Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato

1. Introduction

Being sessile in nature, plants constantly encounter several abiotic and biotic stresses that adversely affect plant growth, development and survival (Smйkalovб et 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 (Wang et al.2013; Velada et 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 temperature via an 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; Chinnusamy et 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 (Chinnusamy et al. 2007).

Overwintering plants have natural ability to resist ice crystal formation in the cell by inhibiting its growth (Pudney et 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(Kumar et al. 2014). Lolium perenne belongs to the Poaceae family, which are adapted to grow in the colder Northern hemisphere (Sandve et al. 2011) and reported to withstand freezing (Kuiper et al. 2001). Superior antifreeze protein responsible for this activity was identified from L. perenne, which possessed two putative opposite-facing sites with surface complimentary to the prism face of ice(Kuiper et al. 2001). The LpAFP gene 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 (Sidebottom et al. 2000; Middleton et 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 (Kuiper et al. 2001). Besides that the AFP was also described as protector of cells from damage during non-freezing conditions (Tomczak et al. 2002) mainly due to their interactions with: i) the integral membrane proteins(Rubinsky et al. 1991); ii) the membrane lipids (Hays et al.1996); and iii) the membrane, which modi fies the acyl chain’s order in the bilayer core (Tomczak et 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 (Fan et 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; Badimon et 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 (Harish et 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) (Zhou et al. 2013; Kumar et al. 2014).

Nevertheless, tomato is a thermophilic crop that makes it more sensitive to chilling conditions (Zhou et 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 (Holmberg et al. 2001;Zhu et al. 2010; Lin et al. 2011; Denga et al. 2014; Sun et 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 the AFP isolated from L. perenne and 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 acclimated L. perenne plants 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.

Southern blot analysis of transgenic tomato lines Genomic DNA from the selected transgenic T1 tomato 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-LpAFP plasmid (1 μg positive control) were digested with restriction enzymes (KpnI and MseI) to release the LpAFP and 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(Sambrook et al. 1989). Hybridization, biotin labeling of probe, membrane washing and detection were carried out as per manufacturer’s instructions (Thermo, USA).

2.2. In vitro propagation and genetic transformation of tomato plants

Fig. 1 Molecular evaluation of transgenic lines. A, schematic vector map of pEarly102-LpAFP showing restriction enzymes used for Southern blot. B, Southern blot analysis. Lane 1, negative control (wild type); lanes 2 and 3, transgenic tomato lines showing the integration of AFP in transgenic lines Tm31 and Tm35, respectively; lane 4, positive control (pEarly102-LpAFP vector digested with KpnI and MseI); lane M, marker. C, RT-PCR analysis showing mRNA expression pattern of AFP in transgenic tomato lines.Lanes 1 and 2, transgenic tomato lines; lane 3, wild type. 26S rRNA was used as an internal reference gene. LB, left border; BAR,basta (herbicide) resistance gene for selection of transgenics; 35S, the Cauli flower mosaic virus 35S promoter; HA, haemagglutinin;OCS, 3´ sequences of the octopine synthase gene, including polyadenylation and termination sequences; RB, right border.

Gene expression analysis of AFP by RT-PCR The 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 using AFP gene 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.

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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 the in vitro T1 transgenic lines. Seeds of T1 tomato lines (Tm5, Tm12,Tm17, Tm24, Tm27, Tm31 and Tm35) were randomly selected and cultivated after surface-disinfection in vitro in 1/2 MS media+20 mg L-1 glufosinate. 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

Seeds of S. 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-1 zeatin and 0.1 mg L-1 indole-3-acetic acid (IAA) (～30 explants per plate in a total of 20 plates). The precultured cotyledons were used as explants for Agrobacterium-mediated genetic transformation. Agrobacterium culture (OD600=0.6)harboring pEARLY102-LpAFP was harvested at 5 000 r min-1 for 10 min. The cells were resuspended in liquid MS solution and the cell density was adjusted to yield a final OD600 of 0.6 and 100 μmol L-1 acetosyringone 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-1 zeatin+0.1 mg L-1 IAA+20 mg L-1 glufosinate+300 mg L-1 carbenicillin). 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-1 carbenicillin).

基于MCGS开发的监控系统要实现的功能包括：实时状态读取；实时监控焊机当前工作状态。正常时，读取焊接电流、焊点计数；异常时，读取显示具体的故障信息。

Southern blot hybridization In 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 of LpAFP gene in all the tomato lines. The hybridization signal observed in the transgenic lines corresponds to the size of LpAFP gene, whereas no signal was observed in WT (Fig. 1-B).The above data demonstrated that the transgene LpAFP was stably integrated in the transgenic tomato.

