T ~ E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 48, Issue of December 2, pp. 30221-30226, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Low Temperature Increases the Abundance of Early Light-inducible Transcript under Light Stress Conditions*

(Received for publication, July 25, 1994)

Iwona Adamska and Klaus KloppstechS From the Znstitut fur Botanik, Universitat Hannouer, Herrenhauser Str: 2, 30419 Hannouer, Federal Republic of Germany

Green pea plants respond to light stress by expression of a nuclear ELIP (early light-inducible protein) gene. Here we report that the accumulation of ELIP transcript in pea plants during light stress is enhanced by low tem- perature treatment. The enhanced level of ELIP tran- script during combined light and cold stress was found to be due to an increased stability of ELIP messenger RNA under these conditions. This transcript is translat- able in vitro. In vivo, however, the amount of accumu- lated protein in the thylakoids declines with the de- crease in the temperature because the translational activity is strongly reduced already at 10 “C.

Plants exposed to light stress at temperatures that do not allow accumulation of ELIP transcript respond by induction of ELIP mRNA and protein during recovery at low light intensity and ambient temperature. The amount of protein that accumulates as a result of this “memory effect” is, however, much lower than that which accumulates as a result of direct light stress. The memory of a perceived light stress persists in plants stored at low temperature for at least 3 h, and the stress response can be released after an increase in tempera- ture. Prolonged cold treatment, however, has a negative effect on the translatability of the ELIP transcript that accumulates during recovery.

Early light-inducible proteins (ELIPs)’ are nuclear encoded chloroplast proteins whose genes are transiently transcribed during the greening process of etiolated plants (Meyer and Kloppstech, 1984; Grimm and Kloppstech, 1987). Only recently it was discovered that ELIPs can be induced in mature green plants exposed to light stress (Adamska et al., 1992a; Potter and Kloppstech, 1993). Analyses of ELIP induction as a func- tion of light quality demonstrated that blue (410-480 nm) and UVA (335-365 nm) light are the most effective wavelengths for expression of ELIP genes in green plants. These data indicate that the photoreceptor cryptochrome is involved in perception of the light stress (Adamska et al., 1992a, 1992b).

ELIP mRNA is short-lived under ambient and light stress conditions. In contrast, the thylakoid-bound ELIP appears to be stable under light stress conditions but is degraded during recovery from light stress at low light intensity (Adamska et al., 1993). The molecular basis for protein stabilization under light

* This work was supported by Deutsche Forschungsgemeinschaft, Bonn, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed. Tel.: 49-511-762- 2633; Fax: 49-511-762-3992; E-mail: adamska8mbox.botanik.uni-han- nover.de.

tein(s1; LHCP, light-harvesting chlorophyll alb protein; E, einstein; p s The abbreviations used are: ELIP(s), early light-inducible pro-

11, photosystem 11.

stress conditions is not yet known. Accumulation of ELIP in the thylakoid membranes correlates with the photoinactivation of photosystem I1 (PS 111, degradation of D l protein, and changes in the level of pigments (Adamska et al., 1993). Based on these results, it was proposed that ELIP is a light stress response protein (Adamska et al., 1992a).

So far no definite function has been described for ELIP in higher plants. Sequencing of ELIP cDNA clones (Kolanus et al., 1987; Grimm et al., 1989) has demonstrated that ELIPs are closely related to the chlorophyll alb-binding (cab) gene family. Proteins that have high sequence homology to the ELIPs and that are related to carotene biosynthesis under light stress conditions have been reported to exist in the unicellular alga Dunaliella bardawil (Lers et al., 1991; Levy et al., 1992). A desiccation- and light-induced protein related to ELIP has been described for the resurrection plant Craterostigma plantag- ineum (Bartels et al., 1992).

