The chilling tolerance divergence 1 protein confers cold stress tolerance in processing tomato

Plant Physiology and Biochemistry 151 (2020) 34–46

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Research article

The chilling tolerance divergence 1 protein confers cold stress tolerance in processing tomato

T

Li Zhang1, Xinyong Guo, Yujie Qin, Bin Feng, Yating Wu, Yaling He, Aiying Wang∗∗, Jianbo Zhu∗ Key Laboratory of Agricultural Biotechnology, College of Life Science, Shihezi University, Shihezi, 832000, China

ARTICLE INFO

ABSTRACT

Keywords: LeCOLD1 Tomato Cold tolerance Overexpression RNA interference Antioxidants

Tomato (Lycopersicon esculentum Mill [Solanum lycopersicum L.].) is an important food material and cash crop, as well as a model plant for genetic evolution and molecular biology research. However, as a cold-sensitive crop originating from the tropics, the growth and development of tomato is often affected by low temperature stress. Therefore, how processing tomatoes resist this type of stress has important theoretical and practical significance. In this study, the LeCOLD1 gene was cloned from processing tomato. Subcellular localization analysis showed that LeCOLD1 was located in the plasma membrane. Real-time quantitative PCR analysis showed that LeCOLD1 was highly expressed in roots. Drought, salt and low temperatures induced the expression of COLD1. Overexpression and RNA interference vectors of LeCOLD1 were constructed and were transformed into tomato by the Agrobacterium-mediated method, and then obtaining transgenic tomato plants. It was found that LeCOLD1 increased the height of processing tomato plants and increased the length of their roots. In addition, overexpression of LeCOLD1 significantly improved the cold resistance of the plants. Overexpressing LeCOLD1 in tomato plants reduced the damage to the cell membrane, accumulation of ROS and photoinhibition of PSII, and maintained the high activity of antioxidant enzymes and the content of osmotic regulators. Further analysis revealed that during low temperature stress, the cells maintained high levels of antioxidant enzyme activity by regulating the transcription of the genes encoding these enzymes. The results show that overexpressing LeCOLD1 in tomato increases the plants’ resistance to low temperatures, and that reducing LeCOLD1 expression makes the plants more sensitive to low temperatures.

1. Introduction Tomatoes (Lycopersicon esculentum Mill.) originated in tropical and subtropical areas but they are currently grown more widely around the world. The plants are sensitive to low temperatures and therefore cold stress causes serious economic losses (Thompson, 1974). China is a leading producer of processing tomatoes in the world, and more than 90% of them in China are produced in Xinjiang which has a unique regional climate. There is always a shortage of raw materials in the early and late stages of production, and a large amount of the raw materials in the middle stage are overstocked and wasted (Wang et al., 2012a,b). After many years of exploration, adopting delayed cultivation has had little effect, and early spring cultivation was found to be more feasible for balancing the supply of raw materials with the demands of production (Oyanedel et al., 2001). Low temperatures are the primary adversity

that early spring brings to cultivation and so research on the cold tolerance of processing tomatoes during the germination and seedling periods has practical significance for guiding the selection of suitable varieties and formulating high quality and efficient cultivation measures (Kruse et al., 2000; Sah et al., 2016). Previous studies have shown that low temperatures activate calcium ion signals and elicit an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in plant cells that activate downstream cold signals and response factors (Knight et al., 1996). The calcium channel protein CNGC is thought to be involved in temperature perception and responses (Finka et al., 2012). Low temperatures activate the ICE1-CBFCOR transcriptional cascade in which the bHLH-like transcription factor ICE1 directly binds to the promoter region of CBF1 (c-repeatbinding factors) to activate the expression of the CBF genes. These genes encode transcription factors that further activate downstream expression of a series of genes called CORs (cold-responsive genes).

Corresponding author. Key Laboratory of Agricultural Biotechnology, College of Life Science, Shihezi University, Shihezi, 832000, China. Corresponding author. Key Laboratory of Agricultural Biotechnology, College of Life Science, Shihezi University, Shihezi, 832000, China. E-mail address: [email protected] (J. Zhu). 1 The first author: Li Zhang. ∗

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https://doi.org/10.1016/j.plaphy.2020.03.007 Received 30 November 2019; Received in revised form 4 March 2020; Accepted 5 March 2020 Available online 10 March 2020 0981-9428/ © 2020 Published by Elsevier Masson SAS.

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Thus, the ICE1-CBF-COR transcriptional cascade mediates the plant's ability to withstand low temperatures (Chinnusamy et al., 2003; Shi et al., 2015). Many aspects of cryogenic signaling pathways remain poorly understood, for example, it is not known how plants use calcium signaling pathways to sense low temperature signals in their environment. Recently, Chong Kang and colleagues used the differences in cold tolerance between japonica and indica rice to construct a near-isogenic line (Ma et al., 2015) from which the COLD1 (chilling-tolerance divergence 1) gene was successfully cloned using QTL fine genetic localization (G.T. Huang et al., 2012; Xu et al., 2012). COLD1 encodes a membrane protein with 9 transmembrane domains that is located in the plasma membrane and endoplasmic reticulum. During cold treatment, COLD1 interacts with RGA1, a n-subunit of a G-protein that is involved in G-protein-dependent signal transduction. This opens a Ca2+ channel, triggering the expression of downstream genes including OsAP2, OsDREB1A, OsDREB1B and OsDREB1C to endow japonica rice with cold resistance (Ma et al., 2015). In addition, cold1-1 mutant rice plants were found to be significantly shorter than the wild type, complementing the mutants with the wildtype COLD1 gene rescued the mutants (Ma al., 2015). More interestingly, Dong's team at the Institute of Crop Science, Chinese Academy of Agricultural Sciences discovered a new heightregulating gene in bread wheat called TaCOLD1 and reported that it interacted with heterotrimeric G protein complexes. Using wheat gene expression profiling, the researchers identified TaCOLD1 as a lightregulated gene that encodes a protein highly homologous to the rice cold sensor COLD1. The TaCOLD1 protein is located in the endoplasmic reticulum and plasma membranes. (Dong et al., 2019). In summary, the COLD1 gene is a major factor regulating cold tolerance in plants that plays an important role in abiotic stress tolerance (Ma et al., 2015; P. Anunathini et al., 2019). COLD1 also affects plant growth and development, especially the height of plants (Ma et al., 2015. Dong et al., 2019). However, it remains unknown whether the LeCOLD1 gene in processing tomato plays a similar role to its homologs in rice and bread wheat. To address this question, we first cloned the LeCOLD1 from processing tomatoes. Next, the biological roles of LeCOLD1 were analyzed by examining its subcellular localization and expression patterns. Finally, we used overexpression and RNAi silencing approaches to investigate the possible role of COLD1 in tomato chilling tolerance.

