Low Temperature Stress Modulated Secretome Analysis and

Article pubs.acs.org/jpr

Low Temperature Stress Modulated Secretome Analysis and Purification of Antifreeze Protein from Hippophae rhamnoides, a Himalayan Wonder Plant Ravi Gupta and Renu Deswal* Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi-110007, India S Supporting Information *

ABSTRACT: Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC−MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (−5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome. KEYWORDS: seabuckthorn, secretome, cold stress, 2-DE, nano-LC−MS/MS, extracellular proteins, antifreeze proteins

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nucleus, and plasma membrane have already been analyzed.5−9 These studies showed that cold stress negatively affects vital processes like photosynthesis and respiration as suggested by degradation of stromal and key matrix enzymes of chloroplast5 and mitochondria,6 respectively. Analysis of nuclear proteome showed that out of 184 identified proteins, 54 were cold responsive.7 Plasma membrane has sensors to sense the temperature and then respond by changing the membrane fluidity. The cold induced plasma membrane targets were mainly responsible for membrane repair, membrane protection, and proteolysis.8 However, more subproteomes/organelles need to be analyzed to know the complete LT responsive/regulatory proteins repertoire and the signaling mechanisms. Apoplast is a dynamic and complex compartment of the cell where ice is formed at subzero temperatures to prevent lethal cell damage. It contains the ice interacting proteins that are involved in inhibiting or stimulating the ice crystal growth. Besides, these extracellular proteins (ECPs) could be involved in signal perception, cell to cell communication, and signaling to provide defense against both biotic and abiotic stress.10 Unfortunately, the molecular mechanisms and components of

INTRODUCTION Low temperature (LT) stress decreases the productivity and restricts the distribution of crops. LT stress can be broadly categorized into cold stress and freezing stress.1 Plant’s responses to freezing stress are entirely different: some can tolerate extracellular ice formation and are referred as freezing tolerant, while others prevent freezing by supercooling their sap and come under the category of freeze avoiding.2 Freezing tolerant plants acquire tolerance to freezing temperatures during cold acclimation (CA), which is the period when they are exposed to low but nonfreezing temperatures. Moreover, freezing tolerance of the plants can be further enhanced by exposure to moderate subzero temperatures following cold acclimation, a process known as subzero acclimation (SZA) or second phase hardening.3 CA in plants has been extensively studied, but reports about the SZA are limited.3 Furthermore, most of the studies on CA have focused on gene expression analysis, which has a relatively limited scope in functional genomics.4 Proteins are the real executors as well as final reflectors of the gene expression. Therefore, it is appropriate to know the proteome rather than the genome. Moreover, subproteome analysis could be very beneficial in resolving the low abundance targets by reducing the complexity of total proteome. Cold modulated subproteomes of chloroplast, mitochondria, © 2012 American Chemical Society

Received: September 19, 2011 Published: April 10, 2012 2684

dx.doi.org/10.1021/pr200944z | J. Proteome Res. 2012, 11, 2684−2696

Journal of Proteome Research

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the signaling in the apoplast during LT stress are poorly understood. Overwintering plants secrete antifreeze proteins (AFPs) in the apoplast to keep a check on the growth of the ice crystals.11 AFPs bind to the ice crystals and prevent accretion of water molecules to the growing crystal planes in a noncolligative manner. Thermal hysteresis (TH) and ice recrystallization inhibition (IRI) are the two independent properties of AFPs, by virtue of which they control the ice crystal size.12 AFPs depress the nonequilibrium freezing temperature of water below the melting temperature, known as thermal hysteresis (TH). TH of plant AFPs is comparatively lower (0.1−0.5 °C) than fish (2 °C) and insect (2−5 °C) AFPs.13 It seems that the main function of these proteins is to inhibit ice crystal growth rather than preventing the ice formation in plants. Ice crystals grow in size because of ice recrystallization (smaller ice crystals join to form bigger ones). AFPs irreversibly bind to the ice crystals and modify their shapes and growth. Ice crystals are hexagonal in the presence of AFPs, while they are disk shaped in their absence. AFPs increase survival of plants by inhibiting the ice crystal growth, slowing down the ice propagation in tissue,13 and cryoprotecting the enzymes necessary for metabolism.14 Some of the plant AFPs are homologous to pathogenesisrelated (PR) proteins.15 Cold induced PR-proteins (chitinases and glucanases) retained their partial hydrolytic activity in addition to the AFP activity.15,16 Most of the AFPs have low molecular weight and are apoplastic except AFP from Solanum dulcamara, which is 67 kDa and is cytoplasmic.17 Interestingly, most of the AFPs are purified from the herbaceous plants; reports about the woody plant AFPs are few.14 Hippophae rhamnoides (seabuckthorn) is a deciduous shrub of Himalayas. It is considered as a “wonder” plant because of its multiple uses in biomedicine, nutraceuticals, cosmetics, and food industries.18 Besides a plethora of medicinal properties, it can also withstand multiple abiotic stress conditions like salinity, drought, UV−B radiation, and cold. Seabuckthorn has a capability to survive at −40 °C and thrives well under cold desert conditions. Therefore, it could be a good system for studying its LT tolerance, and the valuable information generated can be used to develop cold/freezing stress tolerant transgenic crops. Such crops could be introduced in snow-clad land masses, which could then be brought under cultivation. Proteomics and genomics analyses of seabuckthorn for understanding its abiotic stress tolerance traits are still unexplored areas. There are very few reports dissecting the molecular mechanism of its cold hardiness.19,20 A major bottleneck in using seabuckthorn as a research material is the germination of seeds in the laboratory conditions. It takes several months and needs special presoaking treatments.21,22 A rapid and simple seed germination procedure is described in the present report. Relatively fewer studies are reported for secretome analysis in planta because of difficulties in the extraction of pure ECPs.10 Here we describe an extraction procedure for isolating contamination free ECPs. MS identification of LT induced ECPs resolved on 2-DE gels showed the presence of putative AFPs (PR proteins that could have antifreeze activity). Interestingly, the antifreeze activity has not been reported in seabuckthorn to date. To confirm the presence of AFPs, antifreeze activity was tested in the ECPs, and to validate, these were purified using the ice adsorption chromatography (IAC). The goal of this study was to dissect the components and the probable mechanisms of LT stress signaling in the secretome.