Exposure of plants to light stress at chilling or freezing tem- peratures causes damage to the photosynthetic apparatus that is much more severe than that observed at ambient tempera- tures (for reviews see Krause (1988), Oquist et al. (19871, and Hunter et al. (1993)). In circumpolar and alpine habitats such conditions appear to be quite normal and occur for instance when plants emerge from the snow and are exposed to bright and reflected sunlight. Thylakoid membranes have been iden- tified as a main target of the chilling injury (for a review see Hunter et al. (1993)). Several factors contributing to cold-in- duced damage in vivo have been described, such as a reduced ability of inactivated PS I1 for degradation and synthesis of D l protein (Chow et al., 1989; Gong and Nilsen, 1989; Aro et al., 19901, slowed migration of proteins between appressed and stroma-exposed thylakoids (Carlberg et al., 19921, and a de- creased capacity of oxygen scavengers (Richter et al., 1990; Schoner et al., 19891, as well as a lower ability to form zeaxan- thin (Demmig and Bjorkman, 1987).

Very little is known, however, regarding changes in gene expression in cold-susceptible plants exposed to light and cold stress. I t was of particular interest to assess whether exposure of leaves to low temperature during light stress would affect the expression of light-regulated genes. In this work we report that the abundance of ELIP transcript is positively regulated by cold stress. In contrast to other cold-regulated genes known to date, light is the essential factor for the accumulation of ELIP transcript, and low temperature has a regulatory effect on the mRNA level. Furthermore, the response to light stress can be “memorized” in plants stored at low temperatures and released after an increase in temperature.

MATERIALS AND METHODS Growth of Plants and Illumination-Pea plants (Pisum sativum L.

var. Rosa Krone) were grown for 12-14 days on vermiculite at 25 “C at a light intensity of 50 pE/mZs provided by white fluorescent lamps under a light regime of 12 h darWl2 h light (light from 08:OO to 20:OO).

The high light treatment was performed on detached mature leaves floated on water in temperature-controlled water baths. The tempera-

3022 1

30222 Expression of ELIP Gene under Conditions of Stress tures of the water baths were 5, 10, 15, and 2 0 T , respectively. A temperature of 0 "C was achieved by addition of ice to the water bath. High light intensity was provided by homemade lamps with four 1000- watt bulbs (Philips PF811). The plants were protected from excess in- frared radiation by a water bath containing a 3-cm water layer. In various experiments light intensities of 2,000-3,000 pE/m's were used because it has been shown before that ELIP transcription saturates at levels as low as 1,000-1,500 pE/m's (Adamska et dl., 1992a).

Isolation and Assay of RNA-Poly(A+) RNA was isolated as described (Potter and Kloppstech, 1993) using oligo(dT)-cellulose chromatography (Pemherton et al., 1975). I n vitro translation was performed using the wheat germ system according to Roberts and Paterson (1973) in the presence of 1:"Slmethionine (Amersham Corp.). Dot blot hybridization was performed using a '"P-labeled random-primed homologous cDNA insert of ELIP clone p17C2 (Kolanus et al., 1987) or LHCII clone (Otto et al., 1988) according to Amersham protocol.

Isolation of Nuclei and in Vitro Danscription-Nuclei were isolated from pea plants exposed to 1,000 pE/m's for 30 min using the method described by Gallagher and Ellis (1982). The final nuclei pellet was resuspended in 50 mxl Tris-HCI, pH 8.5,5 m>T MgCI,, 10 mhf p-mercap- toethanol, and 50% ( d v ) glycerol a t a concentration of loR nuclei/ml and stored a t -80 "C.

A standard transcription assay mixture (60 pl) contained loG nuclei in 50 mxr Tris-HCI, pH 8.5,5 my MgCI,, 10 mu P-mercaptoethanol, 20% (v/v) glycerol, 50 mxr (NH,,)?SO,,, 2.5 pw creatine phosphate, 25 pg of creatine phosphokinase, 0.5 mxr ATP, CTP, and UTP, and 2 pCi of la-:"PIGTP (400 Ci/mmol). The assay mixture was incubated a t various temperatures (0-20 "C) for 30 min, and transcription was terminated by spotting aliquots onto filter discs. As a control, an aliquot of tran- scription mixture was taken before starting the incubation. The filter discs were washed 5 times for 5 min with 5% trichloroacetic acid, and then twice in ethanol. The discs were then dried and counted in a scintillation counter.

