Involvement of polyamines, abscisic acid and anti-oxidative enzymes in adaptation of Blue Panicgrass (Panicum antidotale Retz.) to saline environments
A hydroponic experiment was conducted to assess the possible involvement of polyamines (PAs), abscisic acid (ABA) and anti-oxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in adaptation of six populations of Panicum antidotale Retz. to selection pressure (soil salinity) of a wide range of habitats. Plants of six populations were collected from six different habitats with ECe ranging from 3.39 to 19.23 dS m−1and pH from 7.65 to 5.86. Young tillers from 6-month-old plants were transplanted in plastic containers each containing 10 l of half strength Hoagland's nutrient solution alone or with 150 mol m−3 NaCl. After 42 days growth, contents of polyamines (Put, Spd and Spm) and ABA, and the activities of anti-oxidative enzymes (SOD, POD and CAT) of all populations generally increased under salt stress. The populations collected from highly saline habitats showed a greater accumulation of polyamines and ABA and the activities of anti-oxidative enzymes as compared to those from mild or non-saline habitats. Moreover, Spm/Spd and Put/(Spd + Spm) ratios generally increased under salt stress. However, the populations from highly saline environments had significantly higher Spm/Spd and Put/(Spd + Spm) ratios as compared to those from mild or non-saline environments. Similarly, the populations adapted to high salinity accumulated less Na+ and Cl− in culm and leaves, and showed less decrease in leaf K+ and Ca2+ under salinity stress. Higher activities of anti-oxidative enzymes and accumulation of polyamines and ABA, and increased Spm/Spd and Put/(Spm + Spd) ratios were found to be highly correlated with the degree of adaptability of Panicum to saline environment.
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Keywords: Bluegrass; Adoptability; Evolution; Soil salinity; Selection pressure
2. Materials and methods
3.1. Growth attributes
3.2. Activities of antioxidant enzymes
3.3. Polyamines and ABA
3.4. Ion accumulation
Soil salinity is one of the major limiting factors for crop and forage production in the arid and semi-arid regions of the world including Pakistan. Although, the recent technological advancement has brought about novel and effective techniques to overcome this problem, their high cost makes them non-practicable for poor farmers particularly living in areas inflicted with severe abiotic stresses. In this situation, exploration and cultivation of salinity tolerant species offer a practical solution for effective utilization of stress-affected soils ([Wu, 1981], [Amtmann et al., 2005], [Ashraf and Harris, 2005] and [Liang et al., 2008]). For example, the species that are found exclusively on saline habitats possess genetic variation necessary to allow the evolution of salt-tolerant populations in response to the selection pressure of the habitats (Ashraf et al., 1989). Undoubtedly, the evolution of any particular character depends on two major determinants running parallel for a sufficient length of time; i.e., the existence of appropriate genetic variation, and the occurrence of appropriate natural selection pressure ([Noble et al., 1984], [Ashraf et al., 1986], [Ashraf et al., 2005], [Ashraf and McNeilly, 1990], [Al-Khatib et al., 1992] and [Ashraf, 2005]). Although during the recent years, a number of advanced techniques including genetic profiling and molecular enhancements have been tried for enhanced stress tolerance, genetically based variation found in natural populations of plants has been rarely attempted, although the potential value of such genetic resources has recently been emphasized in modern research programs ([Rogers and Noble, 1992], [Munns, 2002], [Ashraf, 2004] and[Hameed and Ashraf, 2008]).
Bluegrass offers an excellent source of studying adaptability in response to selection pressure as it can withstand multiple stresses including salinity, drought, fire, etc. Its colonization on highly saline ([Rasul et al., 1994], [Tomar et al., 2003] and [Ashraf et al., 2006]), alkaline (Ryan et al., 1975), drought-hit ([Cope, 1982], [Bokhari et al., 1987] and [Chaudhary, 1989]), and waterlogged (Ashraf, 2004) soils shows its adaptation to a wide range of environmental conditions. However, study of the genetic basis of adaptability is rather difficult because stress tolerance has been shown to be a complex multigenic trait ([Ashraf, 1994], [Niknam and Mccomb, 2000] and [Ashraf and Harris, 2004]) involving multiple mechanisms which makes it more difficult to study the inheritance of stress tolerance. In this situation, physiological and biochemical changes under stressful conditions might offer any easy approach to study inheritance of stress tolerance (Niknam and Mccomb, 2000).
