Leveraging Atriplex hortensis choline monooxygenase to improve chilling tolerance in cotton

Environmental and Experimental Botany 162 (2019) 364–373

Contents lists available at ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Leveraging Atriplex hortensis choline monooxygenase to improve chilling tolerance in cotton

T

⁎

Yanan Wanga,b,1, Chengzhen Liangb, ,1, Zhigang Mengb, Yanyan Lib, Muhammad Ali Abidb, Muhammad Askarib, Peilin Wangb, Yuan Wangb, Guoqing Sunb, Yongping Caia, Shou-Yi Chenc, ⁎ ⁎ ⁎ Yi Lina, , Rui Zhangb, , Sandui Guob, a

College of Agronomy, Anhui Agricultural University, Hefei, 230036, China Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China c State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cotton AhCMO Osmoprotectant Chilling tolerance

Low temperature is a major factor limiting seedling growth and the production of cotton (Gossypium hirsutum L.). However, enhancing chilling tolerance is typically negatively correlated with yield in agricultural production. Here, we demonstrate that transgenic cotton expressing Atriplex hortensis choline monooxygenase (AhCMO) greatly enhanced resistance to chilling stress. The promotion of chilling tolerance is mainly due to an increase in the content of osmoprotectants, especially glycine betaine and proline. The increased chilling tolerance was further verified at the molecular level using genome-wide expression profiling by RNA-sequencing. Further detailed analysis showed that the number of genes involved in scavenging of reactive oxygen species (ROS) was down-regulated and the activity of superoxide dismutase (SOD) and catalase (CAT) were decreased in AhCMO transgenic cotton compared with wild type after low temperture treatment. More importantly, overexpression of AhCMO in cotton moderately improved cotton fiber yield in normal growth condition. These data show that AhCMO transgenic cotton enhances low temperature tolerance via directly accumulating cellular osmoprotectants. Manipulating the expression of AhCMO by biotechnological tools could be a powerful method to enhance chilling tolerance in cotton.

1. Introduction Cotton (Gossypium hirsutum L.) is a major cash crop worldwide but because it initially originated from tropical and subtropical areas, it is sensitive to chilling stress (Pham et al., 2018). In China, cotton is primarily distributed in the northwest inland areas, such as Xinjiang province, with high latitude and high altitude. Low temperature is certainly the major limiting factor for cotton growth and production in these regions (Pham et al., 2018). Chilling stress significantly inhibits seed germination, seedling growth, reduces cotton yield and deteriorate fiber quality due to fertilization failure, boll abscission, and even plant death, the losses may reach up to 30%–40% of lint cotton yield (Groot et al., 2018; Yu, 2007). Plant cold tolerance is a complex trait controlled by a finely-tuned regulatory network. Genes in the response to cold stress can be divided into two categories (Kreps et al., 2002; Yamaguchi-Shinozaki and

Shinozaki, 2006). The first group involves the regulation of signal transduction and downstream stress response genes, such as transcription factors and protein kinase (Liu et al., 2018). The second is the genes encoding proteins directly involved in cold stress response, such as osmotin, antifreeze proteins, and key enzymes for osmolyte biosynthesis. Although several cold regulatory genes have been cloned to functionally analyze specific C-repeat/DREB binding factors (CBFs), the active expression of these genes usually slowed plant growth, resulting in yield losses (Liu et al., 2017; Shi et al., 2018). Currently, there are no genes reported that can enhance low temperature resistance without yield losses. Plants can accumulate osmoprotectants to adjust osmotic homeostasis against chilling stress, such as glycine betaine, proline, soluble sugars, and polyols (Jorge et al., 2016; Kishitani et al., 2010). Glycine betaine (N, N, N-trimethyl glycine), one of the most widely studied osmoprotectants, protects plant cells by stabilizing protein quaternary

⁎

Corresponding authors. E-mail addresses: [email protected] (C. Liang), [email protected] (Y. Lin), [email protected] (R. Zhang), [email protected] (S. Guo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.envexpbot.2019.03.012 Received 29 January 2019; Received in revised form 9 March 2019; Accepted 12 March 2019 Available online 14 March 2019 0098-8472/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

