Drought and salinity induced changes in ecophysiology and proteomic profile of Parthenium hysterophorus

Parthenium hysterophorus is a plant that tolerates drought and salinity to an extremely high degree. Higher expression of stress-responsive proteome contributes for greater defence against abiotic stresses. Thus, P. hysterophorus could be a rich source of genes that encode stress-imparting mechanisms and systems. The present study utilizes comparative physiological and proteomic approaches for identification of key proteins involved in stress-defence of P. hysterophorus. Thirty-days-old plants were exposed to drought (10% PEG 6000) and salinity (160 mM NaCl) for 10 days duration. Both stresses induced oxidative stress estimated in terms of TBARS and H2O2. Levels of both enzymatic and non-enzymatic antioxidants were elevated, more by drought than salinity. Particularly, SOD, GR, CAT and GST proved to be assisting as very commendable defence under drought, as well as salinity. Levels of ascorbate, glutathione and proline were also increased by both stresses, more in response to drought. Comparative proteomics analysis revealed a significant change in relative abundance of 72 proteins under drought and salinity. Drought and salinity increased abundance of 45 and 41 proteins and decreased abundance of 24 and 26 proteins, respectively. Drought and salinity increased and decreased abundance of 31 and 18 proteins, respectively. The functions of identified proteins included those related to defence response (26%), signal transduction (13%), transcription and translation (10%), growth and development (8.5%), photosynthesis (8.5%), metabolism (7%), terpenoid biosynthesis (5.5%), protein modification and transport (7%), oxido-reductase (4%) and Miscellaneous (11%). Among the defence related proteins, antioxidants and HSPs constituted 26% and 21%, respectively. Present study suggests a potential role of defence proteins. Proteins involved in molecular stabilization, formation of osmolytes and wax and contributing to stress-avoiding anatomical features emerged as key and complex mechanisms for imparting stress tolerance to P. hysterophorus.


Estimation of magnitude of oxidative stress
The level of oxidative damage in terms of peroxidation degradation products in the leaf was measured by the method of Heath and Packer [15]. The thiobarbituric acid reactive substances (TBARS) were estimated as indicator of magnitude of oxidative damage in 1.0 g of fresh leaf which was ground in liquid nitrogen and added to 10 ml of 1% (w/v) trichloroacetic acid (TCA). The homogenate was spun at 6,708 x g/10 min. To 1.0 ml of the supernatant 4.0 ml of 0.5% (w/v) thiobarbituric acid (TBA) was added and the mixture was placed in a water-bath for 30 min at 99˚C followed by a quick cooling in ice bath. The volume was then made to 5.0 ml by adding DDW, and spun at 2,795 x g/5 min. The optical density (OD) of the supernatant was read at 532 nm and corrected for any unspecific turbidity by subtracting the OD value recorded at 600 nm on a UV-Vis Spectrophotometer (DU-64, Beckman, USA).

Estimation of hydrogen peroxide (H 2 O 2 )
Content of H 2 O 2 was estimated by the method of Yu et al. [16]. Leaf sample (0.5 g) was ground and mixed with potassium-phosphate buffer (3 ml, 50 mM, pH 6.5)/4˚C. The homogenate was spun at 9,660 x g/20 min/4˚C. Supernatant (1.5 ml) was mixed with 0.5 ml of titanium chloride solution (0.1% w/v TiCl 4 in 20% v/v H 2 SO 4 ) followed by an incubation at room temperature for 10 min. This mixture was again spun at 9,660 x g for 10 min. Optical density of supernatant was read at 410 nm to estimate H 2 O 2 concentration.

