Purification of dual-functioning chitinases with hydrolytic and antifreeze activities from Hippophae rhamnoides seedlings

Chitinases are glycosyl hydrolases which hydrolyse β-1,4-glycosidic bonds between N-acetylglucosamine residues of chitin. Seabuckthorn (Hippophae rhamnoides), a cold desert plant, is a storehouse of many cold-tolerant proteins including dual-functioning antifreeze proteins (AFPs) possessing both hydrolytic and antifreeze activities. Herein, we report the purification and characterization of antifreeze chitinases from seedlings grown in laboratory conditions. Chitin-affinity chromatography led to homogenous purification of two acidic chitinases HrS CHT1a (33 kDa) and HrS CHT1b (38 kDa) from seedlings. Antifreeze activity of purified AFPs was confirmed by the formation of hexagon-shaped ice crystals using nanolitre osmometer. Similarly, sucrose sandwich splat assay also confirmed their ice recrystallization inhibition activity (1.6-fold decrease in mean ice crystals). The chitinase activity of AFPs was confirmed by chitin hydrolytic assay where higher activity (1.8-fold) was observed in HrS CHT1b (500 U/mg) than HrS CHT1a (222 U/mg). MS identification showed homology of HrS CHT1b with provicilin while HrS CHT1a was identified as uncharacterized protein. In silico analysis showed that purified AFPs differ significantly in biochemical properties which suggests their different physiological roles. Protein association network analysis using string showed interaction of HrS CHT1b with enzymes involved majorly in pathogenic protection (pectinesterase, glycosyl hydrolase protein with chitinase domain). However, HrSCHT1a showed interaction with proteins associated with growth and energy regulation (glycine and purine synthesis, vitamin B metabolism) thereby indicating differential functional roles of both the chitinases. Conserved domain analysis also supported that these AFPs are multifunctional and exhibit differential regulatory roles in enabling the plant growth and defense responses. Further validation of these targets may open gates for commercial utilization of this plant growing abundantly in the Himalayan regions of India for protection of freeze-susceptible crops or biomedical applications.


