Ectopic Expression of a Novel Cold-resistance Protein 1 (BoCRP1) from Brassica Oleracea Promotes Tolerance to Cold via Modulating Stress Associated Components

Cold stress is considered as a major environmental factor that adversely affect the plant growth and distribution. Therefore, there arises an immediate need to cultivate effective strategies aimed at developing stress-tolerant crops that would boost the production and minimise the risks associated with cold stress. In this study, a novel cold-responsive protein1 isolated from Brassica oleracea (BoCRP1) was ectopically expressed in a cold susceptible tomato genotype Shalimar 1 and its function was investigated in response to chilling stress. BoCRP1 was constitutively expressed in all the tissues of B. oleracea including leaf, root and stem however, its expression was found to be signi�cantly increased in response to cold stress. Moreover, transgenic tomato plants expressing BoCRP1 exhibited increased tolerance to chilling stress (4oC) with an overall improved rate of seed germination, increased root length, reduced membrane damage and increase in accumulation of osmoprotectants. Furthermore, we observed increased transcript levels of stress responsive genes and enhanced accumulation of ROS scavenging enzymes in transgenic on exposure to chilling stress. These results are therefore strongly in support of the role of BoCRP1 in offering the plant a protective shield and heightened resilience to chilling stress by maintaining osmotic balance, utilising the cellular antioxidant system and enhancing the transcription of cold responsive genes.


Introduction
Cold stress is considered as a pivotal component of environmental stress as it is one of the major contributing factor that severely affects the plant growth, and productivity across the globe.Since plants are sessile in nature, they are immobile and can respond to the cold stress only by changing the pattern of expression of speci c stress-related genes 1 .A microarray analysis by Seki, Narusaka 2 demonstrated that under cold stress conditions a signi cant number of stress responsive genes got differentially expressed. .There are a range of stress responsive genes and transcription factors identi ed and characterised hitherto, across different plant species 3,4 .The speci c interaction between a Crepeat/dehydration response elements (CRT/DRE) present in the promoter region of cold regulated (COR) genes with CRT/DRE binding factors (CBFs) regulates the expression pattern of many downstream genes most of which are stress responsive genes t with a role in cold tolerance, salinity and drought. 5,6,7,8 Th involvement of CBF proteins (CBF1, CBF2 or CBF3) in imparting cold tolerance is strengthened by the fact that transgenic lines constitutively over-expressing CBF proteins have concomitant expression of many cold regulated genes and subsequently improved tolerance to cold 9,10,11,12 such that down-regulation of both CBF1 and CBF3 resulted in a decline in expression of CBF regulatory genes in Arabidopsis which was also marked by decreased tolerance to cold . 13.While some of the COR proteins have role in imparting tolerance to cold by cryoprotection, has been established however, much needs to be studied vis-à-vis role of CBF regulon encoded proteins in cold tolerance and the subject remains open for thorough and elaborate research.COR genes are classi ed and grouped into four gene families such that each family is constituted of two members.These gene families include KIN (cold-induced), RD (responsive to desiccation), LTI (low temperature-induced) and ERD (early dehydration-inducible), Within a particular family of genes, each gene is in tandem association with the other member in the genome 14 .
The CRISPR/Cas9 generated mutants of CBF (single, double and triple) offered enough evidence to state that CBF genes regulates the expression of 414 downstream COR genes, besides the rate of seed germination was signi cantly lower in case of CBF mutants compared to WT 15 .Higher transcript accumulation of Cor/LEA genes has been associated with higher germination index in common wheat 16 .
Researchers have identi ed vast number of cold-responsive genes in Arabidopsis and other plant which include KIN1, KIN2, RD29A, RD29B, DREBs , and DELLA etc. 17,18,19,20,21,22 .These cold responsive genes (KIN1, KIN2, COR15A, COR15B RD29A and RD29B,) are present as on the same chromosome as tandem sequences. 2318,24 Kin 1 and Kin 2 are coordinately regulated in cold.Kin 2 mRNA is accumulated to a higher level during cold acclimation 18 .The fundamental interest in understanding the molecular mechanisms governing the cold stress stems from the thought that such an insight will aid in devising new strategies aimed at engineering the agronomically important crops for enhanced tolerance to cold.In the last few decades, a lot of strategies involving genetics, functional genomics, physiology and biochemistry have been employed to understand the response of plants to different environmental stresses such drought, cold and salinity.Here, we focus on the functional aspect of a novel cold resistant protein 1 from B oleraceae whose expression analysis indicates that BoCRP1 was constitutively expressed in different tissue samples of B. oleracea variety capitata and rapidly induced under cold stress conditions.This BoCRP1 protein is a member of Kin family and shows about 90% homology with other members of KIN gene family (KIN1 and Kin2). Toidentify the possible role of BoCRP1 gene, we looked for the genome data and found that two Kin sequences from A. thaliana Kin1 (At5g15960) and Kin2 (At5g15970) has high level of homology to BoCRP1 gene.There are a number of highly similar DNA sequences in related organisms.The one coding identical protein sequences in other B. oleracea species are annotated as cold-resistant proteins, KIN1 and KIN2.To explore the possible mechanism of cold stress we tried to characterize the function of BoCRP1 by overexpressing BoCRP1 under stress inducible promoter AtRd29A in cold susceptible tomato.Our ndings suggest that overexpressing BoCRP1 in cold susceptible tomato renders it tolerant to cold stress.