Extraction and analysis of antioxidant enzymes
Leaf sample (0.5 g) was homogenized using 5 ml of ice-cold phosphate buffer (100 mM, pH 7.2, 1 mM EDTA, 2.5 mM DL-dithiothreitol, 0.5% (v/v) Triton X-100 and 4% (w/v) insoluble polyvinyl polypyrolidone). The homogenate was centrifuged at 9,660 x g for 30 min, and the supernatant was stored in separate aliquots at -80˚C prior to enzymatic analysis. Enzyme extraction was done at 4˚C and enzyme assays were performed at 25˚C.
Superoxide dismutase assay. Superoxide dismutase (SOD) assay was performed by the method of Dhindsa et al. [17]. SOD's ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) was estimated in the supernatant by reading at 560 nm and SOD activity was expressed as EU min -1 mg -1 protein. Ascorbate peroxidase assay. Ascorbate peroxidase (APX) assay was performed as modified by Qureshi et al. [18]. APX assay was performed in reaction mixture (100 mM K-phosphate buffer, pH 7.4, 0.5 mM ascorbate, 0.3% (v/v) H 2 O 2 and 100 μl enzyme extract in a total volume of 1 ml by following ascorbate consumption at 290 nm. APX activity was expressed as EU min -1 mg -1 protein.
Glutathione reductase assay. Glutathione reductase (GR) activity was assayed by the method of Foyer and Halliwell [19]. The reaction was initiated by adding 100 μl enzyme extract to 0.9 ml of reaction mixture (100 mM K-phosphate buffer, pH 7.2, 0.02 mM GSSG, and 0.2 mM NADPH) performed for 5 min. The assay was initiated with the addition of 0.2 ml of enzyme extract and the activity was determined by monitoring the glutathione-dependent oxidation of NADPH. The activity of GR was calculated by using extinction coefficient 6.2 mM -1 cm -1 . Enzyme activity was expressed as EU min -1 mg -1 protein.
Catalase assay. Catalase (CAT) assay was performed by the method of Aebi [20]. The reaction mixture of final volume 1 ml comprised of 50 mM Na-phosphate buffer (pH 7.0), 20 mM H 2 O 2 and 100 μl enzyme aliquot. A decrease in the absorbance of H 2 O 2 was followed at 240 nm for 5 min. CAT activity was expressed as EU min -1 mg -1 protein.
Glutathione S-transferase assay. Glutathione-S-transferase (GST) activity was assayed by the method of Habig [21] using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The enzyme activity was assayed in a reaction mixture containing 1.65 ml of phosphate buffer (0.1 M, pH 6.5), 0.2 ml 1mM GSH (freshly prepared in 0.1 M phosphate buffer pH 6.5), 100 μl of leaf extract, 50 μl of 1 mM freshly prepared CDNB. The solution was vortex mixed and the volume was made up to 10 ml with distilled water in a final volume of 20 ml. The increase in absorbance corresponding to an increase in CDNB-conjugate formed was recorded at an interval of 30 s for 3 min at 340 nm. Results were expressed as nmol of CDNB conjugated formed min -1 mg -1 protein by using the molar extinction coefficient of 9.6 x 10 3 M -1 cm -1 .

Non enzymatic antioxidants
Ascorbate estimation. Ascorbate was estimated by the method of Law et al [22]. Fresh leaf material (0.1 g) was ground to a powder in a mortar-pestle using liquid nitrogen. The powder was homogenized in 2 ml of extraction buffer and centrifuged at 6,708 x g for 10 min. To 400 μl of supernatant, 10% (w/v) TCA (200 μl) was added. The mixture was vortex mixed and cooled in ice for 5 min and 10 μl of 5M NaOH was added, and spun again at 959 x g for 5 min. The supernatant (200 μl each) was used for the assay of total (ASA + DHA). For the estimation of total ascorbate a 100 μl dithiothretol (DTT) and 200 μl of reaction buffer (150 mM K-phosphate buffer, pH 7.2) was added to the assay mixture and incubated at 25˚C for 15 min. After incubation, 100 μl of 0.5% N-ethylmelamide was added. To other tube 200 μl of reaction buffer (150 mM K-phosphate buffer, pH 7.2) and 200 μl of double distilled water was added. Both samples were vortex-mixed and incubated at room temperature for 30 seconds. To each tube was then added 400 μl of 10% (w/v) TCA, 400 μl of 44% (v/v) H 3 PO 4 , 444 μl of 4% (w/v) bipyridyl and 200 μl of 3% (w/v) FeCl 3 . After vortex-mixing, samples were incubated at 37˚C for 60 min and the absorbance was recorded at 525 nm. The amount of ascorbate (nmol g -1 fresh weight) was estimated against the standard curve of ascorbic acid (10-100 μM).
Glutathione estimation. Glutathione content was estimated by the glutathione recycling method of Anderson et al [23] which measures total glutathione (GSH+GSSG) through a reaction catalysed by glutathione reductase (GR). Leaf (0.5 gm) was homogenized in 2 ml of 5% (w/v) 5-sulphosalcylic acid to control the oxidation of GSH. The assay mixture was read at 412 nm (30˚C for 5 min) after adding the extract. The reaction blank was prepared by replacing the plant extract with 5% 5-sulphosalcylic acid. To the same tube 0.2 units per assay glutathione reductase from yeast and 50 μl of 0.4% (w/v) NADPH was added and the reaction was allowed to run for 30 min at 25˚C and the absorbance at 412 nm was monitored for total glutathione. An actual value of glutathione content was determined against GSH standard curve (10-100 nmol) and was expressed as nmol g -1 FW.
Proline estimation. Proline content in leaf samples was estimated by the method of Bates et al [24] using ninhydrin reagent. The toluene (upper) layer was read at 520 nm. The corresponding concentration of proline was determined against the standard curve of L-proline and expressed as μmol g -1 FW.

