Plant cells exposed to photo-inhibitory conditions respond by accumulation of the early light-induced proteins (Elips) with a potential photoprotective function. Here we studied the expression of Elip in various pea cultivars grown under agricultural or climate-chamber conditions. We demonstrated that the expression of Elip in all cultivars was developmentally regulated and its level decreased during flowering and post-flowering periods. Surprisingly, significant amounts of Elip transcripts, but not proteins, accumulated in senescing leaves already under low light conditions and the exposure to light stress resulted in a 10-times higher induction of Elip transcripts. Furthermore, the expression pattern of Elip transcript and protein significantly differed under field and growth-chamber conditions. First, the expression level of Elip was much higher in field-grown than in chamber-grown cultivars. Second, substantial amounts of Elip transcripts and protein were detected during the night in field-grown plants in contrast to chamber-grown cultivars due to a synergistic effect of light stress occurring during the day and low temperature present during the following night. The expression of the PsbS protein related to Elips and involved in the photoprotection of the photosystem II was relatively constant under all conditions tested.
In order to maintain their physiological functions under light stress plants developed multiple repair and protection strategies. The induction of light stress proteins, Elips (early light-induced proteins), can be considered to be a part of such protective responses (Montané & Kloppstech 2000; Adamska 2001).
All higher plant Elips investigated so far are nuclear-encoded proteins, post-translationally imported into the chloroplast and inserted into the non-appressed regions of the thylakoid membrane. A recent purification of Elips from light-stressed pea leaves revealed (Adamska et al. 1999) that these proteins bind chlorophyll a and lutein, however, the quantity of pigments bound to Elip, as well as their binding characteristics, differed significantly from those reported for Cab proteins (Kühlbrandt, Wang & Fujiyoshi 1994; Green & Durnford 1996). First, an extremely high carotenoid content was calculated for purified Elips and second, a very weak excitonic coupling between chlorophyll molecules was measured (Adamska et al. 1999). Although no define function was found for this group of proteins these data strongly suggest that chlorophylls bound to Elips are not involved in energy transfer but fulfil alternative functions. It was proposed (Adamska et al. 1992; Braun et al. 1996; Montanéet al. 1997) that Elips might play a protective role within the thylakoids under light stress conditions either by transient binding of free chlorophyll molecules and preventing the formation of free radicals (‘pigment-carrier’ function) and/or by acting as sinks for excitation energy. A similar non-light-harvesting function was proposed for another distant member of the Cab family, the PsbS protein (Funk et al. 1995b; Li et al. 2000).
The PsbS protein (Funk 2001) is a structural component of PSII and contains four predicted transmembrane helices (Kim et al. 1992; Wedel et al. 1992). It binds four to six chlorophyll molecules and one carotenoid per protein and exhibits an unusual high chlorophyll a/b ratio (Funk et al. 1995b). However, unlike other known chlorophyll-binding proteins the PsbS protein is stable in the absence of pigments and is present in etiolated plants or chlorophyll-free mutants (Funk et al. 1995a).
In this work the expression of Elip and PsbS protein was investigated in field-grown pea cultivars with different genetic and phenotypic backgrounds and compared with chamber-grown plants. A similar pattern of Elip expression was assayed among cultivars; however, there were significant differences in amounts of the induced Elip. Furthermore, we demonstrated that in contrast to the PsbS protein, the level of which was constant during plant development and not influenced by abiotic stresses, the expression of Elip in the field-grown plants was strongly dependent on the developmental stage of pea cultivars and regulated by a synergistic action of light stress and low temperature. Interestingly, a high expression of Elip transcripts but not Elip protein was observed during the senescence process.
MATERIALS AND METHODS
Growth of plants and stress conditions
To investigate Elip expression under field conditions the experimental pea varieties (Pisum sativum var. x 5, x 8, x 353, x 695 and x 862) were grown in 1999 under agricultural conditions in a field of Svenska Findus AB near Bjuv, Sweden. All genotypes in the investigating breeding material were affected in the r (rugosus) locus (Bhattacharyya et al. 1990) determining wrinkled seeds at full seed maturity.