Isolation and Sequence Analysis of BoCRP1 gene
Previously we have performed proteomic study to compare the gene expression analysis in Brassica oleracea var.capitata under both normal and cold conditions (4°c).Under cold conditions, a novel low molecular weight protein called cold induced protein 1 (BoCRP1) was found to be highly induced.The CDS of of BoCRP1 (Accession no.GQ461800.1 ) consists of 198 bps which encodes a low molecular weight protein of 65amino acids a molecular weight of 6.5 KDa PI approaching to 9.1.Various sequence alignment tools revealed a strong homology between BoCRP1 protein and Kin proteins of B. napus, B. rapa and A. thaliana annotated cold-resistant proteins (Fig. 1a).On performing the phylogenetic analysis, it was clear that BoCRP1 has a close semblance to homologue from A. thaliana, B. rapa and B. napus, (Fig. 1b).As shown in Fig. 1, LEA proteins are also similar stress related proteins which are homologous to BoCRP1 protein.
However, BoCRP1 is closer to Kin1/Kin2 of Arabidopsis than to LEA-related protein from Actinidia chinensis.We found that the closest relative plant to B. oleracea on STRING database is Brassica rapa.It has a close relative (ortholog) of BoCRP1.In B. rapa, the gene (Bra008661) is connected to BRA000263 (COR15B Cold regulated gene), which suggests that Bra008661 (and hence BoCRP1 too) is involved in the cold response.Homology models showed a folded alpha-helix structure of BoCRP1 similar to that of KIN2 of A. thaliana 25 .All the above ndings strongly advocate the involvement of BoCRP1 protein in cold resistance.

Transcript analysis of BoCRP1 in B. oleracea
To examine the tissue speci c mRNA levels of BoCRP1 in B. oleracea var.capitata, plants were exposed to cold (4°C) for varying time periods.Expression studies indicated that the BoCRP1 transcript levels were highly up-regulated and reached a maximum up to 8 fold after 12hrs.After that, the expression shows a gradual decline (Fig. 2b).These results strongly suggest that BoCRP1 plays an important role by offering an early response to cold stress.Tissue-speci c expression analysis exhibited enhanced mRNA levels of BoCRP1 in the leaf tissues when exposed to cold.However, comparatively lower levels of BoCRP1were observed in the stem and the root tissues under cold compared to normal conditions (Fig. 2a).

