreases, being again 20% less at 220 μE m−2 s−1, but at 400 μE m−2 s−1 mutant and wild-type plants are the same (Fig. 3B). At the light intensity of 120 μE m−2 s−1, ELIPs were present in the wild type only in the first day of illumination and then disappeared, in agreement with their transient expression (Casazza et al., 2005); at the higher light intensities, they do not disappear but instead accumulate (Fig. 3D). Furthermore, to evaluate the extent of photoinhibition, the Fv/Fm was measured: in seedlings grown at 120 and 220 μE m−2 s−1, Fv/Fm around 0.8 in wild type and elip1/2 indicates absence of photoinhibition, whereas at 400 μE m−2 s−1 both genotypes were similarly and substantially photoinhibited (Fig. 3C).

Figure 3.
Greening of etiolated seedlings in continuous light. Wild-type and elip1/2 seedlings were grown in the dark for 5 d and then transferred to continuous light of different intensities. A, Chlorophyll content of seedlings exposed to 120 μE m−2 ...

Photoinhibition and Photooxidative Damage in Mature Plants

Wild-type and elip1/2 plants, grown for 21 d at 120 μE m−2 s−1 and 22°C, were exposed to high light (500 μE m−2 s−1) and chilling (8°C) for 7 d. As shown in Figure 4A, ELIPs are not present in wild-type plants before the treatment. Exposure to high light and cold induces in the wild type the accumulation of both ELIPs at an expression level remaining rather constant during all the experiment. As expected, the mutant does not accumulate ELIPs. Figure 4B shows Fv/Fm during the exposure to high light and cold. In the first 3 d of treatment, wild-type plants show a rapid decrease of Fv/Fm (from approximately 0.85 in growing conditions to approximately 0.5) followed by a progressive recovery of the photosynthetic activity (Fv/Fm was around 0.7 after 7 d). elip1/2 shows the same behavior as the wild type during the entire treatment.

Figure 4.
Photoinhibition in high light and cold. Wild-type and elip1/2 plants grown at 120 μE m−2 s−1, 22°C for 21 d were exposed to high light (500 μE m−2 s−1) and cold (8°C) for 7 d. Leaves were ...

Peroxidation of membrane lipids, as an index of photooxidative damage (Havaux, 2003), was determined by thermoluminescence (TL) measurements in both genotypes. Figure 5A reports the high-temperature TL emission band of wild-type plants grown at 300 μE m−2 s−1, 22°C compared to plants exposed for 7 d at 1,300 μE m−2 s−1, 6°C. The treatment leads to an increase of the amplitude of the TL signal with a maximum around 130°C to 140°C. The amplitude of the TL peak, which is well correlated with the lipid peroxidation level (Havaux, 2003), was used to follow the time course of lipid peroxidation during the exposure of wild-type and mutant plants to high light and cold (1,300 μE m−2 s−1, 6°C). Figure 5B shows that both in wild type and in the mutant, the amplitude of the signal at 135°C was at a maximum after 2-d treatment and then decreased slightly. More importantly, the amplitude of the TL signal, i.e. the extent of lipid peroxidation, was the same in both genotypes. Figure 5C shows the appearance of wild-type and mutant plants after 7-d treatment. Again, the two phenotypes are very similar.

Figure 5.
Lipid peroxidation during high light and cold treatment. Plants were grown for 21 d at 300 μE m−2 s−1, 22°C and then exposed for 7 d at 1,300 μE m−2 s−1, 6°C. A, Lipid peroxidation-related ...