Introduction
Plants are constantly exposed to diverse abiotic stresses including flood, drought, temperature fluctuations, salinity, mineral deficiency and toxicity which restrict their growth, productivity, cultivation, survival and geographical distribution. Abiotic stress conditions make the plants more prone to pathogenic infections owing to increased emergence of virulent and broad host range pathogens. Low-temperature stress is one of the major environmental factors affecting the agricultural crop yield worldwide.
Plants have complex cold/freezing stress tolerance mechanisms including antifreeze proteins (AFPs) to protect themselves from cold/freezing stress. Cold-responsive dual-functioning AFPs comprise a diverse family of proteins which interacts with ice and protect the cells from damages due to intracellular and intercellular ice formation. These have been previously reported in freezing-tolerant organisms including fish, insects, bacteria and plants (Griffith and Yaish 2004). In plants, AFPs were first reported in an overwintering freeze-tolerant herbaceous monocot, Secale cereale (winter rye) leaves (Griffith et al. 1992) showing homology with PR proteins which provide protection from pathogens. N-terminal amino acid sequences, immune crossreaction and enzyme activity assays of cold-acclimated apoplastic extracts showed AFPs homologous to PR proteins such as chitinases (35 kDa), β-1,3-glucanases (32, 35 kDa) and thaumatin-like proteins (16, 25 kDa) (Hon et al. 1995;Yaish et al. 2006). Further, chitinase genes CHT 9 and CHT 46 encoding Class I and II chitinases isolated from coldacclimated winter rye also showed antifreeze activity.
Chitinases (EC 3.2.1.14) and chitin-binding lectins (CBLs) are induced during stress and belong to the widely studied class of pathogenesis/defense-related proteins which act by catalyzing the hydrolysis of 1,4-β-N-acetyl-dglucosamine (GlcNAc) linkages. Depending on the catalytic domains, amino acid sequence similarity, substrate specificity, mechanism of catalysis, localisation, signal peptides and inducers, plant chitinases are broadly classified into two glycosyl hydrolase families (GH 18,19) and seven (I-VII) different classes. Classes I, II and IV belong to GH19 whereas Classes III, V, VI and VII belong to GH18 family (Patil et al. 2000(Patil et al. , 2013. In general, plant chitinases exhibit an N-terminal signal region with a structural chitin-binding domain (CBD) and/or a glycoside hydrolase (GH) family-like catalytic domain. Both these domains are connected by linker amino acid sequences which allow independent and efficient working of these domains (Li and Greene 2010). Chitinaselike lectins or proteins belong to GH18 family and share significant sequence and structural similarities with active chitinases but may or may not display chitinase activity (Patil et al. 2013). Many plant CBLs including agglutinins (Urtica dioica, Triticum) and lectins (Solanum tuberosum, Lycopersicon esculentum) contain structural motifs composed of cysteine-rich amino acid sequences called chitinbinding domains (CBDs). Alternatively, some CBLs (Datura stramonium, Solanum lycopersicum) may lack CBDs but possess hydroxyproline (Hyp)-rich domain, similar to the cell wall glycoprotein extensin. Crystal structure analysis of a novel N-acetyl glucosamine-specific chitinase like-lectin from Tamarindus indica showed 41% and 39% sequence identity with tamarinin and chitinase, respectively, but lacks chitinase activity (Patil et al. 2013).
Plants produce multiple chitinase isozymes which are regulated differentially with the developmental stages, have diverse subcellular location and are tissue specific to play differential regulatory role in plant physiological responses (organogenesis, embryogenesis, programmed cell death, abscission zone formation). Expression of these enzymes may either be constitutive or induced in response to multiple abiotic (osmotic, salt, cold, heavy metal) and biotic (wounding or pathogenic attack) stress (Grover 2012) allowing their accumulation in extracellular or vacuolar regions. Differential constitutive expression of apoplastic dual functioning chitinase-like AFPs (33 kDa) exhibiting both hydrolytic and antifreeze activities was observed in overwintering Chimonanthus praecox petals, corolla, flowers, leaves, bark and root showing their multifunctionality to provide protection from freezing injury psychrophilic pathogens and developing cold tolerance (Zhang et al. 2011).
Seabuckthorn (Hippophae rhamnoides), a freeze-tolerant high-altitude (2500-4000 masl) shrub belonging to family Elaeagnaceae is capable of withstanding extreme climatic conditions; however, its abiotic stress tolerance mechanism remains largely unexplored. Transcriptome and EST analyses have shown that seabuckthorn possesses cold-inducible transcripts including freezing stress and disease-responsive AFPs (thaumatins and chitinases) suggesting their probable role in biotic and abiotic stress (Ghangal et al. 2012). In a recent study, cloning and characterization of seabuckthorn recombinant chitinase (HrCHI1) (954 bp, 317 aa) showed its regulation by CBF/ ICE-dependent cold stress signaling pathway (Kashyap and Deswal, 2017).
Additionally, apoplastic AFPs including PGIP and dualfunctioning Class I chitinases were purified from low-temperature stress-modulated seedling secretome indicating their role as survival mechanisms during sub-zero conditions. During cold acclimation, accumulation of these cold-responsive AFPs was shown to increase the survival percentage of seedlings thus, providing cold hardiness. Deswal 2012, 2014). Similarly, antifreeze activity has been confirmed in different parts (leaf and berry) of naturally growing seabuckthorn populations and AFPs have been purified from these populations indicating their role in cold hardiness (Sharma et al. 2016(Sharma et al. , 2018. Therefore, the objective of the current study was to investigate the presence of dual-functioning cytoplasmic chitinases in laboratory-grown seedlings. Here, the main aim was to understand if chitinases in cytoplasm are similar or different from the apoplastic chitinases. Further, comparison of these cytoplasmic AFPs with the seedling secretome chitinases (Gupta and Deswal 2014) may provide an insight into their diverse functions. Moreover, these novel dual-functioning AFPs may be utilized in biotechnological or biomedical sectors and production of freeze-and pathogenic-tolerant transgenic crops, a possible solution to the increasing world food crisis due to constant rise in global warming and frequent fluctuations in temperature.

Plant materials and growth conditions
Hippophae rhamnoides berries were collected from the river side of Rangrik village, Lahaul and Spiti valley, Himachal Pradesh, India. The tissue was collected, washed with double-distilled water, frozen in liquid nitrogen and transferred to the laboratory for storage at − 80 °C. Seedlings were obtained from seabuckthorn seeds isolated from H. rhamnoides berries and were germinated and grown in growth chamber as previously described (Gupta and Deswal 2012). Briefly, the seeds were plated on the germination paper and to obtain uniform growth, incubated for 20 days in growth chamber with 16-h light/8-h dark and a photosynthetic photon flux density (PPFD) of 270 mmol/m 2 /s, 24 °C.

Extraction of proteins from H. rhamnoides seedling
For the extraction of total proteins from seedlings, the tissue was ground to fine powder in liquid nitrogen and 20 mM ammonium bicarbonate (ABC) buffer, pH 8.0, in a ratio of 1:2 was added. Insoluble material was pelleted down at 12,500 rpm, 4 °C for 30 min and the supernatant containing the proteins was used for further experiment. Protein concentration was determined using bovine serum albumin (BSA, Sigma) as a standard (Bradford 1976).