Transformation and molecular Analysis
To validate and characterize the function of BoCRP1 in cold susceptible tomato variety, (Shalimar 1) the plant binary vector pcambia2301 was selected to clone the entire ORF of BoCRP1 gene under a stressinducible promoter of Rd29A gene (Fig. 3a).The transformation of the recombinant vector was executed in tomato cultivator Shalimar 1 (Fig. S1a-d) and obtained 20 kanamycin-resistant tomato lines independently (T 0 generation).
Using NPTII and BoCRP1 speci c primer sequences, a total of 10 transgenic lines were identi ed (Fig. 3b).Among them, four stably transgenic lines, OE1, OE2, OE8 and OE11 were con rmed to contain singlecopy insertion and segregated in 3:1 ratio for antibiotic selection possibly do to single T-DNA insert (Fig. 3c).In order to assess the transgene expression, q-PCR was performed to analyse different transgenic lines.We obtained three putative independent transgenic lines OE1, OE2 and OE11 with signi cant proportion of transgene expression under cold stress and were considered for further investigations (Fig. 3d).These selected transgenic lines were allowed to grown for 2 to 3 generations to obtain homozygous (T3) lines.

Overexpression of BoCRP1 in tomato improved the seedling growth and seed germination
To assess the cold tolerance, it was imperative for us to study the germination rate and seedling growth in both transgenic lines as well as in WT under normal (25˚C) and cold conditions (4˚C).At 25˚C, we observed a similar germination rate both in WT as well as transgenic lines, con rming that both the set of seeds are 100% viable, however, the rate of seed germination went signi cantly up in transgenic lines relative to the WT under chilling stress (4˚C) (Fig. 4a).The germination rate of transgenic lines OE1, OE2, and OE11 was approximately 80%, 79%, and 84% respectively compared to about 30% in WT under cold stress (Fig. 4b).
Furthermore, to understand whether BoCRP1 over-expression in uenced the seedling growth in transgenic lines, seedlings after germination were placed at 10˚C for a period of 2 weeks, following which the hypocotyl length along with main root length was measured with a ruler.In BoCRP1 expressing plants, no obvious difference in the root and hypocotyl length were observed compared to wild-type at 25˚C.Interestingly, at 10˚C both the root as well as hypocotyl length was suppressed in WT as compared to BoCRP1 expressing lines (Fig. 4c).Which at 10˚C displayed a marked increase in root and hypocotyl length.(Fig. 4d & e).The above results declared that the cold-induced expression of BoCRP1 can modulate the ability of a plant to resist low temperatures as was observed in early seedling and germination stage of transgenic tomato.

Ectopic expression of BoCRP1 in transgenic tomato enhances tolerance to cold
To further our understanding, we investigated the functional signi cance and physiological effect of BoCRP1 under chilling stress (4˚C) in both WT as well as transgenic lines.For this we incubated plants at 4˚C in the growth chamber for 4 days and then shifted to recovery at 25˚C.At ambient temperatures (25˚C), we observed no signi cant differences at any stage, neither in transgenic lines nor in WT tomato plants (Fig. 4f).However, transgenic lines showed a survival rate of about 73% (OE1), 68% (OE2), and 80% (OE11), respectively, while only 26% of the WT plants survived during recovery (Fig. 4e), suggesting an increased tolerance in BoCRP1 expression lines under the chilling stress.

BoCRP1 transgenic plants accumulate increased osmoprotectants under cold conditions
Under normal growth conditions, the WT and BoCRP1 expressing lines showed similar content of osmoprotectants (proline and soluble sugar).However, we observed signi cant accumulation of both the osmo-protectants at 4˚C in BoCRP1 lines compared to WT.Further, soluble sugar content increased by 2.4, 2.6 & 2.7 fold in transgenic line OE1, OE2 and OE11 respectively compared to WT under cold stress (Fig. 5a).Similarly, proline content shot up by 1.5 fold in OE1, 1.6 fold in OE2 and OE11 respectively, compared to WT (Fig. 5b).

