J. Plant Physiol. 158. 185 – 197 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Acclimation of chloroplast transglutaminase to high NaCl concentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type Luca Dondini, Stefania Bonazzi, Stefano Del Duca, Anna Maria Bregoli, Donatella Serafini-Fracassini* Dipartimento di Biologia Evoluzionistica Sperimentale – Sede Botanica, Università di Bologna, via Irnerio 42, 40126 Bologna (Italy)
Received April 11, 2000 · Accepted June 27, 2000
Summary The wild type (Wt) and the polyamine-deficient strain (PA – vs) of the halotolerant Dunaliella salina were subjected to stress caused by 3.5 mol/L NaCl concentration. The chloroplasts were isolated and the molecular aspects of their reaction to salt stress were studied together with their recovery response to these hyper-saline conditions. In the Wt, the photosynthetic complexes were found to be severely affected by salt stress under light conditions. Transglutaminases, which are present in chloroplasts as two units of 25 and 50 kDa, were immunorecognized by antibodies raised against rat prostatic gland transglutaminase. The amount, in particular that of the 50 kDa unit, underwent an immediate change following hyper-saline stress. These concentration changes were found to coincide with variations in enzymic activity, which is also affected by the presence or absence of light. The PA – vs has a concentration of proteins and chlorophylls which is much lower than that of the Wt. In addition, the PA – vs appeared to be more severely affected by both salt and subculture stresses. Its recovery time was also longer. Its TGase activity increased after salt stress and was always higher in the light than in the dark, except soon after subculture, showing an additive stress effect of salt and light. In the PA – vs acclimated to high salinity, or immediately after stress application, the chloroplast content of chlorophyll a and b was considerably enhanced, like the TGase activity (by two-fold or more), and these changes exhibited almost coincident behaviours. Some transglutaminase substrates (proteins of 68, 55, 29 and 27 kDa) were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were more markedly labelled by [1,4-14C] polyamines when the transglutaminase assay was performed in the light than in the dark, and much more in algae already acclimated to hyper-saline conditions than in those cultured in the optimal saline medium, or subjected to stress. The amount of 68 and 55 kDa polypeptides was particularly high in the 3.5 mol/L NaCl acclimated cells. The possible role of polyamine conjugation in the assembly of chloroplast proteins in cells affected by salt stress is discussed. Key words: chloroplast – Dunaliella salina Teodor – light – polyamine – protein modification – salt acclimation – salt stress – transglutaminase * E-mail corresponding author:
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Abbreviations: Chl chloroplast. – ChlTGase transglutaminase of chloroplast. – CAB chlorophyll a and b protein. – hmP high molecular mass proteins. – LHC light harvesting complex. – PAs polyamines. – PAGE polyacrylamide gel electrophoresis. – PA – vs polyamine deficient strain. – PS photosystem. – PU putrescine. – Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase. – SD spermidine. – SM spermine. – TCA trichloroacetic acid. – TGase transglutaminase glutaminyl-peptide γ-glutamyltransferase (E.C. 2.3.2.13). – Wt wild type
Introduction Several molecular mechanisms of acclimation to salt stress are known in Dunaliella salina, a halotolerant, unicellular green alga, which contains a large unique chloroplast. Hyper-saline stress inhibits growth and affects cell metabolisms, including photosynthesis (Kirst 1989). As a response to high salt stress, this alga produces beta carotene (concentrated in the interthylakoidal spaces, thus protecting the photosystems) and a high amount of glycerol (up to 7 mol/L) (BenAmotz and Avron 1983, Avron 1992). Acclimation after exposure to 3.5 mol/L NaCl takes four hours to complete (Sadka et al. 1988). In D. salina after hyper-osmotic shock, Weiss and Pick (1990) observed the occurrence of a rapid (within minutes) and transient cytoplasmic acidification manifested by a Na + /H + exchange. Like higher plants under osmotic and salt stress conditions (Boucherau et al. 1999, Erdei et al. 1990), D. salina grown in a saline optimal medium (1 mol/L NaCl) exibits a transitory increase of free polyamines within 8 min from subculture into a hyper-saline medium (3.5 mol/L NaCl). Under these conditions, putrescine, spermidine, and spermine reach a total concentration of 1.5 mmol/L, and their binding to proteins is delayed (Dondini et al. 1994). This concentration of free PAs is exceptionally high when compared to that of D. salina in other phases of the culture (0.05 mmol/L). Moreover, this abrupt increase of free PAs cannot have an osmotic effect: their concentration does not appear sufficient to counterbalance that of the salt. Considering the inhibitory effect that polyamines exert on growth when present in a supra-optimal concentration (Bagni and Serafini-Fracassini 1985), we hypothesize that they may induce a lag period in the cell cycle. Pick (1992) reported that in D. salina, an increase of ammonia and amines causes an alkalinization and a transient stress, which manifests itself as a loss of mobility, a drop in ATP concentration, and an inhibition of photosynthesis. Then, by compartmentation of the aminic compounds in the vacuole, the cell recovers. Turning to bound PAs, among them, the conjugated derivatives are affected by variations of transglutaminase activity (TGase) (E.C. 2.3.2.13) (Dondini et al. 1994). This enzyme covalently links polyamines to endoglutamines of specific proteins, forming mono-(γ-glutamyl)-PAs (mono-PAs) and bis(γ-glutamyl)-PAs (bis-PAs) (Folk 1980). In animals, TGases