Leaves from the latest developed node were collected during plant development (starting from 31 d after sowing and continuing until harvest occurred at between 78 and 95 d) at the afternoon at 1500 h during a cloudy day (200–500 µmol m−2 s−1) that was preceded by at least three sunny days (1000–2000 µmol m−2 s−1). In some of the experiments leaves were collected during the night shortly before sunrise at 0400 h. The temperature during the growth period varied between 6 °C (minimal during the night) and 29 °C (maximal during the day).
To investigate Elip expression under controlled conditions pea plants were grown in a growth-chamber under a light regime 8 h dark : 16 h light at a photon flux density of 100 µmol m−2 s−1 (low light) at 20 °C during the daytime and at 18 or 10 °C during the night. In order to simulate the field conditions, light stress treatment (1000 µmol m−2 s−1) was applied for 6 h for 3 d before collection of leaves. On the day of the sample collection the light intensity was lowered to 700 µmol m−2 s−1. All irradiances were measured with a SKP200 light meter equipped with a SKP215 quantum sensor (Skye Instruments Limited, Llandrindod Wells, Powys, UK). Collected plant material was frozen in liquid nitrogen and stored at −80 °C for further analysis.
Isolation and assay of proteins
Leaves frozen in liquid nitrogen were homogenized in medium containing 300 mm sorbitol, 20 mm Hepes-NaOH pH 7·4, 5 mm MgCl2, 2·5 mm ethylenediaminetetraacetic acid and 10 mm KCl, supplemented with a protease inhibitor cocktail according to the advice of the manufacturer's (Sigma-Aldrich Sweden, Tyrresö, Sweden). The homogenate was solubilized in a sample buffer (Laemmli 1970), proteins denaturated at 70 °C for 5 min and separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970) on 14% polyacrylamide gels using a Hoefer mini gel system. The gels were loaded on an equal protein basis. However, for better quantification of Elip signals 50% less protein were loaded for samples collected from field-grown plants.
Immunoblotting was carried out according to Towbin, Staehelin & Gordon (1979) using a polyvinylidene diflouride PLUS transfer membrane with 45 µm pores (PVDF; Micron Separations Inc., Westborro, MA, USA) and an enhanced chemiluminescence assay (ECL; Amersham Biosciences, Uppsala, Sweden) as the detection system. For quantification, signals linear in intensity with exposure time (A600 < 0·8) were scanned at 600 nm (Personal Densitometer; Molecular Dynamics, Sunnyvale, CA, USA) using the Image-Quant 3·3 program (Molecular Dynamics).
Isolation and assay of RNA
Total RNA was extracted using a RNeasy mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. After separation of 3 µg RNA in 1·2% agarose gel, RNA was transferred to Hybond-N+ membrane (Amersham Pharmacia) prior to the hybridization as described (Sambrook, Fritsch & Maniatis 1989). The cDNA probes were labelled with α-32P-dCTP by using a megaprime DNA labelling kit (Amersham Pharmacia). The signal on the filter was analyzed by using a Phosphorimager FLA3000 (Fujifilm; Fuji, Tokyo, Japan).
Meteorological data including minimal and maximal temperature, an average daily temperature and photon fluency rates for the whole plant cultivation period in 1999 were obtained from the meteorological station at Selleberga, Sweden.
Seventy-five experimental pea cultivars were grown under field conditions and five of them, designated x 5, x 8, x 353, x 695 and x 862, were selected for further analysis of light stress responses using Elip and PsbS proteins as molecular markers. The selected pea cultivars differed in their genetic background, resulting phenotypes and their responses to light stress. They varied in leaf types, pigmentation of cotyledones, resistance to downy mildew, size and yield of seeds, as well as in the accumulation of antioxidant ascorbic acid in leaves and changes in the chlorophyll fluorescence in response to light stress (not shown).
