Srinivasan Balamurugan, Jayan Susan Ann, Inchakalody P Varghese, Shanmugaraj Bala Murugan,Mani Chandra Harish, Sarma Rajeev Kumar, Ramalingam Sathishkumar

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, India

1. Introduction

Being sessile in nature, plants constantly encounter several abiotic and biotic stresses that adversely affect plant growth, development and survival (Smйkalovбet al. 2014).Among them, abiotic stresses are very critical, as they are uncontrollable and unpredictable, which will potentially limit plant survival and thus lead to reduced yield (Wanget al.2013; Veladaet al.2014). The temperature stress imposed on plants has huge effect on agriculture. For instance, it has been reported that decrease of 1°C in the world average temperature might result in 40% reduction in rice production(Hale and Orcutt 1987). Hence, there exists a pressing need to overcome the crop loss due to negative impact of chilling stress on plant growth and survival.

Generally, temperate plants can increase their chilling tolerance by exposing to cold nonfreezing temperatureviaan adaptive response called cold acclimation (Thomashow 1999). Whereas, the plants of tropical and subtropical regions are sensitive to chilling stress and the negative effects of chilling stress are known to be higher in these plants, as they lack the cold acclimation mechanism(Thomashow 1999; Chinnusamyet al. 2007). Moreover,many important food crops such as rice, maize, soybean,cotton and tomato are chilling-sensitive, which are unable to acclimate cold and also they cannot withstand the chilling stress when exposed to low temperature (Chinnusamyet al. 2007).

Overwintering plants have natural ability to resist ice crystal formation in the cell by inhibiting its growth (Pudneyet al. 2003). Proteins isolated from many grass species have shown to possess antifreeze properties that help them to cope up with the cold conditions (Antikainen and Griffith 1997). Antifreeze proteins (AFP) are a class of proteins found in a range of over-wintering plants that protect them from the damage imposed by chilling stress(Kumaret al. 2014).Lolium perennebelongs to the Poaceae family,which are adapted to grow in the colder Northern hemisphere (Sandveet al. 2011) and reported to withstand freezing (Kuiperet al. 2001). Superior antifreeze protein responsible for this activity was identified fromL.perenne, which possessed two putative opposite-facing sites with surface complimentary to the prism face of ice(Kuiperet al. 2001). TheLpAFPgene consists of 354 bp that encodes a protein of about 118 amino acids (13.5 kDa)with a semi-conserved seven-amino acid repeat X-X-N-X-VX-G on its entire length (Sidebottomet al. 2000; Middletonet al. 2009). AFP reduces the freezing point of the solute non-colligative, the process termed as thermal hysteresis(TH), by inhibiting the recrystallization of the growing ice crystals (Kuiperet al. 2001). Besides that the AFP was also described as protector of cells from damage during non-freezing conditions (Tomczaket al. 2002) mainly due to their interactions with: i) the integral membrane proteins(Rubinskyet al. 1991); ii) the membrane lipids (Hayset al.1996); and iii) the membrane, which modi fies the acyl chain’s order in the bilayer core (Tomczaket al. 2002).

Tomato (Solanum lycopersicum) is one of the most consumed vegetables worldwide with the production of 1.70 million tons (http://www.fao.org/statistics/en/) in 2014 and is also one of the most preferred garden crops (Fanet al. 2015). In recent years, the consumption rate of tomatoes has substantially increased worldwide and during last two decades, the production and harvesting of tomato has doubled (Bergougnoux 2014). Across the globe, Asia dominates the tomato market where India ranked the second after China in tomato production. Tomato is one of the preferred vegetables in routine human diet, which achieved a great familiarity over the last century (Harish and Sathishkumar 2011; Badimonet al. 2017). Apart from the dietary source, tomatoes are also considered as an excellent source of antioxidants, phytochemicals such as lycopene, β-carotene, ascorbic acid, lutein, tocopherol,phenolic compounds and it is also cholesterol-free. These compounds are shown to promote positive health bene fits like antitumorigenic effects in human system, reduced risk of cardiovascular and different types of cancer (Harishet al. 2012). Besides its role in human diet, tomato plants are being extensively used in research, mostly due to its simple diploid genetics, short life cycle and relatively simple genome (～900 Mb) (Zhouet al. 2013; Kumaret al. 2014).

