Journal of Experimental Botany, Vol. 52, No. 361, pp. 1711-1719,
August 1, 2001
© 2001 Oxford University Press
Original Papers |
Heat-shock responses in two leguminous plants: a comparative study
Facultad de Ciencias, Universidad de Chile, Departamento de Biologia, Casilla 653, Santiago, Chile
Received 21 August 2000; Accepted 30 March 2001
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Abstract |
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Relative growth rates, basal and acclimated thermotolerance, membrane damage, fluorescence emission, and relative levels of free and conjugated ubiquitin and HSP70 were compared after 2 h of treatment at different temperatures between Prosopis chilensis and Glycine max (soybean), cv. McCall, to evaluate if the thermotolerance of these two plants was related to levels of accumulation of heat shock proteins. Seedlings of P. chilensis germinated at 25 °C and at 35 °C and grown at temperatures above germination temperature showed higher relative growth than soybean seedlings treated under the same conditions. The lethal temperature of both species was 50 °C after germination at 25 °C. However, they were able to grow at 50 °C after germination at 35 °C. Membrane damage determinations in leaves showed that P. chilensis has an LT50 6 °C higher than that of soybean. There were no differences in the quantum yield of photosynthesis (Fv/Fm), between both plants when the temperatures were raised. P. chilensis showed higher relative levels of free ubiquitin, conjugated ubiquitin and HSP70 than soybean seedlings when the temperatures were raised. Time-course studies of accumulation of these proteins performed at 40 °C showed that the relative accumulation rates of ubiquitin, conjugated ubiquitin and HSP70 were higher in P. chilensis than in soybean. In both plants, free ubiquitin decreased during the first 5 min and increased after 30 min of heat shock, conjugated ubiquitin increased after 30 min and HSP70 began to increase dramatically after 20 min of heat shock. From these data it is concluded that P. chilensis is more tolerant to acute heat stress than soybean.
Key words: Heat-shock proteins, thermotolerance, LT50, HSP70, free-ubiquitin, conjugated-ubiquitin.
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Introduction |
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Most cultivated species have wild relatives which exhibit high tolerance to abiotic stress (Nobel, 1988
; Wu and Wallner, 1983). Prosopis chilensis (Mol.) Stuntz (Chilean algarrobo) is a leguminous tree which grows in the arid and semi-arid regions of Northern and Central Chile. This tree has been described as resistant to temperature, drought, salinity, and wounding (Medina and Cardemil, 1993; Rodríguez and Cardemil, 1994; Ortiz et al., 1995; Cazebonne et al., 1999). P. chilensis has a high capability for acquiring thermotolerance since the seedlings are able to survive at 50 °C after germination at 35 °C, or after exposure for 2 h at the sublethal temperature of 40 °C (Medina and Cardemil, 1993). It has also been shown that, under field conditions, P. chilensis expresses heat shock proteins daily at the hours of higher temperatures, which is presumed to be an adaptation to its harsh natural environment (Ortiz et al., 1995). Thermotolerance acquisition and expression of heat shock proteins has also been found in the cultivated leguminous soybean (Lin et al., 1984; Hernandez and Vierling, 1993).
Since P. chilensis could be considered a genetic resource for crop improvement in desert environments, this work attempts to evaluate and compare the heat shock responses of seedlings and plants of two legumes, P. chilensis and cultivated Glycine max (soybean), studying growth, membrane damage, fluorescence emission, and heat shock protein levels in response to heat stress. The level of thermotolerance of P. chilensis could be related to higher amounts of heat shock proteins, induced by thermal stress as compared with the amounts induced in soybean, another leguminous plant able to acquire thermotolerance when subjected to sublethal temperatures (Lin et al., 1984; Hernandez and Vierling, 1993).
The objectives of this work are (1) to quantify thermotolerance between soybean and P. chilensis, (2) to quantify and compare the levels of ubiquitin (in its free and conjugated form), and HSP70 induced by heat shock in these two plants. This information can be used in the near future to improve crops in desert environments.