To test whether light stress responses differ in various phases of plant development we investigated the expression of the Elip and PsbS proteins during the vegetative, flowering or post-flowering stadium of pea cultivars grown under agricultural (Fig. 1a) or controlled growth-chamber (Fig. 1b) conditions. Under both conditions plants encountered light stress at least 3 d before sample collection. Three pea cultivars, x 353, x 695 and x 862 were selected for these analyses. The results demonstrated that Elip expression decreased with plant development under both experimental conditions. The highest amount of Elip protein was induced in leaves collected from plants being in the vegetative phase, the amount was reduced to approximately 50% during the flowering stadium and only traces of Elip were detected in leaves collected from the post-flowering phase in field-grown cultivars (Fig. 1a, upper panel). A similar Elip expression pattern was assayed in chamber-grown plants, although the decrease of Elip amount during progressing development was less pronounced (Fig. 1b, upper panel). In chamber-grown cultivars the level of Elip expression was reduced to approximately 85% during the flowering and to 62% during the post-flowering stadium, respectively, as compared with Elip amounts induced in the vegetative stadium (Fig. 1b, upper panel).
In contrast to Elip, the amount of PsbS protein did not change significantly during plant development, however, a slightly higher amount of this protein was detected in leaves collected during the flowering stadium (Fig. 1a & b, lower panels).
It has previously been reported that the expression of Elips is strongly enhanced during senescence of tobacco leaves (Binyamin et al. 2001). To test the expression of Elip and PsbS protein in senescing leaves, pea cultivars x 353, x 695 and x 862 were grown in a growth-chamber for 130 d, yellowish leaves were collected and the amount of Elip and PsbS protein were assayed under ambient (control) or light stress conditions (Fig. 2a & b). Since progressive loss of chlorophyll is symptomatic for the decline of photosynthesis during leaf senescence (Matile 2001) we measured the chlorophyll content in collected leaves. The results revealed that the chlorophyll content in such leaves was reduced to 50% as compared to mature green leaves (not shown). For comparison, the expression of Elip transcripts (Fig. 2a) and Elip and PsbS proteins (Fig. 2b) were assayed under the same experimental conditions in young leaves collected from 14-day-old-plants. As expected no Elips were detected in young leaves kept under low light conditions but exposure of these leaves to high intensity light resulted in the induction of Elip transcripts (Fig. 2a, left panels) and accumulation of the corresponding protein (Fig. 2b, left panels). No significant changes in the PsbS protein level were assayed under these conditions (Fig. 2b, lower panel). In contrast, a significant amount of Elip transcripts was detected in senescing leaves of cultivars x 353 and x 695 and a residual amount in the cultivar x 862 under low light conditions (Fig. 2a, right panels) but no Elip protein was detected in the thylakoid membranes (Fig. 2b, right panels). Exposure of leaves to light stress resulted in a massive accumulation of Elip transcripts in all three cultivars to a level that was approximately 10-times higher than that assayed in young leaves (Fig. 2a, compare left and right panels). Surprisingly, only a very small amount of Elip protein accumulated in senescing leaves after the light stress treatment (Fig. 2b, right panels). This indicates a post-transcriptional or/and post-translational regulation of the Elip expression during senescence. The amount of Elip protein in senescing leaves was reduced to approximately 30% as compared with the level present in young leaves (Fig. 2b, compare left and right panels). In contrast to Elip no significant changes in the PsbS protein level were assayed in senescing leaves under low or high light conditions (Fig. 2b, lower panel).
