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The involvement of LHCII-associated polyamines in the response of the photosynthetic apparatus to low temperature
Journal of Photochemistry and Photobiology B: Biology 84 (2006) 181–188 www.elsevier.com/locate/jphotobiol
The involvement of LHCII-associated polyamines in the response of the photosynthetic apparatus to low temperature Maria Sfakianaki a
a,1
, Liliana Sfichi
a,1
, Kiriakos Kotzabasis
a,b,*
Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Crete, Greece b Department of Botany, Natural History Museum of Crete, Heraklion, Crete, Greece
Received 27 January 2006; received in revised form 10 March 2006; accepted 13 March 2006 Available online 3 May 2006
Abstract The influence of low temperature on the structure and function of the photosynthetic apparatus was investigated in Phaseolus vulgaris L. Ten-day-old plants (grown at 26 C) have been exposed to low temperature (6 C) for 52 h and, then, transferred to the initial temperature (26 C) for additional 30 h. Biochemical and physico-chemical measurements performed in the low temperature-treated plants showed that the response of the photosynthetic apparatus to low temperature is affected by the changes occurring in the pattern of LHCII-associated putrescine (Put) and spermine (Spm) which adjust the size of LHCII. The decrease of Put/Spm ratio, mainly due to the reduction in the quantity of LHCII-associated Put led to an increase of the LHCII, especially of the oligomeric forms. These alterations in the structure of the photosynthetic apparatus combined with the reduction in the photosynthetic electron transfer rate resulted in the inactivation of active reaction centers and the increase of dissipated energy which diminished the photosynthetic efficiency and the maximal photosynthetic rate. The transfer of plants at 26 C after the low temperature treatment showed that, structurally and functionally, the photosynthetic mechanism recovered quite fast to the initial condition. 2006 Elsevier B.V. All rights reserved. Keywords: LHCII; Phaseolus vulgaris; Photosynthesis; Putrescine; Spermine
1. Introduction The influence of low temperature on plant photosynthesis has been intensively investigated in a multitude of species showing different or similar degrees of cold tolerance. Morphological changes, such as chloroplast expansion and thylakoid deformation [1,2] accompanied by the alteration of photosynthetic pigments [3,4] or inhibition of chloroplast photodevelopment [1,5] have been reported for plants exposed to low temperatures. Additionally, the electron transport rate and the photosynthetic performance, as well as the Calvin cycle enzyme activities have been found to be decreased in low temperature-treated plants [6–9]. Ultimately, these changes lead to increased PSII excitation *
1
Corresponding author. Fax: +30 2810 394 408. E-mail address: [email protected] (K. Kotzabasis). Contributed equally to this work.
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.03.003
pressure [10], mainly at high light intensities [11–14]. This phenomenon induces the generation of reactive oxygen species (ROS) which can cause the degradation of photosystems, lipid peroxidation with the reduction of thylakoid membrane fluidity, and inactivation of Calvin cycle enzyme activities [15–17]. A number of studies conducted in different plant species showed that cold acclimation depends on the ability of the photosynthetic apparatus to increase the dissipation of excess energy as heat [18,19] or the rate of photochemical quenching [20,21]. In addition, recent works indicated that the tolerance degree to low temperatures in several plant species is influenced by polyamine accumulation [22,23]. For instance, putrescine (Put) was accumulated in shoots of a chilling tolerant cultivar of rice during exposure to low temperature [24], whereas high levels of spermidine (Spd) and S-adenosylmethionine decarboxylase (SAMDC) were found in a chilling tolerant cultivar of spinach [25].
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Increased Spd was also effective in the suppression of oxidative damage in a chilling tolerant cucumber [26]. The influence of polyamines on the tolerance degree of the photosynthetic apparatus to low temperatures or other environmental stresses such as ozone [27,28] or UVB-radiation [29,30] may occur not only due to their antioxidant properties [31] but, also, due to their involvement in the structure and functioning of the photosynthetic apparatus. It is well known that polyamines are associated with the thylakoid membranes, especially, the LHCII and the PSII complex of spinach. The tetramine Spm was mainly found in considerable concentrations in highly purified PSII-core antenna and reaction center particles of PSII [32]. In addition, a plastidic transglutaminase catalyses the incorporation of polyamines into thylakoid membrane and stroma proteins [33] contributing to the stabilization of these components of the photosynthetic apparatus. Also, Besford et al. [34] observed that exogenous polyamines supplied to osmotically stressed oat leaves retarded protein degradation, inhibited loss of chlorophyll and stabilized the thylakoid membranes. Recently, it was found that changes in the pattern of polyamines, especially Put and Spm play a regulatory role during exposure to high light intensity [35] or UVB radiation [29,30] and adjust the sensitivity of the photosynthetic apparatus in stress conditions by regulating the size of LHCII [30]. The aim of the present work is to study the changes that occur in the structure and function of the photosynthetic apparatus, especially in the organization of LHCII and in the pattern of LHCII-associated polyamines during exposure to low temperature of Phaseolus vulgaris L. plants. In addition, the capacity of plants to recover from low temperature effects is investigated. 2. Materials and methods 2.1. Plant material and experimental conditions Plants of Phaseolus vulgaris L. (Fagiolo nano Borlotto L.DI Fuoco.) obtained by seed germination in 5 cm plastic pots containing perlit were grown in controlled light (70 lmol m 2 s 1), temperature (26 C) and humidity (70%) conditions. Ten-day-old plants incubated in continuous visible light conditions were exposed to 6 C for 52 h and, then again to 26 C for additional 30 h. To compare the effect of low temperature on the structure and functioning of the photosynthetic apparatus, control plants, permanently maintained at 26 C in continuous light, were used. 2.2. Polarographic measurements Maximal photosynthetic and respiratory rates were determined polarographically at 30 C with a Clark type electrode system (Hansatech Instruments, Kings’s Lynn, Norfolk, UK). The actinic light (470 lmol m 2 s 1) was generated with a lamp (ENX360W/82V) and its intensity measured with a sensitive photoradiometer (International
