Introduction

Perennial pepperweed (Lepidium latifolium Linn.) is an ecologically important plant of Himalayan cold deserts that is used as ‘phytofood’. It belongs to family Brassicaceae. Although, native to southern Europe and Asia (Hultén and Fries 1986; Schultze-Motel 1986; Rollins 1993), it has shown widespread presence in most parts of world including western United States, coastal New England, Mexico, Canada and Australia (Young et al. 1995). It grows between 30 cm and 2 m and has woody stems, waxy leaves and small white flowers arranged in clusters producing a large number of seeds. Of late L. latifolium has gained focus of plant biologists after been recognized as noxious weed in Western Coast of North America (Young et al. 1995; Pimentel et al. 2000). It is extremely competitive in many habitats, especially in wetlands and riparian areas and considered a potential threat to biodiversity and ecosystem function. Pepperweed flowering causes uniform reflectance over the visible range of the electromagnetic spectrum, thus, decreasing the amount of energy absorbed by the canopy and available for photosynthesis (Sonnentag et al. 2011). This affects the overall photosynthetic ability of the ecosystem, specially the herbaceous plants. The mechanism for its successful invasion includes reproduction via seed and root propagules, and its ability to withstand long duration flooding and relatively dry conditions with a large perennial rhizome.

Unlike its aggressive invasive character in riparian plains of North America, its growth is restricted in cold arid zone of Himalayan cold desert and it is present mainly in habited areas. The plant survives harsh conditions that represents unique ecosystem characterized by large temperature variations, sub zero temperatures, low annual precipitation (100–300 mm mostly in form of snow), intense radiation load, varying moisture levels and low partial pressure of gases. Vegetative part of L. latifolium is present for about 7–8 months, unlike other wild plants of the region that shows growth for about 3–4 months (Pal Murugan et al. 2010). In Ladakh Himalayas, leaves of this plant are extensively used as garnish. In a taste and preference study, its local receipe called ‘shangso chonma’ is preferred the most amongst all Ladakhi dishes made from ‘phytofoods’ (Pal Murugan et al. 2010). Apart from edible purposes, it has also been reported to have therapeutic properties as diuretic (Navarro et al. 1994) and anti-hypertensive (Tabassum and Ahmad 2011).

Longer vegetative growth in harsh conditions (temperatures <−20 °C during night), signifies its biochemical adaptability. Recently, studies have been initiated to harness the winter surviving capacity of this plant by identifying cold regulated genes from winter tolerant ecotype of Ladakh (Aslam et al. 2010). Rapid biochemical changes are also expected as the plant is known to have tolerance to flooding and drought (Chen et al. 2002). However, very little is known about the biochemical behaviour of this plant and its response to variable climate. Of the various known adaptive mechanisms working in plant, role of redox status in signalling and cellular homeostasis has been well worked out (Buchanan and Balmer 2005; Foyer and Noctor 2009, 2011). The content of reactive oxygen species (ROS) produced during normal metabolic functions affects this status. Photosynthetic cells contain an array of photosensitising pigments that are prone to oxidative stress, thus, becomes the major source of ROS. Any environmental cue would generate more ROS and this tilts the balance of redox status. Primarily, the responsibility of keeping check on ROS lies with enzymatic and non-enzymatic antioxidant defence. However, the maintenance of internal environment is done by major redox buffers viz., GSH/GSSG, ASC/DHA and pyridine molecules that determine the reduced/oxidized state of the cell. These low molecular weight compounds interact with several components for optimum cellular homeostasis. The role of redox in correct folding and appropriate activity of proteins have been known since long (Levitt 1962). Higher amount of GSH and its reduced state in plants has been considered as a measure of physiological robustness (Noctor and Foyer 1998). NADPH is involved in the maintenance of higher GSH:GSSG ratio through the action of glutathione reductase. Pyridine nucleotides i.e., NAD and NADP play a crucial role in numerous redox reactions and thus virtually influence almost every metabolic process. On the other hand, ascorbate acts as a reductant in the metabolic processes and protects cells from effects of toxic oxygen derivatives.