specific primers LeCOLD1 (Kpn Ⅰ)-CF and LeCOLD1 (Sal I)-CR (Supplementary Table 1), which were designed based on the mRNA sequence of Solanum lycopersicum. The PCR was carried out using PrimeSTAR Max DNA Polymerase (TaKaRa) and the following thermal cycles: 1 cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 61 °C (annealing) for 30 s, and 72 °C for 10 min. The obtained 1411-bp PCR fragment was cloned into pMD19-T vector (TaKaRa, Beijing, China); its sequence was confirmed by DNA sequencing. 2.3. Construction of LeCOLD1-overexpressing and RNA interferenceexpressing plasmids In the construction of LeCOLD1 -overexpressing plasmid, LeCOLD1 gene was PCR-amplified and cloned into pCAMBIA2300 binary vector under the control of 35S promoter at the Kpn Ⅰ and Sal Ⅰ restriction sites. To generate the RNA interference (RNAi) construct, a 220 bp segments of LeCOLD1 gene cDNA (340–560 bp) were selected as RNAi target segments and amplified using PCR, using pGM-LeCOLD1 as the template to design a pair of specific primers. The specific fragment was recovered and inserted into the pGM-T vector. The ligation product was transformed into Escherichia coli DH5α by the CaCl2 method and the correct positive clone was named pGM-S (Bu et al., 2005). The primer sequences are shown in Supplementary Table 1. pGM-S was digested using the Xho I and Bgl II restriction enzymes. The small fragment S1 was recycled and incorporated into pUCCRNAi, an intermediate vector that was also digested and recovered by the same pair of enzymes. The ligation product was transformed into E. coli DH5α competent cells to obtain the recombinant vector pUCS1. Then, pGM-S was digested with Xho I and Bgl II and the small fragment S2 was recovered. Sal I and BamH I were used to digest pUCS1 and the large fragments were recycled and connected to S2. Using the intron of the intermediate vector pUCCRNAi in the interval area, the intermediate vector pUCS1S2 with the reverse repeat sequence was obtained. Finally, pUCS1S2 was digested with the single enzyme Pst I. The small fragments were recovered and connected to the pCAM2300 vector, which was recovered by the same single enzyme digestion to obtain the plant RNAi expression vector pCAS with the reverse repeat sequence. The recombinant plasmid pCAS was transferred into Agrobacterium GV3101 by freezethaw method for transformation into processing tomato (Gallois and Marinho, 1995). 2.4. Sequence analysis of LeCOLD1

2. Materials and methods

The obtained LeCOLD1 sequence was subjected to sequence alignment using DNAMAN (version 8.0). The transmembrane domains of LeCOLD1 protein were predicted using the TMHMM algorithm available online at the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/ services/TMHMM/). Phylogenetic analysis was conducted by MEGA 5.1 software (http://www.megasoftware.net/) using the Neighbor-Joining method and 1,000 bootstrap replicates; bootstrap scores of < 50% were deleted.

2.1. Plant materials and growth conditions Wild-type seeds of tomato plant (L. esculentum Mill) variety ‘Yaxin 87-5’ were provided by Yaxin Seed Co. Ltd. (Shihezi City, Xinjiang, China). Fifteen-day-old tomato plants were initially grown from the seeds in a tissue culture room at 25 °C and 60–70% humidity under a 16 h light/8 h dark cycle and a light intensity of 70 μmol m−2 s−1. After that, the seedlings were transplanted into plastic pots (50 cm long, 35 cm wide and 15 cm deep) containing equal parts of peat and soil (weight 2.4 kg), and were allowed to grow in a naturally lit greenhouse at 22–28 °C and a relative humidity of 60%–70% under a 16 h light/8 h dark cycle, during which, the plants were irrigated with 500 mL of Hoagland's nutrient solution twice a week.

2.5. Determination of subcellular localization of LeCOLD1 protein To investigate the subcellular localization of LeCOLD1, the fulllength open-reading frame (ORF) of LeCOLD1 without a stop codon was amplified by PCR using the primers LeCOLD1 (Kpn Ⅰ)-SF and LeCOLD1 (Sal Ⅰ)-SR (Supplementary Table 1), which contain Kpn Ⅰ and Sal Ⅰ restriction sites, respectively. The PCR fragment was then ligated with pCAMBIA2300-GFP expression vector to generate p35S-LeCOLD1-GFP plasmid, which is controlled by CaMV 35S promoter. The p35S-LeCOLD1-GFP plasmid and PM-rk plasmid (a marker for plasma membrane localization) were co-transformed into Arabidopsis mesophyll protoplasts using the PEG method (Nelson et al., 2007; Hu et al., 2012; Zhou et al., 2012). The protoplasts were cultured at 23 °C for 16 h; after that, their fluorescence signal was observed under a Leica SP8 laser

