JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 399, pp. 1125-1134, May 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Involvement of polyamines in the interacting effects of low temperature and mineral supply on Pringlea antiscorbutica (Kerguelen cabbage) seedlings
Received 13 November 2003; Accepted 10 February 2004
Centre National de la Recherche Scientifique, Université de Rennes 1, UMR 6553 ECOBIO, Campus de Beaulieu, bâtiment 14A, 263 avenue du Général Leclerc, F-35042 Rennes Cedex, France
* To whom correspondence should be addressed. Fax: +33 2 23 23 50 26. E-mail: Ivan.Couee{at}univ-rennes1.fr
Abbreviations: Agm, agmatine; Put, putrescine; RGR, root growth rate; Spd, spermidine; Spm, spermine.
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Abstract |
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Pringlea antiscorbutica, which is the sole endemic crucifer in the subantarctic zone, undergoes seedling development in a harsh and cold environment. Since, at the mature stage, this species exhibits several adaptations linked to cold tolerance such as high polyamine levels, potential adaptations and polyamine response were investigated in seedlings. In order to assess the specificity of responses, P. antiscorbutica was compared with Arabidopsis thaliana, which is characterized by a life cycle preventing cold exposure at seedling stage. P. antiscorbutica and A. thaliana seedlings were found to have strikingly contrasted responses to temperature changes and to mineral nutrition. Whereas A. thaliana seedlings showed the typical growth arrest of chilling-sensitive plants, P. antiscorbutica seedlings showed optimal root growth at low temperature (5/10 °C) and temperate conditions caused the early arrest of root growth. Cold tolerance was associated with increased levels of polyamines or with maintenance of high levels of polyamines. Comparison of both species showed that polyamine levels could be a significant marker of chilling tolerance in seedlings. Treatments with varying mineral supply showed a positive relationship between root growth rate and variations of agmatine and putrescine endogenous contents in roots of P. antiscorbutica. This may be the first demonstration that, even under conditions of accumulation induced by environmental stress, polyamine levels can still be correlated with developmental processes. Com parison of mineral supply and temperature effects strongly indicated a trade-off of polyamine involvement between development and response to stress.
Key words: Agmatine, Arabidopsis thaliana, chilling tolerance, Kerguelen cabbage, Pringlea antiscorbutica, seedling development.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Low, but above-freezing, temperature damages many species of plants, especially those of tropical origin, by causing chilling injuries and thus restricting their growth and development (Lyons, 1973). By contrast, in cold-tolerant plants, responses to low temperature involve regulatory and molecular mechanisms that are triggered to optimize growth under suboptimal temperature (Stitt and Hurry, 2002). Polyamines (Spd and Spm) and their precursors (Agm and Put) are aliphatic amines, which, in higher plants, are associated with developmental processes (Bagni and Torrigiani, 1992; Evans and Malmberg, 1989; Couée et al., 2004) and responses to stress (Bouchereau et al., 1999). Since polyamines can act as free radical scavengers, therefore protecting cellular membranes from oxidation (Besford et al., 1993), their role in cold tolerance has been studied in several plants (Tajima and Kabaki, 1981; Kramer and Yang, 1989; Lee et al., 1995). Growth of excised rice roots at 5 °C was promoted by the addition of Put in culture media (Lee, 1997), and Spd treatment in cucumber improved chilling tolerance of the photosynthetic apparatus (He et al., 2002a). Correlatively, cold-tolerant plants appear to increase endogenous polyamine levels in response to chilling to a much greater extent than sensitive ones (Guye et al., 1986; Lee et al., 1997). Shen et al. (2000) have shown that, during chilling, Spd content in leaves markedly increases in cold-tolerant cucumber cultivars, but not in sensitive cultivars. Cold-tolerance of rice cultivars has also been correlated to the extent of Put accumulation in shoots in response to low temperature (Lee et al., 1995). Besides, He et al. (2002b) have shown in spinach, a cold-tolerant plant, that inhibition of Spd synthesis increased photoinhibition. Pringlea antiscorbutica R. Br (Kerguelen cabbage), the sole endemic cruciferous species of