Nevertheless, tomato is a thermophilic crop that makes it more sensitive to chilling conditions (Zhouet al. 2013).Any modi fications in terms of enhancing the chillingtolerant ability of tomato plants will represent important biotechnological breakthrough in high-altitude farming.Transgenic technologies are presenting promising results in improving plant traits, such as enhanced chilling tolerance,by introducing single or multiple genes involved in response to chilling stress. A number of reports showed that the transgenic plants expressing AFP from various sources proved enhanced chilling tolerance (Holmberget al. 2001;Zhuet al. 2010; Linet al. 2011; Dengaet al. 2014; Sunet al.2014). As an attempt to develop chilling-tolerant tomato plants that could aid in high altitude farming, here we are reporting for first time the development of transgenic tomato plants by expressing theAFPisolated fromL.perenneand successfully demonstrated the chilling-tolerant capability of these transgenic lines.

2. Materials and methods

2.1. Gene isolation, cloning and plant expression using gateway technology

Young leaves from cold acclimatedL.perenneplants were used for total RNA isolation using Qiagen RNeasy RNA Isolation Kit (Qiagen, USA). And 1 μg of total RNA was used for cDNA synthesis using the random hexamer primer and M-MuLV reverse transcriptase (Fermentas, USA) following manufacturer’s instructions.

TheLpAFPgene (GenBank accession number:AJ277399) was amplified of each gene-specific primer(LpAFP-f101 and LpAFP-r102, both flanked by the attB sequences by Phusion DNA polymerase (New England Biolabs, USA) using a My-cycler Thermal Cycler (Bio-Rad,USA). The amplicon generated was puri fied using PCR Puri fication Kit (Qiagen, USA) and cloned into the entry vector pDONR/Zeo using the BP clonase II (Invitrogen,USA) following the procedure described by manufacturer’s protocol. Reaction product was then transformed intoEscherichia colicompetent cells. The sequencing-con firmed plasmid was recombined with the destination vector pEARLY Gate 102 (Earleyet al. 2006) by LR Clonase II Reaction(Invitrogen, USA) according to manufacturer’s instructions,thus enabling the transgene to be driven by the CaMV35S promoter (Fig. 1-A). Recombinant plasmid pEARLEYLpAFPwas electroporated intoAgrobacterium tumefaciensstrain LBA 4404 (Hoekemaet al. 1983) and used for stable plant transformation.

2.2. In vitro propagation and genetic transformation of tomato plants

Seeds ofS.lycopersicum cv.Pusa Ruby (procured from National Seed Corporation, New Delhi, India) were used.These seeds were washed 3–5 times with sterile distilled water and then surface-disinfected by immersion in 4%sodium hypochlorite solution for 10 min with continuous shaking. Subsequently, the seeds were rinsed with sterile distilled water to remove the traces of excess sodium hypochlorite and blot dried before inoculation. Surfacedisinfected seeds were then inoculated in Petri dishes(9.0 cm×1.5 cm) (GenAxy, India) with half-strength Murashige and Skoog (MS) medium (Duchefa, the Netherlands) (Murashige and Skoog 1962). Three days before transformation, cotyledons of previously inoculated seeds were excised and inoculated in the preculture media characterized by MS solid media with 2.0 mg L-1zeatin and 0.1 mg L-1indole-3-acetic acid (IAA) (～30 explants per plate in a total of 20 plates). The precultured cotyledons were used as explants forAgrobacterium-mediated genetic transformation.Agrobacteriumculture (OD600=0.6)harboring pEARLY102-LpAFPwas harvested at 5 000 r min-1for 10 min. The cells were resuspended in liquid MS solution and the cell density was adjusted to yield a final OD600of 0.6 and 100 μmol L-1acetosyringone was added.The explants were blot-dried, inoculated in co-cultivation media and incubated for 3 days. After the co-cultivation period, the cotyledons were blot-dried on sterile filter paper for 15 min and inoculated in selection media (MS with 2.0 mg L-1zeatin+0.1 mg L-1IAA+20 mg L-1glufosinate+300 mg L-1carbenicillin). After 2–3 rounds of selection in the selection media, the regenerated putative transformants with 5–6 cm length were excised and inoculated in rooting medium (1/2 MS media supplemented with 300 mg L-1carbenicillin).