2D proteomics of Parthenium hysterophorus
One of the major objectives of the present study was to explore the changes occurring in P. hysterophorus at the proteome level caused by drought and salinity. An overall strategy adopted for proteomic analysis in this study has been depicted in a schematic chart (Fig 2).
Protein extraction and sample preparation. Protein from leaf tissue was extracted as mentioned in Watson et al [25]. Leaf samples were frozen in liquid nitrogen and ground to a fine powder with the help of pre-chilled pestle and mortar. Leaf powder was homogenised in 40 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.07% (v/v) β-mercaptoethanol, 2% (w/v) PVP, 1% (w/v) PMSF and 1% (v/v) TritonX100. The homogenate was centrifuged at 13,148 x g for 30 min at 4˚C. The supernatant was mixed in 1:2 ratio with 10% (w/v) TCA and 0.07% (v/v) βmercaptoethanol and left at -20˚C overnight. The mixture was centrifuged at 8,150 x g for 15 min at 4˚C to yield a protein pellet. The pellet was washed with chilled acetone containing 0.07% (v/v) β-mercaptoethanol and 2 mM EDTA. Pellet was incubated with 100% (v/v) chilled acetone for 5 h at 4˚C and then vacuum dried. This pellet was then used for subsequent 1-dimensional (isoelectric focussing) and 2-dimensional (SDS-PAGE) gel electrophoretic experiments as described ahead.
For two-dimensional gel electrophoresis, protein pellet was dissolved in a solubilisation cocktail [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM DTT, 40 mM Tris-base and 2% Pharmalyte 1 (v/v) pH 4-7 NL]. After 1 hour of gentle stirring at ambient temperature, samples were centrifuged at 8,150 x g for 15 min at 4˚C and pellet was discarded. Immediately prior to sample loading on IPG strips, concentration of protein was determined using Bradford reagent.
2D IEF/SDS-PAGE and protein staining. Isoelectric focusing (IEF) was accomplished as described by Schlesier and Mock [26]. An equal amount of protein quantified using Bradford reagent for IPG strip was used for 1 st dimensional run. Immobilized pH gradient (IPG) strips (ReadyStrip™, Bio-Rad, USA) of 11 cm (pH 4-7 NL) were passively rehydrated overnight with 200 μg of protein. Isoelectric focusing of proteins was performed using the following programme of electric current flow till 70000 volt hours (Vh) were achieved (S1 Table).
Equilibrated IPG strip was loaded onto 12% (w/v) SDS polyacrylamide gel containing protein ladder (MW 14,300 Da-97,400 Da). The strip was sealed on gels with 0.5% (w/v) agarose containing bromophenol blue as tracking dye. Separation in the second dimension was performed at a constant current of 25 mA per gel and 13˚C using a protean II (Bio-Rad, USA) with upper (containing 1% w/v SDS) and lower tank electrode buffers.
At completion of electrophoretic run, gels were removed from electrophoresis assembly and washed 2-3 times with DDW and incubated in colloidal CBB or Blue Silver [27] stain [containing 10% (v/v) 0-phosphoric acid, 10% (w/v) ammonium sulphate, 0.12% (w/v) CBB (G250) in MeOH overnight for staining of proteins. After staining, the gels were placed in DDW for destaining till the back ground of the gels become clear and protein spots were clearly visible.
Image analysis. Images of the gel were digitised using gel documentation system (Bio-Rad, USA) for further analysis based on spot density, relative abundance and location (for pH and mass). The image analysis was performed with image master PDQuest software (Version 8.0, Bio-Rad, USA). The optimized parameters were as follows: saliency 2.0, partial threshold 4 and minimum area 50. The intensity of the spots was normalized to that of land mark of proteins used for internal standardization. Spots were quantified on the basis of their relative volume, which was determined by the ratio of the volume of a single spot to the whole set of spots. Only those spots which showed significant quantitative changes 1.5 or more fold in abundance used for further analysis.
In-gel digestion and protein identification. The protein spots from 2D gel of interest were pricked with the help of sterilized surgical syringe and micro tips, in the form of small gel pieces containing protein spots for in-gel digestion by trypsin [28]. These pieces were washed with 100 μl ultra pure water and centrifuged at 151 x g for 20 min and washed again in same manner. To this 50-100 μl of 50% (v/v) acetonitrile was added and centrifuged at 151 x g for 20 min at 22-24˚C followed by repetition of this step. To this, 5 μl of 1M DTT and 49.5 μl of ammonium bicarbonate (20 mM) were added and incubated at 56˚C for 45 min in acetonitrile (ACN). Acetonitrile was discarded and to this 40 μl IAA (55 mM) was added and centrifuged again at 151 x g/22-24˚C. Then 50% (v/v) ACN was added by discarding IAA and centrifuged at 151 x g/24˚C. ACN was again discarded and 100% (v/v) ACN was added followed by centrifugation at 176 x g for 20 min. A pellet was obtained which was vacuum-dried. The samples were incubated with 20 μl of 1% (w/v) trypsin prepared in 20 mM ammonium bicarbonate and kept overnight at 37˚C. After overnight protein digestion, samples were centrifuged at 176 x g for 20 min and supernatant was separated in another tube and 1% tetra-fluoroacetic acid (TFA) prepared in 50% (v/v) ACN was added for mass spectrometric analysis.
The digested proteins in terms of peptides or peptide mass finger prints of differentially expressed proteins were collected and analyzed on an ABI 4800 MALDI-TOF/TOF MS Analyzer (Applied Biosystems, USA) and protein identification (ID) was performed using the result dependent analysis (RDA) of ABI GPS Explorer software, version 3.5 (Applied Biosystems, USA). Some of the crucial parameters set were as follows: Digestion enzyme: trypsin with one missed cleavage; MS (precursor-ion) peak filtering: 800-4000 m/z interval, monoisotopic, minimum signal-to-noise ratio (S/N) 10, mass tolerance 50 ppm. MS/MS (fragmentation) peak filtering: monoisotopic, MH + , minimum S/N, MS/MS fragment tolerance 0.2 Da; database used: Viridiplantae (Green Plants)Swissprot database; Contaminants database were also included. During the initial MS scan, data were analyzed as peptide mass fingerprinting (PMF), and preliminary protein ID was done by searching against the database using the MASCOT (Matrix Science, http://www.matrixscience.com/cgi/search_form.pl?FORMVER= 2&SEARCH=PMF) algorithm. To evaluate the protein identification, proteins with maximum scores and nearest to the theoretical molecular weights and pI values were considered and selected for result interpretation.
Protein classification. NCBI (http://www.ncbi.nlm.nih.gov/protein) and UniProt (http:// www.uniprot.org) databases were used to assemble the functional information of identified proteins. On the other hand, Target P (http://www.cbs.dtu.dk/services/TargetP/)/PSORT (http://psort.hgc.jp/) and UniProt databases were used to recognize the sub-cellular location of identified proteins. Using Jiang et al. [29] convention, identified proteins were categorized on the basis of their biological functions.
Correlation analysis. To visualize the overall correlations between different proteins in response to the drought and salinity were performed by MetaboAnalyst 3.0 software. Proteinprotein correlation analysis was carried out on the whole data set of proteins using Pearson's correlation.
Statistical analysis. All experiments in present study were performed from pool of five biological and three experimental replicates. The results were explained as mean ± standard deviation (SD). The significant difference at ÃÃ P 0.05 was determined by Tukey's test.