Synergistic effect of light stress and low temperature on Elip expression in the field
It has previously been shown (Adamska, Scheel & Kloppstech 1991) that the expression of Elip in pea seedlings grown under controlled growth-chamber conditions undergoes diurnal and circadian oscillations. To investigate a diurnal regulation of Elip expression in the field we collected samples during the night shortly before sunrise and during the early afternoon of the following day for both vegetative and flowering stadiums of plant development (Fig. 3a, left panels). In parallel the expression of Elip was assayed during the dark and light phases in pea cultivars grown in a climate-chamber under controlled light and temperature conditions (Fig. 3a, right panels). Immunoblot analysis revealed that there were significant differences in Elip expression under field and growth-chamber conditions. Generally much higher Elip levels were assayed in plants grown in the field than in a growth-chamber, especially during the vegetative stadium of plant development (compare Fig. 3a, left and right panels). The most pronounced difference, however, was the presence of Elip in leaves collected at night from field-grown cultivars and its absence in the corresponding leaves collected from chamber-grown plants. The amount of Elip detected during the night depended on both, the investigated cultivar and the developmental stage of plants but was highest during the vegetative stadium of plant development in all three cultivars (compare with Fig. 1a&b). Residual amounts of Elip protein were assayed in leaves collected during the night from the flowering stadium of the field-grown pea cultivar x 695 after overexposing of immunoblots (not shown). Under both, field and growth-chamber conditions, a significant amount of Elip was detected in leaves collected during the daylight period (Fig. 3a, left and right panels).
To test whether the differences in the protein expression between field- and chamber-grown pea cultivars are restricted to Elip or represent a more general regulation of gene expression we assayed the amount of the PsbS protein, the D1 protein of PSII reaction centre and four antenna proteins of PSI under the same experimental conditions (Fig. 3b). The results demonstrated that there are no significant differences in the expression pattern of these proteins between field- and chamber-grown cultivars (Fig. 3b, compare left and right panels).
Since it was reported that the accumulation of Elip in mature green pea plants grown under controlled conditions depends on light intensity and a duration of light stress (Adamska et al. 1992; Pötter & Kloppstech 1993) the expression of Elip was investigated under field conditions in a correlation to photon fluency rates in five different pea cultivars. A mean value obtained from the quantification of immunoblots was plotted on a graph showing the average daily light intensity (Fig. 4a). The results demonstrated surprisingly that the level of Elip expression did not correlate with the light intensity present on the day of the sample collection. The Elip expression on day 31 was very low despite of a high photon fluency rate during this day or on day 45, where a significant amount of Elip was present in the absence of high-intensity light (Fig. 4a). Similarly to the results shown in Figs 1 and 3 the amount of induced Elip decreased during the flowering and in post-flowering stadium independently of the daily photon fluency rates. Interestingly, a very high level of the transient Elip expression was noticed during the first few days of the flowering phase as shown for day 57 (Fig. 4a). This high expression of Elip did not result from changes of environmental conditions as a similar high Elip expression at the beginning of plant flowering was observed in five cultivars grown under controlled growth-chamber conditions (not shown).
It was previously shown (Adamska & Kloppstech 1994) that the combination of light stress with low temperature increased expression of Elip-mRNA in pea leaves. Furthermore, exposure of non-hardened barley plants to prolonged light stress and cold treatment applied in a growth-chamber under light-dark conditions resulted in a constantly high level of Elips (Montanéet al. 1997). Thus, the high amount of Elip during the night (Fig. 3a, left panel) or during cloudy days (Fig. 4a) in field-grown cultivars could result from the effect of low temperature on the Elip expression.
The data obtained from the climate station over the whole plant cultivation period revealed that night temperature in the field was approximately 8–10 °C lower than that set in the growth-chamber and was on average close to 10 °C for the first 60 d of growth (Fig. 4b). The average daily temperature was close to 15 °C; however, maximal values reached 22–25 °C at the early afternoon between 1200 and 1500 h (not shown).
To determine whether the low temperature influences Elip expression we exposed chamber-grown pea cultivars to two different temperatures, 10 °C (cold-treatment) or 18 °C (control) during the night and Elip transcript and protein levels were assayed by Northern (Fig. 5a, upper panel) or Western (Fig. 5b, upper panel) blotting during the dark or light period. Northern blot results demonstrated (Fig. 5a, upper panel) that different Elip expression patterns were obtained depending on the night temperature. A high level of Elip transcripts was detected during the dark period in leaves from plants exposed to cold treatment and only traces of these transcripts were present in control plants exposed to 18 °C (Fig. 5a, left panel). Furthermore, in cold-stressed plants the amount of Elip transcripts decreased during the day reaching approximately 10% of the night value at 1500 h. In control plants Elip transcripts were induced during the day in response to light stress but their level was significantly lower (Fig. 5a, upper panel). The transcript level assayed for psbS, lhcb2 (encoding the major antenna protein of PSII) and psbA (encoding the D1 protein of PSII reaction centre) genes under the same experimental conditions (Fig. 5a, middle and lower panels) revealed that the expression of these genes was either not significantly influenced by low temperature (as for the psbS gene) or the expression pattern was similar to that of the elip gene (as for lhcb2 or psbA genes) but fluctuations of the transcript level were less drastic.