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Materials and methods |
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Germination and growth determinations
Seeds from multiple individuals of P. chilensis wild collected in Peldehue (Central Chile), in 1997 and 1998 and seeds of G. max, cv. McCall from the Instituto de Investigaciones Agropecuarias (INIA) La Platina, were germinated at 25 °C or 35 °C in plastic trays on paper towels as described earlier (Medina and Cardemil, 1993
). Seedlings derived from seeds germinated at 25 °C, taken 72 h after germination were subjected to 2 h temperature treatments of 25, 30, 35, 40, 45, and 50 °C in incubation chambers. Seedlings germinated at 35 °C were subjected to temperature treatments of 35 °C and above 35 °C, (40, 45 and 50 °C) for 2 h. Thermotolerance during germination was tested at temperatures above the germination temperature. Growth was determined after the treatment by measuring the embryo axis' length. Since P. chilensis grows faster than soybean at all temperatures, even at 25 °C which is the control temperature, the results of these experiments are standardized for both species in terms of relative growth.
Membrane damage
Three mm pieces of fully developed leaves from 3-month-old P. chilensis and one-month-old soybean plants grown at 35 °C were used. The ages of plants were chosen to compensate the developmental differences existing between an annual plant, such as soybean and a perennial plant, such as P. chilensis. About 0.12 g of leaf pieces, were incubated in flasks from 25–60 °C. After tissue incubation, the flasks were cooled to room temperature and 25 ml of deionized water were added to the flasks for incubation at 25 °C for 1 h, under agitation. The measurements were performed in P. chilensis and soybean leaves according to the method described previously (Raymond et al., 1986; Hallam and Tibbits, 1988), but slightly modified. The results were standardized by 1 g unit of weight. Water conductivity was measured using a conductivity meter. To calculate the maximum membrane damage, the flasks containing the pieces of leaves were left at 80 °C overnight or boiled for 15 min. The conductivity of the water was then determined. Conductivity measurements enabled us to determine the lethal temperature (LT50). LT50 is defined as the temperature at which there is 50% membrane damage determined by the conductivity of the water surrounding the tissue treated at different temperatures. The percentage of membrane damage was calculated according to the following equation,
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Fluorescence emission determinations
For fluorescence emission determinations, plants were grown in chambers at 25 °C under an 11/13 h light/dark cycle and treated for 2 h at 25, 35, 40, and 45 °C. The temperatures of the leaves were measured with thermocouples and were always 1–2 °C below the chamber temperature. Initial fluorescence (F0), maximum fluorescence (Fm), variable fluorescence (Fv), and the photochemical efficiency (Fv/Fm) of photosystem II (PSII) were determined with a Plant Efficiency Analyser, (Hansatech, London, UK). Three determinations were performed in fully developed leaves of 3-month-old P. chilensis plants and of 1-month-old soybean plants. A portion of the leaves was maintained in darkness for 30 min using a leaf clip. After the dark period, the leaves were illuminated using light of maximal emission of 650 nm. The light intensity was 2250 µmol m-2 s-1.
In other experiments, plants were subjected to heat shock for 2 h at 35 °C and returned at 25 °C in darkness for periods of 1, 2, 3 or 5 h. The fluorescence was measured at the end of the dark period. These experiments were done to determine if, after heat shock, a longer dark period at normal temperature would be sufficient to reverse ‘state changes’ of PSII.
PAGE and Western blot analysis of proteins
PAGE and Western blot analyses of proteins were performed with seedlings of P. chilensis and soybean after 2 h of temperature treatment at 25, 35, 40, and 45 °C. For this, frozen tissue was ground in buffer (1.5 mM TRIS-HCl, pH 7.5, 2% (w/v) Triton X-100 and 0.1% (v/v) 2-mercapthoethanol), in a ratio 1 : 2 (w/v). The mixture was then heated at 100 °C for 90 s and centrifuged at 15 000 g for 5 min. The supernatant was transferred to a fresh tube and the protein was precipitated with cold acetone in the proportion 5 : 1 acetone : supernatant. The protein was resuspended in 100 mM TRIS buffer (pH 6.8). Protein content was determined according to Bradford (Bradford, 1976). BSA was used as standard. The soluble proteins were analysed by SDS-PAGE as described previously (Laemmli, 1970). Proteins were stained with Coomassie Blue.
For Western blot analyses, three kind of antibodies were used: two polyclonal antibodies raised against free and conjugated ubiquitin from human erythrocytes (Hershko et al., 1982), and a third monoclonal antibody raised against the HSPs of 72 and 73 kDa (purchased from Amersham) located in the nucleoli of HeLa cells (Welch and Suhan, 1986), which detects two HSPs from P. chilensis, one constitutive protein of 69 kDa and another of 71 kDa (Ortiz et al., 1995). The two antibodies raised against free and conjugated ubiquitin were provided by Professor Ciechanover. P. chilensis and soybean proteins were loaded and separated by SDS-PAGE and electrotransferred to a nitrocellulose membrane overnight (Towbin et al., 1979). After protein transfer, the nitrocellulose membranes were blocked in 1% (w/v) BSA and incubated with the primary and secondary antibodies using the same antibody dilutions described earlier (Ortiz et al., 1995).