Antifreeze activity using nanoliter osmometer and sucrose sandwich splat assay
Extracted proteins were assayed for their antifreeze activity following the method described previously (Hon et al. 1994). Based on their ability to inhibit ice recrystallisation (IRI) and to modify the ice crystal morphology, the antifreeze activity was detected. Bovine serum albumin (BSA) and ammonium bicarbonate (ABC 20 mM) buffer were used as negative controls.
Nanoliter osmometer was used to observe ice crystal morphology. In brief, nanoliter volumes of protein sample (0.2 mg/mL) dissolved in ABC (20 mM) were loaded onto the sample-loading disk containing mineral oil and kept on the freezing stage of the phase-contrast microscope (Nikon). The sample was frozen rapidly at -20 °C using osmometer (Otago, New Zealand) to form multiple ice crystals and the temperature was then increased gradually to allow melting, until a single ice crystal is left in the well. The temperature was further decreased and the change in the morphology of the ice crystal was observed. In the presence of AFPs, the ice crystals were hexagonal whereas these were disc shaped in their absence. To confirm that the activity was due to AFPs, BSA (a non-AFP) and ABC (buffer) were used as a negative controls. The disk was overnight stored in organic solvent and vacuum dried to remove any impurities or protein left in the wells which could otherwise give false-positive antifreeze activity.
Sucrose sandwich splat assay was performed as described in Gupta and Deswal (2012). For measuring IRI activity, the protein extract obtained from seedling was dissolved in 30% sucrose in 20 mM ABC and 2 µL of this solution was sandwiched between two round coverslips. The sandwich was snap-frozen in chilled heptane and transferred to a chamber containing heptane maintained at − 6 °C for an hour. Ice crystals were allowed to anneal and their growth was photographed using a stereo-microscope (Leica, Wild M10) fitted with a camera (Nikon) and NIS-element software. For the quantification of the IRI activity, the cumulative diameters of ice crystals from three different fields in an image were measured using Image J software (version 1.51). The assay was performed in triplicates with three biological replicates and their average and standard deviation were calculated to find the percent IRI of each fraction. Student's t test was performed to observe the significant changes (p < 0.05) between the readings of control sample and the protein fractions. The data was compared with negative control (20 mM ABC) (Smallwood et al. 1999).

Purification of AFPs using chitin affinity chromatography
Chitin affinity chromatography was used for single-step purification of chitinases as described previously by Gupta and Deswal (2014). Briefly, colloidal chitin beads in 20 mM ABC (pH 8.0) were added to the protein extract until the optical density at 590 nm reached 0.8-0.9. The mixture was stirred at 4 °C to allow overnight binding of the chitinases with beads which were then packed into a column. The unbound proteins in the supernatant were collected as flow-through. To avoid non-specific binding, the column was washed with four bed volumes of ABC (20 mM) followed by two bed volumes of ammonium acetate (20 mM, pH 5.5). Bound chitinase was eluted using a linear gradient between 10 and 250 mM acetic acid (pH 3.0) for 30 min in ice (Kang et al. 1999).The eluted proteins were neutralized immediately using Tris (pH 9.5). The purity of the chitinase was checked by resolving the eluted proteins using 12% SDS-PAGE (Laemmli 1970) and were assayed for their respective antifreeze and hydrolytic activities.

SDS and native gel electrophoresis
SDS-PAGE was performed on a 12% denaturing polyacrylamide gel (Amersham) according to Laemmli (1970). Molecular marker (GE Healthcare, India) was the standard protein mixture consisting of phosphorylase b (97.0 kDa), BSA (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lysozyme (14.4 kDa). The protein samples (crude and purified) were loaded on gel by mixing these in sample buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 1% bromophenol blue and β-mercaptoethanol) followed by heating up to 100 °C. Electrophoresis was performed at 80 V for stacking gel and 120 V for separating gel, to obtain the protein profile. After electrophoresis, gels were stained with colloidal coomassie brilliant blue or silver stain accordingly (Laemmli 1970). For native-PAGE (10%) the proteins were resolved in non-denaturing conditions at 4 °C without SDS and β-mercaptoethanol in the loading and the running buffers.

Chitinase hydrolytic activity assay
Chitinase activity was measured using a colorimetric assay by measuring the release of N-acetylglucosamine (GlcNAc) from the substrate colloidal chitin regenerated from chitin (Legrand et al. 1987). For the determination of hydrolytic activity, reaction mixture (0.5 mL) containing the purified enzyme (100 µL) was incubated with 2% colloidal chitin (200 µL in 50 mM sodium acetate, pH 5.0) at 37 °C with constant agitation for 30 min. The reaction was terminated by adding potassium ferricyanide solution (0.5 g potassium ferricyanide in 1 L of 0.5 M sodium carbonate) followed by boiling of the mixture for 15 min. The reducing end group produced was determined colorimetrically by measuring the decrease in absorbance at 410 nm (Imoto and Yagishita 1971).

