flavones (FLA) levels were 2.5, 2.8 and 3.0 times greater in CP-4333 in comparison with. HSF-240. ... CAR, as components
ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 723—730
www.elsevier.de/jplph
Possible involvement of some secondary metabolites in salt tolerance of sugarcane Abdul Wahid, Alia Ghazanfar Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan Received 10 May 2005; accepted 19 July 2005
KEYWORDS Cytosol; Ions toxicity; Salt tolerance; Secondary metabolites; Sugarcane
Summary Accumulation of toxic ions in plant tissues modulates the levels of primary and secondary metabolites, which may be related to salinity tolerance. In this study two sugarcane clones, CP-4333 (tolerant) and HSF-240 (sensitive), were exposed to salinity levels at the formative stage, and evaluated three times at 10-day intervals. Although net rate of photosynthesis (Pn), leaf area, length and dry weight of shoots were decreased in both clones, the CP-4333 showed less reduction compared to HSF-240. Both clones displayed a general tendency to accumulate Na+ and Cl and little K+, though CP-4333 accumulated less Na+ and more K+ compared to HSF-240, and thus showed a higher K+:Na+ ratio. The carotenoid (CAR) content remained steady, while total chlorophyll (CHL) was slightly reduced in the tolerant clone and significantly reduced in HSF-240. In contrast, soluble phenolics (PHE), anthocyanins (ANT) and flavones (FLA) levels were 2.5, 2.8 and 3.0 times greater in CP-4333 in comparison with HSF-240. The decrease in Pn and most secondary metabolites demonstrated by the sensitive clone, but not evidenced in the tolerant clones, suggest that the presence of those metabolites is related to increased salt tolerance of sugarcane. The increased synthesis of PHE, ANT and FLA seems to protect sugarcane from ion-induced oxidative stress, probably due to a common structural skeleton, the phenyl group, of those metabolites. CAR, as components of the light harvesting center (LHC) and biosynthesized in chloroplasts, may confer resistance to this organelle. The PHE, ANT and FLA synthesized in the cytosol may protect cells from ion-induced oxidative damage by binding the ions and thereby showing reduced toxicity on cytoplasmic structures. & 2005 Elsevier GmbH. All rights reserved.
Abbreviations: ANT, anthocyanins; CAR, carotenoids; CHL, total chlorophyll; EC50, salt tolerance limit; FLA, flavones; LHC, light harvesting center; MEP, methylerythritol phosphate; PHE, soluble phenolics; Pn, net rate of photosynthesis Corresponding author. Tel.: +92 41 2644219; fax: +92 41 9200764. E-mail address:
[email protected] (A. Wahid).
Introduction Increased salinity affects primary carbon metabolism, plant growth and development by iontoxicity, induced nutritional deficiency, water
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.07.007
ARTICLE IN PRESS 724 deficits and oxidative stress (Sairam and Tyagi, 2004; Flowers, 2004). Moreover, it modulates the levels of secondary metabolites, which are physiologically important particularly in stress tolerance. Some of these metabolites have light absorptive properties, harvest light for photosynthesis and protect the cells from damaging effects of high energy radiation, while others promote defensive action against herbivores and pathogens (Harborne and Williams, 2000; Taiz and Zeiger, 2002). Most secondary metabolites are synthesized from the intermediates of primary carbon metabolism via phenylpropanoid, shikimate, mevalonate or MEP pathways (Buchanan et al., 2000). Among those, chlorophylls, carotenoids (CAR) and phenolics are commonly studied metabolites in plant kingdom. Enhanced synthesis of determined secondary metabolites under stressful conditions is believed to protect the cellular structures from oxidative damage (Buchanan et al., 2000), in addition to osmotic advantage to the plants (Chalker-Scott, 1999; Winkel-Shirley, 2002; Close and McArthor, 2002). Concerning direct involvement in photochemical reactions of leaves, total chlorophyll (CHL) and CAR are the most important secondary metabolites. Decrease in CHL content is associated to enhanced expression of chlorophyllase activity under stress (Majumdar et al., 