Light, Newburyport, MA, 01950) consisting of a control box (IL 1700), a power supply (IL 760) and a photomultiplier (IL 780). The infrared part of the applied irradiation was filtered off by inserting a 2% CuSO4-containing cuvette (4 cm path length) into the light beam. 2.3. Fluorescence induction measurements For the fluorescence induction measurements, plants were dark-adapted at the room temperature for 30 min. Fluorescence was measured with the portable Plant Efficiency Analyser (Hansatech Instruments, U.K.). Several parameters such as the photosynthetic efficiency (Fv/Fm), the electron transport per reaction center (ET0/RC) the antenna size per reaction center (ABS/RC), the rate of energy dissipation per reaction center (DI0/RC) and the density of active photosynthetic reaction centers per unit area (RC/CS) were measured according to the JIP method of Strasser and Strasser [36]. The method is based on the measurement of a fast fluorescence transient with a 10 ls resolution in a time span of 40 ls to 1 s. 2.4. Isolation of chloroplasts and thylakoid membranes Chloroplasts and thylakoid membranes were isolated from leaves randomly collected from plants incubated at 26 C (control), from plants treated at low temperature for 52 h (low temperature) and from those transferred after low temperature treatment to 26 C for 30 h (rewarming). To isolate chloroplasts, the leaf samples (without petiols) were homogenized in a buffer (A) containing 30 mM tricine and 0.3 M sucrose (pH 7.6) and filtered. The filtrate was collected and centrifuged for 7 min at 500g and then the supernatant was centrifuged at 1000g for 15 min. The pellet was resuspended into a buffer (B) containing 10 mM tricine and 0.3 M sucrose (pH 7.6). The samples prepared in this way were loaded in the top of step sucrose gradients (0.75 M, 1 M and 1.5 M) and centrifuged for 1 h at 2400g. After centrifugation, the chloroplasts were collected from the band situated between 1.5 M and 1 M sucrose and redissolved in the same quantity of 10 mM tricine and 0.3 M sucrose (pH 7.6) and then centrifuged at 5800g for 5 min. The pellet containing pure chloroplasts was used for the isolation of thylakoid membranes. For this, the chloroplasts were redissolved in 0.05 M tricine (pH 7.3) and centrifuged at 10,000g for 45 min. After centrifugation, the thylakoids were collected from the top of the pellet (green layer) and stored in 0.05 M tricine (pH 7.3) at 80 C until the use. 2.5. Isolation and quantitative determination of LHCII amount For the isolation of LHCII sub-complexes, the thylakoid membranes were solubilized in 0.05 M Tris and 0.06 M borate by vortexing. To the solubilized samples, 0.5% SDS was added 1 min before their fractionation by ultracentrifugation (18 h, 17,000g at 4 C) in a continuous
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sucrose gradient 5–22%, as previously described [37]. The LHCII comes in as two closed green bands. The upper one represents the trimeric form and the lower one the monomeric form of the LHCII [30,37]. These green bands of the sucrose gradient were harvested with a syringe. The quantitative LHCII amount was determined as the protein amount of the corresponding bands. 2.6. HPLC analysis of polyamines Isolated LHCII forms were suspended in 1 N NaOH. A volume of 0.350 ml suspension was mixed with 36% HCl in a ratio of 1:1 (v/v) and incubated at 110 C for 18 h. The hydrolysis products were evaporated at 70–80 C and resuspended in 0.3 ml of 5% (v/v) perchloric acid. For the qualitative and quantitative estimation of polyamines, the samples were benzoylated according to the method of Kotzabasis et al. [38]. Specifically, 1 ml of 2 N NaOH and 10 ll benzoylchloride were added to 0.2 ml of the hydrolyzate and the mixture vortexed for 30 s. After 20 min incubation at room temperature, 2 ml of saturated NaCl solution were added to stop the reaction. The benzoylpolyamines were extracted three times into 2–3 ml diethylether, all ether phases being further collected and evaporated to dryness. The remaining benzoylpolyamines were redissolved in 0.2 ml of 63% (v/v) methanol. 20 ll aliquots of this solution were injected into a high performance liquid chromatography (HPLC) system for the analysis of polyamines. The analyses were performed with a Shimadzu Liquid Chromatography apparatus (LC-10AD) equipped with a diode array detector (Shimadzu SPD-M10A) and a narrow-bore column (C18, 2.1 · 200 mm, 5 lm particle size Hypersyl, Hewlett-Packard). For the quantitative determination of polyamines the method of Kotzabasis et al. [38] was followed again. 2.7. Pigment extraction and estimation
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initially, assessed by chlorophyll fluorescence induction measurements performed at different time periods, during low temperature treatment and rewarming. For the 10-day-old plants, the polyphasic kinetics of the chlorophyll fluorescence rise from F0 (O level) via J–P phase to Fm (P level) of a Kautsky curve [36] were examined at three-experimental steps: prior to low temperature exposure (control), after 52 h of low temperature exposure (low temperature) and after 30 h of incubation at 26 C (rewarming). As Fig. 1 shows, the low temperature caused the decrease of J–P phase (Fm), which fully recovered after 30 h of rewarming. The Fm decrease resulted in the lowering of the photosynthetic efficiency, expressed as the Fv/Fm ratio. After an initial phase of about 15 h exposure to low temperature, the Fv/Fm ratio progressively decreased from 0.75 up to 0.55 (in the 25th hour) and remained at this level until the end of treatment. The transfer of plants to 26 C (rewarming) resulted in the fully recovery of the photosynthetic efficiency to the initial values (Fig. 2a). In addition to the Fv/Fm ratio, several biophysical parameters (such as ABS/RC, DI0/RC, ET0/RC, and RC/CS) were investigated with the JIP-test (Fig. 2). This test represents a translation of stress-induced shape changes of O–J–I–P transient to changes in biophysical parameters quantifying the energy flow through PSII [36]. The antenna size (ABS/RC) expresses the total absorption of PSII antenna chlorophylls divided by the number of active reaction centers. Although the first hours of exposure to low temperature were followed by the decrease of antenna size, from the 10th hour until the 25th hour of treatment it progressively increased over the corresponding control values (Fig. 2b, Table 1). Similar changes occurred in the dissipation energy rate per reaction center (DI0/RC) (Fig. 2c), whereas the electron transport rate per active reaction center (ET0/RC) decreased gradually from the beginning until the 25th hour of exposure to low temperature (Fig. 2d). In opposition, the density of active reaction
Chlorophyll was extracted and the amount was calculated according to the method of Holden [39].