Glycosylation analysis of purified AFPs using Concanavalin-A affino-blotting
Glycoproteins were detected using Concanavalin-A (Con-A) affino-blotting method (Gupta and Deswal 2014). Briefly, the purified proteins were separated by SDS-PAGE using 12% gel. Electrophoretic transfer to Trans-Blot nitrocellulose paper (Amersham) was done at 4 °C, 400 mA for 1 h. The transferred polypeptides were visualized using Ponceau-S stain followed by blocking in phosphate-buffered saline (PBS) containing 3% BSA, at 37 °C for 1 h, followed by overnight incubation in Con-A solution (10 mg/mL, Sigma-Aldrich) containing 50 mM MnCl 2 , 100 mM CaCl 2 , 100 mM MgCl 2 and 0.5% Triton X-100 in PBST (PBS containing 0.05% Tween-20). The membrane was rinsed twice with PBST for 30 min followed by incubation with horseradish peroxidase (50 mg/ mL, Sigma-Aldrich) for 1 h. The reaction was developed by incubating the blot with 3.4 mM 4-chloro-1-naphthol (Sigma-Aldrich) in 100 mM Tris, pH 7.5, containing 0.3% H 2 O 2 in dark, which was freshly prepared immediately before use. Glycoproteins became visible as purple bands within 5 min of incubation. Once the bands with desired intensity appeared, the reaction was stopped by washing several times with water. The NC membrane was blotted dry, and stored in dark. BSA was used as negative while ovalbumin as positive control in the immunoblots.

Identification of purified AFPs using MALDI-TOF/TOF MS
The purified proteins were excised from SDS-PAGE gel, subjected to in-gel trypsin digestion (Trypsin, Promega) and identified using MALDI-TOF/TOF mass spectrometry as described previously (Sharma et al. 2018). Briefly, the excised polypeptides were washed, destained at RT and incubated in reduction solution (5 mM DTT) for 20 min. The reduced peptides were alkylated in dark followed by complete dehydration and air dried. Trypsin digestion was performed with 20 ng/μL at 37 °C, overnight. Peptides were extracted in buffer containing ACN and trifluoroacetic acid (50 μL) and mixed with R-cyano-4-hydroxycinnamicacid (CHCA) matrix, spotted onto a MALDI sample plate (Opti-TOF 384 well plate, Applied Biosystems, MA) for MS/MS ion search. The resulting digests were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (ABSCIEX MALDI TOF-TOF 5800 system Shanghai, China). For protein identification, the acquired MS/MS spectra were searched against the NCBIprot 20170707 database using the search engine MASCOT V2.1 (Matrix-Science, London, UK). Search parameters were set as follows: taxonomic category-Viridiplantae (5147436 sequences); enzyme-trypsin (cleavage at C-term side of Lys and Arg unless the next residue was Pro); carbamidomethyl as fixed modification; oxidation as variable modifications; unrestricted protein mass with maximum one missed cleavage. Peptide and fragment mass tolerance were set as 1.2 ppm and 0.5 Da, respectively. Confidence threshold was set to 95% and MASCOT protein ion with p < 0.5 was considered significant.

Bioinformatics analysis of purified AFPs for functional classification
Bioinformatic analysis of purified AFPs was performed as described previously in Sharma et al. (2018). Physicochemical characterization was done using ExPASy-Prot-Param software (https ://us.expas y.org/tools / protparam. html). Conserved functional domains were identified using Conserved Domain Database (CDD) web server (www. ncbi.nlm.nih.gov/Struc ture/cdd/wrpsb .cgi). Secondary structural elements were predicted using secondary structure prediction server GOR IV. BLASTP search (https :// blast .ncbi.nlm.nih.gov/Blast .cgi) was performed through Blast2GO against NCBI protein database. Subcellular localization was predicted using Plant-Protein Subcellular Localization Prediction (Hong Kong Polytechnic University) Version 2.0. STRING database (https ://strin g-db.org, Version 10.5) was used for protein-protein interactions.

Results and discussion
Understanding the cold tolerance mechanism of H. rhamnoides, a cold-hardy and abiotic stress-tolerant shrub, is of great interest as the research till date is focused mainly on analyzing its nutraceutical and pharmaceutical traits. Laboratory-grown seabuckthorn seedling secretome analysis has already revealed accumulation of two dual-functioning chitinases (31 and 34 kDa) during CA, suggesting their role in cold tolerance. Therefore, the objective of the current study was to analyze if similar or different cytosolic AFPs are present in H. rhamnoides seedlings and to further understand their functional roles.