2.2. Plasmid construction and plant transformation

structure and regulating cytoplasmic osmotic pressure (Luo et al., 2007; Xu et al., 2018). Endogenous accumulation and exogenous application of glycine betaine can enhance cold stress tolerance of plants (Park et al., 2004). For example, cold stress of alkali grass (Puccinellia tenuiflora) induced the accumulation of glycine betaine (Meng et al., 2016). Exogenous glycine betaine application improved cold tolerance in tomato, which is a cold sensitive plant (Park et al., 2006). Glycine betaine has been reported to protect photosystem II, and mitigate reactive oxygen species (ROS) damage (Chen and Murata, 2010). In addition, proline, which accumulates under cold conditions, can regulate the osmotic potential of cells and hold turgor pressure in stressed cells (Zhou et al., 2018). It is well-established that proline acts as a compatible osmolyte, which is synthesized during Arabidopsis stress response (Hildebrandt, 2018). Soluble sugars have been recognized to preserve woody plants from damage triggered by cold stress, owing to their positive roles in preserving cellular constituents (Zhao et al., 2016). It has been shown that proline and soluble sugar accumulated in cold-tolerant Pinus halepensis seed sources (Taïbi et al., 2018). ROS are products of higher plant metabolic processes (Foyer et al., 2009), and can damage molecules under abiotic stress (Liu and He, 2017; Møller et al., 2007). Many single stress experiments have revealed that glycine betaine, proline, and soluble sugars function in scavenging of ROS and redox buffering (Bartels and Sunkar, 2005; Rivero et al., 2014). Atriplex hortensis, a glycine betaine natural accumulator also called mountain spinach, can tolerate harsh conditions such as cold, drought, and high salinity (Shen et al., 2002; Tao et al., 2018). Since glycine betaine has been shown to have the main osmotic stress-resistant effect in many plants, cloning of key enzymes of the glycine betaine synthesis pathway can be used to establish an effective abiotic tolerance regulation pathway and improve the abiotic tolerance of crops (Saneoka et al., 1995). In higher plants, glycine betaine synthesis is catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), which are localized in chloroplast stroma (Bhuiyan et al., 2007). CMO is a rate-limiting enzyme that catalyzes the first step of glycine betaine synthesis and is a stromal and Fd-dependent monooxygenase with a Rieske-type [2Fe-2S] center (Burnet et al., 1995; Rathinasabapathi et al., 1997; Russell et al., 1998). A transgenic study demonstrated that overexpressing choline monooxygenase from Spinacia oleracea accumulated glycine betaine and enhanced tolerance to low temperature stress at the rice seedling stage (Shirasawa et al., 2006). Glycine betaine contents and tolerance to stress are positively correlated with a functional OsCMO transcript level in transgenic tobacco plants (Luo et al., 2012). The AmCMO promoter region contains the sequence responsive to salt stress and is important for the accumulation of glycine betaine in Amaranthus tricolor (Bhuiyan et al., 2007). In 2002, Shen et al. cloned and characterized the CMO gene from Atriplex hortensis, a GlyBet natural accumulator, and found that overexpression of AhCMO in tobacco significantly increased tolerance to drought and salt stress (Shen et al., 2002). A previous study implied that osmoregulation accumulation is closely related to low temperature response (Artur et al., 2011), which led us to speculate that AhCMO might be a good candidate for genetic engineering of chilling-tolerance in cotton.

The coding sequence fragment of AhCMO was amplified using specific primers that introduced Pst I and Xho I restriction sites. The coding sequence was then sub-cloned into the pUC-19 vector and cloned into the binary vector pBI121-CaMV 35S to construct an overexpression vector (Supplementary Fig. 1). The vector was transformed into cotton using the hypocotyl segments of upland cotton cultivar variety R15 by Agrobacterium-mediated transformation to generate plants for chilling resistance analysis. 2.3. Low temperature assay in cotton Cotton accession R15 and its AhCMO transgenic plants were cultivated in a mixture of vermiculite and nutrient soil (1:2, w/w) at 28 °C/ 25 °C (light/dark) under a 16 h photoperiod. Plants grown for 6 weeks were used in the experiments. For chilling treatment, plants were placed in an artificial incubator set to 12 °C/12 °C (light/dark) under a 16 h photoperiod for 24 h. Plants incubated at 28 °C were used as a control. After low temperature treatments, cotton leaves were immediately frozen in liquid nitrogen, and stored at −80 °C prior to use. The chilling treatment experiment was performed three times. 2.4. Trypan blue staining Wild-type and transgenic cotton leaves were boiled for 5–8 min and stained with a solution of trypan blue for 6–8 h at room temperature. After 95% ethanol decolorization treatment, the distribution of damage in the leaf tissues with low temperature stress was observed. Three leaves were treated per experiment. 2.5. Chlorophyll measurement The chlorophyll and free nitrogen content of the cotton leaves were measured using a handheld chlorophyll meter. Ten leaves were treated per experiment; all analyses had three biological replicates. Data analyses were conducted using SPSS version 19.0 (IBM SPSS Statistics). 2.6. H2O2 measurement H2O2 content was determined using the method described by Deniz et al. (Tiryaki et al., 2018). Fresh cotton leaves (0.1 g) were homogenized in 1 ml of cold acetone. Then, H2O2 content was determined using a hydrogen peroxide assay kit (Solarbio, China) and its absorbance was measured at 415 nm. Data are represented as the amount of H2O2 per gram leaf (μmol/g). All analyses had three biological replicates. 2.7. Measurement of glycine betaine content First, to extract glycine betaine, 1 ml 80% methanol was added to 0.2 g of dried leaf tissues and the mixture was shaken for 30 min in a 60 °C water bath. Samples were then centrifuged at 10,000 rpm at room temperature for 15 min. The supernatant was then held at 70 °C to volatilize methanol. Samples were then diluted to 1 ml with doubledistillation H2O (ddH2O) to obtain a crude enzyme extract. Glycine betaine content was measured using a betaine assay kit (Solarbio, China), and its absorbance was measured at 525 nm.