In order to determine if the primary antibodies were equally reactive against the corresponding ubiquitin and HSP70 family of proteins for P. chilensis and soybean, calibration curves were performed with serial dilutions starting from 0.05–2 µg of the pure corresponding protein from each species. The pure proteins were obtained by preparative purification, electroeluting the bands from the gels after SDS-PAGE. The calibration curves gave very similar immuno-cross reactivity for both plants. To compensate for any difference in the immuno-cross reaction of the antibodies with the corresponding protein of each species, the results have been expressed as relative accumulation of the proteins compared to the control treated at 25 °C.
The densitometry of the bands of the Western blots was performed using a Gelscan CS-4500 scanner and the image processing was done by the Gel-Perfect 5.2 software as described earlier (Medina and Cardemil, 1993; Ortiz et al., 1995).
Immuno dot blot analyses
Kinetic experiments were performed by incubating at 40 °C seedlings 72-h-old germinated at 25 °C. Samples were taken at 5, 10, 20, 30, 60, and 90 m after heat shock began. A Bio Rad chamber was used to load the protein samples (Bio-Dot model). The amount of protein loaded in each well was 12.5 µg or 20 µg depending on the specificity of the antibodies and using the same quantity of protein for each sample in each experiment. Membranes were washed and incubated with the primary and secondary antibodies as described before.
The densitometry of the dot blots was performed with the same scanner and the same image processor described for the Western blot. The results are expressed as the relative amount of each protein accumulated over time of treatment.
Statistical analyses
ANOVA and Duncan Tests were performed to evaluate the significance of differences between the parameters measured at different temperatures or at different times between these two species.
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Relative seedling growth determinations at different temperatures
It was reported that seedlings of P. chilensis have a lethal temperature of 50 °C when they come from seeds germinated at 25 °C (Medina and Cardemil, 1993
). However, they can grow at 50 °C when they are germinated at 35 °C. To determine if similar thermotolerance acquisition was present in soybean as compared to P. chilensis, the relative growth of seedlings was measured at temperatures from 25–50 °C after germination at 25 °C (Fig. 1A) and from temperatures of 35–50 °C after germination at 35 °C (Fig. 1B). When P. chilensis seeds were germinated at 25 °C, and then transferred at higher temperatures: the growth rate was 2.2 times faster at 30 °C; 2.4 times faster at 35 °C and 2.1 times faster at 40 °C than the control. At 45 °C the growth of P. chilensis was 80% of that achieved by control seedlings. Soybean seedlings grew 2.3 times faster at 30 °C and 1.2 times faster at 35 °C than their control seedlings. At 40 °C and 45 °C the growth of soybean seedlings was retarded, becoming 30% and 20%, respectively, of the growth of the control group. When the temperature of germination was 35 °C, P. chilensis and soybean seedlings could grow at 50 °C. P. chilensis grew 2.3, 3.2 and 1.8 times faster than its control group at 35, 40 and 45 °C. At 50 °C the growth of P. chilensis seedlings was 84% of that of its control group. The maximum growth of P. chilensis was at 40 °C as reported previously (Medina and Cardemil, 1993). By contrast, when seedlings of soybean were germinated at 35 °C the growth at this temperature and above was slower than the growth of its own control group at 25 °C. At 50 °C the growth was only 24% of that of its control group (Fig. 1B).
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Membrane damage
The membrane damage caused by heat was compared in these two plants. For this, the LT50 of leaves of both species was measured. There was a difference of 6 °C between the LT50 of both species: 53.3 °C for P. chilensis and 47.2 °C for soybean (Fig. 2).