Uncoupled Chlorophyll Molecules

It was previously suggested that the absence of ELIPs in the chaos mutant, under conditions of light stress, was correlated with the presence of high levels of chlorophylls, apparently energetically uncoupled from the photosystem antenna matrix (Hutin et al., 2003). In principle, uncoupled chlorophylls are expected to have a high triplet yield and hence lead to photoinhibition via singlet oxygen formation (Santabarbara et al., 2001). Their presence may be experimentally demonstrated either by time resolved fluorescence decay measurements (Vasil'ev et al., 1998), due to their nanosecond lifetime, or by steady-state fluorescence measurements due to their blue-shifted emission (absorption) characteristics (Santabarbara and Jennings, 2005). To examine this aspect, we have performed both kinds of measurements on thylakoids extracted from wild type and elip1/2, as well as fluorescence lifetime measurements on intact leaves of wild type and elip1/2. Plants were pretreated under photoinhibitory conditions similar to those used by Hutin et al. (2003; 1,100 μE m−2 s−1, 6°C) for 3 and 4 d, which led, for all genotypes, to a decrease in the Fv/Fm ratio from 0.85 to 0.20 ± 0.03. Data are presented in Figure 6 for the usual sum of exponentials global decomposition, measured near the Fo level, for thylakoids prepared from wild-type (Fig. 6A) and elip1/2 (Fig. 6B) plants that had been exposed to the above photoinhibitory regime for 4 d. The wild-type and elip1/2 decay-associated spectra (DAS) descriptions are very similar, displaying the usual PSII lifetime components of approximately 200, 450 to 500, and 30 to 50 ps (e.g. Vasil'ev et al., 1998; Engelmann et al., 2005). The 70- to 100-ps component is associated with PSI (Engelmann et al., 2005), and the usual low-amplitude, long-lifetime component (approximately 2 ns) also is present. In this study, no 5-ns decay was detectable in any sample, in contrast to previous measurements on the chaos mutant (Hutin et al., 2003), where in photoinhibited leaves this component accounted for >25% of the fluorescence intensity (amplitude) and much less (<2%) in the wild type not subjected to photoinhibition. In Table I, we present photoinhibition time-course data for the amplitude of the nanosecond decay from the global decomposition (multiwavelength) data. At zero photoinhibition time, the 2 ± 0.4-ns (errors are the data spread) decay components, in all cases, lie in the range 1.3% to 1.8%. After several days of photoinhibitory treatment, the amplitudes of wild type and elip1/2 decrease to approximately the same extent and lie in the range 0.5% to 1.0%. As this is the only component that may reasonably be associated with energetically uncoupled chlorophylls, we conclude that photoinhibition does not lead to an increase in this population, and no significant differences are apparent between the wild type and elip1/2. Similar results have been obtained with the chaos mutant under our growth and experimental conditions (data not shown).

Figure 6.
DAS of chlorophyll fluorescence from thylakoids of photoinhibited plants. Plants were exposed for 4 d at 1,100 μE m−2 s−1 at 6°C. Thylakoids were extracted and chlorophyll fluorescence decay measured and globally analyzed ...
Table I.
Amplitudes of the 2-ns decay component during photoinhibitory treatment

As uncoupled chlorophylls are blue shifted with respect to the protein-bound antenna chlorophylls (Santabarbara and Jennings, 2005), we also have performed steady-state emission measurements of thylakoids from plants that had undergone the same photoinhibitory treatment as described above. With an optical multichannel analyzer experimental resolution of less than 1%, we were unable to detect differences between the wild-type and mutant thylakoids (data not shown), in agreement with the above conclusion.

As the experiments of Hutin et al. (2003) were performed on intact leaves, we also investigated the fluorescence decay in intact leaves. Due to significant backscattering, it was not possible to correctly perform the convolution of the instrument response function with the fluorescence measurements. Thus, a global analysis in terms of the DAS was not possible. However, inspection of the fluorescence decay profiles clearly indicates the absence of significant nanosecond components either before or after the usual photoinhibitory treatment (data not presented) in both the wild-type and mutant plants, confirming the more detailed data obtained with thylakoids.