Article

EXPERIMENTAL SECTION

Plant Growth Conditions

H. rhamnoides berries were harvested from Keylong, Spiti Valley of Himachal Pradesh, India. Seeds were removed from the berries, washed, dried, and stored in a desiccator at RT. For germination, seeds were washed with 1% teepol and surface sterilized with 70% ethanol for 5 min. These were soaked for 1−5 days in the deionized water. After incubating for one day in the dark, these were plated in wet germination paper rolls and transferred to a B.O.D. incubator at 24 ± 2 °C under white fluorescent light (270 μmol/m2/s, 16 h light/8 h dark). Seedlings were watered regularly and screened for any infection from time to time. Increasing the seed soaking duration from 1 to 5 days showed a linear increase in the germination. Maximum (62%) germination was observed in 5 days of soaked seeds, while it decreased by 10% after 6 days soaking (Figure S1A, Supporting Information (SI)). Growth of seedlings was best at 20 days. One such roll is shown in Figure S1B (SI). The germination paper was changed twice a week to check for accumulation of inhibitors (alkaloids and phenolics) released by seeds and to avoid any infection (Figure S1C (SI)). Cold Acclimation and Subzero Acclimation

For CA, 20 day old seedlings were kept at 4 °C in same light conditions for 1 day and 5 days. SZA was given by transferring cold acclimated seedlings to −5 °C under dim light (35 μmol/ m2/s, 8 h light/16 h dark) for 24 h. One set of seedlings were given direct −5 °C for 24 h in nonacclimated conditions. For recovery, direct −5 °C treatment was followed by incubation at 24 °C for 24 h. Freezing Survival Test and Relative Electrolyte Leakage Test for Detecting Freezing Tolerance

Seabuckthorn seedlings were subjected to the freezing survival test under nonacclimated, cold acclimated, and subzero acclimated conditions. LT treatments were given in three sets of 10 seedlings each. Survival rates were calculated after allowing the seedlings to recover at 24 °C for 3 days. For REL assay, seedlings were kept in small vials immersed in 10 mL of deionized water. LT treatments were given as described earlier. The initial conductivity (Ci) of the water was measured at room temperature (RT) and the final conductivity (Cf) was measured after boiling for 10 min. REL was calculated as the percentage of conductivity before and after boiling (Ci/Cf × 100) using a conductivity meter (Mettler Toledo). Three biological replicates were performed for both survival test and REL measurements. Statistical analysis was performed by one-way ANOVA and post hoc Tukey HSD test, applied for multiple paired comparisons. A value of p < 0.05 was considered statistically significant. Secretome Extraction

The ECPs were isolated using vacuum infiltration method.11 Shoots of control and stressed seedlings were cut into 1.0− 1.5 cm pieces. These were rinsed several times with deionized water to remove the cytoplasmic proteins. After washing, these sections were vacuum infiltrated at 10 mmHg for 30 min with different buffers (deionized water, 50 mM Tris pH 7.4, 20 mM ascorbic acid with 20 mM CaCl2, 20 mM ascorbic acid with 20 mM MgCl2, and 20 mM ascorbic acid). These were dried and placed in a syringe barrel in a falcon tube. The ECPs were collected by centrifugation at different speeds (2000−6000g) for 10 min. Proteins were acetone precipitated and quantified using Bradford’s method.23 For total protein extraction, tissue 2685



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the differences in protein loading and gel staining. After spot detection in these gels, a second level of matchset was created in which master gels of different treatments were matched and compared. The intensity of a given protein spot was expressed in terms of its volume, which was defined as the sum of the intensities of all pixels constituting the spot in the image. Student’s t-test was performed to observe the significant changes (p < 0.05) between the intensities of the spots.

was extracted in 1:1 (wt/vol) buffer (50 mM Tris pH 7.4, 20 mM EDTA, 30% glycerol, and 5 mM PMSF). Extract was centrifuged at 12 500 rpm at 4 °C for 25 min (Beckman Coulter, Allegra 64R). Supernatant was acetone precipitated after filtering through two layers of muslin cloth. The pellet was dissolved in the glycine buffer pH 8 and used for the glucose-6phosphate dehydrogenase (G6PDH) activity assay. Testing ECPs for Cytoplasmic Contamination

Protein Identification Using Nano-LC−MS/MS

G6PDH activity assay was performed to test cytoplasmic contamination in ECPs of control and stress treated seedlings. For G6PDH assay, 40 μL of protein extract was added in 1 mL of the reaction mixture containing 55 mM Tris pH 7.8, 3.3 mM MgCl2, 6 mM NADP, and 0.1 M of glucose-6-phosphate. Absorbance was taken at 340 nm for 5 min at 25 °C using UV−visible spectrophotometer (DU 730, Beckman Coulter), and enzyme activity was calculated as described by Noltmann et al.24 G6PDH assay was performed in ECPs and crude extract. Percentage cytoplasmic contamination was calculated as the activity in the apoplastic extract over the activity in crude extract. Purified G6PDH from Leuconostoc mesenteroides was used as a positive control.

Protein identification was carried out at The Centre for Genomic Applications (TCGA), New Delhi, India using Agilent 1100 series 2D nano-LC−MS/MS. LT induced polypeptides were excised from the gel and destained. In gel reduction was carried out using 10 mM DTT in 100 mM ammonium bicarbonate at 56 °C for 30 min. Alkylation of reduced proteins was done using 50 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min in the dark. Gel pieces were washed with 1:1 ammonium bicarbonate and acetonitrile (ACN) solution and dehydrated using 100% ACN for 5 min. Gel pieces were digested with 5 μL of trypsin solution (20 ng/μL, gold mass spectroscopy grade, Promega, Madison, USA) in 50 mM ammonium bicarbonate pH 7.8 for 16 h at 37 °C. Tryptic digested peptides were extracted twice with 0.1% trifluoroacetic acid (TFA) and were separated by HPLC using Agilent 1100 NanoLC-1100 system (Agilent, Palo Alto, CA, USA) combined with a microwell-plate sampler and thermostatted column compartment for preconcentration (LC Packings, Agilent). Samples (6 μL) were loaded on the Zorbax 300SB-C18 column (150 mm × 75 μm, 3.5 μm) using a preconcentration step in a micro-precolumn cartridge (Zorbax 300SB-C18, 5 mm × 300 μm, 5 μm) at a flow rate of 5 μL/min. The precolumn was connected with the separating column, and after 5 min, a multistep gradient (3% until 5 min, 15% for 5− 8 min, 45% for 8−50 min, 90% for 50−55 min, 90% for 55− 70 min, then again 3% for 71 min) was started. Formic acid (0.1%) in water and in 90% ACN were used as buffers. An LC− MSD Trap XCT with a nanoelectrospary interface (Agilent) operated in the positive ion mode was used for MS. Ionization (1.5 kV ionization potential) was performed with a liquid junction and a noncoated capillary probe (New Objective, Cambridge, USA). Standard Agilent tune mix was used to calibrate the instrument. Peptide ions were analyzed by the data-dependent method. The scan sequence consists of 1 full MS scan followed by 4 MS/MS scans of the most abundant ions. Data was analyzed using Agilent ion trap analysis software (ver 5.2). The peak lists were submitted to MASCOT (ver 2.1) search engine (http:/www.matrixsciences.com) and searched against the NCBInr database. The search parameters were as follows: mass values, monoisotopic; protein mass, unrestricted; fixed modifications, carbamidomethylation; variable modification, methionine oxidation; peptide mass tolerance, ±1.2 Da; fragment mass tolerance, ±0.6 Da; maximum trypsin missed cleavage, 1; and instrument type, ESI-TRAP. Only significant hits, as identified by the MASCOT probability analysis (p < 0.05) were accepted.