Impact of drought and salinity on oxidative stress in P. hysterophorus
Magnitude of oxidative stress was increased by both drought and salinity in P. hysterophorus. As compared to control plants, TBARS were 96% and 91% higher under drought and salinity, respectively 10 DAT (Fig 3A). The H 2 O 2 content increased significantly in both drought and salt stressed plant. H 2 O 2 content increased 46% by drought and 38% by salinity at 10 DAT as compared to the control ( Fig 3B).

Impact of drought and salinity on antioxidant activities
Both drought and salinity modulated the activities of antioxidant enzymes (SOD, APX, GR, CAT and GST) in P. hysterophorus. As compared to control, activities of SOD were 73% and 68% higher under drought and salinity, respectively. Whereas, increase in activities of APX was 40% and 33%, GR was 216% and 150%, CAT was 41% and 32% and GST was 61% and 33% under drought and salinity, respectively (Fig 4A-4E).
Levels of non-enzymatic antioxidants including glutathione and ascorbate were also found increased under both drought and salinity. As compared to control, total ascorbate (AsA +DHA) and total glutathione (GSH+GSSG) content were 46% and 34% and 60% and 50% more at 10 DAT of drought and salinity, respectively (Fig 4F and 4G).

Impact of drought and salinity on proteome of P. hysterophorus
Per gel around 180 protein spots, of considerable quality, were reproducibly detected (Fig 5A-5C). Most of the proteins migrated to pH values between 4.1 and 6.9 of IPG strips and dispersed in range of Mr 100 kDa to 13 kDa. The per cent volumes (% vol) of the reproducibly detected proteins were determined and mentioned as normalized values for each individual spot against the total volume of all selected spots. Consequently, a total of 72 leaf protein spots showed significant change in response to drought and salt stress. A comparative account of relative abundance showed that 45 and 41 proteins were upregulated by drought and salinity, respectively (Fig 6) at 10 DAT. Drought down-regulated 24 proteins whereas salinity downregulated 26 proteins (Fig 6). In this study, Hsp70-Hsp90 organizing protein 3 (spots 5, and 27), Glutathione S-transferase U4 (spots 29 and 46), 19.0 kDa class II heat shock protein (spots 57 and 90) and Probable WRKY transcription factor (spots 64 and D7) were identified in more than one spot on the same gel. This phenomenon may have resulted from the presence of different protein isoforms or post-translational modification cessation [30].  GST (E) enzymes and total ascorbate (F), total glutathione (G) and proline (H) caused by drought and salinity stress. All enzymes were assayed after being extracted from leaf of P. hysterophorus exposed to drought (PEG6000) and salinity (160 mM NaCl) for 10 days and compared with control. Bars represent means ± standard deviation. (**P 0.05, Tukey's test).