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Fluorescence emission under heat shock
It was reported that the photosynthesis is very much affected by temperature stress (Berry and Björkman, 1980; Weiss and Berry, 1988). The stability of PSII under heat stress was evaluated through the fluorescence emission in plants of P. chilensis and soybean treated at 25, 35, 40, and 45 °C for 2 h. The initial fluorescence (F0) was higher (P
0.05) in soybean than in P. chilensis when the treatment temperature was 35 °C (Fig. 3A). This difference in F0 between species was not detected when plants were returned for 3 h in darkness at 25 °C, after a heat shock of 2 h at 35 °C. In P. chilensis, F0 only increased between 40 °C and 45 °C, while in soybean the F0 began to increase at 35 °C with a difference of 5 °C in the break point of the curve.
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The maximal fluorescence signal (Fm) was higher in soybean than in P. chilensis. In both plants Fm decreased progressively from 35–45 °C (Fig. 3B
). The lowest value of Fm observed was at 45 °C for both species. In both plants, the variable fluorescence (Fv) decreased when the temperature of the treatment increased (Fig. 3C). The slope of Fv was steeper in P. chilensis than in soybean plants.
The quantum yields, expressed as the ratio Fv/Fm, were almost identical in P. chilensis and soybean plants (Fig. 3D). The Fv/Fm values decreased slightly from 35–40 °C. Between 40 °C and 45 °C, there was a drastic decrease in the Fv/Fm.
No differences in F0 or in Fm and, therefore, no differences in Fv/Fm between species were found when plants were returned at 25 °C for 3 h in darkness, after a heat treatment of 2 h at 35 °C.
HSP relative amounts at different temperatures
Since much of the thermotolerance shown by plants could be due to the presence of heat shock proteins (Schöffel et al., 1998; Feder and Hofmann, 1999), the relative amounts and rates of accumulation of free and conjugated ubiquitin, and HSP70 were studied in both P. chilensis and soybean seedlings using antibodies raised against these proteins.
Ubiquitin was quantified as free and conjugated ubiquitin using antibodies raised against these two forms of the molecule. In P. chilensis the relative amount of free ubiquitin increased 20% at 35 °C (from 0.6 to 0.72 µg mg-1 protein), then decreased progressively at 40 °C and 45 °C. In soybean, free ubiquitin levels decreased progressively when the temperature increased (from 0.3 to 0.15 µg mg-1 of protein), the relative amount of free ubiquitin always being lower than the level found in soybean control seedlings (Fig. 4A). At 35 °C ubiquitin level in soybean seedlings decreased, 33% from the level shown at 25 °C. In both plants, the smallest amount of free ubiquitin was detected at 45 °C.
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The levels of conjugated ubiquitin showed an opposite tendency, compared to free ubiquitin levels (Fig. 4B
). In both plants, conjugated ubiquitin increased with temperature (from 0.8 to 1.6 µg mg-1 of protein in P. chilensis and from 0.3 to 0.52 µg mg-1 of protein in soybean). The relative amounts of conjugated ubiquitin shown by P. chilensis seedlings were slightly higher than those shown by soybean for temperatures of 40 °C and 45 °C. The maximum accumulation of conjugated ubiquitin was at 45 °C and the minimum at 25 °C for both species.
Two bands of proteins in P. chilensis immuno-cross reacted with the HSP72 monoclonal antibody. The proteins have molecular masses of 69 and 71 kDa as reported previously (Ortiz et al., 1995). In soybean there are also two bands of proteins which immuno-cross-reacted with this antibody. These proteins have molecular masses of 70 and 73 kDa, the 70 kDa band being expressed constitutively (Fig. 5A).
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The relative increases in HSP70 at 35, 40 and 45 °C were significantly higher in P. chilensis than in soybean seedlings (P
0.05). In P. chilensis HSP70 increased from 120 O.D. units mm2 µg-1 of protein at 25 °C to 250 O.D. units mm2 µg-1 of protein at 40 °C and in soybean it increased from 80 O.D. units mm2 µg-1 of protein at 25 °C to 145 O.D. units mm2 µg-1 of protein at 40 °C. In both plants the maximum accumulation of HSP70 was at 40 °C (Fig. 5B).
Time-course studies of accumulation of ubiquitin and HSP70
A time-course of relative levels of free ubiquitin was studied at 40 °C during the first 90 min of heat shock (Fig. 6A). Free ubiquitin amounts decreased drastically during the first 5 min at 40 °C in both species. In P. chilensis free ubiquitin decreased 70% and in soybean it decreased 90%. After this time, free ubiquitin increased steadily during the next 90 min of treatment at 40 °C in both species.