2.3. T1 seed collection and germination

The transgenic tomato plants (T0) were hardened in the mixture (coir pith:vermiculite:red soil in 1:1:1 (v/v) ratio).The surviving transgenic lines were con firmed by genomic PCR. The PCR con firmed transgenic plants were harvested individually. The transgenic lines were grown to maturity;flowers were self-pollinated and covered with a polythene bag to avoid cross contamination. Seeds were collected and harvested in order to further generate thein vitroT1transgenic lines. Seeds of T1tomato lines (Tm5, Tm12,Tm17, Tm24, Tm27, Tm31 and Tm35) were randomly selected and cultivated after surface-disinfectionin vitroin 1/2 MS media+20 mg L-1glufosinate. Then 4–5 weeks-old plantlets were selected by genomic PCR as mentioned earlier and subjected to chilling treatment and further molecular analysis.

2.4. Molecular characterization of transgenic plants

Gene expression analysis of AFP by RT-PCRThe cDNA was synthesized from the total RNA as described earlier in Section 2.1. The synthesized cDNA was used as template for the RT-PCR usingAFPgene specific primers(LpAFP-f201 and LpAFP-r202). The native 26S rRNA gene was used as reference gene with a forward (Ref-f)and reverse primer (Ref-r). All the oligonucleotide primers employed in this study are provided in Appendix A.

Southern blot analysis of transgenic tomato linesGenomic DNA from the selected transgenic T1tomato lines were isolated using DNeasy Maxi Kit (Qiagen, USA). The genomic DNA (about 30 μg) from transgenic lines, wild type (WT) lines (negative control) and pEARLY-LpAFPplasmid (1 μg positive control) were digested with restriction enzymes (KpnI andMseI) to release theLpAFPand the digested product was electrophoresed at 25 V for 18 h on a 0.8% agarose gel and then transferred to nitrocellulose membrane (BioTrace NT, Pall, USA) by capillary blotting(Sambrooket al. 1989). Hybridization, biotin labeling of probe, membrane washing and detection were carried out as per manufacturer’s instructions (Thermo, USA).

2.5. Physiological characterization of transgenic plants

Physiological indexes such as electrolyte leakage index(ELI), relative water content (RWC) and estimation of total sugar content was performed for the T1transgenic lines under chilling and normal conditions to study the chilling tolerance capability.

Chilling treatmentThe T1transgenic lines (5 weeks old)were subjected to chilling treatment at 4°C in the growth chamber and the leaves were harvested every 24 h up to 5 days. The control samples were taken from the plants grown in the green house at normal conditions. The collected samples were used for further analysis.

Total soluble sugarsTotal soluble sugar content was measured according to the protocol described by Paridaet al. (2007) with slight modifications. Briefly, after chilling stress, the leaf samples (～100 mg) were ground in liquid nitrogen and added with 10 mL of 75% ethanol and incubated overnight in room temperature (RT) at 150 r min-1.Thereafter, the samples were centrifuged at 12 000 r min-1for 5 min and 1 mL of freshly prepared anthrone reagent(0.2%) was added to the supernatant, incubated in boiling water bath for 15 min and cooled to room temperature. The absorbance was measured at 625 nm.

RWCRWC (%) was measured in the T1transgenic and WT leaves as per the protocol described by Balestrasseet al. (2010).