mainly have a structural role (Äschlimann and Paulsson 1994). In plants, they are probably also involved in cytoskeleton reorganization (Del Duca et al. 1997). TGases exist in multiple forms that may have different functions (reviewed by Serafini-Fracassini et al. 1995). In D. salina whole cells, TGase seems to play a role in the acclimation to high salt concentrations, not in the immediate response to the stress, but during the ensuing acclimation phase. Its activity continuously increases till the 24th hour, when the cell parameters stabilise again (Sadka et al. 1988). The enzyme is also responsive to subculture into fresh iso-saline medium, thus allowing the cells to actively resume cell division (Dondini et al. 1994). It is worth noting that a similar TGase response was observed in explants of parenchyma of Helianthus tuberosus stressed by wounding, where it induced the resumption of the cell cycle (Serafini-Fracassini et al. 1989). It has been reported that PU, SD, and SM are present in chloroplasts of algae and higher plants (Bagni and Serafini-Fracassini 1974, Kotzabasis et al. 1993). Moreover, PAs are involved in chloroplast development and photoadaptation (Dörnemann et al. 1996, Kotzabasis et al. 1999). It is known that the presence of PAs prevents chlorophyll loss in senescent leaves and in oat protoplasts (Cohen et al. 1979, Popovic et al. 1979, Galston and Kaur-Sawhney 1990, Shih et al. 1982, Besford et al. 1993). The molecular mechanism of this process has not yet been clarified. TGase is present in the chloroplasts of higher plants, where its activity is modulated by the presence of light. Its substrates are Rubisco and some antenna complexes of thylakoids, like LHCII, CP 29, CP 26, CP 24. This suggests an involvement of TGase in photosynthesis (Del Duca et al. 1994). In the present paper, the role played by chloroplasts in stress response, as originally proposed by Cowan et al. (1992), has been studied, also taking into account for the first time the possible involvement of TGase-induced protein conjugation with PAs. Thus, the chloroplast TGase and its substrates were analysed to verify whether they are responsive to salt stress, either immediately or in the second phase of acclimation. This could also provide a more general insight into the role of PAs conjugation to chloroplast proteins. The effect of light on TGase activity was evaluated because it is stimulatory in higher plants; but it should be noted that its effect can be additive to that of salt stress, which damages D. salina chloroplasts (Canaani 1990).
Stressed Dunaliella plastid TGase Even though chloroplasts of algae inoculated in iso-saline medium were used as the control, it cannot be excluded that the parameters (protein and chlorophyll concentrations and TGase activity) used to characterise the stress reaction are also involved in the resumption of growth. Therefore, to ensure that the effects of salt and subculture stresses are not severely masked by growth phenomena, in this study we have also utilised a variant strain of D. salina isolated in our laboratory. It has a very slow growth rate and is polyaminedeficient (Dondini et al. 2000). Because of the latter property, this strain provides an ideal system for the detection of PAsconjugated proteins since conjugation sites are available for polyamine linkage, a situation which could not occur with the Wt.
Materials and Methods Plant material, organism, culture conditions Dunaliella salina Teodor was collected from Saline di Cervia (Ravenna) and selected by the Laboratory of In vitro Plant Culture of the Dipartimento di Biologia E. S. (University of Bologna). Algae were maintained in Petri dishes on agarised Eddy (1956) medium. The experiments were performed on algae growing in Eddy liquid medium. Usually, 4 × 104 cells were inoculated in 100 mL sterile liquid medium, final pH 7.5, supplemented with 1 mol/L NaCl, the micronutrient solution of Heller (1953), and cultured for one month at 25 ˚C under a 16/8 hour light/dark period (44 µmol photons m – 2 s –1). Growth and sterility were routinely monitored since during four years of culture, a fungal infection was occasionally observed. Repeated subcultures in Petri dishes allowed single colonies to form, growing by binary fission (sexual reproduction is very rare). One of these colonies spontaneously developed phenotypic differences: a restricted capacity for growth and a very pale green colour. This colony was repeatedly sub-inoculated, and the population concentration at the stationary phase always reached only about half the value of those growing normally (1,100,000 cells/mL). Their polyamine and chlorophyll content was much lower than that of normally growing algae and for more than two years showed no reversion to normal levels. Because of the rarity of the occurrence of this new phenotype, its stability over about 25 subcultures and under different conditions (i.e. salt stress), and the fact that these algae multiply by fission, this strain is considered a «variant strain» («PA – vs»). The original strain was denominated «Wt», wild type. Growth rate and protein, chlorophyll and polyamine content were monitored. 1mol/L and 3.5 mol/L NaCl were chosen as the control and stress medium, respectively. Cells were counted using a Burker’s chamber, collected by centrifugation (3,000 RPM for 5 min), and resuspended in the appropriate buffer.