Construction of 2-DE Reference Map of LT Induced ECPs

ECPs were resolved on SDS-PAGE following Laemmli.25 A “cleanup” step was included to remove contaminants. One volume of protein extract was mixed with four volumes of methanol followed by addition of one volume of chloroform. Three volumes of deionized water were added, and the mix was centrifuged at 12 000 rpm (1−15k, Sigma) for 5 min at RT. Supernatant was carefully discarded, keeping the interphase protein layer. To the protein disk, three volumes of methanol were added, and it was again centrifuged at 12 000 rpm for 5 min. After removing the supernatant, the pellet was dried in a vacuum chamber (Millipore). Each step was followed by vigorous vortexing to mix the contents. Protein pellets after “cleanup” step were mixed with 1X sample buffer, boiled for 3 min, and resolved on a 15% SDSPAGE gel (Hoefer MiniVE). Electrophoresis was carried out at 120 V for about 2 h, and the gels were silver stained. For 2-DE, 250 μL of rehydration buffer containing 250 μg of protein was loaded onto 13 cm IPG strips (pH 3 to 10, nonlinear gradient, GE Healthcare) by rehydration loading overnight at RT. IEF was performed on an EttanIPGphore isoelectric focusing system (GE Healthcare) for 27.5 kVh. After IEF, the strips were reduced in an equilibration buffer (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS, and 0.002% BPB) containing 1% DTT as the first step and then alkylated by 2.5% iodoacetamide as the second step. For the second dimension, proteins were resolved on 15% SDS-PAGE using Hoefer SE 600 Ruby (GE Healthcare). The gels were silver stained.26 Five biological replicates were performed for freeze treated samples, and 2-DE gels of other samples were performed with three biological replicates. Image Acquisition and Data Analysis

Enzyme Assays

Gels were scanned using Alpha Imager (Alpha Innotech Corporation). ImageMaster2DPlatinum software (ver 6.0; GE Healthcare, Sweden) was used to analyze the silver stained 2-DE gels. A first level matchset was created in which master gels were developed from five replica gels of freeze treated samples and three replica gels of other samples that have correlation coefficient of at least 0.8. Volume of each spot was normalized in percentage spot volume mode to compensate for

Glyoxylase 1 assay was performed according to Deswal and Sopory with slight modifications.27 In brief, 20 μL of the protein extract was added in 1 mL reaction mixture containing 200 mM sodium phosphate buffer pH 7.6, 3.5 mM methylglyoxal, 1.6 mM glutathione, and 5 mM NiCl2 (instead of 16 mM MgSO4). Absorbance was taken at 240 nm for 5 min. One enzyme unit is the amount of enzyme catalyzing the 2686



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formation of micromole of S-lactoyl glutathione per minute per milligram. Chitinase assay was performed using chitin azure as a substrate.28 Briefly, 50 μL of the sample was added in 950 μL of 200 mM sodium phosphate buffer pH 7 containing 10 μg of the substrate. Reaction mixture was incubated at 37 °C for 24 h with constant shaking. Absorbance was taken at 570 nm after centrifuging it at 16000g for 10 min. One unit of Chitinase is the change in absorbance of 1.0 in 24 h at 570 nm. For SOD assay, 50 μL of protein was added in 1 mL of reaction mixture containing 100 mM triethanolamine (pH 7.4), 100 mM EDTA, 50 mM MnCl2, 7.5 mM NADH, and 10 mM mercaptoethanol. Decrease in absorbance was recorded at 340 nm for 15 min. One unit of enzyme is the amount of SOD capable of inhibiting 50% rate of NADH oxidation observed in the control.29 Three biological and technical replicates were performed for each assay.

proteins, ice binding proteins were eluted in 50 mM Tris pH 7.6 from the 12% preparative native gels.11 Treatment with Glycosidase

For deglycosylation, 4 μg of purified PGIP was treated with 1 unit of N-terminal glycosidase (peptide-N-glycosidase, PNGase-F, Sigma) and incubated at 37 °C overnight following manufacturer’s instructions.

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RESULTS AND DISCUSSION

Analyzing the Effect of LT Stress on Seabuckthorn Seedlings

Effect of freezing stress on nonacclimated, cold acclimated, and subzero acclimated seabuckthorn seedlings was observed by freezing survival test and REL measurement. Survival rates of control, 1 day, and 5 days cold acclimated seedlings were 100%. However, when seedlings were exposed to freezing stress in nonacclimated condition, survival rate was 81.65%. This survival rate of direct −5 °C treated seedlings was much higher than LT50, suggesting that seabuckthorn is able to tolerate direct freezing temperatures as well. Droop test analysis also supported this observation, as seedlings were able to tolerate freezing temperatures in nonacclimated conditions up to 5 days without any visible sign of drooping. One-way ANOVA followed by Tukey post hoc test showed insignificant difference (p > 0.05) in survival rates between the seedlings that were directly subjected to freezing stress and the seedlings that were exposed to freezing stress after 1 day of CA. However, when 5 days cold acclimated seedlings were subjected to freezing stress, survival rates were increased significantly to 94.5% (Figure 1A). These results showed that although seabuckthorn is able to tolerate direct freezing temperatures in nonacclimated conditions, its freezing tolerance can be enhanced further by prolonged cold acclimation. Consistent with the results of survival test, REL measurement showed a similar trend, as freezing injury was significantly less after 1 and 5 days of CA (34.29 and 26.86%, respectively) in comparison with the nonacclimated seedlings exposed to freezing stress (35.15%, Figure 1B).