subantarctic regions, is a useful model of tolerance to low temperature. This species withstands the cold climate of the Kerguelen Archipelago, which shows an annual mean temperature of +4 °C (Hennion and Martin-Tanguy, 2000). At the mature stage, P. antiscorbutica accumulates massive amounts of Agm in all organs, and exhibits a flexible polyamine metabolism, which is responsive to low temperature (Hennion and Martin-Tanguy, 2000). Whereas most plants cannot survive cold exposure at the seedling stage (Battey, 2000), early development of P. antiscorbutica occurs during the austral summer, which is characterized by a low and fluctuating temperature regime. By contrast, Arabidopsis thaliana, a temperate cruciferous species, avoids cold exposure at the seedling stage through a life cycle that allows overwintering as a seed or a rosette (Simpson and Dean, 2002). In order to study polyamine involvement in early development at low temperature, growth and development patterns and polyamine levels were compared between these cruciferous species, which naturally grow either in a temperate climate or in the subantarctic climate. Combinations of mineral supply and temperature treatments showed positive relationships between root growth rate and Agm and Put endogenous contents in roots of P. antiscorbutica. As far the authors know, this is the first demonstration that, even under conditions of large accumulation induced by environmental stress, polyamine levels can still be correlated with developmental processes. These results are discussed in terms of developmental strategy in response to mineral supply and in relation to temperature tolerance.
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Materials and methods |
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Plant material
Seeds were collected from a population of Pringlea antiscorbutica R. Br. (Brassicaceae) in Australia Island within the Kerguelen Archipelago (68–70° E, 48–50° S) during the austral summer in February 2001. After collection, seeds were kept dry at 4 °C in the dark until use. Prior to germination, seeds were surface-sterilized for 1 min in 95% ethanol, then for 2 h in calcium hypochlorite, and rinsed in distilled water. Arabidopsis thaliana L. seeds (ecotype Wassilewskija, Ws) were surface-sterilized for 5–10 min in 50% bayrochlore/50% ethanol, rinsed twice in absolute ethanol, and dried overnight.
Surface-sterilized seeds were plated on square (14x14 cm) Petri dishes under axenic conditions on agar or nutrient-agar medium. Agar growth medium consisted of 0.8% (w/v) agar. Nutrient-agar medium consisted of agar with 1x Murashige and Skoog (MS) basal salt mix (M 5519, Sigma, St Louis, USA), pH 5.7, or with 0.01x or 0.02x dilutions of MS as indicated in the text. Petri dishes were sealed with one layer of Parafilm, which minimizes evaporation, but still permits gas exchange, and stored vertically in the growth chamber. P. antiscorbutica seeds were germinated, up to radicle emergence, in a growth chamber at 22 °C for 4 d (Hummel et al., 2002). A. thaliana seeds were placed in the dark, at 4 °C for 48 h, in order to break dormancy and homogenize germination, and then transferred to 22 °C for 48 h to stimulate germination.
Seedlings were then transferred to two growth chambers, with the same light irradiance (1.3 MJ m–2 d–1), either at low temperature (5 °C/10 °C night/day, 14 h light period, referred to in the text as 5 °C) or under temperate conditions (22 °C/25 °C night/day, 14 h light period, referred to in the text as 22 °C). The low temperature regime was close to the subantarctic summer conditions (Hennion and Martin-Tanguy, 2000). In both growth chambers, Petri dishes were placed vertically, following the same layout and using the same racks and the same density of plates. The constancy of temperature parameters in the growth chambers was checked daily. Seedlings from the low-temperature growth chamber were kept at 5 °C during growth measurements or sample preparation.
Measurement of growth and development parameters
Germination was studied at 22 °C. Germinated seeds were characterized by radicle protrusion. Seedling growth was studied after transfer to controlled growth chambers. Primary root length was measured every 3 d. Measurements were performed until day 50. Growth rate after greening of cotyledons was calculated in the linear part of the curve (between day 3 and day 15) by linear regression. Morphological parameters were calculated as mean (±SEM) of the measurements from 30 seedlings. Each experiment was repeated at least three times.