Chloroplast isolation The method of Pick et al. (1987), based on the osmotic rupture of cells, was utilised. After centrifugation, algae resuspended in a small volume of culture medium were osmotically broken by rapidly ejecting them from an insulin syringe containing the same volume of distilled water. This procedure was repeated 3 – 4 times. Chloroplasts were
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isolated by repeated centrifugation at 3,000 rpm for 10 min and resuspended in three volumes of 50 mmol/L Hepes (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])-KOH pH 8 containing 330 mmol/L sorbitol, 5 mmol/L MgCl2, 2 mmol/L EDTA (ethylenediaminetetra-acetic acid), 0.1 mg mL –1 BSA (bovine serum albumin), 3 mmol/L mercaptoethanol, 5 mmol/L ε-amino-n-caproic acid, and 0.2 mmol/L phenyl-methyl-sulphonyl-fluoride (PMSF) (Gray 1982). Chloroplasts were counted using a Burker’s chamber, and their number compared to that of the initial entire cells.
Chlorophyll and protein determination in isolated chloroplasts Chlorophyll a and b were assayed spectrophotometrically by the method of Porra et al. (1989). Protein concentration was determined by the method of Lowry et al. (1951).
Polyamine determination PAs were extracted from cells or isolated chloroplasts and resuspended in 5 % TCA. Free PAs content was determined according to Dinnella et al. (1992).
Transglutaminase – Biochemical radioassay Chloroplasts were incubated for 1h at 30 ˚C in the light (100 µmol photons m – 2 s –1) or in the dark. The incubation mixture contained: 100 µL of chloroplasts (300–700 µg proteins), 40 µL (56 kBq) of [1,4-14C]PU (4.4 GBq/mmol) (Amersham), or 10 µL (740 kBq) [1,4(n)-3H]PU (1.5 TBq/mmol) (NEN), 100 µL 100 mmol/L Tris buffer pH 8.5, also containing 0.6 mmol/L PU, 3 mmol/L CaCl2, and 660 mmol/L sorbitol. The final volume was adjusted to 300 µL with Tris buffer. After the assay, the mixture was subjected to electrophoresis. Alternatively, the reaction was stopped with 5 % TCA (final concentration) also containing 2 mmol/L cold putrescine; the mixture was precipitated three times with TCA and resuspended in 0.1mol/L NaOH, as previously reported (Del Duca et al. 1994). The solubilized pellet was dissolved in Ultima Gold (Hewlett & Packard, Milano) scintillation liquid, and radioactivity counted in a Beckman LS 1800 scintillation counter.
Gel electrophoresis 1. Monodimensional sodium dodecylsulphate gel electrophoresis (SDS-PAGE) and immunoblotting. Proteins from isolated chloroplasts or chloroplasts subjected to the TGase assay were freeze-dried and dissolved in 60 mmol/L Tris buffer pH 6.8 containing 4 % SDS and 5 % 2-mercaptoethanol and separated by 12 or 14 % acrylamide slab gel electrophoresis (Laemmli 1970). Coomassie Blue R 250-stained gels were incubated with enhancer (1 h in 10 % glycerol, 2 h in 1 mol/L sodium salicylate in the dark) and then dried, exposed at – 80 ˚C to OMAT X-AR Kodak film, and developed after 2 – 3 weeks exposure. Western blot analysis was performed utilising a polyclonal rabbit antirat prostatic gland TGase as primary antibody. Transfer of proteins to nitrocellulose filter (0.2 µm pore size) (Hoefer, Milano, Italy) was performed with a Bio Rad Mini Trans-Blot Transfer Cell (Bio Rad, Milano, Italy). The transfer buffer consisted of 25 mmol/L Tris, 192 mmol/L glycine, 20 % (v/v) methanol, 0.037 % SDS, pH 8.3, and transfer was conducted overnight at 30 V. The nitrocellulose filter was washed for
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1 h at room temperature in saline Tris-buffer (0.02 mol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl) containing 5 % non-fat dry milk, in order to block non-specific binding, and 0.05 % Tween 80. The nitrocellulose filter was incubated overnight at room temperature with the primary antisera diluted 1 : 300 with saline Tris-buffer. After four 10-min washes in TBST (the second of which also contained 0.1% Nonidet P 40), the nitrocellulose paper was incubated for 2 h at room temperature with alkaline phosphatase-conjugated protein A diluted 1 : 500 with saline Tris-buffer. The developing reaction was performed using the BCIP/ NBT substrate system (Sigma fast tablets, Sigma, Italy), following the directions of the manufacturer. 2. Two-dimensional electrophoresis, immunoblotting, and autoradiography. Chloroplasts (25 µg chlorophyll) were solubilized by adding an equal volume of 2 % dodecyl β-D maltoside. After 15 min on ice with occasional stirring on a Vortex mixer, the solution was centrifuged at 15,000 g for 5 min to pellet the insoluble material. The supernatant was loaded into the Deriphat-PAGE gel (‹green gel›). The Deriphat-PAGE (the upper buffer contained 0.1 % Deriphat) was run with a 5–12 % acrylamide gradient (200 × 160 × 1.5 mm), overlayed by a 4 mm stacking gel (3.5 % acrylamide). The electrophoresis was run overnight at 4 ˚C until the front reached 5.5 or 8 cm in the resolving gel. Gel lanes were excised from the Deriphat-PAGE gel and loaded into a fully denaturing SDS-urea PAGE gradient gel (12–17 %) (Santini et al. 1994). The gel was run overnight and was stained with Coomassie-brilliant blue. Spots were identified by immunodecoration of the corresponding nitrocellulose filter with polyclonal antibodies directed against chloroplast proteins, as previously reported (Del Duca et al. 1994).