4000
The total protein content was determined accordingly to the method of Bradford [40]. 2.9. Statistics The experiments were performed at least in triplicate and the data presented here are given as the average of the obtained values. The standard deviations were also calculated.
FLUORESCENCE [mV]
3600
2.8. Estimation of protein content
P
3200
control rewarming
2800
I
2400 2000
J low temperature
1600 1200
O
800 400 0 0.01
0.1
1
10
100
1000
Time [ms]
3. Results The changes induced by low temperature in the structure and function of the photosynthetic apparatus were,
Fig. 1. The polyphasic kinetics of Chl fluorescence induction in control plants (grown at 26 C), in plants exposed to low temperature (6 C) for 52 h and those transferred to rewarming conditions (26 C) for additional 30 h.
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(a)
rewarming
(b)
5 4 3 2 1 0
2.9 2.5 2.1 1.7 1.3 0.9 0.5 0.1
20
40
60
80
0
(c)
ET0/RC
DI0/RC
rewarming
ABS/RC
F v/F m
low temperature 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3
0
20
40
60 350
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4
40
60
80
20
40
60
80
(d)
0
80
20
(e)
RC/CS
300 250 200 150 0
20
40
60
80
Incubation time (h) Fig. 2. The kinetics of the (a) photosynthetic efficiency (Fv/Fm), (b) antenna size (ABS/RC), (c) rate of dissipation energy per reaction center (DI0/RC), (d) electron transfer per reaction center (ET0/RC), and (e) reaction center density (RC/CS) in control plants (grown at 26 C) (closed squares) and those exposed to low temperature (6 C) for 52 h and transferred to rewarming conditions (26 C) for additional 30 h (open squares). The values represent the means ±SD of three to five samples.
Table 1 Structural and functional changes induced after low temperature treatment and rewarming in the photosynthetic apparatus Sample
ABS/RC
LHCII amount (lg protein/thylakoid unit)
Put/Spm (in LHCII)
Chl a/b
Maximal photosynthetic rate (lmol O2 mg Chl 1 h 1)
Control Low temperature Rewarming
3.45 ± 0.15 4.13 ± 0.11 3.37 ± 0.30
499.5 ± 64.2 716.0 ± 85.3 595.7 ± 91.1
2.68 1.64 2.22
2.44 2.28 2.29
18.5 ± 6.0 11.6 ± 2.6 20.2 ± 4.9
30
Oxygen evolution -1 -1 [µmol O2 mg Chl h ]
maximal respiratory rate maximal photosynthetic rate
20
10
0
-10
-20
control
low temperature
rewarming
Fig. 3. Maximal photosynthetic and respiratory rate in control plants (grown at 26 C), in plants exposed to low temperature (6 C) for 52 h and those transferred to rewarming conditions (26 C) for additional 30 h. The values represent the means ±SD of three to five samples and are expressed in lmol O2 mg Chl 1 h 1.
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600 500 400
monomeric forms oligomeric forms
25
300 200 100 0 900
15 10 5 0
700
10
600
9
500 400 300 200 100 0 2.5
8 7 6 5 4 3 2 1 0
2
3 1.5
2.5
1
Put/Spm
LHCII oligomers/monomers
20
800
LHCII-associated Spm (pmole/µg LHCII protein)
Total amount of LHCII proteins (µg protein/thylakoid unit)
time points: prior to low temperature treatment, after 52 h of treatment and after an additional 30 h period of rewarming. Quantitative analysis of the total protein amount in isolated LHCII sub-complexes revealed that the low temperature caused an increase in the oligomeric form concomitantly to the decrease in the monomeric one (Fig. 4). As compared to the control values, the protein amount of LHCII-oligomeric form increased by 120% after treatment, while it decreased by 21% in the monomeric one. These changes led to the increase in the total LHCII protein amount (45% over the control), while the LHCII-oligomer/monomer ratio increased to 180% (Fig. 4). At rewarming, the total protein amount in LHCII oligomeric and monomeric forms, as well as, the LHCII-oligomer/ monomer ratio recovered to the control values (Fig. 4).