Overexpression of BoCRP1 improves membrane stability in transgenic lines
In the context of Abiotic stress(cold, drought and salt) the level of malondialdihyde (MDA) and and relative electrolyte leakage (REL) is considered one of the vital parameter in evaluating the effect on lipid peroxidation and cytomembrane penetrability 26 .While evaluating the levels of MDA and REL in wild-type and BoCRP1 expressing plants, we observed similar no obvious differences in MDA and REL content in wild-type and BoCRP1 lines under control conditions (25 ˚C) (Fig. 5c & d).However, under cold conditions both the WT and BoCRP1 lines displayed an increase in REL and MDA content relative to the controls grown under ambient temperatures (25 ˚C).Cold stressed transgenic seedlings showed signi cantly reduced content of MDA and REL levels.Above results concluded that in WT plants cold treatment induces 2.9 fold increase in MDA content and only 1.8, 2.1 and 1.7 fold increase was observed in BoCRP1 lines OE1, OE2 and OE11 respectively (Fig. 5c) and the REL increased by almost 2.3 fold in WT and only 1.5, 1.5 and 1.3 fold increase in OE1, OE2 and OE3 respectively (Fig. 5d).
Overexpression of BoCRP1 improves the ROS scavenging capacity to enhance tolerance to cold stress.
To evaluate the extent of ROS accumulation under cold and normal temperatures in both WT and BoCRP1 transgenic lines, hydrogen peroxide (H 2 O 2 ) staining of leaves was carried out.The staining pattern was almost similar in WT and transgenic leaves grown under normal temperature (25 o C).However, after 3 days of cold treatment (4˚C) , we observed signi cantly higher staining (dark brown spots) in WT plants relative to transgenic lines (Fig. 6a).The reduced staining pattern observed in transgenic lines depicts improved detoxi cation of H 2 O 2 in the transgenic lines.These attributes were well linked with reduced levels of REL and MDA content in BoCRP1 expression lines, indicating reduced oxidative damage under cold stress.Under cold treatment, the BoCRP1 transgenic lines showed nearly 2.1, 3.0, and 3.1 fold increase in SOD, APX and CAT activity respectively compared to 1.8, 1.3 and 1.7 fold increase of these genes in WT maintained at 25 o C (Fig. 6b-d).These results suggest that BoCRP1 overexpression led to decrease in the levels of MDA and enhanced antioxidant capacity resulting in reduced oxidative injury to the transgenic tomato plants.

BoCRP1 over-expression enhanced Stress Responsive genes in transgenic tomato
To further widen our understanding of the molecular mechanism that governs the enhanced tolerance to cold in BoCRP1 transgenic lines, we analysed the mRNA expression levels of six ROS associated/stress response gene transcripts in both control as well as transgenic lines maintained under cold stress.Following the exposure to cold treatment (4˚C) for 3 days, the mRNA levels of ROS detoxi cation enzymes(POD, CAT and Cu-Zn SOD), signi cant regulatory protein (DREB1), proline Transporter 1 (ProT1), , Lipid transfer protein (LTP1) and stress defensive proteins (EDR15-2 and LEA) was signi cantly upregulated BoCRP1 expressing lines compared to WT (Fig. 7a-h).These ndings therefore, led to the conclusion that the enhanced stress responsive in BoCRP1 lines is a consequence of elevated expression of stress-associated genes.