Influence of drought and salinity protein relative abundance
Proteins identified were classified according to their function in the cellular processes including photosynthesis, defence, protein modification, transport, signal transduction, transcription, translation, growth and regulation, etc. (Table 1).   Table 1. List of proteins identified in P. hysterophorus leaf. Spot IDs corresponds to the labeled 2D gels (Fig 5A, 5B and 5C). The protein names, nearest matching plant species, accession number, molecular weights, and number of matched peptide are shown as fetched through Mascot and UniProtKB and presented with full details in S2 Table. A comparative account of protein relative abundance has been provided in figures at right hand side of the
Functional classification of proteins P. hysterophorus was affected by drought and salt treatments at physiological and biochemical level as seen in the protein phenotyping results. To gain an overview of the processes which were affected by the stress treatment on the protein level, differentially regulated proteins were assigned, when possible, to groups based on their biological functions. The functions included roles in defence response (26%), signal transduction (13%), transcription and translation (10%), growth and development (8.5%), photosynthesis (8.5%), metabolism (7%), terpenoid biosynthesis (5.5%), protein modification and transport (7%), oxido-reductase (4%) and miscellaneous (11%). Major portions of stress related proteins contributed by antioxidant proteins (26%) and heat shock proteins (21%). (Fig 8) (Table 1). Both drought and salinity influenced the abundance of proteins in each functional category (Fig 9). The pattern of relative change (%) of protein abundance in each category varied drought and salinity. However, change in oxido-reductase, signal transduction and growth/ development categories were higher in drought, and miscellaneous category was higher in salinity. The proteins of defence category showed a marked change in drought and salinity (Fig 9).

Discussion
Results of present study indicate that Parthenium hysterophorus certainly has strong mechanisms and better gene regulation so as to combat variety of stresses through strengthening of its proteome. Such mechanisms might help the plant become invasive weed which grows well even under extreme environments. In present study, we first time investigated the proteomic response of P. hysterophorus to drought and salinity in order to identify the possible stress response mechanisms using proteomic 2D-MS approach. The data of biochemical, antioxidant system and proteomics has been discussed in the light of existing literature.

Both drought and salinity elevates oxidative stress and antioxidants
In this study, amount of leaf TBARS was almost doubled by both drought and salinity as compared to control P. hysterophorus. Data indicate towards drought-and salinity-induced elevation in oxidative stress as reported by numerous workers [31-33].

P. hysterophorus accumulates more proline under stress
The second important feature adaptive to abiotic stress is comparatively higher accumulation of proline under negative water potentials and ionic imbalance. Proline is considered to act as an osmolyte, a reactive oxygen species (ROS) scavenger, and a molecular chaperone stabilizing the structure of proteins, thereby protecting cells from damage caused by stress [34-35]. The higher proline levels suggested that compatible solutes contribute to osmoregulation and detoxification of ROS [36].

P. hysterophorus up-regulates cellular antioxidants under drought and salinity
Cellular antioxidants scavenge and control the formation of free radicals thereby preventing oxidative damage to cellular components [18]. Plants possess a complex array of enzymatic antioxidant defence system e.g., SOD, APX, GR, GST and CAT and some non-enzymatic antioxidants such as ascorbic acid, glutathione, carotenoid, and flavonoids. SOD is the major scavenger of superoxide and plays an important role in defence against the cellular damage caused by environmental stress [31]. CAT, together with SOD, is thought to be the most effective antioxidant enzymes in preventing cellular damage [37]. A gradual increase in activity of SOD, APX, GR, GST and CAT has been reported as caused by abiotic stresses [38]. Such stressinduced increase in antioxidant enzymes indicated acclimatization of the plants to counterbalance increased oxidant generation [39]. In this study, SOD activity was increased in drought and salt stress. Similar findings were reported by other workers [40][41]. Thus, activation of antioxidant enzymes under drought and salinity indicates toward possession of strong antioxidant system by P. hysterophorus.
Both ascorbate and glutathione are essential for scavenging ROS and are important in controlling redox status under abiotic stress. Increased levels of both the antioxidants (Glutathione and Ascorbate) indicated a prominent role of these in mitigating the oxidative stress by quenching ROS, enhanced by drought and salt stress conditions in P. hysterophorus. Furthermore, P. hysterophorus showed a good correlation between the enzymatic antioxidant activities and non-enzymatic antioxidant levels which are interdependent for restoring the corresponding pools in stressed cells. Therefore, it can be well inferred that the antioxidative system in P. hysterophorus is well established and modulates efficiently to survive drought and salt stress.

P. hysterophorus modulates its proteome by increasing the abundance of key protein players in response to drought and salinity
In present study, 2D gels provided a holistic view of leaf proteome of control, drought-and salt-treated P. hysterophorus. Differential abundance of proteins in the proteome could be attributed to the proteins which might be directly or indirectly associated with plant defence to said stresses. A diagrammatic illustration of functional and regulatory networks turned on/off or up/down regulated during drought and salinity is described in Fig 10. Important proteins related to stress response are discussed below according to the contribution in stress tolerance.