The present study was therefore envisaged to understand mechanism of redox homeostasis and antioxidant defence as measure of its biochemical adaptation. It is hypothesized that greater tolerance to extreme environment of this plant could only be possible by responsive redox regulating elements. Another pertinent question that has been posed here is whether this expected biochemical plasticity has some role in its ecological plasticity as well. We intend to understand this by comparing its redox and antioxidant mechanism in non-native environment with an aim to identify the elements mediating this response. Our idea is not to demonstrate newer mechanisms of biochemical adaptations but to understand the mechanistic behaviour of this ecologically important plant, which can be further extrapolated to its widespread presence. As may be expected from its closeness to Arabidopsis (both belong to the lineage I clade of the Brassicaceae family), any similarities and variations in known biochemical and physiological changes during adaptation would be interesting.

Materials and methods

Plant material

Seeds were collected from native wild ecotype of Lepidium latifolium Linn. growing around Skara nursery (Badami Bagh) in Leh town (34°10′N, 77°40′E, 3,505 m asl) in 2010. Seeds were grown in the same location for the next season to carry out the experiments. Concomitantly, same seeds were also grown in experimental farm of Indian Institute of Integrative Medicine in Jammu (32°43′N, 74°54′E, 305 m asl). All the measurements on redox were performed on third leaf from the top whereas, other biochemical measurements were performed on the forth leaf from top. Leaf material was collected on a bright sunny day at 10.00 h and immediately stored in liquid nitrogen till further analysis. The study was conducted on plants at both the sites at full vegetative growth (90 days after sowing) during growing period of 2011.

Antioxidative enzyme assays

Enzymes were extracted by homogenizing 100 mg leaf tissue in 1 ml of ice-cold extraction buffer and the homogenate was centrifuged at 14,000 rpm for 15 min. The supernatant was used for various enzyme assays. For enzyme activity of glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) extraction buffer included 100 mM Tricine (pH7.5) containing 1 mM EDTA, 5 % polyvinylpolypyrrolidone (PVPP), 20 % glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM dithiothreitol (DTT). For catalase, ascorbate oxidase (AO) and ascorbate peroxidase (APX), 50 mM phosphate buffer (pH 7.0) was used containing 0.5 % Triton X-100, 1 mM PMSF and 5 % PVPP. For APX, 2 mM sodium ascorbate was added just prior to crushing. Superoxide dismutase was homogenized in 50 mM phosphate buffer (pH 7.8) containing 2 mM EDTA, 1 mM PMSF, 0.5 % Triton X-100 and 5 % PVPP.

SOD was assayed and quantified according to nitro blue tetrazolium reduction assay modified earlier (Vyas and Kumar 2005a). 50 % inhibition in reduction caused by crude extract was considered as one unit of SOD activity. For measuring catalase activity, consumption of H2O2 was monitored at 240 nm for 5 min and expressed as mmol H2O2 decomposed min−1 in the reaction mixture (Abrol et al. 2012). Reaction mixture for APX consisted of 50 mM phosphate buffer (pH 7.0), 20 μL enzyme extract, 10 mM ascorbate, 100 mM H2O2 and decrease in absorbance was continuously monitored at 290 nm for 5 min (ε = 2.8 mM–1 cm–1). APX activity was defined as mmol of ascorbate oxidized min−1. Reaction mixture for glutathione reductase consisted of 50 mM Tris-Cl buffer (pH 7.8), enzyme extract, 50 mM DTNB, 10 mM NaDPH and 100 mM GSSG and A412 was taken after 30 min of incubation at RT (ε = 13.6 mM–1 cm–1) as described by Smith et al. (1988). GR activity was defined as total mmol of DTNB reduced after 30 min. AO activity, defined as mmol of ascorbate oxidized min−1, was determined from the decrease in A265 at 25 °C in a reaction mixture containing 0.1 M sodium phosphate (pH 5.6), 0.5 mM EDTA, and 100 μM of ascorbate (ε = 13.6 mM–1 cm–1) as described by Nakano and Asada (1981). MDHAR assay buffer comprised of 50 mM Tris buffer (pH 7.6) containing 1 mM EDTA, 200 μM NADH, 125 μM sodium ascorbate and enzyme extract. Reaction was started using 5 μL of AO (50 U/ml) and the oxidation of NADH was followed at 340 nm (ε = 6.22 mM–1 cm–1). MDHAR was defined as mmol of NADH oxidized min−1 using crude extract. GSH-dependent DHAR activity was measured in assay mixture containing 50 mM phosphate buffer (pH 7.0), 20 μL crude extract, 0.1 mM EDTA, 2.5 mM GSH and 200 μM dehydroascorbate. Ascorbate formation was followed at 280 nm (ε = 7.83 mM−1 cm−1) and enzyme unit was defined as mmol of ASC formed min−1 in the reaction mixture. Protein content in leaf tissue was calculated following the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard.