1991). The CAR (carotenes and xanthophylls), besides acting as accessory light harvesting pigments, show antioxidant properties (de Pascale et al., 2001). They protect the photosystems by: (a) reacting with lipid peroxidation products to terminate chain reactions; (b) scavenging singlet oxygen and dissipating the energy as heat; (c) reacting with triplet-excited chlorophyll molecules to prevent formation of singlet oxygen; or (d) dissipating excess of excitation energy through the xanthophyll cycle (Rmiki et al., 1999). Steady levels of CAR were tightly correlated with salt tolerance of mungbean (Wahid et al., 2004). Less reduction in CHL and steady levels of CAR are salinity tolerance strategies of sugarcane (Kanhaiya, 1996). The soluble phenolics (PHE) produced by the phenylpropanoid or shikimate pathways are powerful antioxidant in plant tissues under stress (Dixon and Paiva, 1995; Sgherri et al., 2004). They are chemically heterogeneous compounds, mainly flavonoids, lignins and tannins, which play a variety of roles, e.g. defense against herbivores and pathogens, mechanical support, attract pollinators, absorb high energy radiations and reduce the growth of nearby competing plants (Harborne and Williams, 2000; Taiz and Zeiger, 2002). Recently the role of phenolics has been reviewed because of
A. Wahid, A. Ghazanfar their great involvement in the oxidative stress tolerance instead of herbivory (Close and McArthor, 2002). Among flavonoids, the anthocyanins (ANT) are highly water soluble pigments derived from flavonoid precursors via the shikimate pathway that accumulate in the vacuole (Chalker-Scott, 1999). Their accumulation has been seen in various plants including Morus alba (Ramanjulu et al., 1993), Arabidopsis (Mita et al., 1997) and Hedera helix leaves when grown in the presence of sugars (Murray et al., 1994). ANT production is beneficial to the plants in terms of protection of shadeadapted chloroplasts from brief exposure to high solar radiation (Gould et al., 2000; Lee and Gould, 2002), protection of leaves from photo-oxidative damage during senescence (Feild et al., 2001), and reduced damage to photosynthetic systems by absorbing UV-B (Burger and Edwards, 1996). Since the ANT are osmotically active, their enhanced expression may increase hardiness through increased osmotic control (Chalker-Scott, 1999). Kaliamoorthy and Rao (1994) reported up to 40% accumulation of ANT in maize as a salinity stress response. Furthermore, ANT accumulate under UVB (Mendez et al., 1999), drought (Balakumar et al., 1993), low temperature (Krol et al., 1995), nutrient deficiency (Rajendran et al., 1992) and exposure to ozone (Foot et al., 1996). Flavones (FLA), another category of flavonoids, are formed by oxidation of flavanones (Justesen et al., 1997; Buchanan et al., 2000). The enzymes of parsley have been extensively studied for the biosynthesis of FLA (WinkelShirley, 2002). Their biosynthesis is induced by fungal attack or at later developmental stages of plants (Justesen et al., 1997). Like ANT, the FLA glycosides also accumulate in vacuole and possibly H+-antiporters are involved in their sequestration into this cellular compartment (Frangne et al., 2002). However, modulations in their levels under abiotic stresses require investigation. Sugarcane (Saccharum officinarum L.) is a major source of sucrose and therefore ubiquitous in cultivation. Although it is ranked as moderately salt-sensitive (Francoise and Maas, 1999), there are differences in salt resistance (Wahid et al., 1997). Primary metabolism of sugarcane has been well studied under normal or saline conditions; however, there is a lack of information on the biosynthesis and role of secondary metabolites in sugarcane under salinity, although they accumulate in low levels under normal conditions (Franc-a et al., 2001). It is assumed that accumulation of secondary metabolites enhances the sugarcane capacity for salt tolerance. Therefore, the aim of this study was to determine time course changes in the levels of
ARTICLE IN PRESS Secondary metabolites and salt tolerance of sugarcane some secondary metabolites and their physiological implications in salt tolerance of sugarcane.