Xanthophyll-Cycle Pigments

High light-induced xanthophyll-cycle activity and ELIP accumulation show similar transient kinetics (Król et al., 1999); furthermore, it has been suggested that ELIPs may be carotenoid-binding proteins (Adamska et al., 1999). Accordingly, we analyzed the content of xanthophylls involved in nonphotochemical quenching (NPQ) of chlorophyll fluorescence and of α-tocopherol, an antioxidant that quenches 3Chl and 1O2. Measurements were made for wild-type and mutant plants exposed for 7 d to high light (1,300 μE m−2 s−1) and cold (6°C) when both genotypes were recovering from the stress (Fv/Fm ratio around 0.77). In the wild type, the treatment induces a 1.4-fold increase in α-tocopherol and a dramatic increase in antheraxanthin and zeaxanthin (which were practically absent in untreated plants), indicating an activation of the xanthophyll cycle (Fig. 7A). The same pattern was observed in elip1/2, but the accumulation of α-tocopherol was more pronounced (approximately 1.7-fold) and that of zeaxanthin less pronounced than in the wild type (the mutant accumulates only one-half of wild-type zeaxanthin). The dependence of the NPQ parameter on light intensity in wild type and elip1/2 also was determined. The results show that, despite the lower accumulation of zeaxanthin in mutant plants, the NPQ values were virtually identical in the two genotypes at all light intensities assayed (Fig. 7B). The hypothesis that ELIPs are energy-dissipating antennae (Montané and Kloppstech, 2000) is therefore not confirmed here.

Figure 7.
NPQ, photosynthetic pigments, and α-tocopherol. Wild-type and elip1/2 plants grown at 300 μE m−2 s−1 (NT) were exposed to high light and cold (1,300 μE m−2 s−1, 6°C) for 7 d (T). A, Contents ...

SEPs and OHPs in the elip1/2 Mutant

It was shown that not only ELIPs but also ELIP-like proteins, such as SEPs (stress-enhanced proteins) and OHPs (one-helix proteins), are also induced by high-light treatments (Heddad and Adamska, 2000; Jansson et al., 2000; Andersson et al., 2003), and these proteins may have a photoprotective role. To exclude that in the double mutant the absence of ELIPs is compensated by an overexpression of these proteins, we analyzed the transcript and protein levels of Seps and Ohps in wild-type and mutant plants exposed to high light and chilling (1,300 μE m−2 s−1, 6°C) for 7 d (Fig. 8). The treatment of wild-type plants did not induce any change in the amount of Sep and Ohp transcripts, whereas the level of the corresponding proteins exhibited a slight increase. In the double mutant, ELIPs were obviously absent, but the level of Sep and Ohp transcripts and proteins was similar to that of the wild type.

Figure 8.
SEPs and OHPs in wild-type and elip1/2 plants. Wild-type and elip1/2 plants grown in standard conditions (NT) were exposed to high light and cold (1,300 μE m−2 s−1, 6°C) for 7 d (T). A, Transcript and protein level of ELIPs, ...

DISCUSSION

The exposure of plants to photoinhibitory conditions induces the up-regulation of Elips, distant relatives of the Lhc (light-harvesting complex) gene family. Their photoprotective role has been suggested on the basis of the following evidence: a transient expression enhancement related to a number of stress conditions producing photoinhibition and/or photooxidative stress; a preferential localization in the stroma-exposed thylakoids, where the repair of damaged photosystems occurs; a putative binding capacity for chlorophylls and carotenoids; and the observation that chaos, a mutant unable to rapidly accumulate ELIPs, is more photosensitive to high light and chilling than its isogenic wild type and that its photosensitivity is rescued by constitutive ELIPs expression. Up to now, however, a clear-cut proof of the function of ELIPs and of their mechanism of action has not been available. The aim of our work was to gain insight into the ELIP role using an inverse genetic approach, constructing a plant null for Elip genes. We took advantage of the presence of only two Elip genes in Arabidopsis (Elip1 and Elip2), for both of which two independently generated T-DNA insertion mutants were available (characterized in Casazza et al., 2005). By crossing the single mutants, we isolated from the F2 generation double mutant plants bearing homozygous T-DNA insertions in both Elip genes. These plants proved to be completely devoid of ELIPs when exposed to inducing conditions. Notably, plants null for both Elip genes, grown in our standard conditions, were phenotypically indistinguishable from the wild type during the entire life cycle, the only difference being a 10% reduction of the chlorophyll content in mature plants. This finding indicates that knocking out the Elip genes does not substantially perturb the plant phenotype, allowing a meaningful comparison of sensitivity to photoinhibitory treatments between the mutant and the isogenic wild type. From this point of view, the elip1/2 mutant differs from chaos, a mutant in which ELIPs accumulate less rapidly and at a lower level after exposure to excess light (Hutin et al., 2003). chaos, lacking the cpSRP43 subunit involved in the targeting of members of the LHC protein family from the stroma to the thylakoid membrane, has a pale-green phenotype throughout its growth because of a substantial reduction of the LHC level (Amin et al., 1999; Klimyuk et al., 1999).