2.2. Cloning of LeCOLD1 gene Total RNA was extracted from wild-type tomato leaves using RNAisoPlus kit (TaKaRa) containing on-column DNase I according to the manufacturer's instruction. First-strand cDNA was synthesized from the total RNA using oligo (dT) primers and PrimeScript®RTase (TaKaRa). The full-length LeCOLD1 gene (GenBank accession number: XM_004243536.4) was amplified from the obtained cDNA by PCR using 35

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Fig. 1. Sequence analysis of LeCOLD1 from Lycopersicon esculentum. (A)Amino acid sequence alignment; (B) Phylogenetic tree of LeCOLD1 and other COLD1 proteins from Nicotiana sylvestris (XP_009759545.1), Nicotiana attenuata (XP_019249326.1), Nicotiana tomentosiformis (XP_009603306.1), Ipomoea nil (XP_019194057.1), Nelumbo nucifera (XP_010278836.1), Prosopis alba (XP_028752390.1), Cannabis sativa (XP_030501536.1), Olea europaea var. Sylvestris (XP_022882165.1), Erythranthe guttata (XP_012853010.1), Capsicum annuum (XP_016580454.1), Solanum tuberosum (XP_006357657.1), Solanum lycopersicum (XP_004243584.1), Solanum pennellii (XP_015080562.1). The analysis was carried out by MEGA 5.1 using the Neighbor-Joining method. Bootstrap value was obtained from 1000 replicates. (C) Putative transmembrane domains of LeCOLD1.

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Fig. 2. Subcellular localization of LeCOLD1 protein. Arabidopsis mesophyll protoplasts cotransformed with p35S-LeCOLD1-GFP and a plasma membrane marker PM-rk. (A) Schematic representation of p35S-LeCOLD1GFP plasmid used in subcellular localization analysis; (B) Fluorescence images of Arabidopsis mesophyll protoplast expressing LeCOLD1-GFP fusion protein; (C) Fluorescence image of Arabidopsis mesophyll protoplast expressing Pm-rk; (D) Fluorescence image of chloroplast of Arabidopsis protoplast. (E) Bright field image of Arabidopsis mesophyll protoplast; (F) Merged fluorescence images of Arabidopsis protoplast expressing LeCOLD1-GFP and PM-rk marker. (B)–(D) are dark field images. (E) and (F) are bright field images. Scale bars = 7.5 μm. Fig. 3. Expression patterns of LeCOLD1 gene in wild-type tomato (Lycopersicon esculentum) analyzed by qRT-PCR. (A) Expression of LeCOLD1 in different organs, including root, stem, leaf, flower, and fruit, of L. esculentum grown at 25 °C. (B) Expression in leaves of L. esculentum treated with cold stress at 4 °C (B), drought stress using 20% PEG-6000 (C), and salt stress using 200 mM NaCl (D) for 0, 1, 3, 6, 9, 12, 24, and 48 h. Data are means from 3 repeated experiments.

confocal microscope (Leica Microsystems, Germany) operated at excitation wavelengths of 488, 561 and 633 nm.

stress were normalized (Lovdal et al., 2009). The qRT-PCR thermal cycles were set as follows: 2 cycle of denaturation at 95 °C for 30 s, followed by 50 cycles of 95 °C for 5 s, 60 °C for 10 s, and 68 °C for 10 s. The relative gene expression was calculated according to the 2ΔΔCt method (Livak et al., 2001). Three replicate samples were prepared independently and each qRT-PCR was performed three times. All primer sequences used in qRT-PCR are listed in Supplementary Table 1.

2.6. Evaluation of LeCOLD1 gene expression in wild-type tomato plant under different stress conditions The expression of LeCOLD1 gene in different organs including root, stem, leaf, flower and fruit of 80-day-old wild-type tomato was determined by qRT-PCR. Expression LeCOLD1 gene in six-week-old wildtype tomato grown under different stress conditions (cold, drought and salt) was also evaluated. For cold treatment, the plants were treated at 4 °C for 48 h. For drought treatment, the roots of the plants were soaked in 20% PEG 6000 for 48 h. For salt treatment, the roots of the plants were immersed in 200 mM NaCl solution for 48 h. During the treatments, tomato leaves at the same position were collected at 0, 1, 3, 6, 9, 12, 24 and 48 h, respectively, and then subjected to qRT-PCR analysis. In qRT-PCR analysis, total RNA was extracted from the samples using RNAprep Pure Plant Kit (Tiangen, China), from which cDNA was synthesized. One microliter of the synthesized cDNA was then used as a template in qRT-PCR performed using SYBR Green I Master Mix on a LightCycler® 480Ⅱ instrument (Roche Biochemicals, Indianapolis, IN, USA). The tomato EF1 gene (GenBank ID: X53043) was used as an internal control to which the expression levels of genes caused by cold

2.7. Plant transformation and identification of transgenic tomato plants Two transgenic plants, including LeCOLD1-overexpressing transgenic tomato plant lines and RNA interference-expressing transgenic tomato plant lines, were generated and used as plant materials. To generate transgenic tomato plants, the constructed LeCOLD1-overexpressing and RNA interference-expressing plasmids were transferred into wild-type tomato plants using Agrobacterium tumefaciens strain GV3101. The transgenic plants were screened on 1/2 strength MS medium containing 60 mg/ L kanamycin. The kanamycin-resistant T0-generation plant lines were assessed by semi-quantitative reverse transcription PCR using the specific primers for LeCOLD1 (Supplementary Table 1). Different transgenic plant lines were confirmed by qRT-PCR. T2-generation transgenic plant lines that survived MS medium containing 60 mg/L kanamycin were used in subsequent experiments. 37