Native PAGE analysis

Native polyacrylamide gels (10 %) were run for all the different enzymes at 4 °C using Tris-glycine (pH 8.3) as running buffer. The enzymes were extracted as described above, and equal amount of crude protein per fresh weight basis (30 μL) was loaded on to the gels. After completion of the gels, the staining was performed for different enzymes and Rf of each band was calculated. Gels for SOD and APX were stained using standard protocol as described earlier (Vyas and Kumar 2005b). AO was stained essentially as described for APX except that H2O2 was not added to the solution. GR and DHAR were stained exactly as described by Aravind and Prasad (2005). Catalase was stained using potassium ferricyanide (1 %) and ferric chloride (1 %) that forms Prussian blue precipitate (Woodbury et al. 1971).

Redox metabolites

Glutathione was measured using coupled recycling assay as described initially by Tietze (1969) with some modifications. 500 μL acid crushed supernatant was neutralized (pH 5–6) with 0.2 N NaOH. 20 μL of neutralized sample was incubated in assay buffer consisting of 0.2 M NaH2PO4 (pH 7.5), 0.5 mM EDTA, 0.5 mM NADPH and 0.6 mM DTNB. Reaction was initiated by addition of 0.2 units of glutathione reductase and increase in A412 was monitored for 5 min to obtain GSH. To obtain total glutathione, 100 μL of neutralized sample was pre-incubated with 10 μL of 25 mM DTT in 0.1 M NaH2PO4 (pH 7.5) for 30 min at 25 °C. GSSG was calculated by removing GSH from the total glutathione. Alternatively, pre-treatment of extract aliquots with 2-vinylpyridine was used to get the oxidized form. Experimental rates were derived from standard curve made for 0–500 pmol of GSH using initial change of rate. Neutralized samples described above were also used to assay reduced form of ascorbate using ascorbate oxidase as described by Queval and Noctor (2007). Total ascorbate was assayed using pre-incubation of extract using 2.5 mM DTT. Oxidized form of the ascorbate was calculated by subtracting reduced ascorbate from the total ascorbate content.

Different forms of NAD+(H) and NADP+(H) was measured based upon preferential acid–alkali destruction of reduced-oxidized forms using plate reader (Queval and Noctor 2007). For NAD, the assay buffer consisted of 0.1 M Hepes (pH 7.5), 2 mM EDTA, 0.12 mM DCPIP, 2 mM PMS and 25 units alcohol dehydrogenase (ADH). Reaction was started using appropriate volume of absolute ethanol. For NADP, the assay buffer had 1 mM glucose 6-phosphate and 2 units of glucose 6-phosphate dehydrogenase instead of ethanol and ADH.

Calculation of redox potential

Redox potential of individual redox buffers were calculated using Nernst equation as described by Schafer and Buettner (2001).

$${\text{Half cell redox potential }}\left( {E_{\text{hc}} } \right) = E_{\text{m}} - \left( {RT/nF} \right) \, \left( {\ln \, \left( {\left[ {\text{Red}} \right]/\left[ {Oxi} \right]} \right)} \right)$$

where in, R is the gas constant (8.315 J K−1 mol−1), T is the absolute temperature (298.15 K), n is the number of transferred electrons, and F is the Faraday constant (9.648 × 104 C mol−1). Em is the standard midpoint potential of redox buffers for standard conditions at pH 7 (Noctor 2006). The density of water (1 g mL−1) and assumption that leaves contain 100 % water were used for calculations of molar concentrations. In case of glutathione, however, squared value of the reduced form was used for calculations.