In preparative scale assays (1.2 ml), loR nuclei and 150 pCi of In-:"PIGTP were used. Transcription was stopped by the addition of 20 pg/ml DNase (RNase-free), and incubation was continued a t 25 "C for an additional 10 min. Nuclear RNA was isolated by the procedure of McKnight and Palmiter (1979), and the final ethanol precipitate was dissolved in 100 my Tris-HCI, pH 7.6, and 50 mhr EDTA. Hybridization of labeled nuclear transcripts to dot blots containing 600, 300, and 150 ng of ELIP insert was performed according to Amersham protocol. The labeled RNA was heated to 100 "C for 5 min prior to hybridization.

In Vivo Labeling and Isolation of Thylakoid Membranes-For in vivo labeling of proteins, detached leaves were floated in water in the pres- ence of 100 pCi/ml ["Slmethionine (1220 pCi/mmol, Amersham Corp.) during light and temperature treatments. At the end of the incubation the leaves were frozen in liquid nitrogen and further used or stored a t -80 "C. Thylakoid membranes were isolated as described (Adamska et al., 1993).

Electrophoresis and Zmmunoblotting-SDS-polyacrylamide gel elec- trophoresis was performed according to Laemmli (1970) using the Hoef- fer minigel system. Radioactive gels treated for fluorography (Bonner and Laskey, 1974) were exposed to x-ray film a t -70 "C. Immunoblot- ting was carried out according to Towbin et al. (1979) using 45-pm pore size nitrocellulose filters. Blots were incubated with antibodies and detected by the enhanced chemiluminescence method (Amersham Corp.) as described (Adamska et a]., 1993).

Measurenzents of Photosvstem ZIActivit.y-Fluorescence induction ki- netics were measured using a Walz PAh4 fluorimeter. The leaves were placed between two glass plates and exposed to a saturating 1-s light flash through a 5-mm d ) aperture. Variable fluorescence was calculated (F<, = (F,,, - F,,)/F,,) as described (Schuster et al., 1988).

RESULTS Low Temperature Regulates ELIP lhnscr ipt and Protein

Levels under Light Stress Conditions-Detached pea leaves were exposed to high light (3,000 pE/m2s) a t various tempera- tures between 0 and 20 "C for 2 h, and the ELIP transcript level was assayed by in vitro translation of isolated poly(A+) RNA (Fig. L4) and by dot blot hybridization with a "P-labeled insert of an ELIP clone (Fig. 1B). To distinguish between light and low temperature effects on gene expression, control plants were exposed to various temperatures a t low light conditions (Fig. 1, left panel ). The results of such an experiment demonstrate that the steady state level of ELIP mRNA induced by high intensity light increases with decreasing temperature and is maximal

LL HL 0 5 10 15 20 0 5 10 15 20 temp. ["C]

= = W " - - t g ! R

pssu .."""..- A

PLHCP F- " " . u -

- S C Z L - * * - - pELlP

- _. c : E - p Pg RNA

0.1 3 0.25 * 9 ] ELIP 0.5 0 . 0 0

0.13 0 0 L 0 0 0 0

0.25 LHCP 0 0 0 0 0 0 0 0 0 0

B 0.5 I O O O O O O O O O .

FIG. 1. Temperature-dependent accumulation of ELIP tran- script during light stress. Detached leaves were exposed to low (LL; 40 pE/m's) or high intensity (HL; 3,000 pE/m2s) light for 2 h a t various temperatures as indicated. A, in vitro translation of isolated poly (A') RNA followed by fluorography. Bars indicate positions of precursors for three light-regulated proteins: pELIP, pLHCP, and pSSU (small subunit of ribulose-1,5-bisphosphate carboxylase). B, dot blot hybridization with labeled inserts of ELIP and LHCP clones.

around 10 "C. Below 10 "C, the level of ELIP mRNA decreases again; however, the amount of ELIP transcript obtained a t 5 "C is the same as that which accumulates a t 20 "C. When the plants were exposed to light stress at 0 "C, ELIP transcript was undetectable. Since the accumulation of ELIP transcript could not be induced by cold stress alone (Fig. U, left panel), low temperature seems to have a regulatory role superimposed on that of strong light.