HPLC analysis of polyamines
At the end of 15 d growth, seedlings were frozen in liquid nitrogen and then lyophilized. Shoots and roots were separated after lyophilization. Polyamine analyses were performed on pools of tissues or of whole seedlings, corresponding to at least five individual plants. Amine levels were calculated as mean (±SEM) from measurements on three pools. In order to extract free amines, aerial parts, roots or whole seedlings were ground in 0.5 ml of 1 M HCl (Hummel et al., 2002). After extraction for 1 h, samples were centrifuged at 18 000 g for 30 min, and the supernatant, containing free amines, was stored frozen at –20 °C until further use. As previously published (Hennion and Martin-Tanguy, 2000; Hummel et al., 2002), the amine fraction was analysed by HPLC using an LKB 2152 plus an LKB 2150 chromatography system with a HPLC column packed with reverse phase spherisorb ODS-2 (particle size 5 µm; 4.6x250 mm; Waters, Milford, USA). Samples (20 µl) of the amine fractions were applied to the column and eluted with a programmed methanol:water solvent gradient, changing from 60% to 95% over 23 min at a flow rate of 0.8 ml min–1. Elution was completed after 7 min. For detection of dansyl amines, an excitation wavelength of 365 nm was used with an emission wavelength of 510 nm. Results were standardized with equimolar (0.1 nmol) mixtures of dansylated amines. All amine standards were purchased from Sigma (St Louis, USA).
Statistical analysis
Statistical analyses were performed with Minitab release 13.31 statistical software (Minitab Inc., PA, USA). Significant differences among treatments were tested by analysis of variance. Least significant differences (LSD) were calculated at the P <0.05 probability level.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Effects of mineral supply and low temperature on growth and development of P. antiscorbutica seedlings
Germination of P. antiscorbutica was completely achieved 6 d after imbibition. Compared with the MS-free medium, MS salts delayed the beginning of germination without modifying germination rate, nor the final percentage of germination (data not shown). Germinated seeds were then transferred to growth chambers at 5 °C or 22 °C. Figure 1A shows primary root growth under different growth conditions. Greening of the cotyledons and hypocotyl, which began at day 3 and went on until day 6, was not modified by temperature or nutrient levels (data not shown). Whereas higher temperature stimulated root growth rate (RGR) in the linear part of root growth, primary root growth declined after day 15 on MS medium and had completely stopped by day 21 of cultivation (Fig. 1A). At the end of the experiment, roots were about twice as long on MS medium at 5 °C than roots cultivated on MS medium at 22 °C. The same trends were found for growth on MS-free medium at 5 °C and 22 °C. Root growth was significantly slowed down on MS medium during the first 15 d, with about 40% reduction of RGR compared with MS-free medium (Fig. 1A). At 22 °C, root growth arrest occurred on day 28 on MS-free medium, whereas growth stopped 1 week earlier on MS medium. At 5 °C on MS-free medium, root growth was not slowed down over a period of at least 50 d. At this stage, all seedlings were alive and roots did not show any necrosis (data not shown).
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Figure 1B shows shoot phenotype and fresh weight (FW) obtained at day 15 under different growth conditions. Shoot growth was stimulated by temperate conditions on agar and MS medium, and by MS medium when growth occurred at 22 °C (Fig. 1B). This could be related to first leaf emergence on day 9 on MS medium at 22 °C while leaves only emerged at day 15 under other growth conditions. At the end of the experiment, day 48, the differences between shoots were more important (data not shown), with better development of shoots, considering the number of leaves, on MS than on agar medium whatever temperature levels being considered, thus indicating that nutrient supply favoured long-term growth and development of aerial organs.