Antifreeze Activity Assay

IRI and TH activities were analyzed using sucrose sandwich splat assay30 and nanolitre osmometer.11 All antifreeze activities were carried out at a protein concentration of 0.2 mg/mL. For IRI assays, proteins were solubilized in 30% sucrose in 20 mM ammonium bicarbonate pH 8 and were sandwiched between two round coverslips. These coverslips were snap frozen at −80 °C and then transferred to a glass viewing chamber maintained at −6 °C. Ice crystals were allowed to anneal for 2 h and then photographed using a Nikon eclipse 80i microscope with a DSFi1 camera (Nikon) and NIS-elements F-package (ver 3.0) software. For the quantification of the IRI activity, the diameters of the ice crystals in each image were measured using Image J software. TH and the shapes of ice crystals were analyzed using nanolitre osmometer. In brief, nanolitre volumes of the samples were loaded into the wells of the sample holder disk and kept on a freezing stage mounted on the stage of a phase-contrast microscope. Temperature was controlled by a nanoliter osmometer (Otago Osmometers, Dunedin, New Zealand). The samples were flash frozen at −20 °C to form a population of small ice crystals and then thawed until only a single ice crystal remained in the well. Temperature was gradually decreased, and the morphology of the ice crystals was photographed with the 20× objective. The temperature at which this ice crystal grew and shrunk was taken as the freezing and melting point of the sample, respectively. TH was calculated as the difference between the melting and freezing temperatures. ECPs were treated with Proteinase-K (Sigma, 1 mg/mL) overnight at 20 °C. For heat treatment, ECPs were incubated in boiling water for 10 min and then centrifuged to settle the precipitate. Five biological replicates were performed for all antifreeze assays with each assay performed in triplicates.

Extraction of Contamination Free Secretome

Analyzing secretome to understand the mechanism of freezing tolerance is pertinent, as this is the communication channel of the cell with the environment. Also, this is the region of the cell that is most affected by freezing stress due to formation of ice crystals. For secretome analysis, the first and the most crucial step is the extraction of contamination free ECPs. Classical procedures of ECPs extraction involve vacuum infiltration followed by centrifugation (800−2000g).10 However, this method yields low concentration of ECPs. We used combinations of buffers and differential centrifugal speed to improve the yield of ECPs. Out of different buffers used for extraction (as mentioned in experimental section), a combination of ascorbic acid and calcium chloride gave best results, as Rubisco (a chloroplastic protein) was absent and the protein yield was 1.74-fold higher than ascorbic acid alone at 2000g (Figure 2A, lane 3). Extraction in Tris and deionized water showed Rubisco contamination (Figure 2A, lane 5 and 6). For increasing the yield further without compromising on the purity, higher centrifugal force was applied (Figure 2B) with ascorbic acid and calcium chloride. Yield increased (1.9-fold) at 4000g, but it decreased at 6000g (Figure 2B, lane 6). At 5000g and 6000g, a slight greenish extract was obtained, indicating membrane disruption and pigment leakage. Therefore, 4000g was selected for

Purification of Antifreeze Proteins

IAC was done for purification of antifreeze polypeptide.31 ECPs from −5 °C treated seedlings were isolated and acetone precipitated. Pellet was dissolved in 20 mM ammonium bicarbonate pH 8. The cold/brass finger was seeded with a thin layer of ice, and it was immersed in the prechilled protein extract (≈50 mL). Temperature of the coldfinger was gradually decreased using a refrigerated water bath (GP150, Grant Instruments, Cambridge, U.K.) from −3 °C to −7 °C over 30 h with the help of LabWise software. Protein extract was gently mixed on a magnetic stirrer. After completion of the IAC, ice fraction was removed from the coldfinger by increasing the temperature to 1 °C. The ice fraction was melted, and the proteins were lyophillized (Labconco). For identification of antifreeze 2687



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Figure 1. Effect of CA, SZA, and direct freezing on seabuckthorn seedlings. (A) Freezing stress was given to nonacclimated and cold acclimated (1 and 5 days) seedlings, and survival rates were calculated after allowing them to recover at 25 °C. (B) Effect of LT on REL of seabuckthorn seddlings. Error bars represents standard deviation from three biological replicates. Statistical significance was determined by the Tukey−Kramer multiple comparisons test. Values with the same letters are not significantly different (p > 0.05).

Figure 2. Extracting contamination free extracellular proteins from shoot. (A) SDS-PAGE profile of extracellular proteins extracted in different buffers at 2000g. (B) SDS-PAGE profile of extracellular proteins extracted in ascorbic acid and calcium chloride at different centrifugal forces (2000−6000g). Polypeptide labeled as LSU is large subunits of Rubisco. (C) G6PDH assay was performed to test the purity of ECPs. Error bars represents standard deviation from three biological replicates.

the secretome extraction. For the rest of the experiments, ECPs were extracted in ascorbic acid and calcium chloride at 4000g.

ing differential abundance of spots after LT were magnified to get a clearer view of the abundance patterns (Figure 4). The 2-DE gel profile of −5 °C treated seedlings showed that a majority (63%) of the spots were confined to the acidic pI. As ECPs are mainly acidic in nature, this reaffirms the quality of the secretome.