LHCII-associated Put (pmole/µg LHCII protein)
Amount of LHCII sub-complexes (µg protein/thylakoid unit)
centers (RC/CS) decreased after 30 h of treatment (Fig. 2e). At rewarming, the changes induced in all these parameters were fully recovered (Fig. 2b–e). In parallel to the above mentioned effects, the exposure to low temperature led to a decrease in the maximal photosynthetic rate (Fig. 3). At the end of treatment, the maximal photosynthetic rate was 37% lower than the control, but it recovered after rewarming. The biochemical analysis of chlorophylls in fully expanded leaves did not show significant changes in the chlorophyll amount or pattern during the low temperature treatment (Table 1). The monomeric and oligomeric forms of LHCII were separated from the isolated thylakoid membranes at three
185
0.5 0
control
low temperature
rewarming
2 1.5 1 0.5 0
Fig. 4. Quantitative determination of the total LHCII amount, the oligomeric and monomeric forms of LHCII, and the LHCII oligomeric form/monomeric form ratio per thylakoid unit (thylakoid membranes containing 550 lg Chl) from plants prior to low temperature treatment (control), after 52 h of exposure to 6 C and after additional 30 h of rewarming. The values represent the means ±SD of three to five samples.
control
low temperature
rewarming
Fig. 5. Quantitative determination of the LHCII-associated polyamines and the Put/Spm ratio from plants prior to low temperature treatment (control), after 52 h of exposure to 6 C and after additional 30 h of rewarming. The values represent the means ±SD of three to five samples.
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The alterations in the size of LHCII were accompanied by changes in the LHCII-associated polyamines. Under low temperature treatment, the amount of Put associated to the LHCII decreased by 23%, while the LHCII-associated Spm increased by 26%. This resulted in the decrease of Put/Spm ratio that increased again at rewarming, due to the Put increasing and Spm decreasing (Fig. 5, Table 1). 4. Discussion The aim of the present work was to investigate the influence of low temperature on the structure and function of the photosynthetic apparatus. The reduction in the electron transport rate (ET0/RC; Fig. 2d) and the decrease in the photochemical efficiency (Fv/Fm; Fig. 2a) and maximal photosynthetic rate (Fig. 3) indicate that the low temperature treatment increased the excitation pressure of PSII. This is also reflected by the changes occurring in the O–J–I–P transient shape (Fig. 1). Both the Fm quenching and the relative small increase in F0 may result from the inactivation of active reaction centers, which decreased after low temperature treatment, but increased at rewarming (RC/CS; Fig. 2e). Such inactivated reaction centers may act as quenchers of the excitation energy and protect the functional adjacent reaction centers [41,42]. Quantitative analysis of the protein amount in the LHCII oligomeric and monomeric forms isolated from the thylakoid membranes showed an increase in the LHCII size (Fig. 4, Table 1) after 52 h incubation to low temperature. This suggests that the exposure to low temperature simulates low light adaptation responses. In opposition, a prolonged exposure to low temperature can lead to a photosynthetic apparatus that mimics high light acclimation [43,44]. By increasing the LHCII size and the fraction of inactivated reaction centers, the total absorption divided by the active reaction centers (ABS/RC; Fig. 2b) and, subsequently, the rate of dissipation energy (DI0/RC; Fig. 2c) also increased. The formation of such excess energy quenching system may represent the protective means to the excitation pressure of PSII. This is in agreement with previously reported data which indicate the non-photochemical quenching increasing as meaningful way of protection against low temperatures [10,45]. The investigation of polyamines in the isolated LHCII forms showed that the low temperature treatment induced a decrease in the amount of LHCII-associated Put concomitant with an increase in the LHCII-associated Spm (Fig. 5), which led to the decrease of Put/Spm ratio. Recent data obtained for spinach showed that the low temperature induced a decrease in the Put associated to the thylakoid membranes [25]. Since the Spm remained constant, the above data also suggest a decrease in the Put/Spm ratio, which is consistent with our results. The mechanism by which polyamines contribute to the increasing tolerance to low temperatures is not yet understood. In general, it
is believed that polyamines may alleviate the injury induced by low temperatures by reducing the oxidative damage [22– 26]. At the same time, the relative responsiveness to low temperatures of the enzymes involved in polyamine biosynthesis differs with plant species [25]. In addition, the polyamine pattern in stress conditions can show different changes in thylakoid membranes as in chloroplasts and this makes the understanding of mechanisms that are regulated by polyamines more difficult. Previous works indicated that the re-organization of the LHCII, as response to stress, occurs through changes in the polyamine pattern, specifically of the Put/Spm ratio [30,35]. Specifically, an increase in the Put/Spm ratio leads to a photosynthetic apparatus with a smaller antenna exhibiting a high light-adapted photosynthetic behavior. In opposition, a decrease in the Put/Spm ratio leads to a low light-adapted photosynthetic apparatus characterized by a bigger antenna and a lower photosynthetic activity. In this context, it might be possible that the reduction of Put/Spm ratio that we found in the isolated LHCII monomeric and oligomeric forms (Fig. 5) during exposure to low temperature is involved in the triggering of structural changes in the photosynthetic apparatus that led to the increasing of LHCII size (Table 1). The investigation of the plant capability to recover the changes induced by low temperature in the photosynthetic apparatus showed the repair of structural and functional damages at rewarming. Specifically, the LHCII size decreased (Fig. 4), through changes in the polyamine pattern (Fig. 5) and the PSII functionality recovered (Figs. 2 and 3). Although the recovery of changes induced by low temperature was not fully achieved, the photosynthetic activity (Fig. 3) was substantially improved. In conclusion, all these data suggest that, probably, the excitation pressure of the photosynthetic apparatus is the signal for all the above-mentioned changes, since the same experiment performed in darkness (data not shown) showed no significant effect of low temperature on the structure and functionality of the photosynthetic apparatus. The similarity of the photosynthetic apparatus responses to a series of abiotic stresses, like the UV-B radiation [29,30] or enhanced atmospheric ozone [28] and low temperature (present work) support the hypothesis for a common response mechanism of the photosynthetic apparatus against abiotic stress in general. 5. Abbreviations ABS/RC antenna size per reaction center DI0/RC dissipation energy per reaction center ET0/RC photosynthetic electron transport per reaction center Fv/Fm photosynthetic efficiency LHCII light harvesting complex II Put putrescine RC/CS density of active photosynthetic reaction centers Spm spermine