Densitometric analysis of gels The high resolution scanned image of the gels (600 dpi) was analysed using the specific software Phoretix 1D Plus by Phoretix Ltd. (UK). Quantitative determinations were repeated at least three times on separate experiments.
Results A – Dunaliella salina wild type Effects caused by salt stress on the chloroplast. To evaluate the effect of salt stress on chloroplasts, aliquots of algae grown in the medium with optimal NaCl concentration (1 mol/L), were transferred to iso-saline (1 mol/L) and hyper-saline (3.5 mol/L NaCl) media and grown under light and dark photoperiods of 16 and 8 h. In the hyper-saline medium, as shown in Figure 1, algae were found to shrink and undergo morphological changes within 1 min of subculture; a consumption of starch occurred in the chloroplasts. It should be noted that under these experimental conditions the effects of saline stress are superimposed on those caused by subculture. On the basis of previous results (Dondini et al. 1994), the main parameters of the cell reaction to stress were evaluated soon after stress induction (8 min from subculture) and after 24 h.
Figure 1. Time course of shape and size changes of D. salina grown in Eddy medium containing 1 mol/L NaCl after subculture in hypersaline medium containing 3.5 mol/L NaCl. Bar = 10 µm.
A – 1. Chlorophylls The chlorophyll a content of stressed algae started to decline immediately after subculture. In the controls, on the other hand, it showed a transient rise during the first 8 min, followed by a steep decline; consequently after 24 h, it was almost identical in both cultures. Chlorophyll b content exhibited similar, albeit less pronounced changes (Fig. 2).
A – 2. Proteins Subculture in fresh medium caused a slow decrease of Chl proteins, but this effect was much more rapid (8 min) in cells exposed to the hyper-saline medium (Fig. 2). After one day, the Chlprotein values were identical under both conditions. In stressed algae, as suggested by Ben-Amotz and Avron (1983), light could exert a specific degradative effect on Chl proteins. Thus, CAB of chloroplasts extracted from stressed and unstressed D. salina cells (grown for 8 min or 1 day) were analysed by two-dimensional electophoresis. The first dimension was a «green gel» (Deriphat-PAGE) and the second a fully denaturing one (SDS-urea PAGE). The former separates the photosynthetic complexes and the latter the apoproteins (Fig. 3, panels 1– 5). Isolated choroplasts were incubated in light or dark for 1 hour, in order to place them in the same conditions as those of the TGase assay (see below); afterwards, their proteins were analysed. The CAB patterns already showed changes in the samples extracted 8 min after subculture. Figure 3 (panels 2, 4) shows a reduction in the concentration of many proteins in the chloroplasts of stressed cells, particularly in those incubated in the light. However, a more dramatic reduction was observed in the chloroplasts of cells kept under stress conditions for 24 h, especially in those
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level, in the latter it underwent a three-fold increase relative to the initial value. In the unstressed algae, the density of the 25 kDa band showed a similar early fall, albeit much less pronounced, followed by a slow recovery. In the stressed algae, however, it declined to a value which after 24 h was considerably lower than the initial one. The involvement of ChlTGases in stress response was studied under the same stress conditions reported in Figure 3. The TGase assay was performed both in the dark and in the light (Fig. 5). In algae inoculated in the iso-saline medium, ChlTGase activity 8 min after subculture only showed an increase in chloroplasts incubated in the light. In the hypersaline medium, at 8 min, ChlTGase activity only showed an increase in chloroplasts incubated in the dark. After 24 h, ChlTGase activity decreased in all samples.
B – Dunaliella salina PA-variant strain Salt stress effect on chloroplasts
Figure 2. Protein and chlorophyll a and b contents in chloroplasts isolated from D. salina Wt. Algae maintained in optimal (1 mol/L NaCl) medium (0 min) were collected at different times after subculture into the same medium (open symbols) or into a hyper-saline one (3.5 mol/ L NaCl) (closed symbols).
incubated in the light (Fig. 3, panels 3, 5). These changes primarily affected the apoproteins of the photosystem, mainly the PSII antennae, which have a molecular mass of about 30 kDa. These proteins were immunorecognized by antibodies raised against maize PSII LHCII. At 8 minutes from subculture, a relatively high-molecular mass protein appeared in the extracts of cells exposed to both optimal and stress conditions (Fig. 3, panel 4). However, this protein was particularly evident in the extracts of cells kept for 1 day in the hypersaline medium (Fig. 3, panel 5, arrow b).