Purity Assessment of the Secretome

High centrifugal force (4000g) could cause membrane disruption leading to cytoplasmic contamination. Therefore, it was crucial to test the purity of the ECPs. G6PDH assay was conducted to estimate the percent cytoplasmic contamination in ECPs of control, nonacclimated, cold acclimated, and subzero acclimated seedlings. Control ECPs did not show G6PDH activity. However, ECPs obtained from all the LT treated seedlings showed G6PDH activity that was restricted to less than 3% of the total G6PDH activity in the crude, which is within the permissible limit (Figure 2C).32 According to Song et al., up to 10% cytoplasmic contamination is acceptable for secretome analysis.33

Identification and Prediction of the Secretory Nature of the LT Induced Proteins

Spots that showed more than 2-fold increased abundance, student’s t-test (p < 0.05), after LT stress were identified. A total of 31 spots showing 34 proteins were identified by nanoLC−MS/MS (Table 1). Identified proteins with only significant ion scores (p < 0.05) and E-values (E < 0.05) were accepted. However, the difference in theoretical and experimental molecular weights and pIs of some of the identified proteins may be due to lack of protein database for seabuckthorn, protein degradation, protein isoforms, post-translational modifications, and alternative splicing. Extracellular localization, predicted using SignalP and SecretomeP software, showed 75.75% of the identified proteins to be extracellular, while 24.25% were nonsecretory. Out of the 75.75% secretory proteins, 24.25% were classical secretory, i.e., were carrying signal peptide, while the remaining 51.5% were targeted via the nonclassical pathway(s). As nonsecretory proteins are not the resident of apoplast, these might be imported in response to any stimulus like stress condition(s).

LT Stress Differentially Modulated the Shoot Secretome

In order to determine the changes in secretome during CA, SZA, and direct freezing in nonacclimated conditions, ECPs were isolated using a modified vacuum infiltration protocol and were resolved on the 2-DE gels. Image analysis of 2-DE gels of ECPs from the control, CA (1d), direct −5 °C, CA (1d) followed by −5 °C, CA (5d), CA (5d) followed by −5 °C, and recovered seedlings apoplastic extracts reproducibly showed 243, 249, 255, 263, 238, 251, and 227 spots, respectively. The quantitative image analysis in combination with statistical tests showed that a total of 61 (25%) spots changed in abundance by more than 2-fold (p < 0.05) in at least one LT treatment (Figure 3). Out of these 61 differentially expressed spots, 26 spots showed higher abundance, 13 spots showed decreased abundance, and the remaining 17 spots showed a mixed pattern of expression during LT stress treatment. Six gel regions show-

Functional Classification and Clustering of the LT Induced Proteins

On the basis of the biological roles, the identified proteins were classified into 6 categories: redox regulation, stress tolerance, 2688



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Figure 3. 2-DE reference gels of seabuckthorn ECPs obtained from control, CA (1d), −5 °C (1d), recovery, CA (1d) followed by −5 °C, CA (5d), and CA (5d) followed by −5 °C. LT treated seedlings. ECPs (250 μg) were resolved on 3−10 nonlinear IPG strips for the first dimension and 15% SDS-PAGE for the second dimension. Gels were silver stained and analyzed by ImageMaster2D Platinum software (GE Healthcare). Spots showing a more than 2-fold change in abundance due to LT treatments (p < 0.05) are marked by the arrows.

Figure 4. Magnified sections of 2-DE gels of the seabuckthorn shoot secretome showing differential abundance of the proteins after LT stress in a dose dependent manner. Spot numbers in the boxes refer to the spots corresponding to Figure 3.

As the present study examines the abundance patterns of LT responsive proteins by different LT stress conditions, it was very crucial to differentiate the proteins induced by direct

signaling, metabolism, regulation, and others (Figure S2 (SI)). Proteins that fall in the others category were either hypothetical or proteins with unknown functions. 2689



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Table 1. Identification of the Spots That Showed ≥2-Fold Induction after Freezing Stress by Nano-LC−MS/MS functional category

spot no.a

redox regulation

24 15 6 35 36

defense/stress related

signaling

metabolism

regulation

accession no.

protein scoreb

Mc

SCd

th MW/pI

exp MW/ pIe

locf

Lactoylglutathione lyase or Glyoxylase 1 Superoxide dismutase Thioredoxin Aldo/keto reductase Putative lactoylglutathione lyase

XM_002518424.1 P23346 EU056813.1 AC160516.1 Q39366

161 64 62 52 98

2 1 7 2 1

9 3 5 4 4

31.7/7.63 15.2/5.64 20.6/9.63 31.1/6.15 31.74/5.19

24.4/5.5 15.3/5.1 10.4/5.2 29.9/5.3 33.58/5.6

NC NC C NS NC

32

Osmotin-like protein

AF304007.1

83

3

7

27.5/7.41

26.7/5.4

32 12 18 7 37 40 30

Thaumatin-like protein Chitinase Similar to pathogenesis-related protein STH-2 GDSL-motif lipase/hydrolase family protein Dessication related protein Phenylalanine ammonia lyase Late embryogenesis-like protein

P83491 XP_003597296 AB211525.1 NP_177281 AAM65140 ABD42947 AAU29064

64 74 72 56 98 73 53

7 2 3 3 1 1 1

43 3 7 2 5 1 8

11.42/4.4 33.72/5.9 17.34/5.79 41.5/9.1 34.4/8.77 78.2/6 17.32/4.51

26.7/5.4 13.89/5.5 17.9/5.2 11.91/5.9 29.6/7.2 31.91/5.4 21.73/8.8

C C NS NC C NC NC

Calmodulin 1

DQ186609.1

57

3

14

16.9/4.16

14.54/3.1

NC

55 3

Calcium dependent protein kinase 23 GTPase activating protein

XP_002309145 AAQ54568

50

1 1

2 18

60.14/5.91 8.68/4.96

42.3/8.1 12.1/3.5

NC NC

4 41 59 2

C-3 sterol dehydrogenase Sedoheptulose-1,7-bisphosphatase Putative blue light receptor Putative phosphomannomutase

CAL52542 ACQ82818 CAC94940 BAD35746

70 147 52 54

4 6 1 1

1 8 1 3

205.2/7.22 42.53/5.96 81.9/8.67 53.7/6.99

10.9/3.2 36.73/5.3 11.46/3.2

NS NS NS NC

20

ATP-dependent Clp protease ATP-binding subunit clpA homologue Cysteine protease Translation-inhibitor protein Pyrrolidone-carboxylate peptidase family protein

P84565

115

3

51

7.9/4.39

17.04/6.8

NS

AF134152.1 AB082518.1 NP_564721

70 111 48

1 3 1

11 11 6

15.6/4.12 19.8/7.63 24.3/5.98

13.54/5.3 13.54/5.3 26.33/5.9

NC C NS

Thylakoid lumenal 15 kDa protein, chloroplast (Arabidopsis thaliana) Unknown protein (Arabidopsis thaliana) Predicted protein (Physcomitrella patens subsp. patens) Unknown (Populus trichocarpa) Os10g0125700 Unnamed protein product (Vitis vinifera) Predicted protein (Physcomitrella patens subsp. patens) Hypothetical protein OsI_37876 (Oryza sativa Indica Group) Unknown protein 18 Hypothetical protein SORBIDRAFT