Materials and methods Sugarcane clones CP-4333 and HSF-240 were previously tested and confirmed tolerant and sensitive to NaCl salinity with average EC50 of 12.5 and 6.8 dS m1, respectively, at the formative stage (Wahid et al., 1997; Ghazanfar, 2004). Twenty single-noded sets were planted in field plots (measuring 1.5 m 4 m 0.3 m deep) lined with double layer of polythene before refilling with loam soil, and 12 uniform sprouts were finally retained. The analysis of soil revealed the following properties: sand 39%, silt 32%, clay 28%, (textural class loam), organic matter 1.45%, pH 7.3, ECe 2 dS m1, soil saturation 34%, cation exchange capacity 14.2 meq 100 g1 soil, sodium absorption ratio 0.11. The experiment was laid out in parallel in a completely randomized fashion with four replications. One-month-old shoots were gradually exposed to 8 and 12 dS m1 levels of salinity using sodium chloride during 3 days; one plot was kept as control. The plants were irrigated to keep soil moisture at field capacity. Shoots were harvested 10, 20 and 30 days after salt application. Leaf area of intact plants was determined as leaf length leaf width 0.68 (correction factor calibrated for all leaves). Shoot length was evaluated after uprooting. To determine dry weight, shoots were put in paper envelopes and oven dried at 70 1C for 1 week. For ionic analysis, dried ground material was placed in screw capped tubes containing deionized water and heated in a water bath at 100 1C for 1 h, filtered, and diluted to a constant volume. This extract was used for the determination of Na+ and K+ using flame photometry (Jenway, PFP-7, UK) and Cl using a chloride meter (Sherwood Chloride Analyzer 926, Japan). For determination of secondary metabolites levels, fresh excised leaves were wrapped in black plastic bags, kept on ice in a bucket, transferred to the laboratory, and immediately frozen. For the extraction of CHL and CAR, leaf tissue was ground with a mortar and pestle in methanol under CaCO3, vacuum filtered, and analyzed immediately using spectrophotometery (Gitelson et al., 2001). Specific absorption coefficients of CHL and CAR were used as reported by Lichtenthaler (1987). The PHE were extracted from fresh leaf tissue using 80% acetone and the absorbance of the extract was taken at 740 nm using spectrophotometery. Tannic acid was used for constructing the standard curve
725
for PHE (Julkenen-Titto, 1985). To determine ANT, leaves were extracted in acidified methanol (1% HCl v/v), vacuum filtered, and quantified using a spectrophotometer at 535 nm according the method described by Stark and Wray (1989). For FLA evaluation, a sample of the extract prepared for ANT was used and analyzed at 360 nm, using hyperoside as the standard (Justesen et al., 1997). Statistical analysis of the data was performed using COSTAT software. LSD values were determined using Duncan’s new multiple range test and significant differences among genotypes, salinity levels and harvests were determined. Parallels were drawn to determine correlation coefficients for the reduction in net rate of photosynthesis (Pn), ions and secondary metabolites over respective controls of individual clones at all sampling times.
Results Growth, photosynthesis and ionic characteristics Although shoot length decreased in both clones under salinity, it showed maximum reduction in HSF-240 (43%) and minimum reduction in CP-4333 (21%) at final sampling time (Table 1). These changes led to significant (po0:01) differences among treatments, harvests and clones. There was no significant (p40:05) reduction in shoot dry weight, although CP-4333 performed relatively better (30% d.w.) than HSF-240 (48% d.w.) under salinity stress at final sampling time (Table 1). Comparisons among salt treatments and harvests showed significant (po0:01) differences, indicating a salinity effect over time (Table 1). Although HSF240 showed a larger leaf area under controlled condition, it presented a more consistent reduction (71%) compared to CP-4333 (24%) under increased salinity towards the end of the experiment. Salinity levels and harvests produced significant (po0:01) differences in both clones (Table 1). Pn was decreased in both clones, however CP-4333 indicated a lower reduction (37%) compared to HSF-240 (66%) under increased salinity at the final harvest. Both clones accumulated Na+ in the shoots under salinity at all harvests; even so, HSF-240 displayed a higher content (262% expressed over control) of this ion than CP-4333 (202%). Na+ accumulation was linear with time and increased salinity levels (Fig. 1). K+ accumulation was significantly different (po0:01) between clones and salt levels. However, harvests did not affect K+ production, which remained similar during growth periods. CP-4333