2. Materials and methods 2.1. Plant materials and growth conditions Cotton variety R15 and its AhCMO transgenic plants were grown in fields of the Langfang (Hebei province, N39°56′, E116°20′) experimental station. Transgenic homozygous cotton plants from positive T1 lines were selected based on PCR screening. The transgenic homozygous lines were selected and self-fertilized until the agronomic traits were stabilized.

2.8. Measurement of proline and glutamic acid content Amino acids were extracted from 0.1 g of fresh leaf tissues. Proline and glutamic acid contents were measured using a proline content assay kit and glutamic acid content assay kit (Solarbio, China). Both of their contents were determined by comparing the absorbance value with the calibration plot for standard solutions. The absorbance values were 365

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

2.14. Data recording for morphological traits

measured at 520 nm and 340 nm, respectively.

The cotton plants were grown in a standard cotton field at the Experimental Station of the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Langfang (N39°56′, E116°20′), Hebei province. Field planting followed a randomized complete-block design with three replicates. Each tested transgenic cotton lines was planted in an experimental plot of about 22.5 m2 (600 plants for each). The plant to plant distance was 12.5 cm and row to row spacing was 30 cm. All recommended plant protection measures detailed by Gwathmey et al. (Gwathmey et al., 2016) were adopted from sowing to harvesting. Fifty plants from each repeat were randomly selected for important agronomic traits including plant height, number of fruit branches per plant, and number of bolls per plant, which were measured on a single-plant basis. Plant height was determined as the height of the main stem at the boll opening stage. Vegetative shoot and fruiting branch of the main stem were separated manually for measurements of number of fruit branches per plant. All available cotton bolls from a single plant were collected for measurements of number of bolls per plant. All naturallyopened bolls from a single plot were collected and dried at 37 °C in an oven, and 100 randomly picked bolls were used for boll weight measurements. All of the opened bolls before and after frost in a single plot were collected and treated as described above for measurement of actual cotton yield.

2.9. Measurement of soluble sugar content Soluble sugar contents were determined from leaf tissues of 6-weeks old plants grown under normal and chilling conditions. The extraction was prepared from 0.1 g of fresh leaf tissues homogenized with 80% ethanol. Samples were centrifuged at 8000×g for 15 min and the upper solution was purified and used for measurement of erythrose, glucose, sucrose, and raffinose. A High Performance Liquid Chromatography (HPLC) system was used to measure soluble sugar, and external standard solution calibrations of erythrose, glucose, sucrose, and raffinose were used to integrate peaks. Sugar contents were represented in ng mg−1. 2.10. Determination of SOD, POD, and CAT activity Cotton leaf tissues were directly sampled, and the activities of three enzymes were measured under chilling treatment (12 °C). Enzyme activities of the control plants were also measured from seedlings grown in normal conditions (28 °C). Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were respectively measured using a superoxide dismutase assay kit, a peroxidase assay kit, and a catalase assay kit (Solarbio, China). The absorbance values were measured at 560 nm, 470 nm, and 240 nm, respectively.

3. Results

2.11. Measurement of ion leakage

3.1. AhCMO transgenic cotton enhanced the tolerance to chilling stress

Relative ion leakage was measured with a FE38 conductivity meter (Mettler Toledo). Leaves with a diameter of 5–10 mm were equilibrated in 40 ml ddH2O at room temperature and then shaken at 120 rpm for three hours. Electrical conductivity of the solution was measured to get initial conductivity (C1). Then samples were boiled for 10 min, reequilibrated, and remeasured to get conductivity (C2). The ratio of C1 to C2 was used to obtain relative conductivity: (RC), (C1/C2) × 100% (Aihong et al., 2012).