NP_566030

88

3

6

24.1/7.55

13.89/5.8

NC

NP_191832 XP_001763519

101 56

1 1

5 2

35.2/5.27 67.3/6.78

72.37/5.3 11/3.2

C NC

ABK93605 NP_001064077 CBI40282 XP_001784896

179 49 70 73

6 1 1 4

17 1 4 3

21.98/8.76 161.8/8.15 43.6/4.75 42.8.3/5.5

13.54/5.3 26.1/6.3 42.3/8.4 17.71/5.9

NC C NC NC

EEC69045

54

2

1

157.6/6.54

12.5/6.4

NS

P85925 XP_002456647

65 55

1 1

91 8

1.39/5.8 14.8/7.04

12.22/5.9 67.88/5.6

NC

1

11 11 25 others

13 48 5 11 27 57 19 9 8 50

protein identified

C

a

Spot no. refers to the spots labeled in Figure 3. bProtein scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. cNumber of matched peptides. dSequence coverage. eExperimental molecular weight and pI were calculated using ImageMaster2DPlatinum software. fPutative location was predicted using SecretomeP and SignalP servers. C, classical; NC, nonclassical; NS, nonsecretory.

a broader modulation of metabolic and regulatory pathways. Cluster 2 includes proteins that seem to be freezing sensitive, as their abundances were decreased after freezing treatment either in nonacclimated conditions or in cold acclimated conditions. These proteins include putative blue light receptor and unnamed protein product (Vitis vinifera). Cluster 3 represents proteins that are induced by direct freezing. These proteins include GTPase-activating protein, glyoxalase 1, C-3 sterol dehydrogenase, superoxide dismutase (SOD), aldo/keto reductase, hypothetical protein OsI_37876, thylakoid lumenal

freezing, CA, and SZA. For this, the identified proteins were subjected to clustering analysis using cluster 2.1.1 program (http://rana.lbl.gov/EisenSoftware.htm). Six clusters were formed on the basis of the similarities in the abundance profiles (Figure 5). Cluster 1 contains proteins that showed a mixed abundance pattern during LT stress. These proteins include predicted protein (Physcomitrella patens), hypothetical protein SORBIDRAFT, calmodulin, and Os10g0125700. These proteins belonged to different functional categories and thus represent 2690



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Figure 5. Clustering analysis of LT stress modulated proteins of shoot secretome. The cluster was performed using CLUSTER 2.1.1 software (http://rana.lbl.gov/EisenSoftware.htm) and is shown at the top. Differential abundance and functional categories of the proteins in each cluster are depicted in lower left and right panel, respectively.

ascorbate peroxidase and glutathione peroxidase. Although none of the identified plant SOD have signal peptide, MS based identification and significant activity modulation by freezing observed in the present study confirmed its apoplastic localization.35 Role of SOD in freezing tolerance was earlier shown in Medicago.36 Spots no. 36 and 24 were identified as glyoxalase 1 or lactoglutathione lyase. Interestingly, the sizes of two glyoxalase 1 vary, 24.4 and 33.58 kDa, suggesting these could be the two different forms of glyoxalase 1 in seabuckthorn. This enzyme is involved in detoxification of the methylglyoxal that is formed as a byproduct of carbohydrate and amino acid metabolism and thus is indirectly involved in the ROS metabolism. MS based identification of the ECPs showed an increased abundance of glyoxalase 1 after multistress response in poplar37 and dehydration stress in rice,38 hinting at its apoplastic import in stress. In addition to the H2O2 mediated signaling, two other signaling pathways seem to be modulated in the secretome after freezing stress. Increased abundance of the calmodulin (spot no. 1) and calcium dependent protein kinase (spot no. 55) indicates activation of calcium signaling pathway, while increased abundance of GTPase-activating protein (spot no. 3) showed modulation of G-protein signaling. Calmodulin is a ubiquitous calcium sensor in plants. It binds to calcium and stimulates downstream targets. Glyoxalase 1 is activated by calmodulin.39 Thus, accumulation of calmodulin by LT stress may activate glyoxylase 1. It was earlier reported that calmodulin-1

15 kDa protein, and ATP-dependent Clp protease ATP-binding subunit clpA homologue. These proteins seem to be induced by freezing rather than the low temperature effect. Cluster 4 includes proteins that keep on increasing with increasing LT duration. These proteins include unknown protein 18, predicted protein (Physcomitrella patens), calcium dependent protein kinase, and thioredoxin. Proteins of cluster 5 are induced during SZA, as abundances of these proteins are induced only after freezing stress. These proteins include cysteine protease, late embryogenesis-like protein, Chitinase, similar to pathogenesis-related protein STH-2, sedoheptulose-1,7bisphosphatase, thaumatin-like protein, putative phosphomannomutase, and GDSL-motif lipase/hydrolase family protein. Proteins of this cluster are mainly involved in stress tolerance. Cluster 6 includes proteins that maintained a constant abundance pattern in all the LT treatments after an initial increase. These proteins seem to be involved in normal LT stress tolerance response and include putative lactoglutathione lyase and a hypothetical protein. Proteins Involved in the LT Stress Response in Seabuckthorn Secretome

Stress conditions lead to an accumulation of superoxide radicals (O2−) from different sources in the apoplast.34 One of the sources is reduction of NAD+. NAD+ is reduced to NADH by different dehydrogenases (C-3 sterol dehydrogenase, spot no. 4). This reduced NADH is oxidized to superoxide radical by NADH oxidase. SOD (spot no. 15) converts superoxide ions to H2O2, which is finally reduced to H2O either by catalase directly or by 2691



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Figure 6. Enzyme activities of Chitinase (upper panel) and SOD (lower panel) for functional validation of the LT induced targets (A, D). Enzymatic activities were multiplied with the relative intensities of the spots (B, E) to calculate the effective inductions (C, F).

is involved in cold stress signaling cascade in Arabidopsis.7,40 Increased level of calmodulin-1 after cold and high wind was also shown in Nicotiana plumbaginifolia.41 A very encouraging observation was the presence of some putative AFPs like thaumatin-like protein (spot no. 32) and Chitinase (spot no. 12) in the seabuckthorn secretome. Although the main function of these proteins is in biotic stress, their role in cold stress is also well established.15 These proteins acquire antifreeze activity at the time of cold/freezing stress and thus prevent the lethal cell damage caused by the ice crystals. Apart from the antifreeze activity, Chitinase also retains its partial hydrolytic activity at subzero temperatures,16 thus acting as a dually functioning protein. A very high percentage of identified proteins (29%) belongs to the “others” category; this may be due to the scarcity of protein sequences submitted for this plant in the databases. Characterization of these could provide us interesting, unidentified targets for crop manipulation.