Antifreeze activity measurements (nanoliter osmometer and sucrose sandwich splat assay) in seedling crude protein extract
AFPs owing to their characteristic ability to interact with ice crystals and modify their growth and morphology (Antikainen and Griffith 1997) could help in the survival of the plant during freezing conditions. The thermal hysteresis (TH) activity of these AFPs protect the plants by depressing the freezing point while ice recrystallisation inhibition activity prevents the formation of larger ice crystals which could have a detrimental effect (Raymond et al. 1989;Griffith and Ewart 1995;Zachariassen and Kristiansen 2000;Atici and Nalbantoğlu 2003).
To initially investigate the presence of AFPs in H. rhamnoides seedling, antifreeze activity of crude protein extracts prepared in ammonium bicarbonate (20 mM) was determined using both sucrose sandwich splat assay and nanoliter osmometer coupled with phase contrast microscopy. Ice crystal morphology (ICM) using nanoliter osmometer (Otago osmometer, New Zealand) was used for the qualitative analysis of antifreeze activity in H. rhamnoides seedlings as confirmed by the presence of hexagon-shaped ice crystals (0.2 mg mL −1 ). However, control samples (buffer and BSA) were disc shaped in the absence of AFPs which confirmed the specificity of the assays (Fig. 1, upper panel).
Sucrose sandwich splat assay was performed for quantification of the antifreeze activity. IRI activity analysis results were in accordance with the ice crystal morphology. Fig. 1 Antifreeze activity analysis of H. rhamnoides seedling. Ice crystal morphology (upper panel) using nanoliter osmometer and IRI assays using sucrose sandwich splat assays (lower panel), for the detection of antifreeze activity in i ammonium bicarbonate (ABC) buffer ii seedling crude protein samples (0.2 mg/mL) and iii BSA. ABC and BSA used as negative controls did not exhibit antifreeze activity. Magnification scale bar in splat assay represents 100 μm Bright-field images using stereoscopic zoom microscope (Zeiss) also confirmed IRI activity in seedlings as indicated by ~ 1.56-fold decrease in the mean ice crystal size (Fig. 1,  lower panel). IRI endpoint, the concentration below which IRI activity is no longer detected was determined as 65 μg/ mL for seedling AFPs. Daucus carota crude root extract showed IRI activity till 150 μg/mL (Smallwood et al. 1999). Similarly, Japanese radish tuber and leaf crude apoplastic extract showed IRI endpoints of 46 and 16 μg/mL, respectively (Kawahara et al. 2009). Forsythia dehydrin (20 kDa) showed IRI activity at 6 μg/mL (Simpson et al. 2005). However, H. rhamnoides-purified apoplastic AFPs, PGIP (41 kDa) and chitinases (31, 34 kDa) showed endpoints at 12, 60 and 120 μg/mL, respectively Deswal 2012, 2014).
As both ICM and IRI activity analyses confirmed the antifreeze activity in seabuckthorn seedling, efforts were made to purify the dual-functioning AFPs.

Single-step purification of seedling AFPs using chitin-affinity chromatography
Chitinases belong to the family of glycosyl hydrolases which hydrolyses chitin, β-1,4-linked polymer of N-acetyl-D-glucosamine (GlcNAc) and their sizes vary from 20 to 90 kDa. Tissue-specific differential accumulation of chitinase (CpCHT1, 33 kDa) was observed in apoplastic chitinase extracted from the petals, corolla, leaf, bark and root of C. praecox communis L. with flowering (corolla) tissue showing higher expression (Zhang et al. 2011). These proteins are known to provide protection against both psychrophilic phytopathogens and low-temperature stress (Hon et al. 1995;Griffith and Yaish 2004). Chitinases were purified using single-step chitin affinity chromatography as described previously and the dual functionality of these purified AFP chitinases was confirmed by analyzing their antifreeze and chitin hydrolytic activities (Gupta and Deswal 2014).