ELIElectrolytic leakage or ionic leakage index was calculated from both the WT and transgenic lines as described by Wuet al. (2012). Leaf discs of all the samples(transgenic and WT plants) were collected and equilibrated in sterile deionized water for 4 h and initial conductivity was measured. The samples were then boiled and the final conductivity was measured using the formula. ELI(%)=Initial conductivity/Final conductivity×100

2.6. Data analysis

Data were represented as means±standard deviation of mean (SDM). All the experiments were repeated three times. Data were statistically analyzed. The data obtained were subjected to one-way analysis of variance (ANOVA)followed by Duncan’s multiple range test (DMRT) using SPSS ver. 13.0 (SPSS Inc., USA).

3. Results and discussion

3.1. Genetic transformation of tomato using LpAFP

A.tumefaciensLBA 4404 strain harboring the vector pEARLY102-LpAFPunder the control of CaMV35S promoter was used for genetic transformation. After co-cultivation period of 3 days, the cotyledons were placed on the selection media (MS+zeatin (2 mg L–1)+ IAA (0.1 mg L–1)+glufosinate(20 mg L–1)+crbenicillin (300 mg L–1)) to regenerate the transformants. Multiple shoots were induced in selection media within 3 weeks. Individual shoots were separated and inoculated in rooting media. Rooting was observed after 1 week and the plants with well-developed roots were hardened after 3 weeks. The fruits were collected and seeds were harvested to generate T1transgenic lines (Appendix B). Several independent transgenic lines were analyzed in terms of molecular and biochemical analyses (Appendices C, D and E). For clarity of presentation, results from two representative transgenic tomato lines are given.

3.2. Molecular analysis revealed the successful integration, expression and inheritance of transgene in the host genome

Southern blot hybridizationIn order to reinforce the integration of the transgene in the host genome, Southern blot analysis was carried out in the transgenic lines that are preliminary con firmed with genomic PCR (data not shown).The results showed the stable integration ofLpAFPgene in all the tomato lines. The hybridization signal observed in the transgenic lines corresponds to the size ofLpAFPgene, whereas no signal was observed in WT (Fig. 1-B).The above data demonstrated that the transgeneLpAFPwas stably integrated in the transgenic tomato.

RT-PCRThe stable expression ofLpAFPin the T1lines was analyzed by RT-PCR. The expected band size of 354 bp con firmed the transcription ofLpAFPgene in the transgenic plants, whereas no such amplicon was detected in WT. The result showed that the levels of transcription ofLpAFPgene in the transgenic lines were same. This might be due to the presence of constitutive promoter CaMV35S;hence the expression pattern was found to be same in all the lines. The 26S rRNA gene (500 bp) was used as an internal standard (Fig. 1-C).

3.3. Analyses of physiological indices revealed that LpAFP conferred chilling-tolerance in transgenics

Chilling-tolerant ability of transgenic lines was evaluated by phenotypic analysis under 4°C. The results of the chilling treatments con firmed that transgenic lines carryingLpAFPexhibited greater chilling tolerance. The phenotype analysis showed that the WT plants could not survive at 4°C. At the end of the 5th day, the transgenic lines did not show any phenotypic variations and found to be healthy (Fig. 2).

Total soluble sugarChilling stress in plants induces osmotic and oxidative stress that eventually leads to the accumulation of compatible solutes and elevates the antioxidative mechanisms in order to protect the plants from the stress-induced damage. The RWC, ELI and total sugar content were recognized as the important indicators of membrane integrity and chilling-tolerant ability of the plants(Wuet al. 2012). In this study, transgenic tomato lines were subjected to chilling treatment at 4°C and total sugar content was measured in both transgenic and WT plants. The total soluble sugar was increased during the chilling treatment in both WT and transgenic lines. Sugar content was increased up to 3.3-fold after 1 day of chilling treatment. After the 2nd day, sugar content increased gradually till the 5th day of treatment (Fig. 3). significant modi fications in the total sugar content were reported in many of the plants exposed to low temperatures, which was correlated to the increased soluble sugar level (Wanget al. 2013). Congruently, generation of transgenic tomato by overexpressingAtGRXS17resulted in increased total soluble sugar content during chilling stress(Xin and Browse 2000).