To confirm the AhCMO function in enhancing low temperature tolerance in cotton, we developed multiple transgenic cotton plants carrying a CaMV 35S::AhCMO. Quantitative real-time (qRT-PCR) was used to screen for transgenic lines that had high transcript levels of AhCMO. A total of 15 positive independent transgenic lines showed upregulated expression of AhCMO. Among them, two transgenic lines, CMO20 and CMO24, which had significantly increased AhCMO expression, and were chosen for further studies (Fig. 1a). To determine chilling tolerance of the AhCMO transgenic cotton, a low temperature treatment assay was performed. After incubation at 12 °C for 24 h, the two transgenic cottons, CMO20 and CMO24, had significantly enhanced chilling tolerance. The leaves of the wild type seedlings were turned yellow and more necrotic because of low temperature damage, whereas leaves of AhCMO transgenic cotton remained green and vigorous (Fig. 1b and c). Further, trypan blue staining experiments showed that the progression of leaf yellowing, vitrification, and cell necrosis occurred at a much slower rate in both CMO20 and CMO24 compared to their non-transgenic control lines (Fig. 1d). Further the qRT-PCR analysis of the expression of three chilling-related marker genes, C-repeat binding transcription factor1 (CBF1), inducer of CBF expression 1 (ICE1), and calmodulin binding transcription activator 3 (CAMTA3), revealed that these chilling-associated genes were up-regulated significantly at 12 °C in the leaves of AhCMO overexpressing cotton compared with wild type (Fig. 1e–g). These low temperature resistant performances clearly demonstrate effectiveness and practical utility of overexpressing AhCMO for engineering chilling resistant cotton.

2.12. RNA extraction, cDNA preparation, and qRT-PCR analyses Total RNA was extracted using an RNA extraction kit (Yuan Pinghao Bio Co, China) and digested with DNaseI to eliminate genomic DNA. Approximately 2 μg of total RNA were reverse-transcribed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech). Quantitative real-time PCR was performed with a ABI 7500 Real-Time PCR system using KOD SYBR® qPCR Mix. Each sample was collected as three independent biological replicates. The cotton Histone3, with stability expression in the different tissues, developmental stages and environmental conditions (Zhu et al., 2017), was used as an internal control, and expression was measured using the 2−ΔΔCT method (Livak and Schmittgen, 2001). All the primers are shown in Supplementary Table S1. 2.13. RNA sequencing and data analysis

3.2. AhCMO transgenic cotton promoted the accumulation of cellular osmoprotectants

Library construction and sequencing were performed in accordance with the standard experimental procedures provided by the Illumina 2000 system. Gene ontology enrichment (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) were performed using the tools of laboratory data platform cotton-FGD (https://cottonfgd.org/). Cluster and heat map analysis of DEGs was conducted using HemI 1.0 (Wankun et al., 2014). DEGs with log2 > 1 were defined as up-regulated and those with log2 < -1 were defined as down-regulated. DEGs were identified by applying a cutoff p-value < 0.05.

CMO is the key enzyme that functions in the biosynthesis of glycine betaine, which is a neutral chemical compound that protects against osmotic stress, drought, salinity, and low/high temperature. To examine whether the AhCMO enzyme could synthesize glycine betaine in transgenic cotton, the total glycine betaine contents in leaves of CMO20 and CMO24 were measured. Under normal growth condition the CMO20 and CMO24 transgenic lines had 20.5% and 32.1% increases, 366

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

Fig. 1. AhCMO-overexpressing transgenic cotton significantly enhances chilling stress tolerance in cotton. (a) qRT-PCR analysis of the transgenic expression of AhCMO in 6-week-old transgenic cotton plants. The cotton Histone3 gene was selected as a control to normalize transcript levels. (b) AhCMO transgenic cotton plants CMO20 and CMO24 showed higher tolerance to chilling stress compared with wild type cotton. Scale bar: 5 cm. (c) Leaf phenotypes corresponding to (b). (d) Histochemical analysis by trypan blue. The data were obtained with three independent replicates. (e–g) Expression of chilling-related marker genes in wild-type plants and AhCMO transgenic cotton under no treatment and chilling treatment. CBF1, C-repeat binding transcription factor 1; ICE1, inducer of CBF expression 1; CAMTA3, calmodulin binding transcription activator 3. **P ≤ 0.01; Student’s t-test.