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Polyamines (PAs), spermidine (Spd), spermine (Spm) and their diamine obligate precursor putrescine (Put), are small aliphatic amines that are ubiquitous in all plant cells. They play a role as regulators of cell proliferation and differentiation (Bouchereau et al., 1999). Similarly, ABA generally referred to as a stress hormone, is known to mediate a variety of stress responses that help survive plants under different stresses ([Barry et al., 2003], [Zhang et al., 2006],[Khadri et al., 2007], [Maggio et al., 2007] and [Sánchez-Díaz et al., 2008]). In addition, anti-oxidative enzymes (SOD, POD and CAT) have been reported to play a key role in tolerance to environmental stresses in a number of plant species ([Fadrzilla et al., 1997] and [Raza et al., 2007]). The levels of all these bio-molecules are reported to increase soon after exposure to stress conditions and are believed to mediate a variety of responses under stressful conditions ([Iqbal et al., 2006], [Iqbal and Ashraf, 2006] and [Iqbal and Ashraf, 2007]). However, whether their increase under stress condition is an adaptive response is still unclear.
In view of the vital role of polyamines, ABA and anti-oxidative enzymes in stress tolerance, we hypothesized that these bio-molecules could be used as a potential criteria for discriminating the differently adapted populations of bluegrass for their salt-tolerance potential. Since Panicum antidotale has been found growing on a variety of habitats (Ashraf, 2003), there would be a fair chance of evolution of stress tolerance in populations from diverse habitats. Therefore, the principal objective of the present study was to assess whether polyamines, ABA and anti-oxidative enzymes have played a role in the adaptability of P. antidotale populations to saline habitats.
2. Materials and methods
The possible role of polyamines, ABA and anti-oxidative enzymes in adaptation of Blue Panicgrass [Panicum antidotale Retz.] to saline environments was assessed in a hydroponic experiment conducted during 2006. The experiment was laid out in a completely randomized design with four replications. Six populations of P. antidotale Retz. were collected from six different sites with a wide variation in ECe and pH. These populations were collected from (1) salt-affected area of Pakka Anna, Faisalabad (P1) [soil ECe = 19.23 dS m−1; pH 6.75; coordinates = 31°19′26.20″N, 72°45′38.49″E]; the bank of disposal water channel, Rajawala, University of Agriculture Faisalabad (P2) [soil ECe = 14.87 dS m−1; pH 6.45; coordinates = 31°25′30.61″N, 73°03′53.84″E]; the barren area of Punjab Wild Life Research Institute (PWRI), Faisalabad (P3) [ECe = 9.31 dS m−1; soil pH 7.65; coordinates = 31°28′51.27″N, 73°12′46.78″E]; the bank of canal (PWRI), Faisalabad (P4) [ECe = 7.19 dS m−1; soil pH 6.68; coordinates = 31°28′26.21″N, 73°12′13.78″E]; Botanical Garden Research Area, University of Agriculture, Faisalabad (P5) [soil ECe = 4.83 dS m−1; pH 6.10; coordinates = 31°25′43.96″N, 73°04′17.40E] and forest plantation (Eucalyptusspp.) PWRI, Faisalabad (P6) [ECe = 3.39 dS m−1; soil pH 5.86; coordinates = 31°28′36.00″N, 73°12′38.51E]. The climatic conditions of Faisalabad are: altitude = 213 m; minimum temperature = 2 °C; maximum temperature = 47 °C; average temperature 24 °C; average rainfall 300 mm [Climatological Data Processing Centre, Pakistan Meteorological Department, Karachi, Pakistan; http://www.met.gov.pk/cdpc/home.htm].
Plants were collected in four replicates from their parent habitats along with soil samples (500 g at a depth of 30 cm from the surface) that were used for the determination of soil ECe and pH. All the plants were established in pots containing normal soil and allowed to grow for 6 months under natural conditions. The newly grown tillers of comparable size of each population were transplanted equidistant in plastic containers each containing 10 l half strength Hoagland's nutrient solution and they were allowed to establish for 21 days. These populations were exposed to 0 (normal) or 150 mol m−3(saline) NaCl for a period of 42 days. The desired salinity level was obtained by an increment of 50 mol m−3 NaCl on alternate days. The solution was continuously aerated for 6 h daily during the whole experiment period and renewed regularly after 1 week. During the course of experiment, the average day and night temperatures were 37 ± 3 and 24 ± 3 °C, respectively, and photoperiod from 11 to 12 h. The relative humidity ranged from 45.9 to 58.6%.