We compared the photosensitivity of the wild type and of the elip1/2 mutant in deetiolating seedlings and in mature plants. In mutant seedlings, the rate of chlorophyll accumulation was reduced when greening was in continuous light of 120 and 220 μE m−2 s−1 (20% reduction in chlorophyll content after 7 d, to be compared with 10% reduction in mature plants grown in the light/dark cycle). This difference disappeared at higher light intensity (400 μE m−2 s−1) when the chlorophyll content of both genotypes was much less than that present at lower light intensity. More interestingly, although mutant and wild-type plants were not photoinhibited at 120 and 220 μE m−2 s−1, at 400 μE m−2 s−1 both were consistently photoinhibited and to the same extent, and ELIPs were constantly (and not transiently) present in the wild type and, obviously, always absent in the mutant.

Wild-type and mutant mature plants, grown at 120 μE m−2 s−1 until the rosette stage, were exposed to high (500 μE m−2 s−1) or very high (1,100–1,300 μE m−2 s−1) light intensity in the cold for several days, and we evaluated the damage to the photosynthetic machinery by measuring Fv/Fm, lipid peroxidation, presence of uncoupled chlorophyll molecules, NPQ, antioxidants, and pigments of the xanthophyll-cycle content. The response of wild-type plants to the treatments was characterized by an initial phase where a rapid decrease of Fv/Fm and a rapid increase of the level of lipid peroxidation occurred. This phase corresponds to the inactivation of PSII and to the damage of photosynthetic membranes due to light stress-induced photooxidation, a well-known phenomenon occurring in nonacclimated plants (Demmig-Adams and Adams, 1992). Notably, we did not observe the formation of uncoupled chlorophyll molecules in this phase. Thereafter, the recovery began, documented by an increase in the Fv/Fm, in the activation of the xanthophyll cycle and in the accumulation of antioxidant molecules, whereas the level of lipid peroxidation did not increase further. Of most importance, the same behavior was observed in elip1/2 both temporally and quantitatively, indicating that the extent of initial photoinhibition and photodamage, as well as the capacity to recover, were substantially unaffected by ELIPs. In addition, also in the mutant, we failed to observe any increase in uncoupled pigments associated with incompletely assembled or damaged chlorophyll-protein complexes. Thus, the chlorophyll scavenging function previously suggested is not confirmed.

These results therefore indicate that the higher photosensitivity to high light and chilling previously observed in chaos (Hutin et al., 2003) was not exclusively dependent on a slower ELIP accumulation rate after imposure of light stress conditions. Due to the impaired cpSRP targeting pathway, the organization of the photosynthetic apparatus is altered in chaos, and, consequently, one can hypothesize that defects beyond synthesis of the ELIPs also contributed to photosensitivity in this mutant. Apparently, in wild-type Arabidopsis, the presence or absence of one of the two ELIPs (Casazza et al., 2005) or of both during light stress does not modify either the sensitivity to photoinhibition and photodamage or the capacity to recover from the light stress, thus raising some doubts about the photoprotective function of ELIPs. If ELIPs play a role in photoprotection, this role must be marginal and possibly may occur only under more severe stress conditions than those used in this study. Of course, we cannot completely exclude that a possible role of ELIPs in photoprotection was in some way compensated by some unknown mechanism(s). We checked the level of the ELIP-like proteins SEPs and OHPs. Although the function of these proteins in Arabidopsis is not known, we cannot exclude that they are functionally redundant with ELIPs. We did not find any compensatory accumulation of these proteins in the elip1/2 double mutant. However, we cannot exclude the possibility that, under the stress conditions used in this study, a complete loss of ELIPs can be immediately compensated by OHPs and SEPs without requiring a significant change in their concentration. It is not known whether SEPs and OHPs were lowered concomitantly with ELIPs by the mutation of the cpSRP pathway in the chaos mutant. However, it is clear from our experiments that, even in this eventuality, the function of ELIPs is not of primary importance.