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The involvement of LHCII-associated polyamines in the response of the photosynthetic apparatus to low temperature Maria Sfakianaki a
a,1
, Liliana Sfichi
a,1
, Kiriakos Kotzabasis
a,b,*
Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Crete, Greece b Department of Botany, Natural History Museum of Crete, Heraklion, Crete, Greece
Received 27 January 2006; received in revised form 10 March 2006; accepted 13 March 2006 Available online 3 May 2006
Abstract The influence of low temperature on the structure and function of the photosynthetic apparatus was investigated in Phaseolus vulgaris L. Ten-day-old plants (grown at 26 C) have been exposed to low temperature (6 C) for 52 h and, then, transferred to the initial temperature (26 C) for additional 30 h. Biochemical and physico-chemical measurements performed in the low temperature-treated plants showed that the response of the photosynthetic apparatus to low temperature is affected by the changes occurring in the pattern of LHCII-associated putrescine (Put) and spermine (Spm) which adjust the size of LHCII. The decrease of Put/Spm ratio, mainly due to the reduction in the quantity of LHCII-associated Put led to an increase of the LHCII, especially of the oligomeric forms. These alterations in the structure of the photosynthetic apparatus combined with the reduction in the photosynthetic electron transfer rate resulted in the inactivation of active reaction centers and the increase of dissipated energy which diminished the photosynthetic efficiency and the maximal photosynthetic rate. The transfer of plants at 26 C after the low temperature treatment showed that, structurally and functionally, the photosynthetic mechanism recovered quite fast to the initial condition. 2006 Elsevier B.V. All rights reserved. Keywords: LHCII; Phaseolus vulgaris; Photosynthesis; Putrescine; Spermine
1. Introduction The influence of low temperature on plant photosynthesis has been intensively investigated in a multitude of species showing different or similar degrees of cold tolerance. Morphological changes, such as chloroplast expansion and thylakoid deformation [1,2] accompanied by the alteration of photosynthetic pigments [3,4] or inhibition of chloroplast photodevelopment [1,5] have been reported for plants exposed to low temperatures. Additionally, the electron transport rate and the photosynthetic performance, as well as the Calvin cycle enzyme activities have been found to be decreased in low temperature-treated plants [6–9]. Ultimately, these changes lead to increased PSII excitation *
1
Corresponding author. Fax: +30 2810 394 408. E-mail address: [email protected] (K. Kotzabasis). Contributed equally to this work.
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.03.003
pressure [10], mainly at high light intensities [11–14]. This phenomenon induces the generation of reactive oxygen species (ROS) which can cause the degradation of photosystems, lipid peroxidation with the reduction of thylakoid membrane fluidity, and inactivation of Calvin cycle enzyme activities [15–17]. A number of studies conducted in different plant species showed that cold acclimation depends on the ability of the photosynthetic apparatus to increase the dissipation of excess energy as heat [18,19] or the rate of photochemical quenching [20,21]. In addition, recent works indicated that the tolerance degree to low temperatures in several plant species is influenced by polyamine accumulation [22,23]. For instance, putrescine (Put) was accumulated in shoots of a chilling tolerant cultivar of rice during exposure to low temperature [24], whereas high levels of spermidine (Spd) and S-adenosylmethionine decarboxylase (SAMDC) were found in a chilling tolerant cultivar of spinach [25].
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Increased Spd was also effective in the suppression of oxidative damage in a chilling tolerant cucumber [26]. The influence of polyamines on the tolerance degree of the photosynthetic apparatus to low temperatures or other environmental stresses such as ozone [27,28] or UVB-radiation [29,30] may occur not only due to their antioxidant properties [31] but, also, due to their involvement in the structure and functioning of the photosynthetic apparatus. It is well known that polyamines are associated with the thylakoid membranes, especially, the LHCII and the PSII complex of spinach. The tetramine Spm was mainly found in considerable concentrations in highly purified PSII-core antenna and reaction center particles of PSII [32]. In addition, a plastidic transglutaminase catalyses the incorporation of polyamines into thylakoid membrane and stroma proteins [33] contributing to the stabilization of these components of the photosynthetic apparatus. Also, Besford et al. [34] observed that exogenous polyamines supplied to osmotically stressed oat leaves retarded protein degradation, inhibited loss of chlorophyll and stabilized the thylakoid membranes. Recently, it was found that changes in the pattern of polyamines, especially Put and Spm play a regulatory role during exposure to high light intensity [35] or UVB radiation [29,30] and adjust the sensitivity of the photosynthetic apparatus in stress conditions by regulating the size of LHCII [30]. The aim of the present work is to study the changes that occur in the structure and function of the photosynthetic apparatus, especially in the organization of LHCII and in the pattern of LHCII-associated polyamines during exposure to low temperature of Phaseolus vulgaris L. plants. In addition, the capacity of plants to recover from low temperature effects is investigated. 2. Materials and methods 2.1. Plant material and experimental conditions Plants of Phaseolus vulgaris L. (Fagiolo nano Borlotto L.DI Fuoco.) obtained by seed germination in 5 cm plastic pots containing perlit were grown in controlled light (70 lmol m 2 s 1), temperature (26 C) and humidity (70%) conditions. Ten-day-old plants incubated in continuous visible light conditions were exposed to 6 C for 52 h and, then again to 26 C for additional 30 h. To compare the effect of low temperature on the structure and functioning of the photosynthetic apparatus, control plants, permanently maintained at 26 C in continuous light, were used. 2.2. Polarographic measurements Maximal photosynthetic and respiratory rates were determined polarographically at 30 C with a Clark type electrode system (Hansatech Instruments, Kings’s Lynn, Norfolk, UK). The actinic light (470 lmol m 2 s 1) was generated with a lamp (ENX360W/82V) and its intensity measured with a sensitive photoradiometer (International