Developmental plasticity can modify the differential growth of roots and shoots of seedlings in order to fulfil the requirements for nutrients, water, and light (Trewavas, 1986; Bell and Sultan, 1999; Malamy and Ryan, 2001). One of the most commonly observed responses to mineral supply limitation is a shift in the allocation of resources from shoot to root growth (Ericsson, 1995), thus enabling the plant to concentrate its available resources on exploring the soil volume (Forde, 2002). At both temperature levels, P. antiscorbutica exhibited this typical growth response to mineral supply deprivation as shown by the significant enhancement of primary root length on agar medium, compared with the MS salts medium (Fig. 1). Moreover, at 22 °C, root growth of P. antiscorbutica seedlings was slowed down to stop concomitantly with first leaf emergence (Fig. 1), thus indicating that the presence of MS salts resulted in an allocation of resources towards shoot growth.
Whatever the growth medium considered, P. antiscorbutica seedlings exhibited tolerance to low temperature (Fig. 1). Even though low temperature reduced RGR, the seedling roots grew over a longer period of time. The final length of the primary root was thus enhanced at 5 °C, whereas temperate conditions induced growth arrest. Similarly, in winter wheat, low soil temperature reduces overall growth, but tends to increase allocation to root growth in order to counterbalance the reduction of nutrient and water uptake (Gavito et al., 2001). The shoot development of P. antiscorbutica seedlings was slow, and less responsive to temperature than the roots (Fig. 1B). P. antiscorbutica at the seedling stage therefore appeared to be cold-tolerant and sensitive to temperate conditions, through the effects on root development. Much greater sensitivity to 22 °C has been reported for P. antiscorbutica seedlings grown on moist filter paper, with stunted morphology and very slow growth rate of seedlings (Dufeu et al., 2003). This may be due to the combined effects of temperature and water availability (Dorne and Bligny, 1993). The present work thus shows that, even under optimal conditions of humidity, temperate conditions can cause root growth arrest in P. antiscorbutica.
Effects of mineral supply and low temperature on growth and development of A. thaliana seedlings
A comparative study of seedling development was carried out on A. thaliana. Germination occurred within 48 h at 22 °C without any difference between MS-agar and MS-free agar medium (data not shown) and seedlings were then transferred to controlled growth chambers at 5 °C or 22 °C. By contrast with P. antiscorbutica, low temperature modified root growth more drastically than nutrient levels (Fig. 2A). Thus, at 22 °C, MS or MS-free medium did not significantly modify growth of the primary root, which followed the same pattern that was characterized by high RGR followed by a slight decrease (Fig. 2A). By contrast, low temperature reduced RGR compared with growth at 22 °C, in the absence or presence of MS salts in the growth medium (Fig. 2A). However, at 5 °C, MS medium slightly stimulated RGR during the first 15 d of growth and thus led to significantly longer primary roots than those observed on agar medium. Growth at low temperature on MS-free medium resulted in the complete arrest of root growth at day 15 (Fig. 2A), which coincided with the arrest of shoot development. Temperature and nutrient levels led to different numbers of leaves and drastically modified shoot growth and development at day 15 (Fig. 2B). Temperate conditions in the presence of MS led to the most developed aerial parts, whereas MS-free medium and low temperature drastically affected shoots with significantly decreased FW, chlorotic cotyledons, and inhibition of leaf emergence (Fig. 2B). The developmental patterns of A. thaliana seedlings were, therefore, mostly responsive to temperature regime (Fig. 2). Strand et al. (1999) have found that, at the mature stage, A. thaliana leaves could be cold-tolerant. Thus the present work showed that, by contrast, A. thaliana seedlings exhibited a phenotype of chilling-sensitive plants with a reduction in overall growth, and the development of low temperature-induced injury (Allen and Ort, 2001).