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measured in tomato leaves using a portable fluorescence analyzer (DUAL-PAM-100, Walz, Germany). Leaves were allowed to adapt to darkness for 30 min and were then exposed to a flash of saturated light for 1 s. The minimal fluorescence (F0) is fluorescence intensity measured during the dark-adapted state (when all PSII reaction centers are open), and the maximal fluorescence (Fm) is the fluorescence intensity measured when leave was exposed to saturated light (when all PSII reaction centers are closed). The variable fluorescence (Fv) was calculated by the following equation: Fv = Fm – F0 (Krause and Weis, 1991). Content of free proline was measured according to the method described by Bates et al. (1973). Briefly, about 200 mg of ground leaf samples was extracted with 4 mL of 3% sulphosalicylic acid at 100 °C for 10 min. The homogenate was then centrifuged at 12,000 ×g for 2 min; after that, 2 mL of the supernatant was mixed with 2 mL each of acid-ninhydrin reagent and glacial acetic acid. The mixture was boiled for 30 min, and was then soaked in an ice bath to terminate the reaction. The reaction mixture was extracted with toluene (4 mL), and an absorbance at 520 nm of the extracted organic phase was determined. Content of proline was determined by comparing the absorbance of the samples with a standard curve constructed using standard proline with known concentrations. Total soluble sugar content was analyzed by the anthrone method, in which glucose was used as a standard (Fukao et al., 2006). Approximately 200 mg of ground leaf samples was homogenized in 1 mL of distilled water and then boiled for 20 min. After centrifugation at 13,000 ×g for 10 min, 2 mL of supernatant was mixed with 1.8 mL of distilled water and 2.0 mL of 0.14%(w/v) anthrone solution in 100% H2SO4, and the mixture was incubated in boiling water for 20 min. After cooling down, an absorbance at 620 nm (A620) of the mixture was determined. Total soluble sugar content was determined by comparing A620 values of the samples with a standard curve constructed using standard glucose with known content. Each experiment was carried out in triplicate, and three replicate samples were prepared in parallel.

Fig. 4. Expression levels of LeCOLD1 in wild-type (WT), LeCOLD1-overexpressing (OE-2, OE-3), and LeCOLD1-RNAi (RI-1, RI-2) tomato lines. (A) Relative expression levels analyzed by qRT-PCR. (B) Expression levels analyzed by semi-quantitative PCR.

2.8. Determination of phenotypic changes and expression levels of stress response genes T2-generation wild-type and transgenic tomato plants were grown for 3 weeks or 6 weeks in a pot in an illuminated incubation chamber at 25 °C and a relative humidity of 60–70% under a 16-h/8-h light/dark cycle and a light intensity of 200 μmol m−2 s−1. The 3- or 6-week-old tomato plantlets with uniform sizes were subsequently treated with cold stress at 4 °C for 5 d in a biochemical incubator. After that, phenotypic changes of the plants were observed, and their photographs were taken using a Canon 80D camera. Additionally, leaves (the second and third leaves from the top of the plant) were harvested and then subjected to analysis of stress response gene expression levels by qRTPCR, physiological measurement, and antioxidant enzyme activity assay.

2.10. Assay of antioxidant enzymes and reactive oxygen species Fresh leaves (0.5 g) of T2-generation wild-type and transgenic tomato plants exposed to cold and drought stress were collected. The leaves were cut into smaller pieces and were then homogenized with 4 mL of 50 mM sodium phosphate buffer (pH 7.8) containing 1% polyvinylpyrrolidone and 10 mM β-mercaptoethanol in an ice bath. The homogenate was centrifuged at 17426 xg for 15 min at 4 °C; thereafter, the antioxidant enzyme activity of the supernatant was determined. Catalase (CAT) activity was determined according to the method of Cakmak et al. (1992). Superoxide dismutase (SOD) activity was determined by an absorbance at 560 nm according to the method described by Beauchamp and Fridovich (1971). Peroxidase (POD) activity was determined following the method described by Doerge et al. (1997). Absorbance of the samples was recorded on an Infinite M200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). H2O2 and O2− contents were determined following the method of Benikhlef et al. (2013) using a standard curve. Absorbance at 415 nm and 530 nm were determined using a UV-160A spectrophotometer (Shimadzu Scientific Instruments, Japan).

2.9. Measurement of physiological parameters Relative water content (RWC) was determined following the method described by Lara et al. (2003). The RWC was calculated by the following equation: RWC = (FW − DW)/(TW − DW) × 100%, where FW is the fresh weight of leaves, TW is the weight at full turgor (measured after floating the leaves in distilled water at room temperature for 24 h under light condition), and DW is the weight of leaves after being dried at 70 °C until a constant weight was achieved. Malondialdehyde (MDA) content, which can indicate membrane damage, was determined by the modified thiobarbituric acid reaction outlined in Du et al. (1992). Briefly, leaves were excised from the tomato plants and then washed with deionized water. Leaf discs were punched out and then subjected to measurement of MDA concentration using a spectrophotometer (UV-160A, Shimadzu Scientific Instruments, Japan). Relative electrolyte leakage (REL) was determined using an EC 215 conductivity meter (Markson Science Inc., Del Mar, CA, USA) following the method of Du et al. (1992). Leaves were subjected to electrolyte leakage analysis using a conductivity meter. The relative conductance was calculated as follows: REL = (C1 − CW)/(C2 − CW) × 100, where C1 is the conductivity measured before boiling, C2 is the conductivity value measured after boiling, and CW is the conductivity of deionized water. Maximal photochemical efficiency of photosystem II (PSII) was

2.11. Statistical analysis Data were statistically analyzed using GraphPad Prism 7.0 and SigmaPlot 12 (SYSTAT Software). The mean value ± standard deviations of three replicate samples; leaves from three individual seedlings were considered as a replicate sample. The expression level at 0 h (the control time point) was defined as 1.0. Significant differences between wild-type plant and transgenic plant lines were determined using Dunnett's multiple comparisons test, and *P < 0.05 and **P < 0.01 indicate significant differences. 38

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Fig. 5. Biological character analysis of wild-type tomato and transgenic tomato. (A) Growth of wild-type (WT) tomato and transgenic tomato plants overexpressing LeCOLD1 in the field; (B) Fruiting of wild-type and transgenic tomato plants in the field; (C) Plant height; (D) Length of root; (E) Fresh weight of plant; (F) Stem thickness. Data represent the average of three independent biological replicates. Bars represent SDs. *P < 0.05 and **P < 0.01 indicate significant differences relative to wild-type tomato plants.