This conclusion leaves completely unresolved the question of a physiological function for ELIPs, a subject for which at present we have no firm suggestion. Perhaps, on the basis of the reduced rate of chlorophyll accumulation during greening in continuous light of moderate intensity, reflecting the slight reduction of chlorophyll content in mature elip1/2 leaves, a role of ELIPs in the regulation of chlorophyll biosynthesis could be envisaged. An altered chlorophyll level also was observed in leaves of Arabidopsis transgenic lines overexpressing ELIPs (T. Tzvetkova, L. Nussaume, and M. Havaux, unpublished data). Alternatively, considering the reduced level of zeaxanthin in the mutant with respect to the wild type during light stress, a role in stabilizing the level of this xanthophyll can be hypothesized. Very recently, in a study of winter acclimation of bearberry (Arctostaphylos uva-ursi), Zarter et al. (2006) found a correlation between the persistent retention of zeaxanthin and the up-regulation of ELIP-type proteins. They suggested that ELIPs could be involved in long-term acclimation of plants to extreme stress conditions by interacting with this persistent pool of zeaxanthin. Our observation that ELIP synthesis in wild-type leaves is associated with a higher zeaxanthin level relative to elip1/2 leaves is compatible with this hypothesis. The increased level of tocopherols in elip1/2 relative to wild type could be interpreted as a compensation for the decrease in zeaxanthin in the double mutant, as previously observed in zeaxanthin- or tocopherol-deficient Arabidopsis mutants (Havaux et al., 2005). Clearly, a detailed study will be necessary to prove or disprove these suggestions.

MATERIALS AND METHODS

Plant Material, Growth Conditions, and Photoinhibitory Treatments

The Arabidopsis (Arabidopsis thaliana) Columbia-0 line was provided by the Arabidopsis Biological Resource Center (Ohio State University). elip1/2 mutants were obtained by crossing single mutant lines previously characterized by Casazza et al. (2005). Plants were grown in sterilized soil (Technic n.1; Dueemme) on Aratrays (BetaTech) under different light regimes: at 120 μE m−2 s−1, 14-h photoperiod or 300 μE m−2 s−1, 8-h photoperiod. In both conditions, temperature was 22°C (day)/18°C (night).

Etiolated seedlings were obtained by growing sterilized seeds on AIS medium in petri dishes for 5 d in darkness. Thereafter, they were exposed to continuous light in a growth chamber (Microclima MC1750E; Snijders Scientific b.v.) for 7 d at 20°C.

Photoinhibitory treatments were imposed on whole mature plants placed in the growth chamber in high light and low temperature (see “Results”) or on detached leaves floating on water in petri dishes and exposed to high photon flux density provided by a 400-W lamp (Osram HQI-E Power Star). At the level of the dishes, the temperature was maintained at 4°C.

DNA and RNA Isolation and Analysis

Genomic DNA was extracted from leaves using cetyl-trimethyl-ammonium bromide buffer (3% [w/v] cetyl-trimethyl-ammonium bromide, 1.4 m NaCl, 0.2% β-mercaptoethanol, 20 mm EDTA, 100 mm TRIS, pH 8). For the validation of the elip1/2 mutants, PCR analysis was performed using different combinations of the following primers: for Elip1, 5′-primer RTE1F (5′-ggaggacccacgaatgaagactctt-3′)/3′-primer RTE1R (5′-agacgagtgtcccacctttgacgaa-3′); for Elip2, 5′-primer UPE2 (5′-gtttagcgttcaacccaaatatcgat-3′)/3′-primer IIEsr2 (5′-ggtcgagggcacagaaggatctt-3′); and for T-DNA left border, LB (5′-atattgaccatcatactcattgc-3′).