A – 3. ChlTransglutaminase Two TGase bands of 25 and 50 kDa have been immunorecognized in chloroplast extracts of D. salina (Dondini et al. 2000), with the presence of an additional 70 kDa band in whole cell preparations (not reported). Bands of similar mass were immunorecognized by the same anti-TGase antibody in higher-plant chloroplasts (Del Duca et al. 1994). The quantitative densitometric analysis of extracts of D. salina cells harvested from media of different NaCl content revealed a different degree of immunostainability of the two ChlTGase bands (Fig. 4). The density of the 50 kDa band decreased immediately after subculture, both in stressed and unstressed algae; afterwards, while in the former it remained at the same
In the experiments reported above, which were carried out on the wild type, the resumption of growth is superimposed on the stresses induced by: (a) exposure to the hyper-saline medium, (b) to subculture, and (c) light. These factors produce complex effects in which their individual contributions are difficult to ascertain. A much simpler system is provided by the use of PA – vs cells which in addition to containing PAs in only trace amounts, also have a very slow growth rate (Dondini et al. 2000). Their capacity to adapt to hyper-saline conditions was therefore investigated. It should also be stressed that the use of this strain allows better identification of the potential Chlprotein conjugation sites, since lacking PAs, they can be available for the binding of labelled PAs. PA – vs cells were cultured in optimal saline medium or in hyper-saline medium for about one month. To study the effects caused by subculture, algae were then transferred to the same medium: Condition 1 (from optimal to optimal medium), Condition 2 (from hyper-saline to hyper-saline medium). Under Condition 2, algae already being adapted to hyper-saline conditions were not subjected to salt stress. To study the effects caused by the superimposition of subculture and salt stresses, algae grown in the optimal medium were studied after subculture in the hyper-saline medium (Condition 3). To evaluate the capacity for and the timing of stress recovery in the variant strain, the same parameters used for the Wt were studied in isolated chloroplasts.
B – 1. Polyamines At variance with the Wt, in which the polyamine concentration ranges from 1 to 10 nmoles per 107 cells (Dondini et al. 1994), PA – vs algae and their chloroplasts contain only trace amounts of these compounds. The polyamine content of PA – vs was not affected by salt stress at 8 min after subcul-
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Figure 3. Proteins from chloroplasts isolated from D. salina Wt grown in optimal medium (1 mol/L NaCl) (panel 1); inoculated in iso-saline (panels 2, 3) or hyper-saline (3.5 mol/L NaCl) media (panels 4, 5). Proteins were separated in the second dimension of a ‹green› gel and Coomassie stained. Chloroplasts were subjected to the conditions of the TGase assay performed in light (L) or dark (D) for 1h. Panel 1: Control (0 min). Panels 2, 4: 8 min after subculture. Panels 3, 5: 1 day after subculture. Arrows indicate: a) the region of PSII antenna apoproteins. b) the region of the main accumulating protein.
ture, or after 15, 20, 40 min, or 24 h (data not shown), unlike that of whole Wt cell in which at 8 minutes the three PAs already showed a 20 – 200 fold increase in concentration (Dondini et al. 1994).
B – 2. Chlorophylls At subculture, PA – vs chloroplast had a chlorophyll content much lower than the Wt. The chlorophyll content of cells exposed to different culture media varied considerably (Fig. 6). Under Condition 1 (Fig. 6 A), the low amounts of these pig-
ments found at subculture time, and especially that of chl a, increased at day 2. Under Condition 2 (Fig. 6 B), at subculture the chl a content was much higher than under Condition 1. Then, after a marked drop, it reverted to its initial value at day 5. Chl b, on the other hand, did not show any significant variation. Subculture and salt stresses under Condition 3 (Fig. 6 C) caused an immediate increase in the content of chlorophyll a and, to a lesser extent, in that of chlorophyll b, followed by a decline which ended between day 2 and 5. It should be noted, however, that the chlorophyll content at day 5 did not reach the value of the algae adapted to the hypersaline condition (Condition 2). This suggests that complete
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Figure 4. Densitometric measure of the intensity of 25 and 50 kDa bands of D. salina Wt chloroplasts immunostained by the antibody raised against rat prostatic gland TGase. Chloroplasts were isolated from Wt algae grown in optimal (1 mol/L NaCl) medium (0 min) (open symbols) and inoculated for 8 min or 1 day in optimal (1 mol/L NaCl) (open symbols) or hyper-saline medium (3.5 mol/L NaCl) (closed symbols). The same amount of Chlproteins were separated on Laemmli 12 % acrylamide gel, blotted on nitrocellulose filter, and subjected to immunodetection.
Figure 5. TGase activity of chloroplasts isolated from D. salina Wt and incubated with 740 kBq 3H PU in 200 µmol/L unlabelled PU in the light and in the dark. Algae in stationary phase (0 min) grown in optimal medium (1 mol/L NaCl) (open symbols) were collected at different times after subculture in optimal (open symbols) or hyper-saline (3.5 mol/L NaCl) media (closed symbols).
Figure 6. Protein and chlorophyll a and b contents in chloroplasts isolated from D. salina PA – vs. Algae maintained in optimal (1mol/L NaCl) or hyper-saline medium (3.5mol/L NaCl) (0min) were collected at different times after subculture: (A) from optimal medium (open symbols) into the same medium (Condition 1); (B) from hyper-saline (closed symbols) into the same medium (Condition 2); (C) from optimal (open symbols) to hyper-saline medium (closed symbols) (Condition 3).
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recovery requires a longer time span. The behaviour of the chlorophyll a/b ratio between day 1 and 5 under Conditions 1 and 2 suggests that the algae were in a stable adapted state. On the other hand, the ratio observed under Condition 3 is indicative of probable damage to the photosynthetic complexes, an effect which was clearly visible during the first 48 h from subculture.