The LpAFP gene (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 into Escherichia coli competent cells. The sequencing-con firmed plasmid was recombined with the destination vector pEARLY Gate 102 (Earley et 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 pEARLEYLpAFP was electroporated into Agrobacterium tumefaciens strain LBA 4404 (Hoekema et al. 1983) and used for stable plant transformation.

在独生与非独生子女类别上，定向医学生的学习动机及其各维度不具有统计学意义(P>0.05)。且都是在物质追求维度上得分最高。(见表3)

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 T1 transgenic lines under chilling and normal conditions to study the chilling tolerance capability.

Chilling treatment The T1 transgenic 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.

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).

Total soluble sugars Total soluble sugar content was measured according to the protocol described by Parida et 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-1 for 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.

ELI Electrolytic leakage or ionic leakage index was calculated from both the WT and transgenic lines as described by Wu et 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

RWC RWC (%) was measured in the T1 transgenic and WT leaves as per the protocol described by Balestrasse et al. (2010).

2.6. Data analysis

在实际生产车间内布满了生产设备和排气管道等飞行危险壁障，这些生产设备尺寸不一，位置排布没有规则，UAV在从起点飞行到终点的过程中需避开这些危险壁障，确保其始终在安全无碰撞的范围内。因为空间被划分成了m个子空间，所以飞行路径由m个节点构成，需要判断每个节点是否与障碍物保持安全距离。将UAV按照质点来处理[14]，要求UAV在飞行过程中与危险壁障保持一定的安全距离，则危险壁障适应度函数

3. Results and discussion

3.1. Genetic transformation of tomato using LpAFP

A. tumefaciens LBA 4404 strain harboring the vector pEARLY102-LpAFP under 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 T1 transgenic 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

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RT-PCR The stable expression of LpAFP in the T1 lines was analyzed by RT-PCR. The expected band size of 354 bp con firmed the transcription of LpAFP gene in the transgenic plants, whereas no such amplicon was detected in WT. The result showed that the levels of transcription of LpAFP gene 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).

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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 carrying LpAFP exhibited 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 sugar Chilling 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(Wu et 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 (Wang et al. 2013). Congruently, generation of transgenic tomato by overexpressing AtGRXS17 resulted in increased total soluble sugar content during chilling stress(Xin and Browse 2000).

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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 (Sasaki et 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 (Rizhsky et 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.

RWC In 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 (Campos et al. 2003). Similar reduction in the water content during chilling stress was also reported in transgenic tobacco plants (Wu et 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 and vice versa.

学习是一项关系党和国家事业兴旺发达的战略任务。毛泽东关于建设学习型政党理论，为当代中国共产党建设学习型政党进行了开拓性的探索，作出了历史性的伟大贡献，为我们党树立了光辉榜样。党的十七大提出了“建设全民学习、终身学习的学习型社会”的理念，这是我们党与时俱进，具有世界眼光的战略举措。我们要切实推动学习型政党建设，把毛泽东建设学习型政党思想发扬光大，全面推进中国特色社会主义伟大事业。

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.

ELI Plasma 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 (Hu et al. 2015). ELI was found to be a versatile and sensitive assay to study cold acclimation of plants (Yang et 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. Wu et al. (2012) reported that WT lines showed significant increase in electrolyte leakage as compared to transgenic tobacco lines expressing CbCOR15b gene. Similarly, overexpression of transcription factor TERF2/LeERF2 increased 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(Kumar et al. 2014), we have shown that the T0 transgenic tomato plants expressing carrot AFP can grow in the chilling conditions up to 48 h as compared to WT. However, in the present study, T1 transgenic lines expressing LpAFP has 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 of LpAFP under chilling condition, which could be due to the presence of two ice-binding domains as against the single domain present in carrot AFP (Kuiper et al. 2001;Pudney et al. 2003; Kumar et 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(Chinnusamy et 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 (Tomczak et al. 2002). This protective function of AFPs at chilling temperatures is mainly due to the interaction of AFP with the integral membrane proteins(Rubinsky et al. 1991), membrane lipids (Hays et al. 1996)and the membrane itself, which modi fies the acyl chain’s order in bilayer core (Tomczak et al. 2002). Taken together,our study proved that LpAFP confer the chilling tolerance ability to transgenic tomato lines.

5. Conclusion

In the study, we successfully generated transgenic tomato plants expressing LpAFP with enhanced tolerance to chilling stress. Our data proved the protective role of LpAFP in transgenic tomato plants under chilling conditions and also proved that LpAFP is a potential candidate to confer chilling tolerance, probably by upholding the membrane integrity of the transgenic plants. This is the first report of LpAFP expression 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.

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

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