ARTICLE IN PRESS 726 Table 1. times
A. Wahid, A. Ghazanfar Changes in growth and ionic characteristics of sugarcane clones under increased salinity at three sampling
Clones
Sampling time (days after treatment)
NaCl levels (dS m1)
Shoot length (cm plant1)
Shoot dry weight (g plant1)
Leaf area (cm2 plant1)
Net rate of photosynthesis (mmol m2 s1)
CP-4333
10
Cont 8 12 Cont 8 12 Cont 8 12
12.5171.13 11.8370.76 11.1771.44 15.2371.16 13.1371.21 12.3371.15 18.1771.26 16.3370.58 14.3371.15
1.9270.12 1.6770.15 1.5670.17 2.1870.21 1.9370.12 1.7570.10 2.7670.16 2.2370.17 1.9370.14
23.0972.13 22.1671.61 20.5271.84 28.3772.60 24.5371.50 21.7271.56 33.5872.83 30.4572.97 25.4672.17
15.7671.04 15.0771.25 12.8171.31 15.3970.63 13.6471.06 10.8470.99 15.9270.37 11.3471.18 10.7270.55
Cont 8 12 Cont 8 12 Cont 8 12
14.4771.36 10.6771.15 8.6770.58 17.1771.11 12.1770.76 9.3370.58 19.0071.50 12.3371.04 10.8371.04
2.0070.12 1.6670.11 1.4070.09 2.5570.23 1.7870.10 1.5170.10 3.0470.28 1.9770.14 1.6070.12
30.5372.15 18.8671.03 10.6070.77 34.337231 21.3372.02 11.0571.11 39.1072.01 20.9271.66 11.3971.01
15.8770.71 10.8671.28 8.7471.00 15.4270.99 9.1170.82 5.5870.20 15.7470.84 8.4870.24 5.2870.13
Clones Salt levels Harvests
0.602** 0.737** 0.737**
0.085ns 0.104** 0.104**
1.08** 1.33** 1.33**
0.492** 0.603** 0.603**
20
30
HSF-240
10
20
30
LSD of variance sources
**Significant at po0:01; ns, nonsignificant
was superior when compared to HSF-240 for accumulating greater K+ concentrations (31%) under increased salinity during all sampling times (Fig. 1). Salinity increased Na+ and decreased K+ levels of the shoot, resulting in a substantially reduced K+:Na+ ratio (Fig. 1), though this ratio was more reduced in HSF-240 (89%) than CP-4333 (77%). Also, different clones, salt levels and sampling times significantly affected the K+:Na+ ratio. The Cl accumulation in shoots was also linear with time and salinity levels resembling Na+ accumulation (Fig. 1). However, Cl accumulation in CP-4333 was considerably lower (152%) compared to HSF240 (208%), again showing significant difference between clones, salt levels, and harvests.
Secondary metabolites Under increased salinity the CHL level was reduced in both clones, although a greater but consistent decrease was noted in HSF-240 (38%) compared to CP-4333 (21%) at all growth stages (Fig. 2). This reduction was significant for clones and salinity levels but not different in relation to
sampling times. Under salt stress conditions, CP4333 maintained a fairly steady level of CAR (just a 7% decrease) whereas HSF-240 indicated a steep decline (up to 31%) over time (Fig. 2). CAR levels were significantly (po0:01) different among the clones and salt levels. Salinity significantly increased the CAR:CHL ratio at all sampling times, and this ratio was relatively greater in CP-4333 under higher salt level (Fig. 2). Both clones showed increased synthesis of PHE under increased salinity, but this increase was superior in CP-4333 (46%) compared to HSF-240 (16%) (Fig. 2). ANT content was increased in both clones, though it was much greater in CP-4333 (157%) compared to HSF-240 (48%) under increased salinity (Fig. 2), FLA accumulation was also significantly (po0:01) different according to clones, salinity treatments and harvests. CP-4333 produced higher levels of FLA (162%) than HSF-240 (53%) under higher salt stress (Fig. 2).
Correlations Since the intermediates produced during the dark reactions of photosynthesis participate in the
ARTICLE IN PRESS Secondary metabolites and salt tolerance of sugarcane
CAR:CHL ratio of the tolerant clone were nonsignificant while the sensitive clone showed a significant reduction in Pn. These findings revealed that relative modulations in the levels of secondary metabolites are related to greater salinity tolerance of sugarcane.