Purified AFPs exhibited both antifreeze and hydrolytic activities
SDS-PAGE (12%) protein profile of the crude extract showed polypeptides ranging from 110 to 9 kDa (Fig. 2a). Conversely, chitin affinity chromatography led to the purification of two cytoplasmic chitin-binding polypeptides, HrS CHT1a of 33 kDa and HrS CHT1b of 38 kDa from the seedlings indicating the presence of different cytoplasmic isoforms than previously reported apoplastic isoforms (34 and 31 kDa) from H. rhamnoides seedlings (Gupta and Deswal 2014). Like apoplastic chitinases, HrS CHT1a was eluted from the chitin-affinity column using lower molarity of acetic acid (20 mM), while HrS CHT1b was eluted using higher concentration of acetic acid (250 mM) (Fig. 2b) indicating stronger chitin-binding affinity of HrS CHT1b in comparison with HrS CHT1a. Native-PAGE (10%) analysis suggested the monomeric nature of both HrS CHT1a and HrS CHT1b (Fig. 2c).
Antifreeze activity in both HrS CHT1a and HrS CHT1b (0.2 mg/mL) was confirmed by the formation of hexagonshaped ice crystal owing to the presence of these purified chitin-binding AFPs (Fig. 3). Conversely, chitinase hydrolytic assay showed higher (folds) hydrolytic activity in HrS CHT1b (500 U/mg) than HrS CHT1a (222 U/mg). Nevertheless, both the purified chitinases exhibit higher (4.35-and 9.8-fold) hydrolytic activity in comparison with hydrolytic Fig. 2 Colloidal CBB-stained SDS-PAGE (12%) gel showing the protein profiling of a crude seedling extract. Silver stained, b SDS-PAGE and c native gel of cytoplasmic chitinases (HrS CHT1a (33 kDa) and HrS CHT1b (38 kDa) purified from H. rhamnoides seedling activity of crude seedling extract (58 U/mg). Interestingly, differential hydrolytic activity suggests the different roles of chitinases in development-induced defense mechanisms where germinating seeds undergo softening and eventual breakdown of cellular structure, making the tissue susceptible towards pathogen attack. In this case, the enhanced hydrolytic activity of HrSCHT1b might be associated with protection from pathogens during plant growth.
Besides AFPs, various antifreeze glycoproteins (AFGPs) have also been reported from the overwintering plants including Solanum dulcamara (67 kDa), Daucus carota (36 kDa), Ammopiptanthus mongolicus (200,40,39 kDa), Rhodiola algida (29-85 kDa) and H. rhamnoides apoplast (34 kDa) (Table 1). Therefore, these purified AFPs were also tested for the presence of sugar moieties on the protein. Glycoaffinity blot (Con-A-peroxidase staining) indicated only HrS CHT1b to be glycosylated as indicated by a dark purple product formed on providing the substrate of HRPO in the presence of H 2 O 2 while HrS CHT1a was non-glycosylated. Specificity of the reaction was confirmed by ovalbumin  . 3 Antifreeze activity analysis of H. rhamnoides seedling chitinases HrS CHT1a and HrS CHT1b using nanoliter osmometer. Hexagonal ice crystals were observed in purified chitinases while the ice crystals were disc shaped and lacked antifreeze activity in the negative control, i.e., buffer and a non-AFP, BSA (positive control) which stained purple whereas no such color was observed in BSA (negative control) (Fig. 4). Further, MALDI TOF/TOF, structural and string analyses was performed to understand functional significance of these AFPs.

MS identification of purified AFPs
Purified chitin-binding proteins were subjected to MS/ MS identification. Unfortunately, MS analysis of purified proteins neither matched significantly with previously submitted seabuckthorn class I chitinase sequences in NCBI nor with any other plant chitinases suggesting these to be either some novel isoform or non-homologous proteins. Five different chitinase isoforms have been reported in rice, suggesting the presence of multiple isoforms exhibiting different structural domains and functional roles in plants.
Another major constrain which makes the identification of seabuckthorn proteins difficult is the fact that the genome of seabuckthorn is not yet sequenced and the NCBInr database information is confined only to the transcriptome and EST data for seabuckthorn seedlings, with very limited protein information.
Previously, N-terminal sequencing and in silico approaches (comparative sequence homology, and string analyses) were used for the identification of unknown or novel chitinases when MS identification was not successful. Moreover, proteins rather than working as independent entities interact with other proteins to execute their functions. Therefore, these purified proteins were subjected to bioinformatic analysis to analyze the protein-protein interactions and also to have a better understanding of their biological functions.