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Under temperate conditions, the presence of MS salts in the growth medium favoured the development of A. thaliana shoots, thus leading to fully-developed rosettes without any modification of root growth (Fig. 2). A. thaliana seedlings thus exhibited a particular response to mineral supply limitation, which resulted in slowing shoot development, but maintaining root growth. Therefore, commitment of A. thaliana seedling development seemed to require nutritional supply in the growth medium. Germination and seedling growth are probably the most vulnerable stages of plant development as they depend on limited stored reserves (Smith, 2000). A. thaliana seedlings from large seeds survive longer in nutrient deprivation than those from small ones (Krannitz et al., 1991), which has been ascribed to the enrichment of the protein reserves in larger seeds (Andalo et al., 1998). P. antiscorbutica seeds are obviously bigger than A. thaliana seeds. The average volume of A. thaliana seeds is about 0.033 mm3 (Andalo et al., 1998), whereas the pear-shaped seeds of P. antiscorbutica show a mean length of 3.32 mm and a mean width of 2.4 mm (Hennion and Walton, 1997). Since a significant enhancement of root length was found in A. thaliana seedlings emerging from large seeds (Andalo et al., 1998), differences in seed volume might account for the differential responses to nutrient deprivation (Figs 1, 2). Thus, P. antiscorbutica exhibited a higher RGR than A. thaliana under all growth conditions. However, the presence of MS salts only slightly stimulated RGR of A. thaliana seedlings. Nutrient requirement should, therefore, be associated with overall seedling development rather than only root growth. Early development is a two-stage process, beginning with heterotrophic growth, which is only supported by seed reserve mobilization, followed by autotrophic development (Zonia et al., 1995). The constancy of germination rate showed that A. thaliana seeds were rich enough to sustain heterotrophic development without nutrient supply. However, subsequent alterations of A. thaliana phenotypes indicated an irreversible reduction of shoot development in response to nutrient deficiency. Considering the difference in seed volume, the full mobilization of stored reserves might occur sooner in A. thaliana than in P. antiscorbutica. In this regard, the constancy of root growth under deficient nutrient supply might be seen as a foraging strategy of A. thaliana to enhance nutrient supply to the shoots.
Effects of mineral supply on the polyamine response to low temperature in A. thaliana and P. antiscorbutica seedlings
Polyamine levels, whose increase is one of the typical responses of plants resistant to chilling (Tajima and Kabaki, 1981; Kramer and Yang, 1989; Lee et al., 1995; Shen et al., 2000; He et al., 2002a, b), were analysed to assess physiological tolerance to low temperature. Figure 3 shows free polyamine contents in 15-d-old seedlings of P. antiscorbutica (Fig. 3A, B) and A. thaliana (Fig. 3C, D) prior to chilling injury. Polyamine levels in P. antiscorbutica seedlings were generally either decreased or not significantly modified at 5 °C compared with 22 °C in the presence or absence of MS salts (Fig. 3A, B). When considering MS salt treatment, the Agm content decreased in whole seedlings of P. antiscorbutica at 5 °C compared with 22 °C (Fig. 3A). Thus, strikingly, although P. antiscorbutica seedlings were not sensitive to low temperature (Fig. 1), their polyamine contents did not increase in response to cold, except in the case of Spm in seedlings grown on agar medium. In cucumber, adding Spd to the growth medium, prior to cold exposure, significantly enhanced Spd content in all organs and resulted in higher cold tolerance, even if their polyamine pools significantly decreased during chilling treatment (He et al., 2002a). Therefore, chilling damage might be prevented, not only by polyamine accumulation in response to cold, but also when polyamine levels are high prior to chilling exposure and remain high during chilling exposure. Given that chilling tolerance has been associated with an increase of polyamines because of their potential protective effects on membrane and cellular function (Shen et al., 2000), high polyamine contents, even if not enhanced in response to cold, might perform similar protection. By contrast, A. thaliana seedlings showed significantly lower levels of Agm and Put, which, in the case of Put, decreased in response to low temperature in the absence of MS salts (Fig. 3C, D). Polyamine and phenotypic responses, in A. thaliana seedlings, were in accordance with previous studies associating the absence of accumulation or polyamine depletion with cold sensitivity (Tajima and Kabaki, 1981; Kramer and Yang, 1989; Shen et al., 2000). High contents of Agm and Put in whole seedlings were therefore associated with the cold tolerance of P. antiscorbutica, while their low levels were associated with the cold sensitivity of A. thaliana.