To assess whether the changes in Elip transcript level under ambient and cold stress conditions were accompanied by changes in the protein level immunoblotting analyses were performed. The results demonstrated (Fig. 5b, upper panel) that the accumulation of Elip in thylakoid membranes follows the expression pattern of Elip transcripts. A high level of Elip was detected in cold-stressed plants at night and this level was reduced to 60% in samples collected during the day (Fig. 5b, upper panel). Corroborating previous findings reported for chamber-grown pea plants (Adamska et al. 1991) no Elip was detected in leaves harvested during the dark period from plants grown at 18 °C but the exposure of these plants to light stress during the day resulted in Elip accumulation. There were no significant changes in amount of PsbS, Lhcb2 and D1 proteins under all conditions tested (Fig. 5b, middle and lower panels).
The current knowledge about regulation of elip and psbS gene expression is restricted to relatively young plants grown in a growth-chamber under controlled conditions in terms of light, temperature, water or nutrition supply. Only very limited studies were performed on the expression of these proteins under field conditions, where plants are exposed simultaneously to different stresses during their growth and thus partially pre-adapted to environmental changes. This work was dedicated to studies on Elip and PsbS protein expression in field-grown pea cultivars in comparison with chamber-grown plants. The results of our studies have shown that all investigated pea cultivars, independent of genetic background and phenotype, had a similar expression pattern of the elip gene, both under field or growth-chamber conditions. However, there were significant quantitative differences in the level of Elip expression assayed among cultivars.
Comparing the expression pattern of the elip gene between field- and chamber-grown plants we found several substantial differences. Firstly, the expression level of Elip was generally much higher in field-grown than in chamber-grown plants. This effect can be explained by a better pre-adaptation of field-grown plants to high irradiances than plants from the growth-chamber and thus higher level of Elip might offer a better protection against light stress-induced damage.
Secondly, the amount of Elip in thylakoid membranes in field-grown plants was regulated by a synergistic action of light stress and low temperature. It has been demonstrated in the past (Adamska et al. 1991) that in pea plants grown under ambient temperature conditions the accumulation of Elip occurred only during the light period and this protein was subsequently degraded to an undetectable level in the dark. In the present study the combination of light stress during the preceding day with low temperature during the following night resulted in a constant presence of Elip protein in field-grown cultivars. A comparable high level of Elip during the night could be induced in chamber-grown plants by lowering the temperature to 10 °C during the dark phase. A similar effect was reported for non-hardened barley plants during the acclimation to high light fluxes performed at 5 °C (Montanéet al. 1997). The authors suggested that this effect is caused by the uncoupling of the Elip expression from circadian control by light stress and an increased Elip-mRNA stability at low temperature.
An uncoupling of the Elip expression from circadian control and an increased mRNA stability would also explain a high accumulation of Elip transcripts during the night in field-grown cultivars. However, the most probable explanation for a high level of Elip protein in these cultivars is the inhibition of Elip proteolysis by low temperature. We showed previously (Adamska et al. 1996) that the Elip from pea is degraded by a serine-type protease under low light conditions. This protease is an extrinsic 65 kDa protein constitutively present in the stroma lamellae of the thylakoid membranes. Furthermore, the proteolytic activity of this enzyme showed an unusual temperature sensitivity and was reversibly inactivated at temperatures below 20 and above 30 °C (Adamska et al. 1996). Thus, the high level of Elip during the night in field-grown plants is most likely the result of the inactivation of Elip-degrading protease at low temperatures.