3. Results

(Fig. 2C), and co-localized with the GFP signal (green; Fig. 2B and F). This finding shows that LeCOLD1 is a plasma membrane-localized protein.

3.1. Sequence analysis of LeCOLD1 gene from Lycopersicon esculentum The full-length cDNA of LeCOLD1 is a 1410-bp open reading frame encoding a protein of 469 amino acids. The deduced LeCOLD1 amino acid sequence was aligned with the protein sequences from Solanum lycopersicum L., Solanum pennellii, and other 13 plant species obtained from the database. The sequence alignment showed that the amino acid sequence of LeCOLD1 is highly similar to that of SlCOLD1 protein from Solanum lycopersicum L with 98.93% similarity (Fig. 1A). The phylogenetic analysis of the sequences also showed that LeCOLD1 is most closely related to the COLD1 family proteins from Solanum lycopersicum L. (Fig. 1B). Finally, domain analysis revealed that LeCOLD1 contains 9 putative transmembrane domains (Fig. 1C).

3.3. Analysis of LeCOLD1 expression in processing tomato To understand the expression patterns of LeCOLD1 in different organs of processing tomatoes, real-time quantitative PCR was performed to detect the levels of LeCOLD1 expression in the roots, stems, leaves, flowers and fruits. The results showed that LeCOLD1 was expressed in all of the organs with the highest levels of expression in the root and the lowest levels of expression in fruits, representing an 8.43-fold difference in expression (Fig. 3A). This suggests that LeCOLD1 may promote the expression of target genes in some organs of tomato plants. Next, the relative expression of LeCOLD1 under different stress conditions was detected by qRT-PCR. As shown in Fig. 3B, low temperature treatment induced the transcription of LeCOLD1. The expression of the gene peaked at 12 h and then declined. After 20% PEG treatment, the level of LeCOLD1 expression gradually increased to a maximum level at 9 h (a 7.99-fold increase), and then expression decreased (Fig. 3C). After treatment with 200 mm NaCl, it was observed that the expression level of LeCOLD1 increased rapidly to a maximum at 6 h (a 16.17-fold increase). Expression dropped rapidly after 6 h and

3.2. Subcellular localization of LeCOLD1 To find out where the LeCOLD1 protein is found inside cells, the p35S-LeCOLD1-GFP plasmid and PM-rk plasmid (a marker for plasma membrane-localized protein) were co-transformed into Arabidopsis thaliana mesophyll protoplasts (Fig. 2A–F). The PM-rk plasmid was localized in the plasma membrane, as indicated by red fluorescence 39

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Fig. 6. Analysis of low temperature tolerance in young wild-type and transgenic tomato plants. (A) Analysis of the phenotypes of 3-week-old seedlings after low temperature treatment at 4 °C for 0, 3, 5, 7 d, followed by 3 d recovery at 25 °C; (B) Fresh weight; (C) Survival rate. Data represent the average of three independent biological replicates. Bars represent SDs. *P < 0.05 and **P < 0.01 indicate significant differences relative to wild-type (WT) tomato plants. 40

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Fig. 7. Growth performance of 6-week-old wild-type (WT) and transgenic plants grown at 4°C for 0 and 5 d. Fig. 8. Physiological changes of 6-week-old wild-type (WT) and transgenic tomato plant lines (OE-2, OE-3, RI-1 and RI-2) under cold stress. (A) MDA content; (B) Relative electrolyte leakage (REL); (C) Relative water content (RWC); (D) Fv/ Fm values. Data are means ± SDs of three replicate samples. *P < 0.05 and **P < 0.01.

Fig. 9. Changes in osmotic products in 6-week-old wild-type (WT) and transgenic (OE-2, OE-3, RI-1 and RI-2) tomato plants under cold stress. (A) Proline content; (B) Solute sugar content. Data are means ± SDs of three replicate samples. *P < 0.05 and **P < 0.01.

leveled off after 12h (Fig. 3D). These results indicate that LeCOLD1 could be induced by low temperatures, drought and salt stress. Therefore, we speculate that this gene may play a positive regulatory role in stress responses in processing tomato.

temperature stress responses of processing tomato and its influence on the physiology of transgenic plants, we constructed a LeCOLD1-overexpression vector and an RNAi-expression vector and transformed them into processing tomatoes by the Agrobacterium-mediated method. A total of 29 individual kanamycin-resistant T0 tomato transgenic lines were generated from tissue culture (18 plants overexpressing LeCOLD1 and 11 RNAi plants). The progeny obtained from the T0 was named T1. Kanamycin-tolerant T1 plants were checked by PCR (data not shown) using the 35S forward and gene-specific reverse primer pairs. Three

3.4. Generation of transgenic tomato plants with altered levels of LeCOLD1 expression In order to evaluate the biological function of LeCOLD1 in low 41

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Fig. 10. Accumulation of ROS (H2O2 and O2−) and the activities of antioxidant enzymes (SOD, POD, and CAT) in 6-week-old wild-type (WT) and transgenic (OE-2, OE-3, RI-1 and RI-2) tomato plants under cold stress. (A) H2O2 content; (B) O2− content; (C) SOD activity; (D) POD activity; (E) CAT activity. Data are means ± SDs of three replicates. *P < 0.05 and **P < 0.01.