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Growth on MS medium induced a decrease of Put levels compared with agar medium in both P. antiscorbutica and A. thaliana seedlings. Considering absolute pools rather than their relative variations, P. antiscorbutica was still characterized by much higher pools of Agm and Put than A. thaliana under all growth conditions. Even if, on MS medium at low temperature, Put levels decreased down to 2 µmol g–1 DW in P. antiscorbutica, this remained higher than the highest Put levels in A. thaliana, which hardly reached 1.2 µmol. g–1 DW. By contrast, Spd and Spm levels were in a similar range in both species (Fig. 3B, D). The enhancement of the Put levels in both species in response to growth on nutrient-free medium (Fig. 3A, C) was in accordance with Put accumulation observed in response to mineral nutrient deficiency in several plant species (Bouchereau et al., 1999).
Adverse growth conditions, i.e. low temperature or MS-free medium, resulted in reduced development of both shoots and roots of A. thaliana seedlings (Fig. 2). By contrast, roots and shoots in P. antiscorbutica seedlings differentially responded to temperature and nutrient levels (Fig. 1). In order to study their involvement in shoot and root development, free polyamines were separately analysed in roots (Fig. 4A, B) and in shoots (Fig. 4C, D) of 15-d-old P. antiscorbutica seedlings. Polyamine contents in shoots (Fig. 4C, D) were close to those found in whole seedlings (Fig. 3A, B), which could be ascribed to the important contribution of shoot fresh weight in whole seedlings. Figure 4C, D indicated the same trends as in Fig. 3A, B with a reduction of Put level in response to nutrient supply and low temperature. By contrast, whereas Spd levels were similar between shoots and roots of P. antiscorbutica seedlings, Agm and Put levels were almost 10 times lower in roots than in shoots. Moreover, in roots, variations of polyamine contents were highly responsive to growth conditions (Fig. 4A, B). Thus, in the absence of MS salts, low temperature induced the accumulation of all free polyamines in roots (Fig. 4A, B). In the presence of MS salts, low temperature induced the accumulation of Put, Spd, and Spm, but not of Agm (Fig. 4A, B). At 22 °C, the levels of Agm and Put, but not those of Spd and Spm, were decreased in roots by the presence of MS salts (Fig. 4A, B). Therefore, in roots of P. antiscorbutica seedlings, which were highly responsive to temperature (Fig. 1A), and presented a lower basal level of polyamines, the accumulation of polyamines was induced by low temperature (Fig. 4), as occurs in cold-tolerant species with low levels of polyamines in response to chilling (Lee et al., 1995; Shen et al., 2000; He et al., 2002a).
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Interactions between mineral supply and endogenous polyamine levels in P. antiscorbutica
Mineral supply and temperature levels modified the developmental pattern of P. antiscorbutica seedlings (Fig. 1) as well as the polyamine contents in roots (Fig. 4). In order to test the hypothesis that variations of root development can be mediated by changes of polyamine pools, RGR was plotted against the endogenous polyamine contents. Figure 5 shows data obtained from seedlings grown on MS medium, MS-free medium, and 0.01x and 0.02x dilutions of MS medium at 22 °C or 5 °C. Nutrient-driven changes of Agm and Put levels in roots were positively correlated with RGR variation, whatever the temperature considered (Fig. 5A, B). These relationships clearly indicated that Agm accumulation, which is observed in roots grown on agar compared with MS medium at both temperatures (Fig. 4A), is associated with RGR enhancement (Fig. 1A). At 5 °C, high endogenous levels of Agm and Put, which were observed in response to the variations of nutrient levels in the growth media, were also associated with RGR enhancement (Fig. 5A, B). Thus, growth under different nutritional conditions induced variations of endogenous levels of Agm or Put and concomitant changes in RGR, which were correlated in a temperature-dependent manner. By contrast, no correlation was found between nutrient-driven variations of Spd or Spm and RGR (Fig. 5C, D). Spm and Spd contents in roots were similar in agar and MS medium at both temperatures (Fig. 4B), whereas RGR was deeply modified by nutritional conditions (Fig. 1A).