Discussion
The KIN genes are low molecular weight proteins that belong to the COR gene family and serve to offer protective functions to the plant against cold stress.Two of the KIN proteins are arranged tandemly in the genome of A. thaliana whose expression has been seen to go up when exposed to NaCl, ABA,, polyethylene glycol (PEG) and cold 27,28,29 .Many homologues of KIN genes such as BN28a and BN28b, responsive to cold stress, have been identi ed in Brassica napus 30,31 .However, there are still many missing links that need to work so as to offer clear insights into the molecular and physiological mechanism of Kin gene expression and establish its role in stress-responsive mechanism.In this study, we have identi ed a novel cold-resistant protein1 (BoCRP1) from different varieties B. oleracea like acephala (Kale), capitata(cabbage), brotrytis(caulifolower) which was found to be homologous with other KIN proteins from A. thaliana , B. rapa and B. napus, annotated as cold-resistant proteins (Fig. 1a & b) 25 .After analysing the expression of BoCRP1 transcript under both normal as well as cold conditions it was observed that the expression remained more or less constitutive under normal temperatures and got highly induced under cold (Fig. 2b).Furthermore, the comparison of BoCRP1 transcript in different tissue types such as leaves root, and stem revealed higher expression of BoCRP1 in leaves compared to root and stem both under normal as well as stressed conditions with enhanced expression under cold stress treatment (Fig. 2a).This is consistent with what has been observed for other Kin family members such as BN28 of Brassica napus 31 and BoKIN1 of B. oleracea 32 and is also consistent with its proposed role in the process of cold acclimation, wherein the leaf seems to bear the major brunt of cold stress compared to root and stem.These results are therefore suggestive of BoCRP1 role in imparting cold tolerance that was further validated through its functional characterization in cold susceptible tomato.
To explore the physiological and molecular involvement of BoCRP1 in cold stress tolerance, we generated BoCRP1 expressing lines via transformation following the protocol of Arshad, Ihsan-ul-Haq 33,34 .Our ndings showed that BoCRP1 over-expression signi cantly improves the cold tolerance in transgenic tomato along with higher germination rate (Fig. 4a & b), increased root and hypocotyl length relative to WT, when exposed to cold stress (Fig. 4c-e).Moreover, compared to transgenic lines OE1, OE2 and OE11 displayed a marked increase in survival rate when subjected to cold stress for 4 days and returned to 25ºc (Fig. 4f -g ) suggesting a protective role of BoCRP1 against cold stress in tomato.
Under abiotic stress, the accumulation of the two major osmolytes, soluble sugar and 35 proline 36, 37 was observed to improve the osmotic potential by retaining the water in the cells and reduce any loss of water, which prevents the disruption of cellular metabolism.It has been found that proline brings about activation of some some stress-responsive genes 38 and hence relaying its osmoprotective function by causing enhanced expression of stress related genes.Besides relying on the osmoprotectants, plants also utilize some soluble sugars as a nutrient during stress conditions 39 .We also obtained an increase in accumulation of soluble sugars and proline content only in BoCRP1 expressing lines when exposed to cold stress.(Fig. 5a-b).A number of compatible solutes have been reported so far with an ability to confer tolerance against cold stress with proline and soluble sugars playing important role in rendering the plant cold tolerant 40 .
The above data was corroborated by the nding suggesting an enhanced expression of Prot1 transcript in the transgenic lines relative to the WT under cold conditions (Fig. 7d).The Prot1 gene is implicated to have a role in the biosynthesis of proline 41,42 suggesting that transgenic lines expressing BoCRP1 suffered minimum damage due to reduced loss of intracellular water partially by accumulating the elevated levels of intracellular osmolytes 43,44 .Moreover, in transgenic lines OE1, OE2 and OE11 the transcript levels of dehydrogen response protein (LEA) was signi cantly up-regulated under cold stress compared to wild-type tomato plants.The increased expression of LEA proteins under alleviates the osmotic stress and protects the plant from dehydration under abiotic stress conditions 37,45 .
The plant cells under cold stress frequently generate and accumulate a huge load of free radicals that have a damaging effect on the plant cell membranes that may ultimately result in cell death 46,47 .Under cold stress conditions, the plant cellular membranes gets damaged to a large extent which is clearly re ected by intracellular levels of REL and MDA 48,49 .The MDA and REL are indirect but important indicators for evaluating the response of a plant to cold stress as both can serve as markers to the extent of damage that a membrane has suffered 50 .In the study, it was observed that in BoCRP1 expressing lines, the level of MDA and REL were signi cantly lower compared to wild-type plants (Fig. 5c-d) hence, strongly suggesting a pivotal role of BoCRP1 protein in protecting the plant cells from lipid peroxidation.In A. thaliana lines expressing Cor15a in increased amount, tolerance to cold has been observed as there is a marked decrease in freezing induced membrane dehydration in protoplasts when exposed to chilling temperatures 51 .
Plants are well equipped with an effective ROS scavenging defence mechanism that include enzymatic antioxidants such as APX ,CAT, SOD and POD) and some non-enzymatic antioxidants which protect the cellular structures and other macromolecules from damage caused ROS molecules 52,53 .We have observed that the H 2 O 2 levels, as depicted in Fig. 6a.were signi cantly higher in WT under chilling stress compared to BoCRP1 expressing lines.The proteomic investigation by Xu, Li 54 also reported a huge spike in ROS levels and death of leaf cells in frost-sensitive winter wheat cultivars when they were exposed to compared to the frost-tolerant cultivar leaves that accumulated signi cant amount of antioxidant-related proteins.In the present study, we observed BoCRP1 expressing lines displayed increased activity of SOD, APX and CAT compared to the WT (Fig. 6b-d) and that could be attributed to the resilience of transgenic tomato lines to cold stress as compared to WT.The SOD acts as a scavenging enzyme by acting upon superoxide and convert it to H 2 O 2. Consequently, the APX and CAT act on H 2 O 2 and perform its detoxi cation 25 .The higher accumulation of ROS scavenging enzymes in BoCRP1 expression lines is concomitant with reduced accumulation of H 2 O 2 in transgenic lines as depicted in DAB assay.
Based on the above results, we can assume that enhanced expression of BoCRP1 expression imparted the cold tolerance in tomato possibly by activation of genes involved in generating a stress response.To investigate this, we performed qPCR of some stress associated genes including the ROS detoxifying enzymes (SOD, POD, and CAT), lipid transfer protein 1 (LTP1), Dehydration response element-binding protein (DREB1), Proline biosynthesis gene (ProT1) and Late embryogenesis abundant proteins (ERD15-2 and LEA).qPCR analysis of these stress-responsive genes led to several interesting ndings.SOD, CAT and POD involved in ROS detoxi cation mechanism are highly induced under cold in the transgenic lines relative to Wild-type plants (Fig. 7a-c).The observations are in consonance with the increased activity of these enzymes that result in reduced accumulation of H 2 O 2 in BoCRP1 expressing lines on exposure to cold.Furthermore, the up-regulated expression of LTP1 in transgenic lines relative to the WT plants (Fig. 7f) also exhibited consistently reduced MDA and REL.Since LTP1 is involved in lipid metabolism 55 it's up-reregulation in BoCRP1 expressing lines under chilling stress indicates its role in reducing the damage to lipids and membranes.ProT1 which is involved in proline biosynthesis showed enhanced expression under chilling stress in transgenic lines than WT (Fig. 7d) which is concomitant with the enhanced accumulation of Proline in BoCRP1 expressing lines under cold.DREB1 (signi cant regulatory protein) along with EDR15-2 and LEA (stress defensive proteins) showed increased expression under cold stress in over-expression lines compared to WT (Fig. e, g & h).These stress-responsive genes protect the plant cells against different types of environmental stresses by stabilizing the liable enzymes, protecting macromolecules and cellular membranes 56 .This further explains the reduced accumulation of REL and MDA content and increased survival of transgenic lines under chilling stress.Our results are in complete concordance with the study carried out by 37, 43, Liu, Yu 55 with regard to the up-regulation of stress responsive gene under abiotic stress.Based on the results discussed, we can conclude that the BoCRP1 acts as a multifunctional protein that acts through the downstream proteins to combat the cold response in plants.To further investigate the BoCRP1mechanismin modulating the cold tolerance, we need to unravel its regulation and downstream target proteins.