Photosynthesis
Drought and salt stress reduced the ability of plant to obtain water by disturbing the osmotic, nutrient and ionic equilibrium which in turn affected the phosphorylation and activity of enzymes involved in numerous physiochemical processes such as photosynthesis and respiration [42]. Responses of photosynthetic processes under stress were notably revealed by the proteomics approach. Photolysis of water in photosystem II (PSII) is mediated by oxygen-evolving enhancer protein (OEE) consisting of OEE1, OEE2, and OEE3 subunits [43]. The Oxygenevolving enhancer protein 2 was easily detached from the photo system-II under stress conditions [44]. This protein dissociation might be repaired by the increased abundance of OEE2 to maintain photolysis of water in photo system-II [45]. The up-regulation of OEE 2 in our study is in conformity with a similar report of the salt-stress response in soybean genotypes [46].
The integrity of Photosystem I (PSI) and ferredoxin-thioredoxin reductases, both containing Fe-S clusters maintains by Fe-S cluster assembly factor HCF101 (high chlorophyll fluorescence 101) [47] which work as a chloroplast scaffold protein that facilitates the assembling of iron-sulfur (4Fe-4S) clusters and transfers them to the ferredoxin-thioredoxin and PSI [48]. Thus, we can suggest that the increased abundance of Fe-S cluster assembly factor HCF101 is one of the ways to protect the PSI and ferredoxin-thioredoxin reductases under drought and salt stress. Oxygen-evolving enhancer protein 2 (OEE2) was positively correlated with Fe-S cluster assembly factor HCF101 in present study. Thus OEE2 along with Fe-S cluster assembly factor HCF101 improved or helped in retaining the photosynthetic efficiency of plant under stress environment [49].

Cellular metabolism
Our results showed that ATP-dependent 6-phosphofructokinase, a leading enzyme of glycolysis was significantly more abundant under drought and salinity as noted by [50] in cotton root. Since fructose phosphates good scavenging activity, elevated level of phosphofructokinase helps plant in re-adjustment of carbohydrate metabolism and ROS detoxification [51]. Stress conditions lead to the higher accumulation of diverse compatible solutes those participating in osmotic adjustment of cell. Such as sucrose synthase 2 was appreciably increased in the stressed leaves of P. hysterophorus, reflecting presence of high level of sucrose which is further converted to glucose and fructose, serving as compatible solute. This study is in agreement with the finding of Zhao et al [52] in drought stressed cassava leaf. Sucrose acts as a key osmolyte and has a defensive function under stress conditions [53]. Thus increased level of sucrose synthase may possibly help in protein stabilization. Interestingly, mannitol dehydrogenase 1 was decreased in P. hyterophorus leaves under drought and salinity which might elevate level of mannitol in stressed plants and performs osmo-protection.
Malate dehydrogenase is crucial for stomatal movement, pH stability, respiration, β-oxidation of fatty acids and metabolism of C 4 plant [54]. These diverse activities of malate dehydrogenase could be helpful in P. hysterophorus to deal with drought and salt stress via high concentration of malate dehydrogenase. This observation is supported by the strong accumulation of malate dehydrogenase in soybean leaf under drought and heat stress [55].

Cellular defence
Defence proteins are key elements to acquire the tolerance in plants against diverse abiotic stresses. Small heat shock proteins (sHSPs) are big players in stress tolerance by preventing aggregation of denatured proteins [56]. sHSPs of 17.6 kDa, 18.1 kDa and 19.0 kDa were more abundant under drought and salinity and might be associated with stress tolerance [55,57]. Increased Hsp 70 and Hsp 90 organising protein 3 (HOP) might be assisting to HSP 70 and HSP 90 [58]. Activation of antioxidant system is one of the common responses of plants to abiotic stresses [59]. Our proteomic study showed a shift in glutathione S-transferase (GST) and catalase (CAT) abundance which are well known for ROS detoxification [60][61]. http://www.ncbi. nlm.nih.gov/pmc/articles/PMC4490509/-B10-ijms-16-13561 In addition, both drought and salt stress modulated the expression of MHDAR; however its activity was increased in drought and decreased in salt stressed plants. This indicates that during drought condition ascorbate regeneration is maintained by MDHAR whereas under salinity through DHAR. Additionally, trehalose phosphate phosphatase 7 was increased in P. hysterophorus under drought and salt stress that produces free trehalose by the elimination of phosphate from trehalose 6-phosphate. Protein molecules and lipid membrane are stabilized by trehalose under stress [53]. Similarly, in Arabidopsis, increased abundance of trehalose 6-phosphate phosphatase improved tolerance toward salinity stress [62].
Ferritin is a storage form of iron, located in plastids, involved and performs ROS scavenging [63][64]. This study indicates towards similar role of ferritin-1, inhibition of oxidative stress [64][65]. Another protein upregulated by stress, cysteine proteinase inhibitor, has provided a positive connection with abiotic stress responses [66]. The conversion of succinate semialdehyde to succinate is catalyzed by succinate semialdehyde dehydrogenase that may amplify the accumulation of GABA and succinate. Our results showed the upregulation of succinate semialdehyde dehydrogenase in response to drought and salt stress which were supported by the findings of Acevedo et al. [67] in Ilex paraguariensis under drought stress. Two other proteins namely desiccation-related protein and peptide methionine sulfoxide reductase B4, more abundant under drought, have defensive role against oxidative stress [68][69].
Pearson's correlation showed that GST and CAT were strongly and positively correlated with each other and both weakly correlated with MDHAR. GST and CAT were strongly and positively correlated with Hsp 19.0, desiccated related protein, ferritin, cysteine proteinase inhibitor 9, lactoglutathione lyase but negatively correlated with Hsp17.6. Protein-protein correlation of antioxidants, Hsp 19.0 and defence related proteins substantiate the strong correlation between these metabolites during stress conditions (Fig 11).