at the top of wild-type and RNAi plants was inhibited. After 7 days of low temperature treatment, the RNAi lines were almost all prostrate, as were some of the wild-type tomato seedlings. On the other hand, the LeCOLD1 overexpressing seedlings did not collapse and only partially withered. After 7 days of low temperature treatment, the seedlings were moved to 25 °C and allowed to recover for 3 days. We found that almost all of the RNAi seedlings had died, as had some of the wild-type plants. However, most of the LeCOLD1 overexpressing plants had survived. Similar results were obtained for the fresh weight of the plants. Compared with the wild-type plants, the overexpression lines had higher fresh weights and the RNAi plants had slightly lower fresh weights (Fig. 6B). The survival rates of the overexpression lines were significantly higher than that of the RNAi plants (Fig. 6C). These results indicate that overexpressing LeCOLD1 in processing tomato increases resistance to low temperatures. To further analyze the resistance of transgenic tomatoes to low temperatures, wild-type and transgenic tomato seedlings grown under normal conditions for 6 weeks were treated at 4 °C for 5 days and their phenotypes were observed. At 25 °C, the wild-type and transgenic tomatoes grew normally. After 4 °C treatment, wilting was observed in all of the wild-type and transgenic lines. However, the degree of wilting differed between the lines: the RNAi lines were most severely affected and the overexpression lines were the least affected (Fig. 7). These results suggest that LeCOLD1 is crucial for cold resistance in processing tomato plants.

transgenic lines overexpressing LeCOLD1 (OE-1, OE-2 and OE-3) and three RNAi transgenic lines (RI-1, RI-2 and RI-3) were selected for qRTPCR analysis (Fig. 4). Compared with the wild-type, the relative levels of LeCOLD1 mRNA were 4.7, 9.0 and 7.0 times greater in the LeCOLD1 overexpressing lines, and 0.82, 0.67 and 0.66 times lower in the RNAi lines. Therefore, we selected two overexpression lines (OE-2, OE-3) and two RNA interference lines (RI-1, RI-2) as representatives for further studies on cold resistance. 3.5. Biological characteristics of transgenic tomatoes Recently, a new plant height regulator named TaCOLD1 was reported in bread wheat (Dong et al., 2019). To this end, we investigated whether LeCOLD1 expression in processing tomatoes affects the height of the plants and other phenotypes. As shown in Fig. 5, we analyzed the biological trait indexes of 80day-old wild-type and transgenic tomatoes. We found that the plants overexpressing LeCOLD1 were significantly taller than the wild-type tomato plants (15–16.8% increase; Fig. 5A and B). The RNAi plants were shorter than the wild-type plants (18.6–24.1%; Fig. 5C). The roots of all of the transgenic tomato lines were longer than those of the wildtype, but this difference was more pronounced in the RNAi plants (14.6–19.5% increase; Fig. 5D). The fresh weight of the transgenic plants was slightly greater than that of the wild-type plants (Fig. 5E). The LeCOLD1 overexpression lines had thicker stems than the wildtype, but there was no significant difference between the thickness of the stems of the RNAi and wild-type plants (Fig. 5F). This indicates that LeCOLD1 does indeed alter some phenotypes of processing tomato.

3.7. Overexpressing LeCOLD1 reduced cell membrane damage during cold stress As shown in Fig. 8A and B, the accumulation of MDA and REL in all lines increased under low temperature stress. In addition, the MDA content of transgenic tomatoes were 27.6% and 22.9% lower than that of wild-type tomatoes. The MDA content of RI-1 and RI-2 lines were 31.5% and 22.1% higher than that of the wild-type, respectively. Similarly, the REL content for OE-2 and OE-3 were 37.6% and 30.1% lower than those for wild-type, respectively. On the other hand, the REL content of RI-1 and RI-2 lines were 53.1% and 29.1% higher than that

3.6. Overexpressing LeCOLD1 enhanced the low temperature resistance of transgenic tomatoes As shown in Fig. 6A, under normal conditions, both the wild-type and the transgenic plants performed well and there was no significant differences in their growth. After 3 days of 4 °C treatment, wilting was observed in both the wild-type and RNAi plants, but the growth of the LeCOLD1 overexpressing plants was not affected. After 5 days, growth 42

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Fig. 11. Relative expression of stress-related genes in 6-week-old wild-type (WT) and transgenic (OE-2, OE-3, RI-1 and RI-2) tomato plants under cold stress. (A) LeSOD; (B) LePOD; (C) LeCAT; (D) LeICE1; (E) LeCBF1; (F) LeDRCi7. Data are means ± SDs of three replicate samples. *P < 0.05 and **P < 0.01.

of the wild-type, respectively. As shown in Fig. 8C, the RWC of all the lines were no significant differences under normal conditions. Low temperature stress treatment (4 °C) decreased the RWC of wild-type plants by nearly 26.3%, the overexpression lines OE-2, OE-3 by 7.4% and 13.3%, respectively, and the RNAi lines RI-1 and RI-2 by 56.1% and 41.3%, respectively. These results suggest that the overexpression lines experienced a lower degree of membrane damage, and the RNAi plants experienced a greater degree of damage, compared with the wild-type plants. The maximum photochemical efficiency of PSII (Fv/Fm) was used to determine the photoinhibition of PSII. Before cold treatment, there was only a slight difference between the wild-type and transgenic lines. After 5 days of 4 °C stress treatment, the Fv/Fm values of the wild-type and transgenic lines were significantly lower. The LeCOLD1 overexpressing plants experienced only a slight drop in Fv/Fm that was smaller than the decrease observed in the wild-type (Fig. 8D). On the other hand, the RNAi plants had a much bigger decrease in Fv/Fm compared with the wild-type. These results indicate that overexpressing LeCOLD1 alleviates the photoinhibition of PSII in processing tomato. Before low temperature stress, all plants had similar levels of proline and soluble sugar (Fig. 9A and B). The levels of both osmolytes increased in the wild-type and transgenic lines during low temperature stress. The overexpression lines (OE-2 and OE-3) had significantly