Light, Newburyport, MA, 01950) consisting of a control box (IL 1700), a power supply (IL 760) and a photomultiplier (IL 780). The infrared part of the applied irradiation was filtered off by inserting a 2% CuSO4-containing cuvette (4 cm path length) into the light beam. 2.3. Fluorescence induction measurements For the fluorescence induction measurements, plants were dark-adapted at the room temperature for 30 min. Fluorescence was measured with the portable Plant Efficiency Analyser (Hansatech Instruments, U.K.). Several parameters such as the photosynthetic efficiency (Fv/Fm), the electron transport per reaction center (ET0/RC) the antenna size per reaction center (ABS/RC), the rate of energy dissipation per reaction center (DI0/RC) and the density of active photosynthetic reaction centers per unit area (RC/CS) were measured according to the JIP method of Strasser and Strasser [36]. The method is based on the measurement of a fast fluorescence transient with a 10 ls resolution in a time span of 40 ls to 1 s. 2.4. Isolation of chloroplasts and thylakoid membranes Chloroplasts and thylakoid membranes were isolated from leaves randomly collected from plants incubated at 26 C (control), from plants treated at low temperature for 52 h (low temperature) and from those transferred after low temperature treatment to 26 C for 30 h (rewarming). To isolate chloroplasts, the leaf samples (without petiols) were homogenized in a buffer (A) containing 30 mM tricine and 0.3 M sucrose (pH 7.6) and filtered. The filtrate was collected and centrifuged for 7 min at 500g and then the supernatant was centrifuged at 1000g for 15 min. The pellet was resuspended into a buffer (B) containing 10 mM tricine and 0.3 M sucrose (pH 7.6). The samples prepared in this way were loaded in the top of step sucrose gradients (0.75 M, 1 M and 1.5 M) and centrifuged for 1 h at 2400g. After centrifugation, the chloroplasts were collected from the band situated between 1.5 M and 1 M sucrose and redissolved in the same quantity of 10 mM tricine and 0.3 M sucrose (pH 7.6) and then centrifuged at 5800g for 5 min. The pellet containing pure chloroplasts was used for the isolation of thylakoid membranes. For this, the chloroplasts were redissolved in 0.05 M tricine (pH 7.3) and centrifuged at 10,000g for 45 min. After centrifugation, the thylakoids were collected from the top of the pellet (green layer) and stored in 0.05 M tricine (pH 7.3) at 80 C until the use. 2.5. Isolation and quantitative determination of LHCII amount For the isolation of LHCII sub-complexes, the thylakoid membranes were solubilized in 0.05 M Tris and 0.06 M borate by vortexing. To the solubilized samples, 0.5% SDS was added 1 min before their fractionation by ultracentrifugation (18 h, 17,000g at 4 C) in a continuous
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sucrose gradient 5–22%, as previously described [37]. The LHCII comes in as two closed green bands. The upper one represents the trimeric form and the lower one the monomeric form of the LHCII [30,37]. These green bands of the sucrose gradient were harvested with a syringe. The quantitative LHCII amount was determined as the protein amount of the corresponding bands. 2.6. HPLC analysis of polyamines Isolated LHCII forms were suspended in 1 N NaOH. A volume of 0.350 ml suspension was mixed with 36% HCl in a ratio of 1:1 (v/v) and incubated at 110 C for 18 h. The hydrolysis products were evaporated at 70–80 C and resuspended in 0.3 ml of 5% (v/v) perchloric acid. For the qualitative and quantitative estimation of polyamines, the samples were benzoylated according to the method of Kotzabasis et al. [38]. Specifically, 1 ml of 2 N NaOH and 10 ll benzoylchloride were added to 0.2 ml of the hydrolyzate and the mixture vortexed for 30 s. After 20 min incubation at room temperature, 2 ml of saturated NaCl solution were added to stop the reaction. The benzoylpolyamines were extracted three times into 2–3 ml diethylether, all ether phases being further collected and evaporated to dryness. The remaining benzoylpolyamines were redissolved in 0.2 ml of 63% (v/v) methanol. 20 ll aliquots of this solution were injected into a high performance liquid chromatography (HPLC) system for the analysis of polyamines. The analyses were performed with a Shimadzu Liquid Chromatography apparatus (LC-10AD) equipped with a diode array detector (Shimadzu SPD-M10A) and a narrow-bore column (C18, 2.1 · 200 mm, 5 lm particle size Hypersyl, Hewlett-Packard). For the quantitative determination of polyamines the method of Kotzabasis et al. [38] was followed again. 2.7. Pigment extraction and estimation
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initially, assessed by chlorophyll fluorescence induction measurements performed at different time periods, during low temperature treatment and rewarming. For the 10-day-old plants, the polyphasic kinetics of the chlorophyll fluorescence rise from F0 (O level) via J–P phase to Fm (P level) of a Kautsky curve [36] were examined at three-experimental steps: prior to low temperature exposure (control), after 52 h of low temperature exposure (low temperature) and after 30 h of incubation at 26 C (rewarming). As Fig. 1 shows, the low temperature caused the decrease of J–P phase (Fm), which fully recovered after 30 h of rewarming. The Fm decrease resulted in the lowering of the photosynthetic efficiency, expressed as the Fv/Fm ratio. After an initial phase of about 15 h exposure to low temperature, the Fv/Fm ratio progressively decreased from 0.75 up to 0.55 (in the 25th hour) and remained at this level until the end of treatment. The transfer of plants to 26 C (rewarming) resulted in the fully recovery of the photosynthetic efficiency to the initial values (Fig. 2a). In addition to the Fv/Fm ratio, several biophysical parameters (such as ABS/RC, DI0/RC, ET0/RC, and RC/CS) were investigated with the JIP-test (Fig. 2). This test represents a translation of stress-induced shape changes of O–J–I–P transient to changes in biophysical parameters quantifying the energy flow through PSII [36]. The antenna size (ABS/RC) expresses the total absorption of PSII antenna chlorophylls divided by the number of active reaction centers. Although the first hours of exposure to low temperature were followed by the decrease of antenna size, from the 10th hour until the 25th hour of treatment it progressively increased over the corresponding control values (Fig. 2b, Table 1). Similar changes occurred in the dissipation energy rate per reaction center (DI0/RC) (Fig. 2c), whereas the electron transport rate per active reaction center (ET0/RC) decreased gradually from the beginning until the 25th hour of exposure to low temperature (Fig. 2d). In opposition, the density of active reaction
Chlorophyll was extracted and the amount was calculated according to the method of Holden [39].