Fig. 2 Phenotypic analysis of 5-week-old tomato lines after 5 days under chilling-treatment. A-G, transgenic lines; i.e., Tm5,Tm12, Tm17, Tm24, Tm27, Tm31, and Tm35, respectively. H and I, wild type (WT) lines.

Fig. 3 Measurement of total sugar content in transgenic plants under chilling treatment. Each bar value represents mean±SD, n=3. significant difference between wild type(WT) and transgenic lines (Tm31 and Tm35) are indicated by＊, P＜0.05 level.

Sugars are known to act as cryoprotectants and osmolytes that protect the cell by stabilizing the membrane,thus prevent the cellular dehydration during chilling conditions (Sasakiet al. 1996). Among the various physiological responses during freezing tolerance, sugar content in plants is one of the major factors that determine the extent of freezing tolerance. Earlier studies showed that the exposure of plants to low temperatures led to fructan synthesis (Antikainen and Pihakaski 1994), then led to the accumulation of sucrose, fructose or glucose inside the cell; however, the type of sugar accumulation varies among different plant species (Rizhskyet al. 2004). The accumulation of sugar in turn protects the plasma membrane and other macromolecules from the effects of freezing or dehydration although the mechanism is still unclear. During deacclimation, sugar content of cold-acclimated plants was reduced, which proved that sugars were possibly one of the major factors in chilling tolerance (Antikainen and Pihakaski 1994). These results indicated that the excess content of sugar in both the transgenics and WT plants under chilling treatment might play a vital role in development of cold hardiness in plants and also suggested that total sugar content alone was not responsible for the cold tolerant ability of the transgenic plants.

RWCIn the present study, RWC was found to decrease in both the WT and transgenic lines. There was no significant difference for RWC among the plants just before chilling treatment. Interestingly the transgenic lines were found to have higher RWC level when compared to that of WT lines throughout the chilling treatment. Just before chilling treatment, i.e., the water contents of WT lines and transgenic lines were found to be 84.0 and 88.0% at 26°C, respectively. After 5 days of chilling treatment, water content of the WT lines was observed as 24.0%, while the transgenic lines were found to maintain the higher water content when compared with the WT lines (Fig. 4).Thus the observed reduction could be correlated to the increase in the solutes such as RNA, sugars, proteins and accumulation of osmoprotectants, which helps the plant defense mechanism (Camposet al. 2003). Similar reduction in the water content during chilling stress was also reported in transgenic tobacco plants (Wuet al. 2012).Thus, the RWC in plants was inversely proportional to the freezing injury induced by low temperature, i.e., the presence of large amount of water inside the cell is prone to be more susceptible for chilling injury andvice versa.

Fig. 4 Measurement of relative water content (RWC) in transgenic plants under chilling treatment. Each bar value represents mean±SD, n=3. significant difference between wild type (WT) and transgenic lines (Tm31 and Tm35) are indicated by ＊＊, P＜0.01 and ＊＊＊, P＜0.001 levels, respectively.