Thiol estimation

Thiols were estimated using DTNB-based assay that allows measurement of resultant 2-nitro-5-thiobenzoic acid (TNB) at 412 nm. In order to expose the internal thiol groups, sample was first denatured with 0.1 M HCl and, further neutralized using 0.2 N NaOH before use. Samples were incubated for 30 min at RT in assay mixture consisting of 0.2 M phosphate buffer (pH 7.5), 6 mM EDTA and 0.6 mM DTNB. Total thiol content was calculated from standards prepared by taking GSH concentrations from 0 to 5,000 nmol. Free thiols were estimated by supernatant with equal volume of 20 % TCA, followed by the centrifugation at 14,000g for 10 min at 4 °C for the removal of bound thiols. The supernatant was then neutralized and estimated for free thiols as mentioned above.

In vitro feeding experiment

To study the effect of GSH mediated redox changes on induction/expression of antioxidant enzymes, detached leaves were incubated for 16 h in 10 mM MOPS buffer (pH 6.0) containing 10 mM GSH or 5 mM H2O2. Native staining for SOD and GR were performed on the treated leaves for their expression as described above. The densitometry analysis of the gels was performed using Alpha DigiDoc 1201 software (Alpha Innotech Corporation, CA, USA) and total integrated density values of prominent visible bands for each gel were calculated.

Other biochemical estimations

In order to corroborate the redox status of the plants to two different environments, DPPH radical scavenging assay and reducing power assay were performed as described earlier by our group (Guleria et al. 2011). The damage incurred to cells by radical mediated lipid peroxidation was also assessed using malondialdehyde content following exposure to thiobarbituric acid (Heath and Packer 1968). Other biochemical parameters like sugars, chlorophyll and phenols were measured using standard procedures as described by Sawhney and Singh (2009). Elemental carbon, hydrogen, sulphur and nitrogen in soil samples were estimated using Vario EL Ver III from elemental analysensysteme, GmbH, Germany using thermal conductivity detector. All the experiments were repeated independently for at least three times with different plants. Experimental data of all parameters were analyzed using two-way classification method and significant differences at p ≤ 0.05 were determined (Snedecor and Cochran 1989).

Results

Environmental conditions found at two places were remarkably different (Table 1), specifically in terms of temperature and light intensities. The light intensities in Leh were 500–700 μmol m–2 s−1 higher than their corresponding values in Jammu (1,100–1,600 μmol m–2 s–1). Similarly, the maximum temperature during the growing season in Leh was 21.6 °C, whereas, in Jammu it was found to be 34.6 °C. Out of all, soil elemental profile from two locations showed distinct difference in sulphur content that was found to be 0.003 % in Jammu as compared to 0.058 % in Leh. Plants grown in Jammu were found to contain 71.79 % lower amount of H2O2 (Fig. 1b) and therefore, formation of MDA associated with lipid peroxidation was also found lower by 34.44 % than in Leh (Fig. 1a). DPPH radical scavenging activity and reducing power equivalents were found 22.02 and 62.5 % higher at Leh than Jammu (Fig. 1c, d).

Table 1 Environmental and edaphic conditions at two experimental sites where Lepidium latifolium plants were grown for adaptability studies
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Fig. 1
figure 1

Effect of two contrasting growth environment at Leh (3,505 m) and Jammu (305 m) on MDA content (a), hydrogen peroxide generation (b), radical scavenging activity (c), and reducing power (d) in leaf extract of Lepidium latifolium. All the values are mean of 3–5 independent readings. *statistically significant values at p ≤ 0.05 as determined by least significant difference test