4000
The total protein content was determined accordingly to the method of Bradford [40]. 2.9. Statistics The experiments were performed at least in triplicate and the data presented here are given as the average of the obtained values. The standard deviations were also calculated.
FLUORESCENCE [mV]
3600
2.8. Estimation of protein content
P
3200
control rewarming
2800
I
2400 2000
J low temperature
1600 1200
O
800 400 0 0.01
0.1
1
10
100
1000
Time [ms]
3. Results The changes induced by low temperature in the structure and function of the photosynthetic apparatus were,
Fig. 1. The polyphasic kinetics of Chl fluorescence induction in control plants (grown at 26 C), in plants exposed to low temperature (6 C) for 52 h and those transferred to rewarming conditions (26 C) for additional 30 h.
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(a)
rewarming
(b)
5 4 3 2 1 0
2.9 2.5 2.1 1.7 1.3 0.9 0.5 0.1
20
40
60
80
0
(c)
ET0/RC
DI0/RC
rewarming
ABS/RC
F v/F m
low temperature 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3
0
20
40
60 350
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4
40
60
80
20
40
60
80
(d)
0
80
20
(e)
RC/CS
300 250 200 150 0
20
40
60
80
Incubation time (h) Fig. 2. The kinetics of the (a) photosynthetic efficiency (Fv/Fm), (b) antenna size (ABS/RC), (c) rate of dissipation energy per reaction center (DI0/RC), (d) electron transfer per reaction center (ET0/RC), and (e) reaction center density (RC/CS) in control plants (grown at 26 C) (closed squares) and those exposed to low temperature (6 C) for 52 h and transferred to rewarming conditions (26 C) for additional 30 h (open squares). The values represent the means ±SD of three to five samples.
Table 1 Structural and functional changes induced after low temperature treatment and rewarming in the photosynthetic apparatus Sample
ABS/RC
LHCII amount (lg protein/thylakoid unit)
Put/Spm (in LHCII)
Chl a/b
Maximal photosynthetic rate (lmol O2 mg Chl 1 h 1)
Control Low temperature Rewarming
3.45 ± 0.15 4.13 ± 0.11 3.37 ± 0.30
499.5 ± 64.2 716.0 ± 85.3 595.7 ± 91.1
2.68 1.64 2.22
2.44 2.28 2.29
18.5 ± 6.0 11.6 ± 2.6 20.2 ± 4.9
30
Oxygen evolution -1 -1 [µmol O2 mg Chl h ]
maximal respiratory rate maximal photosynthetic rate
20
10
0
-10
-20
control
low temperature
rewarming
Fig. 3. Maximal photosynthetic and respiratory rate in control plants (grown at 26 C), in plants exposed to low temperature (6 C) for 52 h and those transferred to rewarming conditions (26 C) for additional 30 h. The values represent the means ±SD of three to five samples and are expressed in lmol O2 mg Chl 1 h 1.
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600 500 400
monomeric forms oligomeric forms
25
300 200 100 0 900
15 10 5 0
700
10
600
9
500 400 300 200 100 0 2.5
8 7 6 5 4 3 2 1 0
2
3 1.5
2.5
1
Put/Spm
LHCII oligomers/monomers
20
800
LHCII-associated Spm (pmole/µg LHCII protein)
Total amount of LHCII proteins (µg protein/thylakoid unit)
time points: prior to low temperature treatment, after 52 h of treatment and after an additional 30 h period of rewarming. Quantitative analysis of the total protein amount in isolated LHCII sub-complexes revealed that the low temperature caused an increase in the oligomeric form concomitantly to the decrease in the monomeric one (Fig. 4). As compared to the control values, the protein amount of LHCII-oligomeric form increased by 120% after treatment, while it decreased by 21% in the monomeric one. These changes led to the increase in the total LHCII protein amount (45% over the control), while the LHCII-oligomer/monomer ratio increased to 180% (Fig. 4). At rewarming, the total protein amount in LHCII oligomeric and monomeric forms, as well as, the LHCII-oligomer/ monomer ratio recovered to the control values (Fig. 4).