Physicochemical characterization, secondary structure prediction and conserved domain analysis of seedling AFPs
HrS CHT1b was identified as provicilin [Pisum sativum] a storage protein involved in the allocation of nutrient reservoir or energy metabolism during stress and germination. Similar storage proteins including Urtica dioica agglutinins, Solanum tuberosum lectin, Lycopersicon esculentum lectin and wheat germ agglutinins (WGA) possessing chitin-binding domains have been purified from multiple plant species (Table 1). Interestingly, HrS CHT1b was found to be an N-glycosylated protein and literature studies have shown that glycosylation of provicilin (P. sativum) allows transport of this storage protein from endoplasmic reticulum to protein-storage vacuoles (Jiang and Rogers 1999). Subcellular localization analysis using Plant-mPLoc software confirmed the secretory nature of HrS CHT1b localized in vacuole, indicating these to be imported from other parts in  response to external signal. Surprisingly, vacuolar localization of HrS CHT1b, supported the role of PTMs (glycosylation) in protein targeting. Similar regulatory role of PTMs in protein targeting is also reported in Coffea arabica (Guerra-Guimaraes et al. 2009). Interestingly, physicochemical characterization (ExPASy-ProtParam software) showed that the theoretical molecular weight (M.W) of HrS CHT1b (39,800) related closely to the M.W determined experimentally (38,000). Moreover, HrS CHT1b seedling chitinases (pI 4.9) like their apoplastic counterparts are acidic (pI 4.6) in nature (Gupta and Deswal 2014).
String analysis to identify protein-protein interaction network of HrS CHT1b showed its interaction with GH 19 family protein with N-terminal chitin-binding domain and C-terminal GH catalytic domain which might be responsible for its stronger chitin (substrate) binding affinity and higher catalytic (hydrolytic) activity than HrS CHT1a. Another interesting interacting partner includes pectinesterase, a PR protein which besides providing protection from pathogens, also facilitates plant cell wall modification to manipulate growth and development processes. Association with these interacting partners indicates the probable role of HrS CHT1b during seed germination via degradation of chitooligosaccharides of seed coat to facilitate emergence of radicle. Higher hydrolytic activity of HrS CHT1b might be involved in providing protection to the exposed inner seed tissues against microbial attack (Rawat et al. 2017) (Fig. 5). Similar expression of chitinases prior to radical emergence was also observed in the endosperms of tomato seeds (Wu et al. 2001).
To our surprise, a glycine-rich protein (AT1G04660) associated with cold/salt stress tolerance (Mangeon et al. 2010) was also observed as interacting partner of HrS CHT1b suggesting its role in cold acclimation. Similar glycine-rich AFPs possessing higher antifreeze (hexagon ice crystals) and thermal hysteresis (5.8 C) activities have been purified from Snow fleas (Graham and Davies 2005). Additionally, interaction of HrS CHT1b with gene for late embryogenesis abundant protein 6 (GEA) which accumulates during embryo development at the onset of seed desiccation and water deficit in vegetative plant tissues suggest its probable role during growth of seedlings. Interaction with other storage family proteins such as cruciferin (CRU 3), albumin (SES 4), cupin (PAP 85) and oleosin (AT3G01570) shows regulation of energy metabolism and nutrient allocation during growth period (Fig. 5). Previous studies have also supported that constitutively expressed chitinases in storage tissues such as seeds, fruits and tubers might contribute a storage form of nitrogen (Rawat et al. 2017). However, interaction of HrS CHT1b with export proteins (AT5G47480) involved in ER to Golgi apparatus transport supports their vacuolar targeting (Table 2).
Sequence and structure analyses of purified AFPs may provide better understanding of their functional roles. Previous structural domain-based categorization classified AFPs into different families including leucine-rich repeat (LRR), PR, IRI, hemagglutinin-related, pleckstrin homology, and WRKY-domain-containing families. LRR domains containing AFPs (Daucus carota) are rich in both alpha and beta sheets while IRI regions predominantly contain coils and sheets. AFPs from Deschampsia antarctica, Lolium perenne, Triticum aestivum, and Hordeum vulgare contain both LRR and IRI domains (Muthukumaran et al. 2011a, b).
Secondary structure prediction using GOR IV server showed the highest percentage of random coils in HrS CHT1b (48%) along with moderate content of alpha helices (32, 39%) followed by extended sheets (26, 13%) ( Table 3). The results were in accordance with previous studies showing S. cereale AFPs similar to PR proteins also exhibiting higher coil-like secondary structure with both antifreeze and hydrolytic activities. Interestingly, higher alpha helices without disulfide bridges might allow unrestricted passage to AFPs for proper functioning and prevention of freezing. Further, conserved functional domain analysis (NCBI Conserved Domain Database server) confirmed that HrS CHT1b belongs to glycosyl hydrolase (GH) 1 superfamily and contains substrate binding N-terminal cupin, and C-terminal catalytic GH domains separated by AraC domains which corresponded well to the string analysis showing its similarity with GH 19 family proteins (Fig. 6). GH1 family contains a wide range of β-glycosidases including β-galactosidases, β-mannosidases, phospho-β-galactosidases, phospho-βglucosidases, and