higher levels of proline and soluble sugar than the wild-type plants (proline, 32.6% and 26.5% higher; soluble sugar, 41.2% and 32.5% higher, respectively). The proline and soluble sugar levels in the RI-1 and RI-2 lines were significantly lower than those in wild-type plants (proline, 23.9% and 16.6% lower; soluble sugar 33.1% and 21.5% lower, respectively). Therefore, overexpressing LeCOLD1 increased the levels of proline and soluble sugar in tomato plants during the cold treatment and reduced the degree of damage to the plants. 3.8. Overexpressing LeCOLD1 alleviated ROS accumulation under low temperature stress Low temperature stress often leads to the production of excessive reactive oxygen species (ROS), which can cause serious damage to plant cells. In other words, the difference in physiological indicators between wild-type and transgenic plants during osmotic stress is reflected in the accumulation and clearance of ROS in cells. We investigated how two common ROS in cells – hydrogen peroxide (H2O2) and superoxide (O2−) – accumulated in the transgenic and wild-type lines. Fig. 10A and B shows that, under normal conditions, the levels of H2O2 and O2− in wild-type and transgenic tomatoes were very low and there were no significant differences between the lines. After the low temperature treatment, however, the levels of H2O2 and O2−in the 43

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wild-type lines were higher than those of the overexpression lines (H2O2, 0.53- and 0.34-fold higher; O2−, 0.41- and 0.27-fold higher, respectively). In addition, the RNAi lines accumulated higher levels of ROS than the wild-type: H2O2 levels were 0.33 and 0.23 times higher, and the O2− levels were 0.35 and 0.26 times higher, respectively. The results showed that overexpressing LeCOLD1 reduces the accumulation of H2O2 and O2− under low temperature stress. Plants have evolved a complex system to remove excess ROS that includes the enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). These enzymes may play important roles in removing ROS and alter the balance of ROS in cells. We determined the activities of these three enzymes in the transgenic plants. Wild-type and transgenic tomato lines grown under normal conditions had similar levels of SOD, POD and CAT activity. After low temperature treatment, the activities of these enzymes increased in the wild-type and all of the transgenic tomato lines. However, the enzymes were significantly more active in the overexpression lines, and less active in the RNAi lines, compared with the wild-type (Fig. 10C–E). These results show that overexpressing LeCOLD1 reduces the accumulation of reactive oxygen species in cells under low temperature stress, possibly by protecting the basal metabolic accumulation of LeCOLD1, and by increasing the activities of SOD, POD and CAT.

against these stresses (Anunathini et al., 2019). Our results revealed that LeCOLD1 is expressed in the root, stem, leaf, flower and fruit tissues of tomato and is up-regulated in response to cold, drought and salt stress, suggesting that this gene is also involved in the regulation of abiotic stresses. Furthermore, the leaves of interference (RNAi) transgenic plants showed more obvious drooping and wilting phenotypes, while the leaves of LeCOLD1 overexpressing lines displayed a lesser degree of wilting, under low temperature stress compared with the leaves of the wild-type. These results indicated that increasing the expression of LeCOLD1 may improve the ability of tomatoes to tolerate low temperatures. Changes in membrane structure and function caused by low temperature lead to increased membrane permeability, which in turn leads to electrolyte exosmosis and increased relative conductivity (Jiang et al., 2017). The levels of MDA, a lipid peroxidation product, in cells reflects the degree of lipid peroxidation and damage to cells (Wang et al., 2012a,b; Maki et al., 2005). After 2–3 days of cold acclimation at 4 °C, the membrane protein content of alfalfa increased (Mohapatra et al., 1988). Like COLD1, WPI6 is a plasma membrane protein with two transmembrane domains that play a protective role in maintaining plasma membrane function in wheat during cold acclimation (Imai et al., 2005). Therefore, plasma membrane proteins help maintain the integrity and stability of plant membrane systems in order to resist cold stress. We found that overexpressing LeCOLD1 reduced damage to the membrane system of processing tomato caused by cold stress, while interference inhibition of this gene had the opposite effect. Low temperature stress destroys almost all of the major components of photosynthesis (Allen and Ort, 2001). It inhibits PSII activity in a variety of ways, for example, by slowing down the rate of photosynthesis, blocking the photosynthetic electron transport of PSII, and damaging membrane lipids and photosynthetic pigment complex systems (Feng et al., 2013). Our results show that, overexpressing LeCOLD1 alleviates the photoinhibition of PSII and allows cells to maintain a high photosynthetic capacity. Many studies have reported that ROS production is triggered when plants are subjected to abiotic stress. Low temperature stress leads to the production of a large number of reactive oxygen species, mainly including O2− and H2O2, which damage DNA, proteins and membrane lipids through oxidative reactions (Mignolet-Spruyt et al., 2016). In addition, osmotic regulatory substances participate in ROS clearance (Xiong and Zhu, 2002; Miller et al., 2010; Rejeb et al., 2014). Free proline and soluble sugar are two important osmotic regulators and are closely related to plant resistance to low temperature stress. Our results indicate that the higher proline and soluble sugar levels in plants overexpressing LeCOLD1 play important roles in ROS clearance. Therefore, the low ROS levels maintained by the overexpression lines at low temperatures may be due to the high accumulation of osmotic regulatory materials. To get rid of excess ROS, plants have evolved complex antioxidant enzyme and non-enzyme systems (Miller et al., 2010). Therefore, we measured the activities of three major antioxidant enzymes (SOD, POD and CAT). The results showed that the LeCOLD1 overexpression lines maintained high SOD, POD and CAT activities under low temperature stress. Furthermore, the expression levels of the genes encoding these enzymes (LeSOD, LePOD, and LeCAT) were higher in the LeCOLD1 overexpression lines than in the wild-type and RNAi plants. This indicates that the activities of the SOD, POD and CAT enzymes in plants overexpressing LeCOLD1 under low temperature stress may depend on how highly their corresponding genes are expressed. Ectopic expression of tomato SlCBF1 in A. thaliana has been shown to increase freezing tolerance (Zhang et al., 2004). SlICE1 enhances the cold resistance of tomato during low temperature stress and increases the expression of SlCBF1 and SlDRCi7 (Miura et al., 2012). Our study found that LeCOLD1 increases the expression of the LeICE1 gene in processing tomato, thus regulating the transcription of LeCBF1 and