B – 3. Proteins At subculture, PA – vs chloroplasts had a protein content much lower than the Wt. One-month old subcultures in both the optimal and the hyper-saline medium had very similar Chlprotein contents (Fig. 6 A and B). Their response to subculture stress consisted of a fall in protein content which was very rapid under Condition 1 (Fig. 6 A) and somewhat slower under Condition 2 (Fig. 6 B) and 3 (Fig. 6 C). Under all Conditions, full recovery occurred by day 5 (Fig. 6 A, B, C).
B – 4. ChlTransglutaminase Both ChlTGase 25 and 50 kDa bands were present in the PA – vs strain, but in amounts 4 and 6 times, respectively, lower than in Wt (Dondini et al. 2000). Since the results reported above suggest that PA – vs recovers from stress more slowly than Wt, ChlTGase activity was monitored by incubating, in the light and in the dark, chloroplasts extracted from algae collected from day 0 to 5 after subculture (Fig. 7A, B, C). Under Condition 1 (Fig. 7A), enzymic activity showed a small drop immediately following subculture. Thereafter, it increased slowly when assayed in the light. After one month of subculture in the hyper-saline medium of Condition 2 (Fig. 7 B), ChlTGase activity was twice that measured under Condition 1. This observation suggests that acclimation to a high level of salinity may require that the algae maintain a high level of ChlTGase activity. After subculture in fresh hypersaline medium, ChlTGase activity decreased and then slowly rose again, especially in the light, but without reaching its initial value within 5 days. Under Condition 3 (Fig. 7 C), ChlTGase activity showed a rapid increase at 8 min, attained its maximum value at day 1 in the dark, but decreased in the light. Thus, as in Wt, TGase activity of PA – vs was usually higher when assayed in the light, except soon after stress application (the maximum being delayed). These results, obtained only after addition of 200 µmol/L exogenous PU, confirm that the enzyme is present and exhibits a different level of activity depending on the stress condition of the cells. In summary, ChlTGase activity, like the chlorophyll content, was significantly higher under early stress conditions and in the salt-adapted algae than in cells cultured in the optimal saline medium.
Figure 7. TGase activity (incubated with 740 kBq 3H PU in 200 µmol/L unlabelled PU in the light or in the dark) of chloroplasts isolated from D. salina PA – vs. Algae maintained in optimal (1 mol/L NaCl) or hypersaline medium (3.5 mol/L NaCl) (0 min) were collected at different times after subculture: (A) from optimal medium (open symbols) into the same medium (Condition 1); (B) from hyper-saline (closed symbols) into the same medium (Condition 2); (C) from optimal (open symbols) to hyper-saline medium (closed symbols) (Condition 3).
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B – 5. ChlTGase substrates The TGase-catalysed binding of PAs to Chlprotein substrates was investigated by incubating chloroplasts with radioactivelylabelled PAs under the same conditions as those of the in vitro enzyme assay reported in Figure 7. The labelled TGase substrates were subsequently identified by fluorography of the SDS-blotted gel. The label incorporation was more marked when the assay was performed in the light, and increased during the days which followed subculture. In all samples, polypeptides covalently linked to labelled PU had molecular masses of 68, 55, 29, and 27 kDa. As an example, the fluorographs of chloroplast proteins from algae adapted to the hyper-saline medium (Condition 2), which were the most intensely labelled, are shown in Figure 8. The wells, the boundary region between the stacking and running gels, and the gel front also contained labelled material. A detailed analysis of the amount (evaluated as Coomassie stainability) of the main proteins which undergo PU-conjugation, was performed between day 0 to 5; the changes were similar in trend to those of the total proteins shown in Figure
Figure 9. Densitometric analysis of Coomassie-stained SDS-PAGE of proteins extracted from chloroplasts of D. salina PA – vs incubated with PU for the TGase assay performed in the light, as reported in Fig. 8. Chloroplasts were extracted from algae collected five days after subculture from optimal medium (1 mol/L NaCl) into the same medium (continuous line) (Condition 1) and from hyper-saline (3.5 mol/L NaCl) into the same medium (dotted line) (Condition 2).
6, and thus have not been reported. However, it is worth nothing that under Condition 1, the 55 kDa band was detectable only at day 5, and that bands 68 and 27kDa, although always present, increased considerably at day 5. The densitometric profiles of Chlproteins in Conditions 1 and 2 on day 5 are shown in Figure 9. The Coomassie stainability of the two very close bands of molecular mass around 68 kDa, and, to a lesser extent, that of the 55 kDa band, was markedly higher in algae adapted to hyper-saline conditions than in algae grown under optimal conditions. This data indicates that TGase substrates vary in amount according to the salinity of the culture medium and increase after subculture. The presence of light during the assay caused a weak enhancement of several bands in the sample isolated from the hyper-saline medium (results not shown), in particular of the 68 kDa band. In summary, PA – vs appears to be more severely affected by salt and subculture stresses and to have a longer recovery time than Wt; its free PA content did not change. As a consequence of the subculture in fresh medium, the concentration of TGase substrates was markedly altered, especially in hyper-saline-adapted algae. In addition, when supplied with exogenous PAs, the capacity of ChlTGase to modify these substrates also changed in response to different stress conditions.