Transcription and translation associated proteins
Stress response and tolerance is a fine-tuning of plant genetic processes. The DNA-dependent RNA polymerase is the essential enzyme for the synthesis of RNA. In this study, DNA-dependent RNA polymerase was upregulated under drought and salinity. In the same way, basic leucine zipper 6 (bZIP) protein, auxin responsive protein IAA7 and LOB domain containing protein 32 were upregulated which were also crucial in stress defence due to their involvement in stress and hormone signaling pathways [70][71][72].
The photosynthesis, stress response, seed germination, flower induction, and light-mediated circadian rhythms are important events of plant life cycle. Regulation of such processes are mediated by a plant-specific transcription factor, DNA-binding one zinc finger (Dof) protein, which was up-regulated in salinity and down-regulated in drought stress, suggesting that cells make efforts to continue the above named processes during stress. WRKY 74 is a member of WRKY group III transcription factors, functions with W box (5'-(T) TGAC [CT]-3'), an elicitor-responsive cis-acting element. WRKY 74 has been known as regulator of cold stress, phosphorus and iron homeostasis [73]. In present study, expression of WRKY 74 was downregulated by both stresses however one isoform of WRKY transcription factor as WRKY 20 was observed only in drought stressed plant. WRKY 20 was recognized as a member of IId group that work together with mitogen-activated protein kinases [74]. In addition, WRKY 20 regulates ABA mediated signaling and increased the expression of genes related to wax biosynthesis of cuticle [75]. Thus we can suggest that WRKY20 works as a key player in P. hysterophorus to adapt especially in drought condition. Different transcription factors identified in current study were positively correlated with each other (Fig 11).

Signal transduction
Plants exhibit diverse molecular mechanisms in response to drought and salinity including modulation of regulatory molecules of signal transduction cascade. Such alterations possibly alleviate negative impact of stress by retaining the equilibrium between interconnected elements of physiological processes [76]. Alteration of phospho-regulation is an important adaptive management of plant during stress regulated by kinases and phosphatases. For instance, many physiological processes in plants including growth, light dependent activities, biological clock, cell cycle and hormone signaling instructed by casein kinase II [77]. In this study, casein kinase II subunit alpha-2 was up-regulated by drought and salt stress indicating signal transduction mediated homeostatic equilibrium during stress response. In this study, calmodulinlike protein 1 and probable calcium-binding protein CML15 were decreased whereas calmodulin-like protein 7 was increased in drought and salt stress. These proteins appear to help in balance of Ca 2+ concentration as per need of P. hysterophorus under stress conditions. Calmodulin-like proteins 7 was positively correlated with different identified kinases and negatively correlated with different identified phosphatases. However, calmodulin-like proteins 7 was negatively correlated with calmodulin-like proteins 1 (Fig 11).

Proteins of oxido-reductase group
Upregulation of thioredoxin H4-1 under drought and salinity may be attributed to maintain metabolism of plant. Another protein of this group, probable aldo-keto reductase has been identified as a detoxificant that effectively removes the number of lipid peroxidation products and cytotoxic aldehydes [78]. Thioredoxins H4-1 was positively correlated with GST, CAT and negatively correlated with MDHAR suggesting coordinated contribution in redox signaling in response to drought and salinity (Fig 11).

Terpenoid biosynthesis
Sesquiterpenoids show involvement in many biological properties as a component of plant secondary metabolic pathways [79]. These volatile compounds act as a precursor in the synthesis of important signaling molecules such as ABA and contribute in drought, salinity and thermo-tolerance of plants. The germacrene D synthase 1 is a main enzyme in the biosynthesis of sesquiterpenoid germacrene D which increase the sesquiterpenoids content in plants. Alpha copaene synthase enzyme was upregulated in drought and downregulated in salt treatment. These changes could be related to lipid maintenance in P. hysterophorus under stress conditions.