ELIPlasma membrane plays a vital role in the structure and function of all the cells. During low temperature, it undergoes phase transitions from liquid crystalline to rigid gel phase and streaming of intracellular water and electrolyte through plasma membrane resulting in increased electrolyte and conductivity (Huet al. 2015). ELI was found to be a versatile and sensitive assay to study cold acclimation of plants (Yanget al. 2011). There was a significant increase in electrolyte leakage in the WT lines during the chilling treatment, whereas the transgenic lines maintained the lower level of electrolyte leakage during the chilling treatment up to the 5th day of treatment, which was 50% lower than the WT lines (Fig. 5). This results proved the protective nature of AFP in transgenic lines. Wuet al. (2012) reported that WT lines showed significant increase in electrolyte leakage as compared to transgenic tobacco lines expressingCbCOR15bgene. Similarly, overexpression of transcription factorTERF2/LeERF2increased electrolyte leakage in transgenic plants and conferred freezing tolerance through ethylene signaling pathway in both transgenic tomato and tobacco (Zhang and Huang 2010). In our previous report(Kumaret al. 2014), we have shown that the T0transgenic tomato plants expressing carrotAFPcan grow in the chilling conditions up to 48 h as compared to WT. However, in the present study, T1transgenic lines expressingLpAFPhas shown to tolerate chilling treatment even after 5 days and did not show any phenotypic abnormalities as compared to that of WT. This result clearly proved the better protective ability ofLpAFPunder chilling condition, which could be due to the presence of two ice-binding domains as against the single domain present in carrot AFP (Kuiperet al. 2001;Pudneyet al. 2003; Kumaret al. 2014).

Fig. 5 Measurement of electrolyte leakage index (ELI) in transgenic plants under chilling treatment. Each bar value represents mean±SD, n=3. significant difference between wild type (WT) and transgenic lines (Tm31 and Tm35) are indicated by ＊＊＊, P＜0.001 level.

Once the temperature is reduced, the transgenic lines retained the stamina as against WT lines and no phenotypic abnormalities were observed in the transgenic lines.Even though function of AFPs showed that, this protein would inhibit the ice recrystallization at subzero conditions(Chinnusamyet al. 2007), function of this proteins may differ with respect to the chilling tolerance ability of the plant system (Zhang and Huang 2010). Besides the freezing condition, AFP was reported to protect the cells from chilling injury (Tomczaket al. 2002). This protective function of AFPs at chilling temperatures is mainly due to the interaction of AFP with the integral membrane proteins(Rubinskyet al. 1991), membrane lipids (Hayset al. 1996)and the membrane itself, which modi fies the acyl chain’s order in bilayer core (Tomczaket al. 2002). Taken together,our study proved thatLpAFPconfer the chilling tolerance ability to transgenic tomato lines.

5. Conclusion

In the study, we successfully generated transgenic tomato plants expressingLpAFPwith enhanced tolerance to chilling stress. Our data proved the protective role ofLpAFPin transgenic tomato plants under chilling conditions and also proved thatLpAFPis a potential candidate to confer chilling tolerance, probably by upholding the membrane integrity of the transgenic plants. This is the first report ofLpAFPexpression in transgenic tomato lines to increase the chilling tolerance. The outcome will significantly help the high altitude farming of economically important food crops in long term.

Acknowledgements

The research was supported by the Senior Research Fellowship from the Council of Scienti fic and Industrial Research-Human Resource Development Group (CSIRHRDG), New Delhi, India (09/472(0164)/2012-EMR-I), and the funds from the University Grants Commission-Special Assistance Programme (UGC-SAP) and the Department of Science and Technology-Fund for Improvement of S&T Infrastructure (DST-FIST), Bharathiar University, Tamil Nadu, India.

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

Antikainen M, Grif fith M. 1997. Antifreeze protein accumulation in freezing-tolerant cereals.Physiologia Plantarum, 99,423–432.

Antikainen M, Pihakaski S. 1994. Early developments in RNA,protein, and sugar levels during cold stress in winter rye(Secale cereale) leaves.Annals of Botany, 74, 335–341.

Badimon L, Vilahur G, Padro T. 2017. Systems biology approaches to understand the effects of nutrition and promote health.British Journal of Clinical Pharmacology,83, 38–45.

Balestrasse K B, Tomaro M L, Batlle A, Noriega G O. 2010.The role of 5-aminolevulinic acid in the response to cold stress in soybean plants.Phytochemistry, 71, 2038–2045.

Bergougnoux V. 2014. The history of tomato: From domestication to biopharming.Biotechnology Advances, 32, 170–189.

Campos P S, Quartin V, Ramalho J C, Nunes M A. 2003.Electrolyte leakage and degradation account for cold sensitivity in leaves ofCoffeasp. plants.Journal of Plant Physiology, 160, 283–292.