Humbeck and colleagues (Humbeck, Kloppstech & Krupinska 1994; Humbeck, Quast & Krupinska 1996) reported that the light stress-dependent Elip expression in the field was independent of the developmental stages of barley flag leaves. Developing, mature and senescing leaves of barley had the same capacity to synthesize Elip in response to light stress. In contrast, our data demonstrated that the expression of Elip in all pea cultivars was clearly developmentally regulated, both under field and growth-chamber conditions and was decreasing during the flowering and post-flowering stadium. This pattern of Elip expression might result from a lower requirement for the photoprotection of the photosynthetic apparatus with increasing plant age, especially after seed formation. Interestingly, a transient increase of Elip expression independent of environmental conditions was assayed at the beginning of the flowering phase. However, the physiological importance of this phenomenon is not yet known.
Recently, it was shown (Binyamin et al. 2001) that Elip-mRNA and protein were strongly up regulated during natural senescence of tobacco leaves at low intensity light but no Elip induction was assayed during dark-induced senescence. A high level of Elip-mRNA and protein was reported to accumulate after onset of senescence on bright days in spring and winter barley varieties grown under agricultural conditions (Humbeck et al. 1994). Our data demonstrate that a significant amount of Elip transcripts was present in senescing pea leaves under low light conditions and the exposure of these leaves to light stress resulted in a massive induction of these transcripts. The corresponding protein, however, did not accumulate to the same extent. The Elip protein was not detected in senescing leaves under low light conditions and its level was relatively low in light-stressed leaves in comparison with young leaves. Thus, the reported induction of Elip in senescing tobacco leaves (Binyamin et al. 2001) might represent a plant species-specific response or be related to the degree of light stress-induced damage caused by the loss of protective pigments during the senescence process. On the basis of our data we assume that the induction of Elip transcripts during senescence of pea leaves had no physiological significance and could be related to a factor that is induced during senescence and involved in light stress signalling. Identification of such a signal is under current investigation.
Our previous data showed that the light stress treatment did not influence the PsbS transcript and protein level in tobacco (Funk et al. 1995a) or spinach (Lindahl et al. 1997) plants. The results presented in this work corroborate these findings for all pea cultivars. Furthermore, the amount of the PsbS protein did not change significantly during various developmental stages of pea plants including senescence and was not influenced by low temperature. In contrast, it was reported recently that the level of PsbS-mRNA in Arabidopsis is increased several-fold in response to high light (Li et al. 2000). Furthermore, chilling treatment of both cold-tolerant and cold-sensitive potato varieties induced a transient accumulation of the PsbS transcripts in the light at room temperature and this accumulation was strongly enhanced by low temperature treatment (Rorat et al. 2001). However, no changes in the protein abundance were noticed under these conditions. This discrepancy in reports on the PsbS expression under light stress or cold stress conditions might result from plant specific responses or be related to different experimental setups.
A light stress-protective function has been proposed for Elips and the PsbS protein (Montané & Kloppstech 2000; Adamska 2001; Funk 2001). Interestingly, both proteins are located in different populations of PSII. Whereas the PsbS protein is constitutive present in the appressed region of grana stacks (Funk et al. 1995b), where the functional PSII complexes are located, Elips accumulate only transiently in non-appressed regions of thylakoid membranes (Adamska & Kloppstech 1991), where photodamaged PSII complexes migrate in order to be repaired. Thus, the protective function of both proteins seems to be complementary and the expression pattern of these proteins in field-grown plants is in agreement with this function.
We thank Matilda Andersson, Anna Stegmark, Annette Olofsson, Ingrid Johansson, Anna Kerstin Arvidsson and Marianna Wiren for skillful assistance. This work was supported by research grants from the Swedish Natural Research Council, the Swedish Strategic Foundation, the Carl Tryggers Foundation and Findus R & D AB.
Received 15 April 2002;received inrevised form 26 July 2002;accepted for publication 29 July 2002