LHCII-associated Put (pmole/µg LHCII protein)
Amount of LHCII sub-complexes (µg protein/thylakoid unit)
centers (RC/CS) decreased after 30 h of treatment (Fig. 2e). At rewarming, the changes induced in all these parameters were fully recovered (Fig. 2b–e). In parallel to the above mentioned effects, the exposure to low temperature led to a decrease in the maximal photosynthetic rate (Fig. 3). At the end of treatment, the maximal photosynthetic rate was 37% lower than the control, but it recovered after rewarming. The biochemical analysis of chlorophylls in fully expanded leaves did not show significant changes in the chlorophyll amount or pattern during the low temperature treatment (Table 1). The monomeric and oligomeric forms of LHCII were separated from the isolated thylakoid membranes at three
185
0.5 0
control
low temperature
rewarming
2 1.5 1 0.5 0
Fig. 4. Quantitative determination of the total LHCII amount, the oligomeric and monomeric forms of LHCII, and the LHCII oligomeric form/monomeric form ratio per thylakoid unit (thylakoid membranes containing 550 lg Chl) from plants prior to low temperature treatment (control), after 52 h of exposure to 6 C and after additional 30 h of rewarming. The values represent the means ±SD of three to five samples.
control
low temperature
rewarming
Fig. 5. Quantitative determination of the LHCII-associated polyamines and the Put/Spm ratio from plants prior to low temperature treatment (control), after 52 h of exposure to 6 C and after additional 30 h of rewarming. The values represent the means ±SD of three to five samples.
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The alterations in the size of LHCII were accompanied by changes in the LHCII-associated polyamines. Under low temperature treatment, the amount of Put associated to the LHCII decreased by 23%, while the LHCII-associated Spm increased by 26%. This resulted in the decrease of Put/Spm ratio that increased again at rewarming, due to the Put increasing and Spm decreasing (Fig. 5, Table 1). 4. Discussion The aim of the present work was to investigate the influence of low temperature on the structure and function of the photosynthetic apparatus. The reduction in the electron transport rate (ET0/RC; Fig. 2d) and the decrease in the photochemical efficiency (Fv/Fm; Fig. 2a) and maximal photosynthetic rate (Fig. 3) indicate that the low temperature treatment increased the excitation pressure of PSII. This is also reflected by the changes occurring in the O–J–I–P transient shape (Fig. 1). Both the Fm quenching and the relative small increase in F0 may result from the inactivation of active reaction centers, which decreased after low temperature treatment, but increased at rewarming (RC/CS; Fig. 2e). Such inactivated reaction centers may act as quenchers of the excitation energy and protect the functional adjacent reaction centers [41,42]. Quantitative analysis of the protein amount in the LHCII oligomeric and monomeric forms isolated from the thylakoid membranes showed an increase in the LHCII size (Fig. 4, Table 1) after 52 h incubation to low temperature. This suggests that the exposure to low temperature simulates low light adaptation responses. In opposition, a prolonged exposure to low temperature can lead to a photosynthetic apparatus that mimics high light acclimation [43,44]. By increasing the LHCII size and the fraction of inactivated reaction centers, the total absorption divided by the active reaction centers (ABS/RC; Fig. 2b) and, subsequently, the rate of dissipation energy (DI0/RC; Fig. 2c) also increased. The formation of such excess energy quenching system may represent the protective means to the excitation pressure of PSII. This is in agreement with previously reported data which indicate the non-photochemical quenching increasing as meaningful way of protection against low temperatures [10,45]. The investigation of polyamines in the isolated LHCII forms showed that the low temperature treatment induced a decrease in the amount of LHCII-associated Put concomitant with an increase in the LHCII-associated Spm (Fig. 5), which led to the decrease of Put/Spm ratio. Recent data obtained for spinach showed that the low temperature induced a decrease in the Put associated to the thylakoid membranes [25]. Since the Spm remained constant, the above data also suggest a decrease in the Put/Spm ratio, which is consistent with our results. The mechanism by which polyamines contribute to the increasing tolerance to low temperatures is not yet understood. In general, it
is believed that polyamines may alleviate the injury induced by low temperatures by reducing the oxidative damage [22– 26]. At the same time, the relative responsiveness to low temperatures of the enzymes involved in polyamine biosynthesis differs with plant species [25]. In addition, the polyamine pattern in stress conditions can show different changes in thylakoid membranes as in chloroplasts and this makes the understanding of mechanisms that are regulated by polyamines more difficult. Previous works indicated that the re-organization of the LHCII, as response to stress, occurs through changes in the polyamine pattern, specifically of the Put/Spm ratio [30,35]. Specifically, an increase in the Put/Spm ratio leads to a photosynthetic apparatus with a smaller antenna exhibiting a high light-adapted photosynthetic behavior. In opposition, a decrease in the Put/Spm ratio leads to a low light-adapted photosynthetic apparatus characterized by a bigger antenna and a lower photosynthetic activity. In this context, it might be possible that the reduction of Put/Spm ratio that we found in the isolated LHCII monomeric and oligomeric forms (Fig. 5) during exposure to low temperature is involved in the triggering of structural changes in the photosynthetic apparatus that led to the increasing of LHCII size (Table 1). The investigation of the plant capability to recover the changes induced by low temperature in the photosynthetic apparatus showed the repair of structural and functional damages at rewarming. Specifically, the LHCII size decreased (Fig. 4), through changes in the polyamine pattern (Fig. 5) and the PSII functionality recovered (Figs. 2 and 3). Although the recovery of changes induced by low temperature was not fully achieved, the photosynthetic activity (Fig. 3) was substantially improved. In conclusion, all these data suggest that, probably, the excitation pressure of the photosynthetic apparatus is the signal for all the above-mentioned changes, since the same experiment performed in darkness (data not shown) showed no significant effect of low temperature on the structure and functionality of the photosynthetic apparatus. The similarity of the photosynthetic apparatus responses to a series of abiotic stresses, like the UV-B radiation [29,30] or enhanced atmospheric ozone [28] and low temperature (present work) support the hypothesis for a common response mechanism of the photosynthetic apparatus against abiotic stress in general. 5. Abbreviations ABS/RC antenna size per reaction center DI0/RC dissipation energy per reaction center ET0/RC photosynthetic electron transport per reaction center Fv/Fm photosynthetic efficiency LHCII light harvesting complex II Put putrescine RC/CS density of active photosynthetic reaction centers Spm spermine
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