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All antioxidative and redox regulating enzymes except SOD showed significantly (p ≤ 0.05) higher activities at Leh than at Jammu (Table 2). In gel assay of these enzymes suggest that there is differential expression of various isozymes at both the places (Fig. 2). Out of six isozymes of SOD, at least two (with Rf 0.20 and 0.27; Fig. 2a) showed expression in either one or the other location. These two isozymes belong to Mn class of SOD’s as they were found to be active even after 5 mM H2O2 treatment. H2O2 metabolizing enzymes, catalase and ascorbate peroxidase, showed only one isozyme at Rf 0.2 and 0.43, respectively, and higher level of expression at Leh (32 % over Jammu) without any qualitative change (Fig. 2b, c). However, the enzymes that affect the redox status of main buffers i.e. ascorbate and glutathione showed significant qualitative changes. Glutathione reductase that converts GSSG into GSH showed induction of a new isozyme at Leh with Rf 0.49 (Fig. 2d). The expression level of other isozymes was also significantly higher in Leh than Jammu. Similarly, DHAR that converts DHA to ASC also showed induction of a new band with Rf 0.57 (Fig. 2e). Two new isozymes of ascorbate oxidase with Rf 0.70 and 0.78, were also seen to be induced at Leh (Fig. 2f). In a separate in vitro feeding experiment, SOD (Fig. 4b) and GR (Fig. 4c) isozymes also show increased intensity during GSH treatment as shown by integrated density values.

Table 2 Activities of major redox regulating, antioxidant enzymes and other biochemical parameters from leaves of 90 days old L. latifolium grown at two contrasting environments
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Fig. 2
figure 2

Zymogram pattern of various redox regulating and antioxidant enzymes in leaves grown at Leh (3,505 m) and Jammu (305 m). Arrow indicate the isozymes and their corresponding Rf. Panel represent superoxide dismutase (a), catalase (b), ascorbate peroxidase (c), glutathione reductase (d), dehydroascorbate reductase (e), and ascorbate oxidase (f). Equal amount of protein per unit fresh weight was loaded in each sample well for the purpose. These are the representative pictures of triplicate gels run for the purpose

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Fig. 3
figure 3

Metabolic content of different redox buffer in various states of oxidation and reduction. Panel represents GSH (a), ASC (b), NAD (c), and NADP (d) content in leaves of L. latifolium grown at two different environments. Closed circles represents plants grown at Leh whereas, solid triangles represents plants grown at Jammu. All the values are mean of 3–5 independent readings. *statistically significant values at p ≤ 0.05 as determined by least significant difference test

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Fig. 4
figure 4

PAGE analysis of (a) total protein under denaturating conditions, (b) SOD staining under native conditions and (c) GR staining under native conditions. Equal amount of protein per unit fresh basis (30 µl) was loaded into each well. 16-h treatment of 10 mM GSH or 5 mM H2O2 was given to the detached leaf in 10 mM MOPS buffer (pH 6.0)

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When metabolic content of all four buffer pairs were estimated, glutathione and NADP showed significant changes at these two locations (Fig. 3). Total glutathione in Jammu decreased by 37.7 %, whereas, ascorbate decreased by 16.1 % over their corresponding values in Leh (Fig. 3a, b). Pyridine nucleotides increased in their total content, with NADP showing 177.7 % increase over its content in Leh (Fig. 3c, d). Apart from pyridine nucleotides, the change in total metabolic content is primarily due to decrease in the reduced form of the corresponding buffer. The reduced form decreased by 13.1 % in case of ascorbate whereas, it decreased by 42.1 % in glutathione. Table 3 shows that the reduction state of glutathione and NADP was found 7.7 and 23.7 % higher at Leh than Jammu, respectively. Similarly, physiological redox potential of glutathione in plants grown at Leh was found to be about −145.95 mV, which was 12 mV higher than its corresponding value at Jammu (−133.99 mV). Plants showed higher absolute value (3.80 pmol mg−1) of total thiols at Leh which was 39.3 % higher than in Jammu (Fig. 5). However, the percentage of bound to free thiol was significantly (p ≤ 0.05) higher at Leh (0.65 %) than Jammu (0.33 %).