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After 42 days, excised culms and roots were washed with distilled water, oven dried at 65 °C for 7 days and their dry weights recorded. Superoxide dismutase (SOD) activity was assayed by nitroblue tetrazolium (NBT) method following Giannopolitis and Ries (1977), whereas, catalase (CAT) andperoxidase (POD) activities were measured following the method of Chance and Maehly (1955) with some modifications as reported by Raza et al. (2007). The activity of each enzyme was expressed on protein basis measured by the method of Bradford (1976).
The extraction, purification and quantification of polyamines were done from fresh leaves by benzoylation method as described by Flores and Galston (1982) with some modifications as reported by Iqbal et al. (2006). ABA contents were also determined from fresh leaves (3rd leaf from top) following the methods of Guinn et al. (1986) with some modifications as reported by Iqbal and Ashraf (2006). Both polyamines and ABA were determined using HPLC (Sykam GmbH, Kleinostheim, Germany) equipped with a S-1121 dual piston solvent delivery system, S-3210 UV-vis detector and equipped with a Hypersil ODS reverse-phase (C18) column (4.6 mm - 250 mm, 5-μm particle size: Thermo Hypersil GmbH, Germany). The peak areas for polyamines and ABA were recorded and calculated by a computer package [SRI PeakNT version 2.66[MS] chromatography data acquisition and integration software (SRI Instruments, Torrance, CA, USA]. The values were compared with the authentic standards run through for the whole procedure and the concentrations of PAs and ABA calculated.
The oven-dried plant material of roots, culm and leaves was finely ground to pass through 2 mm sieve. The dried material (0.1-0.5 g) was digested in a digestion mixture (sulphuric acid-hydrogen peroxide) according to the method of Wolf (1982). Concentrations of Na+, K+ and Ca2+ in the digests were determined with a flame photometer (Jenway, PFP7). For Cl− determination, the ground material (0.1-0.5 g) was extracted in 10 ml DW at 80 °C for 6 h and Cl− concentration determined with a chloride analyzer (Model 926, Sherwood Scientific Ltd., Cambridge, UK).
Analysis of variance (ANOVA) of the data was computed using a COSTAT computer package (CoHort Software, 2003, Monterey, California). The mean values were compared with the least significance difference test following Snedecor and Cochran (1980).
3.1. Growth attributes
Analysis of variance of the data for culm and root dry weights revealed highly significant differences for populations, treatments, and population - treatment interaction terms. Salinity stress significantly reduced dry weights of both culm and root in all populations under saline conditions. Both parameters were the highest in population P1 followed by P2 that were collected from areas with ECe = 19.23 and 14.87 dS m−1, respectively. Populations P3 and P4, collected from mild saline soil [ECe = 9.31 and 7.19 dS m−1, respectively] were intermediate in stress tolerance and showed almost equal culm and root dry weights. An important observation in this study was that populations, P5 and P6 collected from soils with very low salinity, ECe = 4.83 and 3.39 dS m−1, respectively, were unable to withstand the applied salinity and showed severe growth inhibition at higher levels of salt stress (Fig. 1a and b).
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Fig. 1. Growth attributes of six Panicum antidotale populations grown hydroponically for 42 days under normal or saline conditions. Vertical lines on bars represent SE values. Means sharing same letters do not differ significantly at 5%. ***Significant at 0.001 level. ECe in dS m−1.
3.2. Activities of antioxidant enzymes
The activities of antioxidant enzymes (SOD, POD, CAT) generally increased in all populations of Panicum antidotale upon exposure to salt stress. Statistical analysis of the data for the activities of all three enzymes revealed significant differences for populations, treatments and population - treatment interaction terms. The populations from highly saline habitats (P1 and P2) showed the highest increase in SOD, POD and CAT upon exposure to salt stress. On the other hand, the populations from mild saline habitats (P3 and P4) showed less increase in the activities of antioxidant enzymes as compared to those from non-saline habitats (P1 and P2) (Fig. 2).