thioglucosidases (Opassiri et al. 2006). HrS CHT1b GH domain showed structural similarity with cyanogenic p-glucosidase from white clover, belonging to GH1 superfamily containing (α/β)8 TIM barrel fold similar to chitinases (Barrett et al. 1995). Meanwhile, the cupin domain showed similarity with wheat germins or agglutinins belonging to chitinases like lectin superfamily. GH (EC 3.2.1) enzyme is involved in carbohydrate catabolic process and catalyzes the hydrolysis of glycosidic bond thereby showing its correlation with corresponding higher hydrolytic activity. However, AraC is a transcriptional regulator with DNA-binding domain and helix-turn-helix structure which includes both metal-dependent and metal-independent enzymes, as well as catalytically inactive seed storage proteins. Cupin 2 family is a functionally diverse class of proteins including a variety of enzymatic as well as nonenzymatic seed storage proteins with conserved beta-barrel fold with one or two cupin domains showing regulation of energy metabolism and nutrient allocation during growth period. Similarly, novel function for globulin-containing cupin domains has been reported in Wrightia tinctoria for sequestering plant hormone (Kumar et al. 2017). These results correlated with the interaction of HrS CHT1b with other storage family proteins such as cruciferin, albumin, cupins and oleosins (Fig. 6, Table 3). String and conserved domain analyses thus supported the dual functionality of HrS CHT1b exhibiting both hydrolytic and antifreeze activities.
On the contrary, limited information was available for HrS CHT1a identified as uncharacterized protein from Glycine max. Physicochemical characterization showed that the theoretical (12750) and experimental (33000) M.W of HrS CHT1a did not match suggesting that the identified protein might be a fragmented portion or partial part of the actual protein. Further, HrS CHT1a chitinases are acidic (pI 4.9) in nature and localized in chloroplast unlike their neutral apoplastic counterparts (pI 7.0) (Gupta and Deswal 2014) ( Table 3).
String analysis showed interaction of HrS CHT1a with bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) involved in denovo glycine/purine synthesis and folate metabolism associated with the synthesis of biomolecules (NADPH, amino acids, nucleic acids, lipids, proteins and vitamin B5) (Fig. 5). Recently, it has been reported that DHFR-TS is involved in the synthesis of secondary metabolites particularly flavonoids thereby helping in maintaining redox status during stress. These results correlated with previous studies where chitinases indirectly trigger the defense reactions in plant via accumulation of secondary metabolites including phenolics, flavonoids and lignins (Grover 2012). These interactions suggest that HrS CHT1a might be involved in the regulation of nitrogen, secondary metabolites and energy metabolism to allow proper growth of seedlings (Gorelova et al. 2017). Conserved domain analysis revealed that HrS CHT1a belongs to pepsin-like aspartate protease superfamily showing structural homology with xylanases inhibitor family proteins (Fig. 6, Table 3). Previous studies have also shown that some chitinase like proteins including XIP-I, a xylanase inhibitor protein I from Triticum aestivum and XAIP, xylanase and alpha-amylase inhibitor protein from Scadoxus multiflorus exhibit specific physiological functions (Patil et al. 2013). Xylanases of fungal and bacterial pathogens are key enzymes involved in the degradation of xylans in the cell wall. Plants secrete proteins which inhibit these degradation glycosidases, including xylanase suggesting the role of HrSCHT1a in providing protection against pathogenic attack. Xylanases are known to act as elicitors for the accumulation of secondary metabolites thus showing its correlation with string analysis. These observations suggest that besides providing pathogenic protection, seedling chitinases might have novel regulatory roles in nutrient allocation, regulation of secondary metabolites/energy metabolism and cold stress acclimation which maintain the overall growth of seedlings.

Conclusion
In the present investigation, antifreeze proteins were purified using chitin-affinity chromatography from seabuckthorn seedlings. Two acidic chitinases (HrS CHT1a, 33 kDa and HrS CHT1b, 38 kDa) exhibiting both antifreeze (hexagonshaped ice crystal morphology) and hydrolytic activities were purified from seedlings. HrS CT1a showed interaction with proteins associated with folate metabolism and nucleotide synthesis indicating their role majorly in growth and Fig. 6 Structural analysis and functional annotation of purified AFPs using NCBI Conserved Domain Search to predict the conserved domains leading to categorization of proteins to different functional categories development regulation during germination. On the contrary, HrS CHT1b showed interaction with proteins involved in seed storage, pathogenic protection (pectinesterases, glycosyl hydrolases family proteins) and transporters indicating their role majorly in defense responses thereby confirming the diverse functional roles of both the chitinases.
Future efforts would be focused on mining the complete repertoire of cold-stress regulated proteins, purification of other AFP candidates as well as validation of these targets which could help in providing better insights into the cold hardiness mechanism of this cold desert shrub. These findings indicate that overexpression of these dual-functioning AFPs could have promising biotechnological applications.