The transcript level of other predominant nuclear encoded chloroplast proteins was neither positively nor negatively af- fected by low temperature treatment under low light conditions (Fig. 1, A and B, left panels). Under conditions of light stress, however, the level of mRNA for light-harvesting chlorophyll a/b protein (LHCP) decreased by 50% with an increase in the tem- perature from 0 to 20 "C (Fig. 1, right panel 1. In contrast to that, the level of mRNA for the small subunit of ribulose-1,5-bisphos- phate carboxylase remained constant under these conditions.

Low temperature could modulate transcriptional activity of specific genes or could control mRNA abundance posttranscrip- tionally at the level of mRNA stability. To test whether cold stress affects accumulation of other light-inducible transcripts to the same extent as ELIP mRNA, dark-preadapted plants were used for further experiments. After exposure of young plants to the darkness for 3 days, the abundance of light-regu- lated transcripts is considerably lowered, and such plants can be used to study the re-accumulation of these transcripts after exposure to light (Fluhr et al., 1986). Since ELIPs are distant relatives of the cab gene family (Grimm et al., 1989; Green et al., 1991), temperature-dependent accumulation of LHCP tran- script was tested in dark-preadapted plants exposed to light stress and compared with that of ELIP mRNA (Fig. 2). The pattern of accumulation of ELIP mRNA in dark-exposed plants was very similar to that observed in light-grown plants (com- pare Fig. 1). Also in this experimental system the accumulation of ELIP transcript was found to be maximal a t 10 "C. In dark- preadapted plants only traces of cab transcript could be de- tected. In contrast to ELIP, however, during illumination of such plants an almost constant steady state level of cab tran- script was observed at the temperatures of 5-20 "C. At a tem-

Expression of ELIP Gene under Conditions of Stress 30223 J/i:rI C 0 5 10 15 20 tamp. PC]

0 - ] ELlP

a 0 . 0 .

[ i" ] LHCP * * e o 0

FIG. 2. Different regulation of gene expression by low temper- ature in dark-preadapted plants exposed to light stress. Pea plants were grown for 5 days under lightldark conditions and then transferred to darkness for 3 additional days. Detached leaves of such plants were harvested in darkness (lane C) or exposed for 2 h to light stress (2,000 pE/m's) a t various temperatures. Poly(A+) RNA was iso- lated and used for dot blot hybridization with labeled inserts of ELIP and LHCP clones.

perature of 0 "C, no light-induced accumulation of LHCP tran- script could be observed (Fig. 2).

The high abundance of ELIP transcript in plants exposed to light stress a t 10 "C might be a consequence of slower degra- dation of ELIP mRNA at lower temperatures. To test this pos- sibility, ELIP transcription was induced in leaves exposed to light stress a t ambient temperature, and subsequently the deg- radation of ELIP transcript was assayed a t various tempera- tures after transfer of plants to low light conditions. The results of this experiment (Fig. 3) show that the rate of ELIP mRNA degradation is indeed temperature-dependent. While no signif- icant degradation of ELIP transcript was detected during the 3 h of recovery a t 0 "C, ELIP transcript was completely degraded in leaves exposed for the same period of time to temperatures of 15-20 "C. In contrast to ELIP mRNA, the LHCP transcript was stable a t all temperatures tested.