Plant material
B. oleracea (Capitata) seeds were sown in a mixture of vermiculite, peat moss and soil prepared in the ratio of 1:2:1 and were allowed to grow in a greenhouse, with a photoperiod cycle of 16/8h (day/night), for six weeks.The plants, when required were given cold stress in a chamber maintained at 4 o C for a maximum period of 3 days.At different intervals samples were collected and stored at -80 o C. Seeds of L. esculentum (Shalimar 1) were thoroughly rinsed in sterile distilled water for about 10 minutes followed by surface disinfection with 70% (v/v) ethanol for 3-min .The seeds were then washed for 15min in a solution containing sodium hypochlorite (4 % (v/v)) and few drops of Tween-20 and rinsed 3 times with sterile water for 3 minutes and allowed to germinate on solidi ed MS medium with half strength 57 mixed with 15 gL -1 sucrose with a resultant pH of 5.8 and the plates were maintained at 25 o C in the dark for 2 days.Post germination the seedlings were grown at at 25 o C for 7-10 days maintained at 16/8h (day/night).Cotyledonary explants of 10-day-old seedlings were cut from proximal as well as on distal sides and cultured on pre-culturing medium for 2 days followed by co-cultivation with Agrobacterium in dark for 3 days.Further, to check the combined effect of plant growth regulators, we carried out three independent experiments containing nearly 150 explants in each experiment.