Discussion Figure 8. Fluorography of 14 % SDS-PAGE, according to Laemmli, of proteins extracted from chloroplasts of D. salina PA – vs incubated with 56 kBq 14C PU in 200 µmol/L unlabelled PU for the TGase assay performed in the light (right) or in the dark (left). Algae were collected at day 2 after subculture from hyper-saline (3.5 mol/L NaCl) into the same medium (Condition 2).
The data presented above shows that several Chlproteins modified by PAs are affected by hyper-saline stress (3.5 mol/ L NaCl) and are involved in the acclimation to stress. The quantitative parameters examined suggest that the Wt chloroplast recovery from stress is almost complete within the first 24 h. However, during this time interval the CAB appear to undergo dramatic changes. The reduction in intensity of
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their spots, separated by gel electrophoresis, could either be due to their partial degradation or to their aggregation under stress. Trezzi et al. (1965) reported that in salt-stressed Dunaliella salina chloroplasts, membrane lamellar pairs under the electron microscope appear to stick closely together, causing an almost total disappearance of the inner space. This observation supports the idea that under these experimental conditions, thylakoids are not disrupted but compacted. Hypersaline stress severely affects CAB, causing mass changes which, in turn, can lead to a decline in photosynthetic activity (Kirst 1989). The role of PAs in the salt stress acclimation, already studied by several authors, (Boucherau et al. 1999, Erdei et al. 1990), has been further analysed in this paper with particular emphasis on the conjugation of certain proteins of the chloroplast. This organelle is particularly important, from a metabolic point of view, since it allows Dunaliella to successfully recover from stress conditions which are lethal for most living organisms (Canaani 1990, Cowan et al. 1992). Using PA – vs cells, it has been possible to unambiguously identify the Chlproteins which are labelled by radioactive PU when algae are exposed to salt stress. The intensity of this modification, which is enhanced by light, increases during the subsequent phases of acclimation to stress. These proteins coincide, as far as their molecular mass is concerned, with those previously observed in the chloroplasts of Helianthus tuberosus leaves and of maize callus (Del Duca et al. 1994, Bernet et al. 1999) and identified as Rubisco LS, CAB, namely: LHCII, CP 29, CP 24, and CP 26 (Del Duca et al. 1994). Catalysed by TGase, the conjugation of Chlproteins with PAs was demonstrated by the identification of glutamylderivatives (Del Duca et al. 1995). It is of considerable interest to note that the acclimation to hyper-saline conditions induces the appearance or enhances the content of precisely those Chlproteins that are modified by PAs. Identifying the proteins modified by PAs can shed some light on possible roles of this modification. At present, the only clear-cut function of polyamination is a structural one, in that it favours the assembly, among others, of cytoskeletal (Del Duca et al. 1997) or other structural proteins (Waffenschmidt et al. 1999). The formation of large protein aggregates, observed in chloroplasts, was reported to be accompanied by their posttranslational modification by PAs in extracts of whole Dunaliella cells (Dondini et al. 1994) and other plant and animal tissues (Del Duca et al. 2000, Grandi et al. 1992, Äschlimann and Paulsson 1994). In chloroplasts, the role of the PAs is believed to be part of a protective mechanism against senescence. Dörnemann et al. (1996) suggested that PAs may play a key role in the assembly of both the photosynthetic membrane complexes and the photosynthetic apparatus. Thus, we can advance the hypothesis that the protective effect against senescence exerted by PAs on several chloroplast components (Cohen et al. 1979, Popovic et al. 1979, Shih et al. 1982, Galston and Kaur-Sawhney 1990, Besford et al.