Detection and Identification of Antifreeze Proteins

Some of the PR-proteins play very crucial roles in LT by switching their PR-activity to antifreeze activity.15 As putative AFPs were identified in the LT treated secretome, to confirm their presence, the AFP activity was analyzed by a nanolitre osmometer coupled with a phase contrast microscope and by sucrose sandwich splat assay. Activity assays showed freezing stress induced antifreeze activity, which inhibits ice recrystallization (average ice crystal diameter was reduced from 23.6 to 16.3 μm), forming hexagonal ice crystals with 0.13 ± 0.02 °C TH (Figure 7A,B). ECPs when treated with proteinase-K lost antifreeze activity (Figure 7A,B), confirming the proteinaceous nature of the antifreeze activity. Heat treatment of ECPs obtained from −5 °C treated seedlings resulted in loss of 68% of the activity (Figure 7A,B), suggesting that 32% of the antifreeze activity was due to heat stable proteins. As antifreeze proteins were detected in the freeze modulated secretome, an effort was made to purify these.

Enzymatic Activities for the Functional Validation of the Identified Proteins

Purification and Characterization of Antifreeze Proteins

Activities of three enzymes, glyoxalase 1, SOD (induced by freezing), and Chitinase (induced by SZA), showing more than 20-fold accumulation by LT stress were analyzed. Both Chitinase and SOD activities were increased during LT stress. In SOD, there was a generalized increment in the activity with increasing LT duration, and maximum activity (1.4-fold higher in comparison with the control) was recorded when freezing stress was given to 5 days cold acclimated seedlings. Chitinase activity was maximum in freezing stress whether the seedlings were acclimated or nonacclimated (Figure 6). Glyoxalase 1 activity was not detected in ECPs probably due to the presence of a different (inactive) form. Apoplastic glyoxalase 1 may perform some different function than cytoplasmic glyoxalase 1. LT induced fold change in enzymatic activities were multiplied with the fold induction in abundance (calculated by ImageMaster), to calculate the effective induction of these enzymes. Increase in the amount of the protein as well as its activation would have a synergetic effect. Chitinase and SOD showed 60- and 25.6-fold effective inductions after freezing stress, justifying their high requirements.

AFPs were enriched using IAC. Out of 50 mL of ECPs in ammonium bicarbonate, nearly 35 mL was bound to the ice fraction after completion of IAC. Lyophilized ice bound proteins showed a major polypeptide of 41 kDa (Figure 7C). However, three other minor polypeptides were also observed in the SDS-PAGE of IAC fraction. Binding of other three proteins to the ice column was not consistent; therefore, these were not analyzed further. Efforts are underway to enrich other AFPs by modifying the IAC procedure. MS identification of 41 kDa ice bound protein showed it to be polygalacturonase inhibitor protein (PGIP). This protein belongs to Leucine-rich-repeat (LRR) protein family, and its main function is to protect the plant from fungal attack by inhibiting the polygalacturonase secreted by pathogenic fungi. Bioinformatic analysis of this protein using SignalP software showed its targeting in the apoplast via the classical secretory pathway. However, in our 2-DE experiments, PGIP was not identified. This may be due to low levels of PGIP in the secretome. To confirm the LT inducible nature of PGIP, IAC was performed using ECPs obtained from control seedlings. PGIP was not detected in the IAC fraction of control seedlings 2692



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Figure 7. Analysis of antifreeze activity in freezing stress induced seabuckthorn secretome. (A) Ice crystal morphologies, IRI assays, and (B) quantification of these activities in buffer, BSA, control ECPs, −5 °C treated ECPs, heat treated, and proteinase K treated ECPs, analyzed by phase contrast microscope coupled with nanoliter osmometer and sucrose sandwich splat assay. (C) SDS-PAGE and (D) native gel profile showing purification of polygalacturonase inhibitor protein by IAC. The band marked with an asterisk is PGIP. Magnification bar in nanoliter osmometry shows 10 μm, while in splat assay it represents 50 μm. Error bars show standard deviation from five replicates.

Figure 8. (A) SDS-PAGE profile showing IAC fractions of control and −5 °C treated ECPs. PGIP was not identified in IAC fraction of control seedlings, while it is present in the IAP fraction of −5 °C treated seedlings. (B) SDS-PAGE profile of PGIP showing its glycosylation and heat stability. The band marked with an asterisk is PGIP, and the band marked with “+” showed PNGase-F. Ice crystal morphology, IRI assays (C), and their quantification (D) in presence of PGIP, deglycosylated PGIP, heat, and Proteinase-K treated PGIP. (E) Determining IRI end point. PGIP was serially diluted and tested for antifreeze activity. AFP activity was completely lost at a concentration of 0.012 mg/mL. Magnification bar in nanoliter osmometry shows 10 μm, while in splat assay it represents 50 μm. Error bars show standard deviation from five replicates.

showing that PGIP binds to the column only after freezing treatment because of its enrichment after freezing (Figure 8A). Cold inducible nature of PGIP was earlier shown in cotton,42 Arabidopsis,43 Brassica napus,44 and Chorispora bungeana.45

Association of antifreeze activity with PGIP is well established in carrot.30 PGIP identified in seabuckthorn showed a high degree (93%) of sequence similarity with carrot PGIP. PGIP was resolved at 41 kDa in both SDS and native-PAGE, 2693