+
-1
Na content (mg g DW)
60
CP-4333
50
HSF-240
40 30 20 10 0 0
80
120
0
10 DAT
80
120
0
20 DAT
20
80
120
30 DAT
Harvests
Fig. 19. Na in two sugarcane clones under increased salinity
15 10
+
-1
K content (mg g DW)
25
5 2.0
0
80
120
0
80
120
0
80
120
10 DAT
20 DAT 30 DAT Harvests Fig. 21. K in two sugarcane clones under increased salinity
1.0
+
+
K :Na ratio
1.5
0.5
0.0 100
Cl- content (mg g-1 DW)
727
0
80
120
0
10 DAT
80
120
0
20 DAT Harvests
80
80
120
30 DAT
Fig. 14. K/Na ratio in two sugarcane clones under 60 increased salinity 40 20 0 0
80
120
10 DAT
0
80
120
20 DAT
0
80
120
30 DAT
Sampling time
Figure 1. Changes in some ionic content in the shoots of two sugarcane clones under increased salinity at different time intervals. DAT, days after treatment application.
biosynthesis of secondary metabolites, relationships of relative changes in growth, ions and secondary metabolites with Pn were determined for both clones separately (Table 2). Reduction in Pn was positively related to length and dry weight of shoot, leaf area, K+ and K+:Na+ ratio, whilst negatively to Na+ and Cl levels of both clones. Decreased levels of secondary metabolites except
Discussion Glycophytes exposed to saline conditions usually show enhanced levels of ions under which they are grown. Excess of toxic ions modulates the levels of both primary and secondary metabolites as assessed from apparent growth and tissue analysis (Sairam and Tyagi, 2004; Winkel-Shirley, 2002; Flowers, 2004). Sugarcane clones under this study were affected by salinity as displayed by elongation and dry weight of shoots and expansion of leaves. The growth inhibition was associated to the reduction in Pn and leaf area, even though this reduction was greater in sensitive clone (Table 1). It was observed that the primary effect caused by salinity was the reduction of photosynthetic area or inhibition of CO2 assimilation. Regarding the ionic parameters of shoots it was noted that although both Na+ and Cl accumulated in both clones, their accumulation was significantly less in CP-4333 compared to HSF-240 (Fig. 1). Despite the fact that shoots-Cl content was greater than Na+, the toxicity of both ions was similar on shoot growth. Accumulation of K+ was greater in CP-4333 clone compared to HSF-240, thus resulting in an enhanced K+:Na+ ratio of the former clone at all sampling times, presenting an important manifestation of salinity tolerance (Sairam and Tyagi, 2004; Flowers, 2004; Wahid, 2004; Ahmad et al., 2005). From the correlation of K+ with Pn (Table 2), it was revealed that minimal reduction of shoot K+ levels is an important manifestation of salinity tolerance in sugarcane. Stress-induced changes in primary metabolism lead to varied levels and classes of secondary metabolites including photosynthetic pigments (CHL and CAR) flavonoids, phenolics, alkaloids, etc. (Close and McArthor, 2002; Gould et al., 2000; Taiz and Zeiger, 2002). Modulations in the levels of CAR, PHE and flavonoids (ANT and FLA) are of greater importance in the prevention of stressinduced oxidative damage or maintenance of osmotic balance (Chalker–Scott, 1999; Gould et al., 2000; Sgherri et al., 2004). Minimal reduction in the content of photosynthetic pigments is important, as it has a direct relationship with salinity tolerance (Ahmad et al., 2005). CAR have
ARTICLE IN PRESS 728
A. Wahid, A. Ghazanfar 0.5 HSF-240
0.9
0.6
80 120
0
0
20 DAT
80 120 30 DAT
0.4 0.3
0
80 120
0
10 DAT
80 120
0
20 DAT
80 120 30 DAT
45
30
15 80 120
0
80 120
0
80 120
10 DAT
20 DAT 30 DAT Harvests Fig. 14. Chl/Car ratio in two sugarcane clones under 60 increased salinity 80
40 20 0
12
-1
0
Flavones (µg g FW)
-1
0.1
60
0.2
Anthocyanins (µg g FW)
0.2
-1
10 DAT
80 120
Soluble phenolics (µg g FW)
Carotenoid:chlorophyll ratio)
0
0.5
100
0.3
0
0.3 0.6
0.4
-1
CP-4333
Carotenoids (mg g FW)
-1
Chlorophylls (mg g FW)
1.2
0
80 120
0
80 120
0
80 120
10 DAT
20 DAT 30 DAT Harvests 8 Fig. 9. Soluble phenolics in two sugarcane clones under increased salinity 6
10
4 2 0
2
8
12
10 DAT
2
8
12
20 DAT
2
8
12
30 DAT
Sampling time (days after treatment)
2
8
12
10 DAT
2
8
12
20 DAT
2
8
12
30 DAT
Sampling time (days after treatment)
Figure 2. Changes in the levels of some secondary metabolites in the shoots of two sugarcane clones under increased salinity at different time intervals. DAT, days after treatment application.