Table 3 Observed reduction state and calculated redox potential of GSSG/GSH and NADP+/NADPH buffer in L. latifolium grown at Jammu (305 m) and Leh (3,505 m)
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Fig. 5
figure 5

Status of total, free and protein bound thiols in leaf extracts of L. latifolium. Line with solid circles represent plants grown at Leh (3,505 m) whereas, solid bars represents plants grown at Jammu (305 m). All the values are mean of 3–5 independent readings. *statistically significant values at p ≤ 0.05 as determined by least significant difference test

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Discussion

Plants require controlled input and withdrawal of energy and reducing equivalents for biomass production and consumption. This imparts a key role to redox status in regulation of metabolism in plants especially during stress (Scheibe et al. 2005; Kornas et al. 2010). It is therefore hypothesized that plants with increased low temperature tolerance would have efficient redox mechanism that caters metabolism and signalling response for optimum growth and development. First objective of this study was, therefore, to evaluate environmental effect on plant growth and development during growing season at both the locations. 96.2 % higher formation of MDA at Leh than Jammu suggested that Lepidium plants growing at higher altitudes indeed undergo secondary ‘photo-oxidative stress’. As H2O2 content in Leh showed 3.5 times higher value, this has been linked to enhanced availability of ROS. Increased free radical content would be a consequence of a compromised radical scavenging activity of the plant, which results in an oxidized cellular environment. On the contrary, both DPPH radical scavenging activity and reducing power equivalents were higher at Leh (Fig. 1); evident of an efficient antioxidant machinery and redox metabolism that keep environment highly reducing.

Differential expression of isozymes is an adaptive feature of L. latifolium

Higher activities of antioxidative and redox regulating enzymes at Leh suggest that plants experience stress during those conditions. Superoxide dismutase that acts on first product of free radical generation (O2 .−) showed relatively higher activities at Jammu (Table 2). Since H2O2 is inhibitor of Cu/Zn SOD, its accumulation is bound to have inhibitory effect on the total activity of SOD at Leh. In fact, we observed lower intensity in band of Rf 0.55 at Leh supporting this (Fig. 2a). Induction of specific isozymes of various antioxidant enzymes has been suggested as the mechanism of adaptation to various stresses (Karpinski et al. 1997; Anderson et al. 1995; Vyas and Kumar 2005a). It is now well established that apart from increased expression of constitutive isozymes, plants also induce new isozymes that have altered kinetics to counter the new environment (Rao et al. 1995; Chronopoulou et al. 2012). Similarly, in L. latifolium two key redox regulating isozymes are induced in Leh that may well be an adaptive strategy during oxidizing environment. Metabolic trade-offs have been earlier reported in plants to conserve the metabolic energy especially during stress (Fendt et al. 2010; Abrol et al. 2012). Increase in phenolics and carotenoids in plants growing at Leh could also be the result of trade-off of energy towards their synthesis as a strategy of countering high light intensities (Table 2).

L. latifolium has a responsive NADP-Glutathione interaction for redox toning

Low temperatures in combination with high light intensities provide ample opportunity of photo-reduction of O2 through Mehler reaction (Mehler 1951). Higher photon flux radiation and temperature variation (Table 1) could play an important role in limiting NADP+. Significant decline (74.6 %) in the availability of NADP+ was observed at Leh than Jammu (Fig. 3d). Since electron transport is coupled with ATP production, the concept of ‘redox poising’ in improving photosynthetic productivity has now been established by several studies (Kramer and Evans 2011; and references thereof). Any change in ATP requirement is mainly met by activation of cyclic electron flow around PSI (Golding et al. 2004). The activation of PSI, has been linked to decreased ratio of NADP+/NADPH (Rajagopal et al. 2003). This ratio is almost half in Leh (0.46) as compared to Jammu (0.81) (Table 3). This low ratio indicates that as an alternative strategy more number of PSI centres is getting activated at higher altitude in L. latifolium, which acts as its protection mechanism during high light intensities. Activation of PSI has associated advantage in generation of proton gradient utilizing PGR5 pathway that helps in inducing thermal dissipation when Calvin-cycle activity is reduced, thereby preventing PSI inhibition (Munekage et al. 2004). Acidification of lumen by buildup of proton motive force also activates photo protective energy-dependent excitation quenching (qE) response (Muller et al. 2001).