Isolation of BOCRP1 and multiple sequence alignment
Total RNA was isolated from B oleracea leaf sample using trizol reagent (Invitrogen).To synthesize the rst-strand cDNA, Super Script™ VILO™ (Invitrogen) was used as per the manufacturers protocol.The fulllength open reading frame (ORF) was ampli ed from leaf cDNA of B oleracea using BoCRP1 speci c primers (Gene Bank accession no.GQ461800.1).To clone the ampli ed fragment, we used pGEM®-T Easy Vector Systems (Promega) and consequently the clones were sequenced for identi cation of desired seqence.A tblastp search was executed on www.ncbi.nih.gov.edu and the proteins with low Evalues were obtained from the sequence database.A multiple sequence alignment of the obtained sequences was executed using ClustalX to nd domains conserved across the sequences.A phylogenetic tree was also constructed by the neighbor-joining method to determine how these aligned proteins are related to each other.
Cloning of BoCRP1 in plant binary vector and Agrobacterium-mediated transformation of tomato plants.
To construct BoCRP1 recombinant vector, the coding sequence of BoCRP1 (cold resistant protein 1) accession number GQ461800.1 was ampli ed from B.oleracea and cloned into BamH1 and SacI site downstream of stress inducible AtRd29A in PCAMBIA2301 plasmid.The recombinant vector harbouring the BoCRP1gene was introduced into super-virulent strain of Agrobacterium, GV3101 using the freezethaw method.Transformation of recombinant vector was con rmed by PCR and restriction digestion.10 days old cotyledons of tomato seedlings were co-cultivated with Agrobacterium.After selection the transformants were regenerated on shoot regeneration medium (SRM) containing MS agar mixed with 2 mg L -1 zeatin, 0.1 mg L -1 IAA and 250 mg L -1 cefotaxime and 50 mg L -1 kanamycin.Regenerated shoots were transferred to the root regeneration medium (RRM) and hardened in a mixture containing 1:1 ratio of soil and vermiculite in the PVC pots and maintained in greenhouse at Kashmir University Botanical Garden.

Validation of BoCRP1 expressing transgenic lines through PCR
For this, total DNA DNA was isolated from the leaf tissues by CTAB method 58 .The insert was con rmed by performing PCR, using primers speci c to NPTII and gene speci c primers.The PCR reaction was performed in a tube containing 100ng of genomic DNA, 1× PCR buffer (MgCl 2 included), 0.25mM dNTP mix (sigma) and 0.05 U Taq DNA polymerase (sigma) with a reaction volume of 20 20µl The reaction conditions were set with an initial denaturation(95°c) for 10min.Following this, for 35 cycles, the following reaction parameters were set as: 95°c/1min, 60°c/30sec, 72°c/ 40sec and nal extension at 72°f or 10 min The amplicons were con rmed on 1% agarose gel and visualized under a UV illuminator.

Evaluation of transgenic tomato lines using Southern blot
For copy number insertion, we performed Southern-blot analysis as per Southern 59 .Initially 15 µg of genomic DNA was digested using BamH1 enzyme and allowed to run overnight on 0.8% agarose gel.The DNA was then transferred onto an Amersham Hybond TM -N + membrane (manufactured by GE Healthcare).This was followed by cross-linking of DNA by exposing the membrane to UV (1200 µJ for 5 min) as explained by Russell and Sambrook 60 .These membranes with cross linked DNA fragments were hybridized with probe directed against NPTII that was labelled with dioxigenin using DNA labelling kit (Roach Sigma) and later exposed to X-ray lm (Amersham Hyper lm TM ECL GE Healthcare).Hybridization, membrane washing, DNA probe preparation, were performed as per the mentioned protocol (DIG Nonradioactive Labelling and Detection system Roch).