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Conjugated ubiquitin showed the opposite time-course of relative accumulation to that of free ubiquitin for both species (Fig. 6B
). During the first 20 min, the initial level of conjugated ubiquitin remained unchanged in P. chilensis. The levels increased progressively between 20 and 60 min of treatment, decreasing afterwards. In soybean, relative amounts of conjugated ubiquitin decreased 25% in the first 20 min of treatment and increased after this, to reach a maximum after 60 min, declining afterwards.
The time-course of relative amounts of HSP70 at 40 °C was similar in P. chilensis and in soybean seedlings (Fig. 7). During the first 20 min of heat shock the relative amount of HSP70 decreased 40% in soybean while it remained unchanged in P. chilensis. After 20 min of heat shock, the relative amounts of HSP70 began to increase drastically, reaching its peak in both plants 30 min after heat shock had begun.
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Discussion |
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Basal and acclimation thermotolerance
After germination at 25 °C, the growth of seedlings of P. chilensis at 45 °C was 80% of that of the 25 °C control group. Soybean seedlings in comparison were able to grow only 20% of their control group at this temperature, indicating that 45 °C is a more stressful temperature for soybean seedlings than it is for P. chilensis. Indeed, soybean seeds were unable to germinate at 45 °C (data not shown), while seeds of P. chilensis can germinate at this temperature, but at a slower rate (Medina and Cardemil, 1993
). For P. chilensis germinated at 25 °C, the optimal temperature for seedling growth was 35 °C, which was 5 °C higher than the optimal temperature for growth of soybean seedlings. The optimal temperature for P. chilensis seedling growth was 40 °C when seeds were germinated at 35 °C. The optimum temperature was therefore, displaced 5 °C in P. chilensis under these conditions. If the same experiment is performed with soybean seedlings germinated at 35 °C, the growth was drastically reduced at all temperatures above 35 °C.
LT50 determinations in leaves
A difference of 6 °C was found between P. chilensis and soybean when heat shock lethality was determined in leaves through membrane damage. The LT50 found was 53.3 °C for P. chilensis which implies that foliar tissue in this plant is more tolerant to heat stress than soybean with an LT50 of 47.2 °C and many other plants (Martineau et al., 1979; Schaff et al., 1987). Plants rarely survive over 45 °C temperatures (Nobel, 1988). In general, over 50 °C, only succulent desert plants and some unicellular organisms survive (Nobel et al., 1986; Nobel, 1988; Ougham and Howarth, 1988). Cacti and agaves survive at 73–74 °C for 1 h. However, the thermotolerance of these plants at these temperatures was evaluated by a different method (Nobel, 1988).
Fluorescence emission as an indicator of heat stress
The exceptional thermotolerance of P. chilensis might be related to photosynthetic machinery if this is less affected by heat shock than that of other plants. Fluorescence emission was used as an indirect indicator of the physiological condition of the photosynthetic process under stress (Kautsky and Hirsch, 1934; Havaux and Lannoye, 1983; Krause and Weiss, 1991). According to Georgieva and Yordanov (Georgieva and Yordanov, 1993), an increase in F0 under stress could be interpreted as a decrease in the absorption efficiency of photons by chlorophyll a in the LHC and of the reaction centre of PSII. It has been suggested that high temperature treatments could promote dissociation between the light-harvesting complex (LHC) and PSII reaction centre, causing an increase of F0 (Schreiber and Armond, 1978), while other authors considered that increases of F0 could also be related to changes in the redox state of plastoquinone A (QA) (Ducruet and Lemoine, 1985). In P. chilensis, F0 increased when the temperature was raised from 40 °C to 45 °C, while in soybean F0 increased from 35 °C to 45 °C. This study's results indicate that the dissociation between LHC and PSII reaction centre occurs during heat shock in soybean at a temperature 5 °C lower than in P. chilensis. The difference in F0 between species caused by heat shock was not observed when plants were returned for 3 h in darkness to 25 °C. This suggests that dissociation between LHC and PSII reactions centres reverses in both plants in 3 h after heat shock. Therefore, the flux of energy from the LHC to the reaction centre of PSII is apparently less affected by heat in P. chilensis than in soybean. Lycopersicon esculentum has two chloroplastic HSPs, HSP60 and HSP24, the latter of which localizes in the thylakoid membranes. Both HSPs are protecting but not repairing PSII from the stress. Another chloroplast HSP of 70 kDa repairs PSII during and after photoinhibition (Preczewski et al., 2000). Such chloroplastic HSPs may be protecting and repairing PSII better during heat stress in P. chilensis than in soybean.