Unlike ascorbate whose oxidized form DHA is largely confined to apoplastic space, glutathione plays a crucial role in maintaining redox potential of cytosolic fraction in plant cell (Noctor 2006). For maintaining the reduced environment in cytosol especially under extreme conditions, the redox potential needs to be more negative (Noctor et al. 2012). In the present study, it was found that plants grown at Leh had 12 mV more negative value than Jammu. These values are achieved by large changes in absolute concentration of glutathione, which was about 60 % more at Leh than Jammu (Fig. 3a). Higher negative potential at Leh also explains the more reducing power equivalents (Fig. 1d). There is large variation in literature on the redox potential values (Kranner et al. 2006), which largely depends upon the pH, ratio of reduced/oxidized forms, developmental stage and the physiological condition of the plant. Lower values of glutathione redox potential obtained in this study may have been the result of a particular developmental stage and assumed water content. It has been suggested that rather than the absolute value, ±8–30 mV change in redox potential is important in propagating a cellular signal (Hancock et al. 2004, Jubany-Mari et al. 2010) and could have large physiological significance.

In accordance with the available literature that suggests increase of ascorbate content under high irradiances (Grace and Logan 1996; Smirnoff 2000), L. latifolium also showed its higher metabolic content (Fig. 3b). Although the overall increase or that of the reduced form may not be to the tune of glutathione, it has been observed that ASC/DHA ratio is more important in terms of regulation (Foyer and Noctor 2011). There is a possibility that in Leh, significant amount of ascorbate has been used as cofactor for enzymes such as violaxanthin de-epoxidase, which is involved in xanthophylls cycle-mediated photoprotection (Eskling et al. 1997). Also, enzymes for antioxidative defence (ascorbate peroxidase), biosynthesis of plant hormones (ABA, GA, ethylene) and anthocyanin pathway utilize ascorbate for their activity. Quality and period of light is also suggested to have effect on synthesis and regeneration of ascorbate (Bartoli et al. 2009). There is no significant difference in the reduction state of ascorbate pool at both the locations (Fig. 3b), as was also seen for total content and reduction state of NAD (Fig. 3c).

Biochemical plasticity is maintained by varying thiol concentrations in L. latifolium

Higher intracellular glutathione in L. latifolium (Fig. 3a) at Leh could induce antioxidant status and other related gene expression through ascorbate–glutathione cycle as reported in Arabidopsis mutants and tobacco (Gomez et al. 2004; Colville and Smirnoff 2008). In a separate experiment, GSH feeding has been shown to cause higher expression of proteins and antioxidative enzymes. When isolated leaves of L. latifolium, were treated with GSH or H2O2, GSH increased the amount of total protein as observed with SDS-PAGE (Fig. 4a). SOD (Fig. 4b) and GR (Fig. 4c) isozymes also showed increased activity during GSH treatment than H2O2 as shown by integrated density values. These results suggest that increase in the activities of antioxidant enzymes is not a direct consequence of H2O2 accumulation, but is mediated through GSH in intracellular environment of L. latifolium (Foyer and Noctor 2011). Earlier, significant role of GSH status is observed in transmitting a subset of signals derived from intracellular H2O2 as observed by gr1 mutation in cat2 background (Mhamdi et al. 2010). One of the reasons for its preference over ascorbate, which is most abundant soluble couple, could be the requirement of significant amount of carbon source in ascorbate synthesis (Yabuta et al. 2007). Few high molecular weight SOD’s do not appear in isolated leaves after 16 h, even in control. The newly induced/expressed isozymes are expected to function under changed redox environment, and therefore, found to have different thiol status. Difference in the protein bound thiols at two locations suggests that the differentially expressed proteins (isozymes of different antioxidative enzymes in this case) could act as redox input elements, targets and signals.

Our results suggest that L. latifolium has a responsive redox homeostasis which is achieved through GSH and/or NADPH redox couples. This helps the plant against low temperature-mediated photo-oxidative damage by activating more PSI reaction centres and channelizes their metabolic energy through the most efficient route. The adjustment of redox status, leads to induction of proteins with higher thiol content suggesting their increased dependence on redox regulation. There could be other factors such as soil quality, seed dispersal, their germination and other biotic considerations (associated microbiome, pests and herbivores) that are important for long-term adaptation under different environments. Significant variations were observed in the properties of soil of the two locations (Table 1). This could probably explain its restricted distribution in at least this part of Himalayas. Further, research is required to identify each of these interactions to understand its geographical distribution.

Author contribution

D.V. and S.K. designed the research; T.K., H.A.B. and A.R. performed the research; T.K. and D.V. analyzed data, and D.V. wrote the paper.