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suggesting its monomeric nature. It was eluted from the native gel and tested for AFP activity. Inhibition of ice recrystallization (average ice crystal diameter was reduced from 23.6 to 2.6 μm) and formation of hexagonal ice crystal by PGIP confirmed it to be an antifreeze protein (Figure 8C,D). A TH of 0.19 ± 0.03 °C was observed at 0.2 mg/mL. This TH of seabuckthorn PGIP was higher than Lolium AFPs (IAP 2, IAP3, and IAP5), which did not produce any detectable TH activity at 0.15 mg/mL.46 Carrot and winter rye AFPs had a TH of 0.35 and 0.33 °C at relatively much higher concentrations of 1 and 60 mg/mL, respectively.30,47 Although at similar concentrations, carrot AFP had slightly lower TH (0.14 °C) than seabuckthorn.48 IAC purified PGIP was found to be N-glycosylated as identified by a gel shift from 41 to 39 kDa after glycosidase treatment (Figure 8B). Antifreeze activity analysis with glycosidase treated PGIP showed similar antifreeze activity, suggesting that glycosylation is not required for antifreeze activity (Figure 8C). To date, only three plant AFPs are known to be glycosylated, out of which only one AFP from S. dulcamara requires glycosylation for its activity.49 The AFP detected in this study showed glycosylation independent antifreeze activity as observed for the other two glycosylated AFPs from carrot and Lolium.49 Glycosylation of AFPs could have some other roles either in targeting or in protein stabilization. Sensitivity of purified antifreeze activity to proteinase-K confirmed its proteinaceous nature (Figure 8C). Heat treatment of PGIP resulted in complete loss of its activity, showing its heat labile nature. However, heat stability studies on antifreeze activity in ECPs showed 32% of the activity to be contributed by the heat stable proteins. The AFP purified in this study (PGIP) is heat labile, suggesting existence of other heat stable AFPs in secretome. Heat stable antifreeze proteins have been previously identified from Lolium,50 Deschampsia antartctica,51 carrot,30 and winter wheat grass.52 The end point of IRI (lowest concentration of AFPs that still blocks the recrystallization) for PGIP was determined as 12 μg/mL by its serial dilutions (Figure 8E). However, this IRI activity exhibited by purified PGIP was comparatively lesser than other AFPs purified from Lolium (3 μg/mL for IAP 3 and IAP 5, and 0.6 μg/mL for IAP 2),46 carrot (1 μg/mL),30 and Forsythia suspensa (6 μg/mL).53 Seabuckthorn PGIP showed least IRI activity in comparison with other plant AFPs, while its TH is comparable or higher. Yu et al. showed that IRI and TH activity are two independent properties of AFPs, and there is no direct correlation between the two.9 Earlier, it was shown that Lolium AFPs (IAP 2, IAP 3, and IAP 5), which showed maximum IRI activity, did not have any detectable TH activity.46 Characterization of seabuckthorn PGIP showed a weak TH activity, which is a characteristic of plant AFPs. However, during purification, no significant increase in the TH activity was observed from total ECPs (0.13 °C) to the purified PGIP (0.19 °C). Similar results were observed in S. dulcamara17 and carrot.30 Smallwood et al. suggested that this may be due to the absence of a cofactor required for the antifreeze activity, which is probably lost during the purification.30

To the best of our knowledge, this is also the first report of antifreeze activity analysis in seabuckthorn. A 41 kDa glycosylated, heat labile protein, identified as PGIP, was purified. High degree of similarity (93%) of seabuckthorn and carrot AFPs suggests a common function/role for both. Sharing of 18% LT responsive proteins in secretome with other subproteomes (mitochondria, plastids, nucleus, and plasma membrane) confirmed that the identified proteins are indeed LT responsive (Table S1 (SI)). Interestingly, defense/stress related proteins were unique to the secretome except thaumatin like protein and phenylalanine ammonia lyase, which were also identified in the plasma membrane proteome. Chitinase, one of the defense related proteins, showed 60-fold effective induction after freezing stress, validating relevance of this defense related protein in LT stress in secretome. Enzymes involved in the ROS metabolism were identified in the secretome as well as in the plastidome, suggesting that antioxidant battery of enzymes would participate in cold stress signaling in both of the compartments. This was further supported by 25.6-fold effective induction of SOD by freezing stress in the secretome. Calmodulin-1, a calcium sensor, was another interesting target identified in the nuclear proteome, also suggesting similarity and continuity in calcium mediated LT signaling between the two organelles. Cold induced nuclear proteome had highest percentage of signaling components, followed by secretome and plasma membrane, suggesting their active participation in LT signaling. Surprisingly, these were absent in plastid and mitochondria, indicating relatively insignificant retrosignaling in LT. Overall, these observations suggest the secretome to be a dynamic player in LT signaling involving different proteins, including antifreeze proteins. We expect that these results will provide a better understanding of the involvement of secretome in LT signaling in plants. Our future efforts would be to purify and catalogue the complete repertoire of AFPs from H. rhamnoides and other two species, H. salicifolia and H. tibetana, present in India. Few efficient candidates would be exploited for their agricultural as well as biomedical applications.

CONCLUSIONS This is the first report of LT induced secretome analysis in plants using a 2-DE-MS approach. LT stress changed the abundance of 25% ECPs. On the basis of the functions of the freeze induced proteins, a LT induced signaling network is proposed (Figure S3 (SI)).

Notes

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ASSOCIATED CONTENT

* Supporting Information S

Figure S1: Optimization of seabuckthorn seed germination and growth procedures. Figure S2: Functional categorization of cold induced identified proteins in secretome. Figure S3: A putative signaling network of LT induced proteins in shoots secretome. Figure S4: Phylogenetic tree of polygalacturonase inhibitor protein (PGIP) identified from different organisms. Figure S5: Histograms of the functional classifications of the cold responsive proteins identified in different organelles. Table S1: A comparative analysis of cold induced targets with targets identified in other subproteomes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: 91-011-27662273.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant (BT/PR10799/NDB/51/ 171/2008) from Department of Biotechnology, Government of 2694



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India and a research grant provided by University of Delhi to R.D. R.G. thanks DBT for a research fellowship.

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ABBREVIATIONS

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REFERENCES

LT, low temperature; CA, cold acclimation; SZA, subzero acclimation; AFPs, antifreeze proteins; REL, relative electrolyte leakage; G6PDH, glucose-6-phosphate dehydrogenase; MS, mass spectrometry; TH, thermal hysteresis; IAC, ice adsorption chromatography; IRI, ice recrystallization inhibition; ECPs, extra cellular proteins; PGIP, polygalacaturonase inhibitor protein

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