Chinnusamy V, Zhu J, Zhu K. 2007. Cold stress regulation of gene expression in plants.Trends in Plant Science, 12,444–451.

Deng L Q, Yu H Q, Liu Y P, Jiao P P, Zhou S F, Zhang S Z, Li W C, Fu F L. 2014. Heterologous expression of antifreeze protein geneAnAFPfromAmmopiptanthus nanusenhances cold tolerance inEscherichia coliand tobacco.Gene, 539,132–140.

Earley K W, Haag J R, Pontes O, Opper K, Juehne T, Song K,Pikaard C S. 2006. Gateway-compatible vectors for plant functional genomics and proteomics.The Plant Journal,45, 616–629.

Fan S S, Li Q N, Guo J C, Wang X X, Guo Y M, Synder J C, Du Y C. 2015. Identification of microRNAs in two species of tomato,Solanum lycopersicumandSolanum habrochaites, by deep sequencing.Journal of Integrative Agriculture,14, 42–49.

Fan Y, Liu B, Wang H, Wang S, Wang J. 2002. Cloning of an antifreeze protein gene from carrot and its in fluence on cold tolerance in transgenic tobacco plants.Plant Cell Reports,21, 296–301.

Hale M G, Orcutt D M. 1987.The Physiology of Plants Under Stress. John Wiley and Sons, New York.

Harish M C, Balamurugan S, Murugan S B, Sathishkumar R. 2012. Influence of genotypic variations on antioxidant properties in different fractions of tomato.Journal of Food Science, 77, 1174–1178.

Harish M C, Sathishkumar R. 2011. Antioxidant potentials of skin, pulp, and seed fractions of commercially important tomato cultivars.Food Science and Biotechnology, 20,15–21.

Hays L M, Feeney R E, Crowe L M, Crowe J H, Oliver A E. 1996.Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.Proceedings of the National Academy of Sciences of the United States of America, 93, 6835–6840.

Hoekema A, Hirsch P R, Hooykaas P J J, Schilperoort R A.1983. A binary vector strategy based on separation of virand T-region of theAgrobacterium tumefaciensTi-plasmid.Nature, 303, 179–180.

Holmberg N, Farrés J, Bailey J E, Kallio P T. 2001. Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco.Gene, 275, 115–124.

Hu Y, Wu Q, Sprague S A, Park J, Oh M, Rajashekar C B,Koiwa H, Nakata P A, Cheng N, Hirschi K D, White F F,Park S. 2015. Tomato expressingArabidopsisglutaredoxin geneAtGRXS17confers tolerance to chilling stressviamodulating cold responsive components.Horticulture Research, 2, 15051.

Kuiper M J, Davies P L, Walker V K. 2001. A theoretical model of a plant antifreeze protein fromLolium perenne.Biophysical Journal, 81, 3560–3565.

Kumar S R, Kiruba R, Balamurugan S, Cardoso H G, Birgit A H, Ahmed Z, Sathishkumar R. 2014. Carrot antifreeze protein enhances chilling tolerance in transgenic tomato.Acta Physiologia Plantarum, 36, 21–27.

Lin X, Wisniewski M E, Duman J G. 2011. Expression of two selfenhancing antifreeze proteins from the beetle dendroides canadensis inArabidopsis thaliana.Plant Molecular Biology Reporter, 29, 802–813.

Middleton A J, Brown A M, Davies P L, Walker V W. 2009.Identification of the icebinding face of a plant antifreeze protein.FEBS Letters, 583, 815–819.

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiologia Plantarum, 15, 473–497.

Parida A K, Dagaonkar V S, Phalak M S, Umalkar G V,Aurangabadkar L P. 2007. Alterations in photosynthetic pigments, protein and osmotic components in cotton genotypes subjected to short-term drought stress followed by recovery.Plant Biotechnology Reports, 1, 37–48.