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Fig. 2. Antioxidative enzymes (units/mg protein) of six Panicum antidotale populations grown hydroponically for 42 days under normal or saline conditions. Vertical lines on bars represent SE values. Means sharing same letters do not differ significantly at 5%. ***Significant at 0.001 level. ECe in dS m−1.
An important observation in this study was that the activities of all three antioxidant enzymes increased up to three-fold in the populations from highly saline habitats as compared to those in the populations from mild saline habitats (two-fold increase). On the other hand, the populations from non-saline habitats showed a less increase in the enzyme activities upon exposure to salt stress. Generally, the increase in SOD activity was greater as compared to those of POD or CAT in the leaves of stressed plants of all populations of P. antidotale (Fig. 2a-c).
3.3. Polyamines and ABA
A significant increase in polyamines (putrescine, spermidine and spermine) and ABA concentrations was observed in all six P. antidotale populations under salt stress. The populations from highly saline habitats (P1 and P2) exhibited a greater increase as compared to those from mild saline environments (P3 and P4). On the other hand, the lowest increase in polyamines was observed in the populations from non-saline environments (P5 and P6) (Fig. 3a-d). A similar trend was observed for Spm/Spd and Put/(Spd + Spm) ratios which generally decreased under stressful conditions. The populations from highly saline habitats (P1 and P2) exhibited less decrease in both ratios as compared to the populations from mild or non-saline habitats (Fig. 3e and f).
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Fig. 3. Polyamine and free ABA in six Panicum antidotale populations grown hydroponically for 42 days under normal or saline conditions. Vertical lines on bars represent SE values. Means sharing same letters do not differ significantly at 5%. ***Significant at 0.001 level. ECe in dS m−1.
3.4. Ion accumulation
All populations of P. antidotale showed a considerable increase in Na+ contents in different plant parts under salt stress. The populations from highly saline habitats (P1 and P2) accumulated significantly lower Na+ contents in the leaves, roots and culm as compared to those from mild saline habitats (P3 and P4). In contrast, the populations from non-saline habitats were the highest of all population in Na+ contents in all plant parts (Fig. 4a-c).
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Fig. 4. Na+ and K+ contents in root, culm and leaves of six Panicum antidotale populations grown hydroponically for 42 days under normal or saline conditions. Vertical lines on bars represent SE values. Means sharing same letters do not differ significantly at 5%. *, **, ***Significant at 0.05, 0.01 and 0.001 levels, respectively. ns = non-significant. ECe in dS m−1.
Although K+ contents in the leaves, roots and culm generally decreased in all populations of P. antidotale upon exposure to salt stress, the populations did not differ significantly for root K+ contents. In contrast, the populations differed significantly for culm or leaf K+ content. Highest K+ contents were observed in the leaves followed by culm and roots. Although all populations collected from diverse habitats did not show a large variation in K+ contents in all organs studied, the response of the populations from highly saline habitats was comparatively better as they showed less decrease in K+ contents under salt stress as compared to the other populations from mild or non-saline habitats (Fig. 4d-f).
A general decreasing trend in Ca2+ content was observed in all organs of Panicum populations upon exposure to salt stress. The populations from highly saline habitats showed a less decrease in Ca2+ contents in all organs as compared to those from mild saline habitats. Highest decrease in Ca2+ contents in all organs was observed in the populations from non-saline environments (Fig. 5a-c).
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Fig. 5. Ca2+ and Cl− contents in roots, culm and leaves of six Panicum antidotale populations grown hydroponically for 42 days under normal or saline conditions. Vertical lines on bars represent SE values. Means sharing same letters do not differ significantly at 5%. *, **, ***Significant at 0.05, 0.01 and 0.001 levels, respectively. ns = non-significant. ECe in dS m−1.
A considerable increase in Cl− content was observed in root, culm and leaves of Panicum populations under salinity stress. The populations from highly saline environments showed comparatively low Cl− contents as compared to those from mild saline habitats. In addition, the populations from non-saline habitats were the highest of all populations in Cl− contents of all organs. Cl− contents were more or less equally distributed in all organs under normal or saline conditions (Fig. 5d-f).