the ability to scavenge the reactive oxygen species (ROS) generated under prevailing stress conditions (Rmiki et al., 1999; de Pascale et al., 2001; Netto, 2001). In this study, the tolerant clone (CP-4333) was capable of maintaining two- and threefold greater CHL and CAR contents, respectively (Fig. 2). Moreover, in contrast to the sensitive clone (HSF-240), in the tolerant clone the reduction of CHL and CAR levels was not associated with Pn (Table 2). A supposed reduction of CAR in the tolerant clone and increased CAR:CHL ratio revealed that, in addition to acting as accessory light harvesting pigments, the CAR provide a protective advantage against salt-induced oxidative damage. Since CAR are the components of light harvesting complex and biosynthesized in the chloroplast, it is likely that their protective role is specific to the chloroplasts.
The role of phenolics has been recently reconsidered more relevant in oxidative stress tolerance than protection from herbivory (Close and McArthor, 2002; Moyer et al., 2002; Sgherri et al., 2004). They show accumulation under water and salt stresses as well (Dixon and Paiva, 1995; Ali and Abbas, 2003). Flavonoids are known to protect the sensitive tissues against the damaging effects of UV-radiation (Singh et al., 1999), but their roles have also been envisaged in salinity, as they are ion chelators (Taiz and Zeiger, 2002; Winkel-Shirley, 2002). They are metabolically active when present in glycosylated form (Frangne et al., 2002). It was found that the salt tolerant clone has a three-fold increase in the biosynthesis of PHE, ANT and FLA (Fig. 2). Reduction in their content (relative to control) by salinity indicated no relationships with that of Pn, whilst these relationships were negative
ARTICLE IN PRESS Secondary metabolites and salt tolerance of sugarcane Table 2. Correlations coefficients (r) of the saltinduced reductions in growth, ionic and secondary metabolite levels with Pn cumulative of all sampling times (n ¼ 6) Plant characteristics
CP-4333
HSF-240
Shoot length Shoot dry weight Leaf area Na+ K+ K+:Na+ ratio Cl CHL CAR:CHL ratio CAR PHE FLA
0.802* 0.883* 0.772ns 0.949** 0.463ns 0.884* 0.948** 0.809ns 0.748ns 0.810ns 0.788ns 0.453ns
0.930** 0.953** 0.864* 0.897* 0.944** 0.880* 0.878* 0.931** 0.247ns 0.969** 0.888* 0.871*
Significant at *po0:05; **po0:01 and ns, nonsignificant.
for sensitive clone (Table 2). This has great implications for salinity tolerance of sugarcane. Since the metabolism of phenolics and flavonoids takes place in the cytosol, we believe that PHE themselves are the scavengers of ROS (Moyer et al., 2002). The ANT and FLA, in addition to alleviating ion-induced oxidative damage, could bind the toxic ions (Taiz and Zeiger, 2002; Winkel-Shirley, 2002), protect cytoplasmic structures and chloroplasts from adverse effects of salinity, and enable the leaves to display substantially less reduction in photosynthetic rate. Obtained results are supported by the findings reported by Franc-a et al. (2001), who found that abiotically stressed sugarcane expressed enhanced levels of chalcone synthase, a key enzyme in the flavonoid biosynthesis pathway. In conclusion, this study strongly supports the hypothesis that secondary metabolites play significant physiological role in sugarcane salinity tolerance particularly against oxidative damage. Such roles of these metabolites are confined to the compartment where they accumulate; CAR in chloroplast and PHE, ANT and FLA in cytosol. The presence of phenyl ring in phenolics such as flavonoids is of key importance in the environmental stress tolerance (Senaratna et al., 2003). Further research is imperative for broad spectrum understanding the roles of those metabolites in various plant species under saline conditions.
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