In order to test the extent of ELIP transcription more di- rectly, a run-off transcription was carried out at the various temperatures. For this purpose nuclei were isolated from plants that were pretreated a t a light intensity of 1,000 pE/m2s for 30 min for the induction of ELIP transcription. Such nuclei were used for the transcription assays, which were incubated for 30 min at various temperatures between 0 and 20 "C as indicated (Fig. 4). The rate of total transcription roughly doubled with a stepwise increase in temperature by 5 "C. In contrast to that, the transcription rate specific for ELIP gene, measured by dot blot hybridization, remained very low between 0 and 5 "C, increased rapidly between 5 and 10 "C, and stayed almost constant between 10 and 20 "C.

The accumulation of ELIP protein within the thylakoid mem- branes during exposure of plants to light stress at various temperatures was assayed by Western blotting (Fig. 5A). As previously reported (Adamska et al., 1992a) ELIP accumulates only under high light conditions; however, the amount of pro- tein detected in the thylakoids was clearly related to the ap- plied temperature (Fig. 5). The amount of ELIP declined almost linearly with decreasing temperature between 20 and 5 "C. No ELIP was detected in leaves exposed to light stress at 0 "C, which is in accordance with the finding that at this tempera- ture ELIP transcript was not induced (compare Fig. 5A with Fig. 1, A and B) . In parallel experiments measurements of PS I1 activity were performed. It was found that the degree of PS I1 inactivation was almost temperature-independent and that the PS I1 activity was reduced to 308 of the initial value in leaves exposed to light stress for 2 h (Fig. 5B) .

It was reported previously (Kahn and Walbot, 1989) that prolonged cold stress can suppress the synthesis of some pro- teins in plants and increase the synthesis of others. Thus i t was of interest to determine whether a similar situation might oc-

FIG. 3. Temperature-dependent degradation of ELIP tran- script during recovery from l ight stress. Detached leaves were exposed to light stress (HL; 2,000 pE/m2s) for 2 h a t 20 "C and then transferred to low light conditions for recovery (Rec. ) a t various tem- peratures for 1-3 h. Poly(A+) RNA was isolated and used for dot blot hybridization with labeled inserts of ELIP and LHCP clones.

100

= a0 0

a) 40 .- > m c.

0

0°C

5°C

10°C

15°C

20°C

ELlP transcript

0°C 5°C 10°C 15°C 20°C Temperature

lated nuclei. Nuclei isolated from preilluminated (30 min a t 1,000 FIG. 4. Temperature-dependent synthesis of transcripts in iso-

pE/m2s) pea plants were used for run-off transcription assays at various temperatures for 30 min as described under "Materials and Methods." The overall transcription rate (W) was estimated by scintillation count- ing of the incorporated [a-"PIGTP into trichloroacetic acid-precipitable material. The maximum value (102 x 10" cpm) was set to 100%. Tran- scription rate specific for ELIP gene (0) was determined by hybridiza- tion of isolated labeled transcripts with the different concentrations (600, 300, and 150 ng) of ELIP gene-specific probe bound to nylon membrane. After hybridization and exposure, the dots were scanned, and the maximum value that was reached a t 20 "C was set to 100%.

cur during brief cold treatment applied in combination with light stress. For this reason detached leaves were radioactively labeled during exposure to high light treatment at various tem- peratures. The results of this experiment (Fig. 5C) show that low temperatures between 0 and 5 "C significantly inhibit the synthesis of both cytosolic and chloroplast proteins independ- ent of light intensity. The effect of light stress on protein syn- thesis was pronounced only a t temperatures above 10 "C, as follows from the amount of radioactivity incorporated into ELIP, LHCP, and D l proteins (Fig. 5C, compare panels LL and HL ).

Based on these results we can conclude that the abundance of ELIP transcript induced by light can be positively regulated by low temperature. The cold stress, however, prevents accu- mulation of the correspondent protein due to a general sup- pression of the translational activity.