The Fm instead, decreased less in soybean compared with P. chilensis suggesting that the electron flux from the QA to the electron transfer chain is slightly more affected in P. chilensis than in soybean under heat stress. As a consequence, the fluorescence results did not show significant differences in Fv between both plants, because the increase in F0 in soybean was compensated with a faster decrease in Fm caused by temperature in P. chilensis. Therefore, the changes in Fv/Fm with temperature were similar in both species. As in the case of F0, the difference in Fm between species was not detected when plants were returned for 3 h in darkness at 25 °C, after a heat shock of 2 h at 35 °C. The reversion at normal values of F0 and Fm in this period of time is in agreement with the transient response of HSP accumulation and gene expression for these proteins. They are back to normal levels after 3 h of heat shock, and when temperature is back to normal temperature (Cooper et al., 1984; Marimoto, 1993).
Stress situations affecting PSII function reduce Fv/Fm, the maximum quantum yield of photosystem II (Klinkovsky and Naus, 1994; Krause and Weiss, 1991). In both species, the temperatures affecting photochemical efficiency are rather high, over 35 °C, as compared to Atriplex sp. (Berry and Björkman, 1980) and pea (Havaux, 1992; Georgieva and Yordanov, 1993).
The protective roles of ubiquitin and HSP70
Ubiquitin is a 76-amino-acid-HSP (Bond and Schlesinger, 1985). It is constitutively expressed in eukaryotic organisms and involved in proteolysis (Ferguson et al., 1990; Jentsch, 1992; Belknap and Garbarino, 1996). The results of this study suggest that the ubiquitinated-proteolytic pathway may be active in both species during heat stress. However, the higher increase in relative amounts of free and conjugated ubiquitin of P. chilensis as compared with soybean after 2 h of heat shock at 40 °C and 45 °C, may indicate that the ubiquitin-mediated proteolysis might be more active in P. chilensis than in soybean. Since seedlings of P. chilensis grew faster at 30, 35 and 40 °C than the control seedlings, the increase in free and conjugated ubiquitin in P. chilensis may not be necessarily related to general protein degradation that occurs as a consequence of heat stress (Finley et al., 1987; Gropper et al., 1991; Ferguson et al., 1994), but rather to its natural heat tolerance.
It may well be that ubiquitin-mediated proteolysis in P. chilensis and soybean eliminates proteins which misfunction under heat stress because elevated temperatures may inactivate enzymes, change peptide conformation, disrupt membrane complexes, and/or accumulate unprocessed peptides (Ferguson et al., 1990). In this way, ubiquitin-mediated proteolysis may protect organisms from heat stress by removing improperly functioning proteins (Finley and Chau, 1991; Boavida-Ferreira et al., 1995). However, from these data it is not possible to tell if there is proteolysis at 40 °C in soybean and in P. chilensis seedlings. Future determinations of protein breakdown, as has been done in wheat roots (Ferguson et al., 1990), will determine if there is ubiquitin-mediated proteolysis in these species.
In most eukaryotic organisms, synthesis of heat shock proteins is induced within 10–20 min following acute temperature increases (Ougham and Howarth, 1988). The results show that this is the case for free and conjugated ubiquitin and HSP70. All of them begin to increase after 20 min of heat shock at 40 °C. The results on free ubiquitin also show that the levels of free ubiquitin decrease immediately after heat shock starts, probably because the cellular pool of ubiquitin molecules immediately begins to conjugate with the heat-damaged proteins to initiate proteolysis (Ferguson et al., 1990; Belknap and Garbarino, 1996). This is coincident with the time-course of accumulation shown by conjugated ubiquitin, which begins to increase soon after heat shock starts. The kinetic results also suggest that there is de novo synthesis of ubiquitin molecules under high temperature stress in both species, because free ubiquitin levels begin to increase after 20 min of heat stress together with high levels of conjugated ubiquitin. This speculation needs to be confirmed by Northern blot analyses using an appropriate probe or by radiolabelling of the newly synthesized protein identified with the ubiquitin antibody.