transcripts of wild-type plants, CMO24 under natural growth conditions (28 °C) had 854 up-regulated genes and 467 down-regulated genes, whereas chilling-treated (12 °C) CMO24 lines had 785 up-regulated genes and 1530 down-regulated genes (Supplementary Fig. 2 and Supplementary Table 2). There were 93 genes commonly up-regulated and 263 genes commonly down-regulated in CMO24 plants at both 28 °C and 12 °C (Supplementary Fig. 2 and Supplementary Table 2). To validate these differentially expressed genes (DEGs), qRT-PCR was performed for 12 randomly selected genes. Comparison of the results from qRT-PCR analysis revealed the regression slope for RNA-seq versus qRT-PCR is close to 1, suggesting a strong positive correlation between the two sets of data, therefore suggesting the credibility of the RNA-seq data (Supplementary Fig. 3). We retrieved genes involved in the cotton glycine betaine biosynthesis pathway, based on a previous report (Lv et al., 2007), to analyze their expression patterns in CMO24 transgenic lines. We found that the transcript level of choline kinase decreased 1.59-fold in CMO24 cotton after 24 h low temperature treatment (Supplementary Fig. 4), while no significant change was observed in other genes, including cotton CMO and BADH (betaine aldehyde dehydrogenase). The key synthesis gene for proline, P5CR (Δ1-pyrroline-5-carboxylate reductase), was clearly up-regulated in CMO24 under both normal and chilling conditions (Supplementary Fig. 5). Previous reports have shown that the accumulation of proline protects plant cells from chilling damage (Zhou et al., 2018). Therefore, the total content of proline, glutamic acid, and arginine were quantified. As shown in Fig. 3a and b, significant increases in proline and glutamic acid were observed in both CMO20 and CMO24 lines at 12 °C. Further, the conductivity was significantly lower in the AhCMO transgenic lines, CMO20 and CMO24,

Fig. 2. Glycine betaine content in AhCMO-overexpressing transgenic cotton. Values are means ± S.D. of 10 biological replicates. DW, dry weight. **P ≤ 0.01. Student’s t-test.

respectively, in glycine betaine levels compared to wild-type cotton (Fig. 2). After 24 h of 12 °C chilling treatment, the glycine betaine concentrations in the CMO20 and CMO24 plants were 1.67- and 1.85fold higher, respectively, than that of the wild-type cotton plants (Fig. 2). After observing similar chilling tolerance in both CMO20 and CMO24 lines, the transgenic line CMO24 was used for physiological and biochemical analysis. To systematically analyze the contribution of AhCMO to low temperature tolerance in cotton, we performed RNA-Seq using CMO24 and wild type cotton leaves. Compared with the 367

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

Fig. 3. Amino acid content and relative conductivity in AhCMO transgenic cotton. Proline (a), glutamic acid (b), and conductivity (c) in leaves of AhCMO transgenic cottons. Values are means ± S.D. of 10 biological replicates. FW, fresh weight. *P ≤ 0.05, **P ≤ 0.01. Student’s t-test.

biological process category among the three groups (Fig. 4a and Supplementary Table 3). Consistently, the genes associated with oxidation/ reduction activity were also enriched by the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Supplementary Fig. 6 and Supplementary Table 4). Among 234 DEGs related to oxidation/ reduction activity in CMO24 under chilling conditions, the number of downregulated genes was about 5.2-times the number of upregulated genes (Fig. 4b and Supplementary Fig. 7 and Table 5). Seventeen DEGs involved in “Glutathione metabolism” were down-regulated, and three “Glutathione metabolism” genes were upregulated (Supplementary Table 6). These data imply that the transcript level of genes associated with ROS scavenging was lower in CMO24 lines than wild-type cotton, which led us to speculate that the level of oxidative stress is much lower

than in the non-transgenic cotton under low temperature treatment (Fig. 3c). These data demonstrate that overexpression of AhCMO could trigger physiological responses via increasing the contents of osmoprotectants including glycine betaine and proline, and thus improving the chilling stress resistance of AhCMO-transgenic cotton. 3.3. The accumulation of H2O2 was significantly inhibited in AhCMO transgenic cotton To further clarify the changes in gene expression in CMO24, we constructed a gene ontology (GO) term network for DEGs in CMO24 with low temperature treatment. GO terms correlated with response to oxidation-reduction and oxidative stress were highlighted in the

Fig. 4. Global gene expression changes of CMO24 under chilling stress conditions. (a) Gene ontology analysis in response to chilling stress in CMO24. (b) The heat map of differentially expressed genes involved in the oxidation-reduction process. The annotation information of genes can be found in Table S3. URGs, upregulated genes; DRGs, downregulated genes. 368