30224 Expression of ELIP Gene under Conditions of Stress

LL HL

€LIP - "- A , - A

0 5 10 15 20 0 5 10 15 20

TEMPERATURE ['C]

LL HL o 5101520 0 5101520 temp. ["Cl

Flc. 5. Temperature-dependent accumulation of ELIP in the thylakoid membranes under light stress conditions. Detached leaves were exposed to low (LL; 40 uE/m's) or high (HL; 3,000 pE/m's) intensity light for 2 h a t various temperatures as indicated. A, Western blot; B , activity of photosystem I1 assayed by measurements of variable fluorescence. The control (100%) value measured prior to illumination

tein, LHCP, and ELIP were identified by Western blotting with the was 2.8. C , fluorography of in vivo labeled thylakoid proteins. D l pro-

corresponding antibodies.

Accumulation of ELIP Dunscript and Protein during Recov- ery from Light and Cold Stress-Detached leaves were exposed to light stress a t 0 and 10 "C for 2 h, and changes in ELIP transcript (Fig. 6) and protein (Fig. 7) levels were analyzed during 1-3 h of recovery at low light and ambient temperature. Control plants were exposed to light stress at 20 "C prior to recovery. In agreement with earlier findings (Adamska et al., 1992b), in control plants the ELIP mRNA that accumulated during light stress is rapidly degraded under low light condi- tions as shown by in vitro translation of isolated poly(A+) RNA (Fig. 6 A ) and dot blot hybridization (Fig. 6B). In this particular experiment small amounts of ELIP transcript were induced in leaves exposed to light stress a t 0 "C. However, in clear contrast to the control plants, the ELIP mRNA in such plants accumu- lates during recovery. When plants were exposed to high light treatment a t 10 "C, the amount of ELIP mRNA induced during light stress remained stable during the first hour of recovery and decreased thereafter. These observations indicate that the light stress can be "memorized" in plants kept a t low tempera- tures and that the response to light stress can be released after return of plants to ambient temperatures. As opposed to ELIP transcript, the levels of both small subunit of ribulose-1,5-bi- phosphate carboxylase and LHCP mRNAs did not change sig- nificantly during recovery (Fig. 6, A and B) .

To assess whether the ELIP mRNA that was induced during recovery from light and cold stress is also translated in vivo and whether the protein accumulates in the thylakoid membranes, Western blotting analyses were performed. The results of this experiment (Fig. 6A) show that ELIP accumulates in leaves during recovery from light and cold stress when the light stress was applied a t 0 or 10 "C. Cold stress, however, reduces the total amount of accumulated protein as compared with leaves exposed to light stress a t ambient temperature (Fig. 6B). No

B

FIG. 6. Changes in ELIP transcript level during recovery from light and cold stress. Detached leaves were exposed to light stress (HL; 2,000 pE/mYs) for 2 h at the indicated temperatures and then transferred to recovery conditions a t 25 "C and a light intensity of 40 pE/mYs (Rec. 1 for 1 or 3 h. A, in vitro translation of isolated poly(A') RNA followed by fluorography. Burs indicate the positions of precursors as described in Fig. 1. I?, dot blot hybridization with labeled inserts of ELIP and LHCP clones.

0°C' 1 3 ' 10OC'l 3' 20°C'1 3 ' hours

ELW 1 4 1 " 0" A r100

FIG. 7. Accumulation of ELIP in the thylakoid membranes dur- ing recovery from light and cold stress. Detached leaves were ex- posed to light stress (HL; 2,000 pE/m's) for 2 h at the indicated tem- peratures and then transferred to recovery conditions for 1 or 3 h at 25 "C and a light intensity of 40 pE/m's (Rec. ). A, Western blot; B, recovery of photosystem I1 activity measured as an increase of variable fluorescence. The 100% value measured before exposure of leaves to the stress conditions was F,. = 2.8.

further increase in ELIP content during recovery was observed when light stress was applied at 20 "C.

We have reported before (Adamska et al., 1993) and con- firmed in this work (Fig. 7, A and B ) that ELIP is degraded during recovery from light stress a t ambient temperature when PS I1 activity is restored. In the present case the increase in PS I1 activity during recovery from light and cold stress is accom- panied by an accumulation of ELIP in the thylakoid mem- branes (Fig. 7, A and B) . After 3 h of recovery, 80% of photo- synthetic activity was recovered, and no degradation of ELIP was observed.