In P. chilensis and soybean, a repairing mechanism for heat-shock-denatured proteins seems to be added to the ubiquitin-mediated proteolysis, as suggested by the accumulation of the chaperone HSP70. The accumulation of HSP70 was chosen from among the other HSPs, because it is the major protein induced by heat and has been linked to the acquired thermotolerance (Guy and Li, 1998). The physiological role of this protein is well documented during environmental stress (Flynn et al., 1989; Boston et al., 1996; Schöffel et al., 1998) and several functional forms of it are localized in all the major subcellular compartments (Guy and Li, 1998). Since the relative levels of HSP70 increased 100% and 70% in P. chilensis at 40 °C and 45 °C, respectively, HSP70 might be involved in the renaturing of proteins at these temperatures (Yalovsky et al., 1992; Georgopoulos and Welch, 1993). These data demonstrate that there is a correlation between increased amounts of HSP70 and thermotolerance in P. chilensis.
The Western blot results of HSP70 in P. chilensis and soybean seedlings showed that there is a basal level of expression of HSP70 protein at 25 °C. Two forms of HSP70 proteins in P. chilensis have been reported (Medina and Cardemil, 1993), one inducible of 71 kDa and another constitutive of 69 kDa (Ortiz et al., 1995). In soybean the same antibody also detects two bands, one of 70 kDa which is slightly detected at 25 °C and another weaker inducible band of 73 kDa.
It has been suggested that HSP70 may work as a signal transducer for the synthesis of other HSPs (Sorger, 1991). If HSP70 is considered a sensor protein for stress and cell damage, the results of this work would imply that P. chilensis might sense better the heat stress than soybean seedlings do. Furthermore, these kinetics studies show that the levels of HSP70 accumulation is higher in P. chilensis than in soybean at 20 min of treatment at 40 °C as well as after 60 and 90 min of heat shock, indicating that there is also a better protection and for a longer time against heat shock stress by the HSP70 in P. chilensis than in soybean.
However, it cannot be concluded that the cause of the natural thermotolerance in P. chilensis is due only to higher levels of HSP70. At 25 °C the basal levels of free and conjugated ubiquitin were also higher in P. chilensis than in soybean. Therefore, the natural thermotolerance of P. chilensis might be related to the basal levels of all these proteins (Feder and Hofmann, 1999).
From the early seedling growth, LT50 results, and initial fluorescence emission of photosystem II, it is possible to conclude that P. chilensis is a more tolerant plant to acute heat stress than soybean. The quantum yield of PSII was, however, similar in soybean and P. chilensis under heat stress. P. chilensis showed a higher level of accumulation for free ubiquitin, conjugated ubiquitin and HSP70 than soybean during 2 h of treatment at 40 °C and 45 °C. These results suggest that after 2 h of heat stress the metabolism of P. chilensis is better protected by both systems: ubiquitin-mediated proteolysis and HSP70 chaperone mechanisms, as has been reported for other organisms such as the intertidal mussel Mytilus trossulus (Hofmann and Somero, 1995) and the hybrid poplar (Populus nigra (L.) CharkowiensisxP. nigra (L. incrassata) (Wisniewski et al., 1997). The kinetics of accumulation also proved to be slightly faster in P. chilensis than in soybean at 40 °C.
Both systems, ubiquitin-mediated proteolysis and HSP70 chaperone mechanism, could be partly responsible for the higher level of basal thermotolerance of P. chilensis as compared with soybean. This has been suggested by genetic engineering experiments in Arabidopsis where dominant regulatory mutations were generated to show a constitutive synthesis of HSPs. The higher level of HSPs accumulation of these transgenic plants is correlated with a higher level of basal thermotolerance (Lee et al., 1995; Prändl et al., 1998).
Future molecular work with appropriate probes to study the expression of these HSPs or the identification of new genes expressed under heat shock could give an answer about the difference found in thermotolerance acquisition between these two species of leguminous plants.
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Acknowledgments |
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The antibodies against free and conjugated ubiquitin were a kind gift from Professor Aaron Ciechanover, Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. We thank Dr Luis Corcuera, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Concepción, Chile, for critical reading of this manuscript. The assistance of Angélica Vega is acknowledged. The research was supported by grants FONDECYT No. 2950043 given to Claudia Ortiz and FONDECYT No. 1950401 given to Liliana Cardemil and Dirección de Investigación y Desarrollo, Universidad de Chile, Project No. EDID 98-006.
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Notes |
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1 To whom correspondence should be addressed. Fax: +56 2 271 7580. E-mail: lcardemi{at}abello.dic.uchile.cl
2 Present address: Facultad de Ciencias Naturales, Universidad de Atacama, Copiapó, Chile.
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