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

Fig. 5. Oxidative stress was significantly reduced in AhCMO transgenic lines after chilling treatment compared to wild type plants. (a) Total extractable leaf H2O2 contents in AhCMO transgenic plants and wild-type cotton. Values are mean ± S.D. of 20 measurements. (b–d) SOD activities (b), POD activities (c), and CAT activities (d) of AhCMO transgenic lines and wild-type plants. Values are means ± S.D. of three replicates. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase. FW, Fresh weight. **P ≤ 0.01. Student’s t-test.

under normal growth condition (Fig. 6a–c). These results showed that AhCMO transgenic cotton enhanced photosynthesis-related processes under low temperature conditions.

in CMO24 transgenic lines than in non-transgenic control after low temperature treatment. To this end, the level of H2O2 contents in CMO24 lines and wild type grown at 28 °C and 12 °C was quantified. As shown in Fig. 5a, the accumulation of H2O2 was significantly inhibited in CMO24 after 12-h low temperature treatment compared with wild-type cotton. However, this phenomenon was not observed between CMO24 and wild type under normal growth condition. Accordingly, the activities of reactive oxygen species (ROS)-scavenging enzymes, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were increased in both the wild type and transgenic lines (Fig. 5b–d). However, AhCMOoverexpressing cotton exhibited slightly lower SOD, POD, and CAT activity compared to the wild-type plants (Fig. 5b-d). These results were consistent with the DEGs expression pattern that AhCMO transgenic cotton had a higher rate of downregulated oxidation/reduction activity genes.

3.5. AhCMO transgenic cotton affects metabolism of carbohydrates under chilling stress We also noticed that the synthesis genes for the carbohydrate metabolic process were enriched in CMO24 under both normal and low temperature treatment (Fig. 4a and Supplementary Table 2). The expression of multiple genes in sugar biosynthesis and metabolism changed in CMO24 lines as compared to wild type (Supplementary Table 8). For example, under chilling stress, the transcript level of sucrose synthases 4 (GhSuS4) was upregulated about 2.1-fold in AhCMO transgenic cotton compared to wild type (Supplementary Table 2). In contrast, the transcript levels of three galactinol synthases (GhGolS2) were significantly downregulated under chilling stress in CMO24 cotton (Supplementary Table 2). We further measured four soluble sugar contents in the seedling leaves of CMO24 and wild type under both 28 °C and 12 °C. AhCMO transgenic lines demonstrated an increase of more than 3.1-fold in erythrose and 1.5-fold in sucrose concentration in comparison with wild-type plants after 24-h chilling treatment (Fig. 7a and b). Consistent with the results of RNA-Seq data, AhCMO transgenic lines retained lower glucose and raffinose as wild-type plants after 24 h of low temperature treatment (Fig. 7c and d).

3.4. AhCMO transgenic cotton improves photosynthesis-related processes under chilling stress We found that the transcript levels of enzymes involved in photosynthetic reaction processes, including 13 photosystem I and 24 photosystem II genes, were all upregulated in CMO24 lines under chillingstress conditions, compared with wild-type cotton (Fig. 4a and Supplementary Table 7). Consistently, under low temperature treatment, the leaves of AhCMO transgenic cottons maintained their chlorophyll pigments for a longer period, had higher levels of functional photosynthetic capacity (Fig. 6a), higher chlorophyll content (Fig. 6b), and higher total nitrogen content (Fig. 6c), compared to wild type. However, no significant differences of physiological indicators mentioned above were observed between AhCMO transgenic cotton and wild type

3.6. AhCMO transgenic cotton improved agronomic performance Besides the improved resistance to low temperature stress, AhCMO transgenic plants also showed improved agronomic performance that led to an improvement of cotton yield compared with the non369

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

Fig. 6. AhCMO transgenic cotton enhances photosynthesis-related processes under chilling stress as compared with wild-type plants. (a) Net photosynthesis rate (400 μmol CO2/m2/s) measured at 24 h after 12 °C treatment in a culture room. Values are means ± S.D. from at least ten independent leaves. (b–c) Chlorophyll and total N content of the leaves corresponding to (b) Values are mean ± S.D. of ten measurements. FW, fresh weight. **P ≤ 0.01. Student’s t-test was used to generate P value.

multiple tolerance activities of AhCMO, which effectively reduced the losses of yield by other abiotic stressors, including drought, high salt, and high/low temperature.