Total RNA was isolated from frozen leaves using the Trizol protocol (Invitrogen). Transcript levels were analyzed by reverse transcription (RT)-PCR (Access RT-PCR system; Promega) using the following as primers: for Elip1, RTE1F/RTE1R (see above); for Elip2, bisE2F (5′-tattgactacacgcaacatcagaa- 3′)/bisE2R (5′-gttttctccctttgataactccat-3′); for Ohp1, 5′-Ohp1 (5′-gctcgtcgccgttatcttcat-3′)/3′-Ohp1 (5′-ggaagatcgagtcctttccca-3′); for Ohp2, 5′-Ohp2 (5′-atgtcaagtagtttcaccgatt-3′)/3′-Ohp2 (5′-taccaactgcgaaaccaaaca-3′); for Sep1, 5′-Sep1 (5′-agtgtctgcgtctctcgcct-3′)/3′-Sep1 (5′-aaaccgcagctagaacaccaa-3′); and for Sep2, 5′-Sep2 (5′-gctatggctacgcgagcgat-3′)/3′-Sep2 (5′-agaccaatcactaggcttggt-3′).

Lengths of amplification fragments were deduced by comparison with 1-kb ladder (GIBCO-BRL) after electrophoresis in agarose gel.

Protein Isolation and Analysis

Crude protein extracts were prepared as described by Pötter and Kloppstech (1993). Proteins were separated by SDS-PAGE using 15% polyacrylamide gels in 6 m urea (Laemmli discontinuous buffer system) and transferred on polyvinylidene difluoride membrane (BioTrace; PALL Gelman Laboratory). The anti-ELIP polyclonal antibody was produced in rabbit by Primm S.r.l. using the recombinant fusion protein glutathione S-transferase-ELIP1 as antigen (Casazza et al., 2005). Antibodies against OHP2 and SEP2 were kindly provided by Prof. I. Adamska and described by Andersson et al. (2003). The secondary antibody was a peroxidase-conjugated goat anti-rabbit immunoglobulin (DakoCitomation). Signals were detected with the SuperSignal West Pico chemiluminescent substrate (Pierce-CELBIO S.r.l.).

Determination of Photosynthetic Pigments and Tocopherols

Chlorophyll content was calculated from the absorbance at 664, 647, and 750 nm (V-530 Jasco spectrophotometer; Sintak S.r.l.) of an N,N-dimethylformamide extract, according to Porra et al. (1989), while the chlorophyll concentration of crude protein extracts was measured according to Arnon (1949). Carotenoids and tocopherols were extracted in methanol and quantified by HPLC as described elsewhere (Havaux et al., 2005).

Spectroscopy

The extent of photoinhibition was measured as the ratio Fv/Fm, with a plant efficiency analyzer (Hansatech). NPQ of chlorophyll fluorescence was measured with a PAM-2000 fluorometer (Walz) and was calculated as (Fm/Fm′) − 1. Photooxidative damage of membrane lipids was measured by TL as described previously (Havaux and Kloppstech, 2001; Havaux, 2003). Samples were composed by leaf discs of 8-mm diameter picked up from different plants and heated from 25°C to 150°C.

The chlorophyll fluorescence decay measurements were performed as described previously by Engelmann et al. (2005) using fresh thylakoids prepared as described by Casazza et al. (2001).

Acknowledgments

We thank Prof. I. Adamska for her generosity in providing the antibodies against SEP and OHP proteins. We also thank Dr. L. Nussaume (CEA/Cadarache), who initiated the collaboration between the two laboratories involved in this study, and the members of the GRAP laboratory (CEA/Cadarache) for skillful technical assistance.

Notes

1This work was supported in part by MIUR (FIRB project no. RBAU01E3CX) and in part by the Institute of Biophysics, Division Research Milano, CNR, Italy.

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: Carlo Soave (ti.iminu@evaos.olrac).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.083055.

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