While measuring different growth attributes it was evident that the populations (P1 and P2) collected from highly saline habitats had better growth and survival under saline conditions as compared to those collected from less saline (P3 and P4,) or non-saline (P5, and 6) habitats. The degree of salt tolerance shown by the populations P1 and P2 can be related to the intensity of soil salinity (selection pressure) that may have been one of the important determinants responsible for the evolution of salt tolerance trait in P. antidotale ([Ashraf, 2003] and [Hameed et al., 2008]).
All Panicum populations showed multi-fold increase in anti-oxidative enzymes (superoxide dismutase, peroxidase and catalase) upon exposure to salt stress. It is now widely accepted that reactive oxygen species (ROS) are produced under salt stress, which can destroy the normal metabolism through oxidative damage of lipids, proteins and nucleic acids (McCord, 2000) thereby ultimately damaging cell structure ([Mittler, 2002] and [Vranová et al., 2002]). Therefore, ROS must be scavenged by enzymatic and/or non-enzymatic antioxidant systems for the maintenance of normal growth in stressful environments. In view of different researchers, salt tolerance is often correlated well with a more efficient anti-oxidative system ([Sreenivasulu et al., 2000],[Ashraf and Harris, 2004], [Demiral and Turkan, 2004], [Juan et al., 2005], [Demiral and Türkan, 2005], [de Azevedo Neto et al., 2006], [López-Gómez et al., 2007], [Masood et al., 2006], [Yazici et al., 2007], [Tuna et al., 2008] and [Ashraf, 2009]). In the present study, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were increased in the leaves of stressed plants of all populations. Activities of all three anti-oxidative enzymes were greater in the populations from habitats with high salinity as compared to those from low saline or non-saline environments. These findings show that genetic variability was present in different Panicum populations that had been triggered in populations as a consequence of exposure to high selection pressure.
Increased levels of polyamines under stressful environments play an important role in stress tolerance of crop plants ([Iqbal and Ashraf, 2006], [Iqbal and Ashraf, 2007], [Iqbal et al., 2006] and [Mutlu and Bozcuk, 2007]). Most widely accepted and experimentally proved viewpoint is that PAs exert their protective action on bio-molecules due to their chemical structure as organic polycations (Kuznetsov et al., 2006). In addition, their functions include stabilization of structure and function of membranes especially that of thylakoid, DNA stabilization, regulation of enzyme activity, maintenance of cell division/cycle and elongation, buffering of cellular pH, delay in senescence, function as second messengers and regulation of other multiple cellular responses ([Krishnamurthy and Bhagwat, 1989], [Galston et al., 1997], [Bouchereau et al., 1999], [Kakkar and Sawney, 2002], [Alcazar et al., 2006], [Kuznetsov et al., 2006] and [Xiong et al., 2006]). Thus, it can be postulated that hyper-production of Put in combination with Spd and Spm regulated growth and development of tolerant populations under stressful conditions and are key physiological determinants of salt tolerance in P. antidotale.
An interesting observation in this study was that Spm/Spd ratio generally decreased under salt stress. The population P1 exhibited the highest Spm/Spd ratios followed by P2, P3 and P4, respectively. In contrast, the populations P5 and P6 showed the lowest Spm/Spd ratio. It is now well established that Spm and Spd have a common biosynthetic pathway ([Friedman et al., 1989], [Bortolotti et al., 2004] and [Kuznetsov et al., 2006]). Thus, the greater increase in Spm/Spd ratio in the populations from highly saline environment under salt stress indicated that the biosynthetic pathway might have been more directed towards spermine biosynthesis.
Similarly, Put/(Spm + Spd) ratio was the highest in P1 and P2 as compared to all the other populations that showed almost equal Put/(Spm + Spd) ratio (Fig. 3e and f). Moreover, an increase in this ratio was greater in the populations from highly saline environments [P1 and P2] as compared to those from mild or non-saline environments which had almost equal increase in Put/(Spd + Spm) ratio. These results support that tolerance to salt-stress was associated with a rise in Put and an apparent impairment in the capacity to synthesize Spd from Put. Consequently, the Put accumulation observed in the stress-tolerant plants in response to stress might have resulted from an enhancement of Put synthesis or, alternatively, from an inhibition of the activity of the enzyme SAM decarboxylase, thus allowing Put to accumulate in plant tissues ([Santa-Gruz et al., 1997] and [Bouchereau et al., 1999]).