3.9. LeCOLD1 positively regulates cold response gene expression in processing tomato In order to further explore why the overexpression lines had higher SOD, POD and CAT enzyme activities, the key genes encoding these enzymes were detected by qRT-PCR. Fig. 11A–C shows that, the LeSOD, LePOD and LeCAT genes were more highly expressed in the LeCOLD1 overexpression lines, but less highly expressed in the RNAi lines, than in the wild-type after low temperature treatment. The change in expression of each gene before and after the cold treatment was similar to the change in activity of the corresponding enzyme. These results show that after low temperature treatment, the increase in the activities of SOD, POD and CAT in the transgenic lines may be caused by the up-regulation of their corresponding genes. To further investigate how LeCOLD1 regulates cold tolerance at the molecular level, we used qRT-PCR to analyze the expression levels of two genes in the ICE-CBF pathway (LeICE1 and LeCBF1) and their regulatory gene LeDRCi7. As shown in Fig. 11D–F, these genes were slightly more highly expressed in the LeCOLD1 overexpression lines than in the RNAi lines under normal conditions. After cold stress treatment, the expression levels of these genes were significantly higher in the overexpression lines compared with the wild-type tomato plants. In addition, the expression levels of these genes were lower in the RNAi lines compared with the wild-type tomato plants. These results suggest that overexpressing LeCOLD1 helps transgenic plants resist cold stress by inducing the expression of several stress response genes. 4. Discussion Membrane proteins play many roles in transmitting external signals in response to stress (Steponkus, l984; X. Huang et al., 2012). Previous studies have shown that some membrane-localized proteins play roles in plants’ response to cold (Lin et al., 1992). During low temperature stress, the G protein signaling regulator COLD1 interacts with GPA1 and activates Ca2+ signaling, activating GTPases in the signal transduction pathway and co-participating in the transduction of rice low temperature stress signals (Ma et al., 2015). Like the COLD1 proteins in rice (Ma et al., 2015) and wheat (Dong et al., 2019), LeCOLD1 in tomato is a typical membrane protein that has nine transmembrane domains and is targeted to plasma membrane in plant cells. The expression of COLD1 genes in different species is significantly increased in response to several different abiotic stresses and this gene appears to play an important role in the development of resistance 44

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inducing the expression of its downstream regulatory gene LeDRCi7. These results suggest that LeCOLD1 may regulate cold tolerance of tomato plants through the ICE-CBF pathway.

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5. Conclusion Taken together, these results indicate that overexpressing LeCOLD1 may improve the low temperature resistance of transgenic tomatoes. Under cold stress, overexpressing LeCOLD1 improved cell membrane integrity, prevented the accumulation of excessive membrane lipid peroxidation products, decreased ion leakage rate, improved cell membrane stability, and increased the expression of antioxidant enzyme genes, thereby increasing the activity of corresponding antioxidant enzymes and the efficiency of photochemical electron transport. As a result, the LeCOLD1 overexpression lines had lower levels of ROS than the wildtype plants. The low temperature tolerance of processing tomatoes was enhanced by maintaining relatively high levels of permeable proline and soluble sugar to protect cells from damage. Therefore, we propose that LeCOLD1 is a multifunctional protein that regulates the activities of target proteins to control plant responses to low temperature stress. Further studies examining LeCOLD1 binding to downstream target proteins are needed to better understand the role of this regulatory protein in tomato. Funding This work was supported by the National Science Foundation Project (31360053). CRediT authorship contribution statement Li Zhang: Conceptualization, Methodology, Writing - original draft. Xinyong Guo: Visualization. Yujie Qin: Supervision. Bin Feng: Software. Yating Wu: Validation. Yaling He: Data curation. Aiying Wang: Investigation. Jianbo Zhu: Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.plaphy.2020.03.007. References Allen, D.J., Ort, D.R., 2001. Impacts of chilling temperatures on photosynthesis in warm climate plants. Trends Plant Sci. 6, 36–42. Anunathini, P., Manoj, V.M., Sarath Padmanabhan, T.S., Dhivya, S., Narayan, J.A., Appunu, C., Sathishkumar, R., 2019. In silico characterisation and functional validation of chilling tolerant divergence 1 (COLD1) gene in monocots during abiotic stress. Funct. Plant Biol. https://doi.org/10.1071/FP18189. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 1, 205–207. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Benikhlef, L., Haridon, F., Aboumansour, E., Serrano, M., Binda, M., Costa, A., Lehmann, S., Métraux, J.P., 2013. Perception of soft mechanical stress in Arabidopsis leaves activates disease resistance. BMC Plant Biol. 13, 1–12. Bu, Q.Y., Liang, W., Yang, S.H., Wan, J.M., 2005. Cloning, characterization and expression vector construction of potato proteas-inhibitor II gene (PIN II-2x) from diploid potato (Solanum phurejia). Yi Chuan 27, 417–422. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M., Zhu, J.K., 2003. ICE1: a regulator of cold‐induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 17, 1043–1054.

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