Growth and development
Drought and salinity primarily influence the growth and development of plant. The tolerance ability of plant depends on its genetic potential that varies to species [34]. During adverse growth conditions cell cycle and cell division play a key role for maintaining cell communication and cell integrity [80]. In our study, several proteins showed noticeable changes related to growth of P. hysterophorus in drought and salt treatment. Up-regulation of kinesin 3 might help in keep-up of cells from stress by renovation of cytoskeletal structures and morphogenesis. The WPP domain containing protein 3 is a functional association of nuclear envelops protein and cell division of plant [81] which was found upregulated in drought and downregulated in salt stress. Besides this, cytokinin riboside 5'-monophosphate phosphoribohydrolase LOG 3 which is recognized as a regulator of cytokinin biosynthesis [82] was downregulated in our investigation; thus, indicating towards growth inhibition in order to improve stress tolerance at the cost of growth.

Protein degradation and transport
The ubiquitin/26 proteosome organization is an essential equipment of cell for controlling the concentration of regulatory proteins [83]. The process of ubiquitination is mediated by three enzymes (E1, E2 and E3). In which E3 (ubiquitin ligase) has shown a significant role in transcription factors-mediated tolerance to drought and high temperature [84]. Our results showed upregulation of E3 ubiquitin ligase in drought and salinity, which might be for effective elimination of denatured and misfolded proteins. ATP-dependent CIp protease proteolytic subunit (CIpP) is a crucial component of caseinolytic protease system that works with ATPase subunits (CIpA/CIpX). It is critical in the removal of misfolded polypeptides as well as synthesis of new peptides. In our study, this CIpP was slightly down-regulated in drought and up-regulated in salinity. A protein recognized as coatomer subunit alpha-2, which covered the non clathrin coated vesicles and involved in transportation of vesicles from endoplasmic reticulum to Golgi apparatus [85] induced by drought and salinity perhaps; assisting in intracellular trafficking of cytosolic proteins. We identified an important protein, proton pump interactor 2 that controls the proton pump mediated activity of plasma membrane ATPase which was significantly upregulated by drought and especially salt stress. This proton pump ATPase (H + -ATPase) itself acts as a prime transporter of H + and establishes the differences in potential and pH across the cell membrane which facilitates the stimulation of secondary transporters [86]. Such secondary transporters could be a part of tolerance strategy of P. hysterophorus under stress especially in salinity. ABC transporter B family 21 (ABCB 21), which is also reported as a transporter protein (involved in scavenging of xenobiotics) [87] was found downregulated by drought and salinity. E3 ubiquitin ligase was positively correlated with ATP-dependent CIp protease proteolytic subunit. Proton pump interactor 2 was negatively correlated with ABC transporter B family 21 in present study (Fig 11).

Miscellaneous
Catalytic subunit of molybdopterin synthase cofactor biosynthesis for production of abscisic acid (ABA) [88]. Upregulated by salinity and down-regulated by drought indicates crucial role in differential stress response in plants [89]. Moreover, pentatrico-peptide repeat-containing protein increased its abundance under salinity which helps in RNA processing and editing [90]. RNA pseudouridine synthase 1 was upregulated by both stresses which is a member of RluA family and involved in pseudouridine synthesis and RNA modification [91]. This change is crucial for the structure and function of small RNAs.

Conclusions
The proteomics data clearly indicates that Parthenium hysterophorus adopts a complex strategy involving the modulation of numerous molecular and metabolic networks to serve favor under drought and salinity stresses. The plant has shown a high degree of threshold against both drought as well as salinity via strengthening cellular antioxidant system and increasing the abundance of proteins mainly involved in signaling, osmoregulation, anti-desiccation and morphological acclimation. Notably, P. hysterophorus strengthens its anti-plasmolysis processes by increasing the abundance of related proteins such as trehalose phosphate phophatase, sucrose synthase 2, enormous increase in desiccation related protein and lignin biosynthesis (only under drought) etc. and decreasing the abundance of mannitol dehydrogenase (oxidizes mannitol to mannose) to retain mannitol level. Data also indicates that besides up-regulating many antioxidant proteins, P. hysterophorus combats oxiradicals by glutathione S-transferase, cysteine proteinase inhibitor, peptide methionine sulfoxide reductase B4, etc. Under drought and salinity the plant perform a better organization of PSI through Fe-S cluster assembly factor HCF101. Proton pump interactor (under drought) seems to play crucial role. Other potent mechanisms include increased length of fatty acid chains (through acyl carrier protein 2), higher accumulation of calmodulin like protein 7 (Ca-sensor) and suppression of early auxin response through auxing responsive protein IAA7. Thus, this study suggest that P. hysterophorus adopts diverse range of proteomic strategies to combat drought and salinity stress.
Supporting information S1