The Response to Light Stress Persists in Plants Stored at Low Temperature-To answer the question of whether the potential to induce ELIP transcription can be preserved for a longer period of time in plants subjected to light stress at low temper-

Expression of ELIP Gene under Conditions of Stress 30225

A 0.13 P9 RNA

0.25

0.5

0.1 3 0.25

0.5

-1 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

] ELlP

1

J LHCP

B - " _ - ELIP FIG. 8. Delayed response to light stress during recovery at am-

bient temperature. Detached leaves were exposed to light stress (HL; 3,000 pE/m's) for 2 h at 0 "C and then transferred to low light conditions (LL; 40 pE/m's) for 1-3 h. Either the temperature was raised to 25 "C immediately after transferring the leaves to low light conditions, or leaves were incubated for 1 or 3 h at 0 "C and low light prior to transfer to growth temperature for an additional 1-3 h.A, dot blot hybridization with labeled inserts of ELIP and LHCP clones. B, Western blot. Lanes marked C in all panels represent poly(A+) RNA or proteins isolated before exposure of plants to light stress.

ature, the following experiment was performed. Detached leaves were exposed to light stress at 0 "C for 2 h and then transferred to low light and normal growth temperature for recovery. While one set of leaves was transferred to ambient temperature immediately after cessation of light stress, the other sets of leaves were incubated for another 1-3 h at 0 "C before recovery. The results of this experiment are shown in Fig. 8. In accordance with previous data (Figs. 1 and 2), no ELIP transcript was induced during exposure of plants to light stress at 0 "C. However, ELIP mRNA accumulated gradually during prolonged exposure of leaves to low temperature even after cessation of light stress. The maximal ELIP mRNA level was reached after 3 h of cold treatment (Fig. 8A ). Rewarming of leaves results in very fast accumulation of ELIP transcript. ELIP transcript that accumulated under these conditions ap- pears to be stable and is not degraded during up to 3 h of low light treatment.

Interestingly, longer cold treatment appeared to influence mRNA translatability in vivo. This was tested in vitro by trans- lation of isolated poly(A+) RNA that was translatable (data not shown) and in vivo by Western blotting (Fig. 8B). Significant amounts of ELIP accumulated in the leaves only when cessa- tion of light stress was accompanied by an increase in temper- ature. Traces of ELIP were still detectable in leaves when re- warming took place 1 h after alleviation of light stress, while a 3-h exposure to cold stress caused the ELIP to be no longer detected by Western blotting.

DISCUSSION The results presented in this work demonstrate that low

temperature positively influences the abundance of ELIP tran- script under light stress conditions. At a temperature of 10 "C, the steady state level of ELIP mRNA is almost 4 times higher than at 20 "C. Light stress, however, is an essential factor in ELIP induction, because cold stress alone did not have any effect on accumulation of ELIP transcript. Thus the low tem- perature seems to have a regulatory role that is superimposed on that of light stress. A combined effect of light and low tem- perature was also reported to be required for maximal expres-

sion of Wcsl9 gene (Chauvin et al., 1993). The function of this gene might be related to cold acclimation of wheat; however, in this case an effect of light stress was not investigated.

Also, concerning other aspects, our findings are clearly dis- tinct from those reported for gene expression during cold stress. Many of the transcripts analyzed so far were reported to be either tightly suppressed or unaffected by long-term cold treat- ment (Hahn and Walbot, 1989). The most strongly cold-sup- pressed mRNAs were either encoded by chloroplast DNA (psaB, pshB, rbcL, a t p E ) or by nuclear genes coding for chlo- roplast proteins (rhcS, cab). The investigated nuclear tran- scripts, such as those encoding p-tubulin, chalcone synthase, ubiquitin, and histone 3, remained either unchanged or were only slightly reduced. The exception was rub-21, which was induced by cold stress (Hahn and Walbot, 1989). Thus, the combined effect of light and cold stress on the expression of ELIPs that is described in this paper represents a rather pe- culiar type of regulation, especially because it involves simul- taneously two types of regulation. ELIP transcription activity is almost temperature-independent between 10 and 20 "C, whereas total transcription shows a positive temperature con- trol. It appears likely that part of the ELIP promoter is respon- sible for this temperature control, as this effect could be ob- served in vitro. In addition, the ELIP transcript appears to be stabilized at lower temperatures, as we observed maximal ac- cumulation at around 10 "C.