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Polyamine involvement in the regulation of root growth has been previously demonstrated in P. antiscorbutica seedlings (Hummel et al., 2002). In the present work, under constant temperature, a strong positive correlation was found between variations of Agm amount and RGR. Inhibition of arginine decarboxylase activity, which catalyses Agm synthesis, has been associated with a decrease of root growth in several species (Martin-Tanguy and Carré, 1993; Biondi et al., 1993; Watson et al., 1998). When inhibitors of polyamine biosynthesis are used to modulate Agm levels on MS medium at 5 °C, the Agm level is associated with an enhancement of RGR (Hummel et al., 2002). In the present work, Put accumulation, but not that of Spd or Spm, was correlated with RGR enhancement (Fig. 5). By contrast, inhibition studies have shown that Spd and Spm, but not Put, levels were positively associated with RGR (Hummel et al., 2002). Such discrepancies may be ascribed to the wide variation of Put, Spd, and Spm levels in response to the inhibition-induced modulation of biosynthesis, associated with deeply contrasted RGR responses (Hummel et al., 2002). By contrast, the physiological variations of endogenous polyamine pools induced by environmental cues were less severe than those obtained through inhibitor-induced modulations of polyamine metabolism.
Mineral supply deprivation induced the enhancement of P. antiscorbutica RGR and resulted in the accumulation of Agm and Put amounts in roots after 15 d of cultivation (Fig. 5A, B). Therefore, the accumulation of polyamines in roots in response to nutrient starvation might be directly involved in root growth, thus facilitating nutrient foraging. However, whereas polyamine levels were higher at 5 °C than at 22 °C at day 15 (Fig. 5A, B), no acceleration of RGR was observed. These high levels of polyamine in roots at day 15 might also be associated with longer period of root growth, since the cessation of root growth was observed at 22 °C, but not at 5 °C (Fig. 1A). The relationships between the Agm and Put levels, measured at day 15, and RGR were positive in response to nutrient levels, but only at constant temperature (Fig. 5A, B). Thus, cold exposure induced the accumulation of Agm and Put in roots on day 15 (Fig. 4A, B), but this accumulation was not associated with RGR enhancement compared with 22 °C (Fig. 1A). Moreover whereas, at 22 °C, variations of RGR and Agm, or Put, contents were consistent with the extent of mineral supply, this relationship was less coherent at 5 °C. On one hand, this showed that, even under conditions of polyamine accumulation at low temperature, the polyamine response to mineral supply limitation remained functional and was at least partially invested in root growth. However, it also suggested the differential use of polyamine accumulation. Thus, mineral-deficiency-dependent accumulation of polyamines appeared to be directed towards the enhancement of root growth, hence the positive correlation between polyamine level and RGR (Fig. 5). By contrast, cold-dependent accumulation of polyamines did not result in enhanced development, and was likely to be directed towards protection against chilling injury.
In conclusion, physiological modifications of endogenous Agm and Put levels by mineral supply were shown to correlate positively with enhanced root growth in P. antiscorbutica seedlings. Thus, the regulation of Agm and Put levels may be directly involved in the response to mineral supply limitation and in improved foraging by enhanced root growth. However, comparison of these relationships at different temperatures strongly suggested that polyamine accumulation in the roots in response to low temperature was not directly invested in root growth, and that polyamines may participate in a trade-off between root growth and a response to stress. The mechanisms underlying this differential investment of polyamines remain to be elucidated.
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Acknowledgements |
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Seeds of Pringlea antiscorbutica were obtained from field programme No. 340 of the Institut Polaire Français Paul-Emile Victor (IPEV, CNRS, Plouzané, France). This work was supported in part by a fellowship (to IH) from the Ministère de l’Education et de la Recherche (France).
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