The ABA contents in all Panicum populations generally increased significantly under salt stress. The populations P1 and P2 had significantly higher increase in ABA contents under salt stress as compared to P3 and P4. The lowest increase in ABA content was found in the populations P5 and P6 which were collected from the habitats with low soil salinity (Fig. 3d). ABA is now well known to rapidly accumulate in response to stresses and is involved in the mediation of many stress responses that help plant survival under stressful environments ([Pierce and Raschke, 1980], [Jia and Zhang, 2000], [Jia et al., 2001], [Jia et al., 2002a], [Jia et al., 2002b], [Mills et al., 2001] and [Zhang et al., 2006]). This is known to occur in a number of monocot plants including wheat ([Mumtaz et al., 1997], [Naqvi et al., 1997], [Aldesuquy and Ibrahim, 2001] and [Iqbal et al., 2006b]), rice ([Henson, 1984], [Kishor, 1989], [Bohra et al., 1995] and [Moons et al., 1995]), barley ([Stewart and Voetberg, 1985], [Kefu et al., 1991] and [Gómez-Cadenas et al., 2003]), sorghum ([Amzallag et al., 1998] and [Saneoka et al., 2001]), and maize ([Benson et al., 1988] and [Liu et al., 2005]). A substantial evidence suggests that increased ABA levels limit water loss by stomatal closure and thus it plays a crucial role in plant adaptation to stresses. ABA has been reported to regulate the process of adaptation in two interacting steps. Firstly, ABA acts via differential signal transduction pathways on cells under imposed stresses. Secondly, ABA may regulate through some genes/gene products, which control the expression (up-regulation or down-regulation) of stress adaptative-specific genes ([Zeevaart and Creelman, 1988], [Bray, 1991] and [Hetherington and Quatrano, 1991]), thereby resulting in overall synthesis of genomic products which may play a role in plant survival under different environmental conditions ([Mundy and Chua, 1988], [El-Enany, 2000], [Chandler and Robertson, 1994], [Barry et al., 2003],[Khadri et al., 2007] and [Maggio et al., 2007]). Thus, the higher production of ABA in the populations (P1 and P2) from highly saline areas may suggest its role in adaptability and evolution of salt-tolerance trait in these populations. On the other hand, the populations from the habitats with mild soil salinity (P3 and P4) accumulated less ABA and thus were less adaptive to salt stress. Populations form non-saline environment (P1 and P3) that had the lowest ABA content did not show any adaptation to salt stress.
The uptake and accumulation of ions in plants is one of the potential indicators of salinity tolerance, because they are genetically regulated, though also affected by the environment (selection pressure) (Chaubey and Senadhira, 1994). In this study, Na+ and Cl− contents generally increased in all plant parts studied of all populations, whereas, K+ and Ca2+ contents decreased significantly. The populations from highly saline habitats showed a less increase in Na+ and Cl− contents and a less decrease in K+ and Ca2+ contents thereby maintaining high K+/Na+ and Ca2+/Na+ ratios as compared to those from habitats with mild or normal salinity levels (Graifenberg et al., 1995). On the other hand, the comparison among populations revealed that the tolerant populations accumulated less Na+ as compared to the sensitive ones. Thus, it can be concluded that ion exclusion mechanism might have been the premier adaptive component of salt tolerance in Panicum populations.
Our results clearly show that intensity of selection pressure (soil salinity) played a key role in the adaptation of Panicum populations to salinity stress. Overall, all polyamines (Put, Spm and Spd), ABA and anti-oxidative enzymes increased under salt stress in all populations. Similarly, the populations adapted to high salinity accumulated less Na+ and Cl− in culm and leaves, and showed a less decrease in leaf K+ and Ca2+ under salinity stress. The populations from highly saline areas accumulated more polyamines, ABA and showed higher activities of anti-oxidative enzymes as compared to those from mild or non-saline habitats. Increased Spm/Spd ratio in the populations from highly saline habitats indicated the efficiency of Spm biosynthetic pathway while increased Put/(Spm + Spd) ratio indicated the efficiency of Put biosynthetic pathway. Thus, both polyamines may have played a role in P. antidotale to tolerate salt stress.