1993) could be due to their TGase-induced conjugation to some thylakoid and stroma proteins. It is likely that the 55 kDa band of D. salina is the Rubisco LS, as previously observed in the chloroplasts of higher plants (Margosiak et al. 1990, Del Duca et al. 1994). A polypeptide of this molecular mass was also found to be the protein most extensively labelled by PU in whole D. salina cells adapted to high salinity (Dondini et al. 1994). The post-translational modification of purified Rubisco LS by PAs favours the polymerisation to the active octameric form and its stabilisation (Margosiak et al. 1990). The two labelled bands of about 30 kDa could correspond to the antenna proteins already identified in chloroplasts of higher plants (Del Duca et al. 1994). A feature peculiar to the PA-deficient Dunaliella strain is the parallel increase of its chlorophyll a and b content and its TGase activity when the cells are acclimated to high salinity, or immediately after stress application. In the acclimated cultures, changes of protein concentration are also superimposable on those of the chlorophylls, of Chl a in particular. It should be noted that in the chloroplast, these molecular species are related to each other since LHC apoproteins are associated with pigments to form monomeric complexes (Dreyfuss and Thornber 1994). In this respect, it is particularly intriguing to note that some chlorophyll molecules are linked to two glutamine residues of the LHCII that could also be covalently modified by PAs. In Helianthus tuberosus, the trimeric LHCII is more intensely labelled in the light by 3H-PU and -SD than its monomeric unit, suggesting that TGase is involved in the formation of its active form (Del Duca et al. 1994). Thus, ChlTGase could play a role in the assembly or polymerisation of these proteins. On the other hand, the phosphorylation of this CAB is known to affect, by the addition of negative charges, its position in the thylakoid membrane, and, consequently, affects its stacking (Allen 1995). It is conceivable that the addition of positive charges by ‹polyamination› may have an effect opposite to that of phosphorylation. These modifications probably affect the energy transfer between photosystems. Gilmour et al. (1985) reported that PSII and associated antennae in Dunaliella tertiolecta are so severely damaged by hyper-saline stress, especially in the presence of light, that cell death can occur; PSII inactivation is associated with a state-2 transition. In algae, high salinity affects the photosynthetic apparatus in at least two sites (the reducing side of PSI and the water side of PSII) as well as the transfer of energy among the pigment complexes (Kirst 1989). PSII inactivation is one of the major factors of photosynthesis inhibition, but its mechanism is not clear (Lu and Zhang 1999). Canaani (1990) reported that in Dunaliella salina chloroplast PSII inactivation is due to a ∆pH increase across the thylakoid membrane. The last main substrate, the 68 kDa band, could tentatively be identified as the 68 kDa dynamine-like protein of Arabidopsis chloroplasts, a GTP-binding protein possibly involved in the biogenesis of the thylakoid and in the assembly of LHCII and CP 29. This protein is present in membranes as a
Stressed Dunaliella plastid TGase high molecular mass complex, and its conformation and association to membranes are Ca2 + -dependent and inhibited by GTP (Park et al. 1997). These properties have many analogies with those of TGase, some isoforms of which in animals are also active as GTPases (Lai et al. 1998). In light of this data, we can hypothesize that the higher than normal concentration of the 68 kDa protein in D. salina chloroplasts acclimated to hyper-salinity, which also exhibits a high TGase activity and chlorophyll content, could be interpreted as an attempt by the cell to repair the effects of the high salt concentration on the thylakoid membrane. Similar effects on the thylakoid membrane were also reported by Ben Amotz and Avron (1983) and by Trezzi et al. (1965). The involvement of light in the PA modification of Chlproteins in D. salina, is supported by the changes of ChlTGase activity observed as a function of a switch in the conditions of illumination. Normally, in the whole Wt alga (Dondini et al. 1994), and in higher-plant chloroplasts (Del Duca et al. 1994, Bernet et al. 1999) TGase activity is stimulated by light. However, under salt stress, the lower activity exhibited by TGase in conditions of light as compared to darkness is suggestive of an additive salt and light stress effect, even though the light intensity used in the ChlTGase assay is rather low, at least compared to what is considered low for whole cells (Harrison and Allen 1993). It is known that when algae are exposed to a hyper-saline medium, light appears to subject the chloroplasts to an additional stress, referred to as photoinhibition. Ben-Amotz and Avron (1983) suggested that in stressed algae, the light could have a specific degradative effect on Chlproteins. Our data shows that, especially in stressed algae, light has a small but evident effect on both the amount of apoproteins detected by gel electrophoresis and the ChlTGase activity. While in unstressed algae after subculture, the isolatedchloroplast TGase activity measured in the light shows a trend which strictly recalls that observed in whole cells (Dondini et al. 2000), in stressed algae it differs substantially from that of the whole cell. This suggests that, under stress conditions, differently compartmented TGases may have different functions or may be influenced by different factors. The stress caused by salt on the photosynthetic apparatus might affect the energy supply to the growing cell. In fact, unstressed algae divide, whereas those subjected to stress resume cell division only after a lag period. Evidence supporting the hypothesis of a relationship among photosyntheticrelated processes, growth, and PAs (whether or not TGasemediated) is also provided by other studies on the PA – vs (Dondini et al. 2000). Its low growth capacity seems to derive from disorders of the photosynthetic machinery, as suggested by the low and differentially altered chlorophyll a and b content. When the variant strain is supplied with exogenous PU, the synthesis of both chlorophyll and proteins is activated in the chloroplasts, the ChlTGase activity undergoes a 9-fold increase in light, and the cell division rate becomes similar to that of the unstressed Wt. It is known that salt-stressed Wt
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cells resume growth following resumption of PAs synthesis (Dondini et al. 1994) and of photosynthesis (Avron 1992). Taken together this data led us to suggest that the saltstressed D. salina could adapt, modifying the chloroplast structures and thus reactivating the photosynthetic process by «polyaminating» key chloroplast proteins in a manner similar to that of the PU-supplemented PA – vs algae observed by Dondini et al. (2000). Acknowledgements. Prof. Serafini-Fracassini’s research supported by C.N.R. Contributo N. 97.04274.CTO4 and by Ministero dell’ Università e della Ricerca Scientifica e Tecnologica (ex 60 %). Antibodies were a kind gift of Carla Esposito, University Federico II, Dipartimento di Biochimica e Biofisica, Napoli (Italy) and of Prof. R. Bassi, University of Verona, whom we also thank for their suggestions. The authors are grateful to Henkel S.p.A. (Fino Mornasco, Como) for the kind gift of Deriphat C 160 KPC. We are very indebted to Prof. A. SerafiniFracassini (Emeritus Professor of the University of St. Andrews, Scotland) for the revision of the English manuscript.
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