The experiment presented in Fig. 2 shows that the reaccu- mulation of LHCP mRNA can occur at temperatures as low as 5 "C. This experiment again substantiates that the transcrip- tional activity in pea plants is sustained at this low tempera- ture. After exposure of plants to 0 "C for 3 h, ELIP transcript accumulates gradually. This observation indicates that adap- tation of plants to low temperature is a rapid process.

The fact that the transcript level is high at 10 "C but the protein cannot be detected by Western blotting can be ex- plained if low temperature preferentially affects the translat- ability of existing transcripts in vivo. The integration of ELIP into thylakoid membranes appears not to be affected by low temperature, because it was temperature-independent be- tween 0 and 30 "C in an in vitro system (Kruse and Kloppstech, 1992). In contrast, the ELIP mRNA induced by light stress during short-term cold treatment was efficiently translated in vitro. Interestingly, however, the translatability of ELIP mRNA declined with the time of incubation of plants at low tempera- ture prior to isolation of the mRNA. Thus, at temperatures below 15 "C, the rate of translation or processing of ELIP pre- cursor appears to represent rate-limiting steps.

One of the major findings of this work is the fact that the plant cells are able to "memorize" a light stress that occurred under conditions that did not allow the plant to respond by transcriptional activity. In this particular case accumulation of ELIP transcript is induced in the absence of light stress during recovery under ambient temperature conditions. This tran- script is translated in vivo, and the corresponding protein ac- cumulates in the thylakoid membranes even under conditions when photosynthetic activity is almost completely recovered. To our knowledge this is the first report that a memory of light stress exists in plants that can influence gene expression after cessation of the stress. The molecular mechanism of this memory is yet not known. One possible explanation is that the expression of light stress-related genes during recovery could be connected to photooxidative damages within the plastid that occurred during exposure of plants to light stress at low tem- peratures. Due to low temperature, such damages of various components within thylakoid membranes might not be re- paired immediately. These persisting defects could then influ-

30226 Expression of ELIP Gene under Conditions of Stress

ence gene expression in the chloroplast and nucleocytosolic compartments after a temperature increase. In this context one should mention that photooxidative damage to the chloroplast was reported to influence expression of some light-regulated nuclear genes coding for plastid proteins (for reviews see Oelmuller (1989) and Taylor (1989)). In clear contrast to the investigated genes that were reported to be down-regulated, the ELIP mRNA level increases as a result of light stress. An alternative explanation might be envisaged. The signal per- ceived by the blue light receptods) might be stored due to an interference of low temperature with some steps in the signal transduction chain. The latter assumption is supported by the observation that the phosphorylation capacity of a 120-kDa protein presumably involved in a blue light signal transduction pathway can be retained for approximately 4 h when the mem- branes are stored at 0 "C (Short et al., 1992). Moreover, mem- branes stored at -80 "C could "remember" the signal even after 1 week and respond by phosphorylation after an increase of temperature to 30 "C. More detailed experiments, however, are required to decide between the alternatives.

Acknowledgments-We are grateful to Prof. B. Andersson (Depart- ment of Biochemistry, Stockholm University, Sweden) and Prof. I. Ohad (Department of Biological Chemistry, The Hebrew University of Jeru- salem, Israel) for comments on this manuscript. We thank Prof. F. Herzfeld (Institute of Botany, University of Hannover, Germany) for the anti-ELIP antibodies.

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