Pudney P D A, Holt C B, Buckley S L, Roper D, Sidebottom C M, Telford J H, Twigg S N, McArthur A J, Lillford P J. 2003.The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne).Archives of Biochemistry and Biophysics, 410, 238–245.

Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R. 2004. When defense pathways collide. The response ofArabidopsisto a combination of drought and heat stress.Plant Physiology, 134, 1683–1696.

Rubinsky B, Arav A, Fletcher G L. 1991. Hypothermic protection:A fundamental property of “antifreeze” proteins.Biochemical and Biophysical Research, 180, 566–571.

Sambrook J, Maniatis T, Fritsch E F. 1989.Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.

Sandve S R, Kosmala A, Rudi H, Fjellheim S, Rapacz M,Yamada T, Rognli O A. 2011. Molecular mechanisms underlying frost tolerance in perennial grasses adapted to cold climates.Plant Science, 180, 69–77.

Sasaki H, Ichimura K, Oda M. 1996. Changes in sugar content during cold acclimation and deacclimation of cabbage seedlings.Annals of Botany, 78, 365–369.

Sidebottom C, Buckley S, Pudney P, Twigg S, Jarman C, Holt C, Telford J, McArthur A, Worrall D, Hubbard R, Lillford P.2000. Phytochemistry: Heat-stable antifreeze protein from grass.Nature, 406, 256.

Smékalová V, Dosko?ilová A, Komis G, ?amaj J. 2014.Crosstalk between secondary messengers, hormones and MAPK modules during abiotic stress signalling in plants.Biotechnology Advances, 32, 2–11.

Sun W H, Liu X Y, Wang Y, Hua Q, Song X M, Gu Z, Pu D Z.2014. Effect of water stress on yield and nutrition quality of tomato plant overexpressingStAPX.Biologia Plantarum,58, 99–104.

Thomashow M F. 1999. Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms.Annual Review of Plant Physiology, 50, 571–599.

Tomczak M M, Hincha D K, Estrada S D, Wolkers W F,Crowe L S, Crowe L M, Feeney R E, Tablin F, Crowe J H.2002. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.Biophysical Journal,82, 874–881.

Velada I, Ragonezi C, Arnholdt-Schmitt B, Cardoso H. 2014.Reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions inHypericum perforatumL.PLoS ONE, 9,e115206.

Wang K, Shao X, Gong Y, Zhu Y, Wang H, Zhang X, Yu D, Yu F,Qiu Z, Lu H. 2013. The metabolism of soluble carbohydrates related to chilling injury in peach fruit exposed to cold stress.Postharvest Biology and Technology, 86, 53–61.

Wu L, Zhou M, Shen C, Liang J, Lin J. 2012. Transgenic tobacco plants over expressing cold regulated protein CbCOR15b fromCapsella bursa-pastorisexhibit enhanced cold tolerance,Journal of Plant Physiology,169, 1408–1416.

Xin Z, Browse J. 2000. Cold comfort farm: The acclimation of plants to freezing temperatures.Plant Cell and Environment,23, 893–902.

Yang S, Tang X F, Ma N N, Wang L Y, Meng Q W. 2011.Heterology expression of the sweet pepperCBF3gene confers elevated tolerance to chilling stress in transgenic tobacco.Journal of Plant Physiology, 168, 1804–1812.

Zhang Z, Huang R. 2010. Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factorTERF2/LeERF2is modulated by ethylene biosynthesis.Plant Molecular Biology, 73, 241–249.

Zhou B, Deng Y S, Kong F Y, Li B, Meng Q W. 2013.Overexpression of a tomato carotenoid ?-hydroxylase gene alleviates sensitivity to chilling stress in transgenic tobacco.Plant Physiology and Biochemistry, 70, 235–245.

Zhu B, Xiong A S, Peng R H, Xu J, Jin X F, Meng X R, Yao Q H. 2010. Over-expression ofThpIfromChoristoneura fumiferanaenhances tolerance to cold inArabidopsis,Molecular Biology Reporter, 37, 961–966.