transgenic control. Transgenic cotton expressing AhCMO showed slightly reduced plant height, and about 2 cm shorter internodes compared with wild type (Table 1). AhCMO transgenic lines had 9–11 fruit branches for each plant, while 8–10 fruit branches were observed in wild-type plants. Moreover, the AhCMO transgenic lines had more than two bolls per plant on average compared to non-transgenic plants. Importantly, the AhCMO transgenic lines had a moderate improvement of fiber yield and lint percentage compared with the non-transgenic cotton plants (Table 1). An examination of the agronomic traits related to fiber yield in CMO24 lines showed about a 9.2% and 13.9% increase in unginned cotton yield and lint cotton yield, respectively, compared with our non-transgenic controls (Table 1). This may be the result of the

4. Discussion In agricultural production, chilling stress has the characteristics of irregularity, uncertainty, and unpredictability. Chilling resistance is a very complex trait (Kenji and Tsuyoshi, 2013). Currently, several coldrelated genes, particularly CBF transcription factors, are known to play a crucial role in enhancing cold stress tolerance and have been characterized in plants (Liu et al., 2017; Shi et al., 2018). However, plant

Fig. 7. Soluble sugar content in AhCMO transgenic cotton. Total erythrose (a), sucrose (b), glucose (c), and raffinose (d) concentrations in 6-weeks old plant leaves. Values are means ± S.D. of 10 biological replicates. FW, fresh weight. *P ≤ 0.05, **P ≤ 0.01. Student’s t-test. 370

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

Table 1 Agronomic traits of CMO24 plants in normal field conditions in Langfang, Hebei province in 2018.

Transgenic Notransgenic P value

PH (cm)

NoBr

NoBo

BW (g)

LCY (g/boll)

UCY (g/plant)

LCY (g/plant)

LP (%)

UCY (kg/plot)

LCY (kg/plot)

69.2 ± 2.8 67.0 ± 14.5 0.24

9.0 ± 1.0 10.0 ± 1.0 < 0.01

11.6 ± 1.8 13.7 ± 1.5 < 0.01

3.5 ± 0.4 3.9 ± 0.4 < 0.01

1.28 ± 0.2 1.34 ± 0.1 0.09

40.8 ± 5.9 48.2 ± 6.6 < 0.01

15.9 ± 2.6 18.3 ± 2.5 < 0.01

35.8 ± 3.2 37.5 ± 4.8 0.15

88.5 ± 11.5 96.7 ± 14.6 < 0.01

31.6 ± 4.7 36.0 ± 6.3 < 0.01

Traits monitored included plant height (PH), number of branches (NoBr), number of bolls per plant (NoBo), boll weight (BW), lint cotton yield per boll (LCY per boll), unginned cotton yield per plant (UCY per plant), lint cotton yield per plant(LCY per plant), lint percentage (LP), unginned cotton yield per plot (UCY per plot), and lint cotton yield per plot (LCY per Plot). The significant differences between the CMO24 and wild-type cotton plants were assessed with Student's t-tests.

biosynthesis of glycine betaine was accelerated, and more choline was used for synthesizing glycine betaine in AhCMO-transgenic cotton. Proline, which is known to contribute to osmotic adjustment, is synthesized by glutamate or ornithine; the glutamate pathway dominates under abiotic stress (Delauney et al., 1993). Glutamate was catalyzed by Δ1-pyrroline-5-carboxylate synthase (P5CS) (Yamada et al., 2005) and Δ1-pyrroline-5-carboxylate reductase (P5CR) catalyzed the final step of proline biosynthesis (Forlani et al., 2015). In our RNA-Seq data, P5CS was significantly down-regulated while P5CR was up-regulated in AhCMO transgenic cotton (Supplementary Fig. 5). As expected, the accumulation of proline was significantly higher in AhCMO cotton under low temperature stress (Fig. 3a). Accordingly, the concentration of glutamate was also significantly higher in transgenic cotton than wild type under both normal and chilling conditions (Fig. 3b). In addition, photosynthesis-related genes were altered in AhCMO transgenic cotton plants (Fig. 4a and Supplementary Table 7). Genes related to photosystem I and photosystem II were up-regulated in CMO24 lines. The better results of chlorophyll content, total nitrogen content and functional photosynthetic capacity in CMO24 are consistent with the RNA-Seq data under chilling treatment (Fig. 6). Moreover, a number of sugar metabolism-related genes were also changed in AhCMO overexpressing cotton (Fig. 4a and Supplementary Table 8). Soluble sugars, such as sucrose and glucose, play an essential role in metabolism at cellular levels (Couée et al., 2006). It is well known that the cell membrane is affected by chilling and is damaged at low temperature. The accumulation of sucrose in AhCMO transgenic plants may promote membrane stability and enhance low temperature tolerance. The upregulated expression of sucrose metabolism enzymes GhSuS resulted in a higher level of sucrose (Fig. 7b and Supplementary Table 2). In contrast, downregulated expression of GhGolS resulted in a lower level of raffinose family oligosaccharides in AhCMO plants under chilling stress (Fig. 7d and Supplementary Table 2). Thus, the improvement of low temperature tolerance of AhCMO transgenic cotton led to efficient plant cell biological function. We also noticed that the accumulation of H2O2 was inhibited in AhCMO transgenic lines compared to wild type after chilling treatment (Fig. 5a). This result was also supported by the RNA-seq data showing that a large proportion of down-regulated DEGs were involved in oxidation reduction (Fig. 4a–b and Supplementary Table 5). The overexpression of AhCMO in plants can promote the biosynthesis of glycine betaine to protect the cells from damage and maintain a lower level of oxidative stress, thereby preventing plants from chilling-induced cell death.