qPCR Analysis
For the quantitative analysis, total RNA was extracted from the leaf tissue of transgenic tomato plants by TRIZOL reagent (Invitrogen) and subjected to DNase I (New ENGLAND BIOLABS).After checking the integrity of RNA, nearly 2µg of puri ed RNA was used to synthesize the rst cDNA strand using Super Script™ VILO™ (Invitrogen).Primers qPCR analysis were performed to detect the transcript levels of BoCRP1 across different transgenic lines, that were either exposed to cold stress (4°C) or maintained at normal temperature conditions (25°C) using β-Tubulin as internal control.qPCR was also performed to analyse the transcripts corresponding to both ROS related as well as stress responsive genes in WT plants and the transgenic lines.In order to calculate the relative expression levels, 2 -∆∆CT method was employed 61 ) using β-Tubulin as internal control to normalize the expression levels of target genes.Both the real-time and full length primers were designed using primer-3 online bio-informatics tool.The primers designed were also crossed checked using Gene Runner tool.For qPCR analysis primer e ciency was calculated for each primer set using 10-fold dilution.In each experiment three biological replicates were used.

Evaluation of Response of Transgenic tomato plants to Cold Stress Germination and seedling growth assay
To determine the effect of cold stress on germination, seeds from homozygous T3 transgenic lines (OE1, OE2 and OE11) and WT were used.For evaluating the cold stress tolerance, approximately 26 seeds were plated on MS media and placed in a growth chamber which was maintained at 25/4°C with 16/8 h (day/night) light cycle.The rates of seed germination were evaluated after a period of one week.For the seedling growth assay, the transgenic and wild-type lines after germination were transferred to 10°C for nearly two weeks.The main root length was measured with a ruler.Pictures were taken after treatment at 10°C.Each experiment was repeated thrice.
Electrolyte leakage (REL) was evaluated using the protocol described by Hu, Huang 62 with certain modi cations.For this the leaves were excised into ne strips and incubated at 28°C in 10 mL of distilled water for approximately 8 h.we measured the initial conductivity (C1) using the conductivity meter (Systronics, India).The samples were boiled in a water bath for 10 min and then cooled to room temperature to measure the electrolyte conductivity (C2).The extent of REL was evaluated using the equation: EL (%) = C1/C2 × 100.The levels of Proline and Malondialdehyde were assessed according to protocol described by Choudhury, Chowdhury 63 while the content of soluble sugar was determined following the outline of 45

Histochemical detection of H 2 O 2
The relative content of H 2 O 2 was detected visually in both transgenic as well as WT tomato leaves by utilizing 3, 39-diaminobenzidine (DAB) staining method described byBindschedler, Dewdney 64, 65

Determination of enzymatic antioxidants
For evaluating the activities of various antioxidant enzymes (such as SOD, APX, and CAT), we observed the protocol outlined by Liu, Yu 55 .

Statistical analysis
The data was statistically analyzed using GraphPad Prisim 5.Each experimental data represents the mean± SD of three Biological replicates such that each sample from the replicate was a combination of leaves corresponding to 10 different seedlings.Tukey's Multiple Comparison Test were performed to calculate signi cant differences in transgenic lines compared to WT. Asterisks represents signi cant differences at *P < 0.05; ** P < 0.01; ***P< 0.001.

Conclusion
As a whole, our ndings indicates that the over-expression of BoCRP1 increased the adaptability of tomato plants to cold stress.Besides there are many other manifestations of cold tolerance such as minimalistic damage to membrane, with increased scavenging potential of antioxidant enzymes (such as CAT SOD, POD and APX, reduced accumulation of MDA, REL and H 2 O 2, enhanced expression genes involved in stress and increased accumulation of osmo-protectants (proline and soluble sugars).This study would help us in gaining new insights for manipulating the chilling tolerance in cold susceptible crop plants without hampering the overall growth and development of the plant.

Figure 1 Sequence
Figure 1

Figure 2 Expression
Figure 2

Figure 4 Low
Figure 4