growth is significantly reduced in transgenic low temperature-tolerant plants under normal growth condition, resulting in yield penalties for crops (Agarwal et al., 2017; Ma et al., 2015; Morran et al., 2011); therefore these genes have few applications in actual agricultural practice. Thus, the ideal low temperature tolerance-related genes must confer strong chilling stress resistance to the crops without any reduction of grain yield in normal conditions. In this study, we demonstrated that transgenic cotton expressing AhCMO significantly enhanced resistance to chilling stress (Fig. 1). The introduction of AhCMO significantly promoted synthesis of the osmoprotectant glycine betaine under chilling conditions, thereby protecting cells from chilling stress damage (Figs. 1 and 2). More importantly, CMO gene family show multiple types of tolerance to different abiotic stresses, such as low temperature, drought, and salinity (Bhuiyan et al., 2007; Luo et al., 2012; Shirasawa et al., 2006). Therefore a large proportion of cotton yield losses caused by occasional chilling, drought, and salinity stress could be mitigated by growing AhCMO transgenic cotton (Table 1). These results indicated that AhCMO transgenic cotton is a potentially valuable variety for improving chilling tolerance in cotton breeding. In plant biological systems, naturally occurring glycine betaine serves as an organic osmolyte, substances synthesized or taken up from the environment by cells for protection against osmotic stress, drought, high salinity, or high/low temperature (Holmstrom et al., 2000; Mansour, 1998). Intracellular accumulation of glycine betaine, which is non-perturbing to enzyme function, protein structure, and membrane integrity, permits water retention in cells, thus protecting them from the effects of dehydration (Wang et al., 2019). Our experimental results also confirmed that, except for changes of specific biochemical or physiological indexes in the AhCMO-transgenic cotton, there was almost no change in growth and development indicators compared with non-transgenic lines in normal conditions. In addition, our RNA-seq data also support the idea that ectopic expression of AhCMO contributes to abiotic stress tolerance without apparent inhibition of growth and development or yield (Fig. 1 and Table 1). Previous studies have shown that overexpression of CMO genes significantly increased drought and salt tolerance in tobacco and rice (Luo et al., 2012; Shen et al., 2002). Different abiotic stresses may share partly similar regulation mechanisms. Our research data of AhCMO-transgenic cotton has not only broadened the perspective of the function of AhCMO in the chilling stress response, but also provided a powerful candidate for genetic engineering to enhance cotton chilling tolerance. Environmental stresses deplete osmotic potential thereby causing osmotic stress and yield losses of crops (Ahuja et al., 2010; Rivero et al., 2014). Therefore, synthesis and accumulation of osmoprotectants has become an important means of plants to cope with osmotic pressure and membrane stability (Nuccio et al., 1999). In plants, choline is the necessary precursor for glycine betaine biosynthesis (Supplementary Fig. 4). In chloroplasts, CMO catalyzes synthesis of glycine betaine aldehyde by choline, followed by the production of glycine betaine by BADH catalysis (Xu et al., 2018). In addition, part of the choline is transferred into the vacuole, or catalyzes synthesis of P-choline by choline kinase (Mcneil et al., 2000). In our RNA-Seq data, the transcript level of genes encoding choline kinase were significantly reduced in AhCMO transgenic cotton (Supplementary Table 2), suggesting that the

5. Conclusion In summary, the present study demonstrated that overexpression of AhCMO conferred high chilling tolerance to cotton and did not lead to any yield losses. The transgenic plants mainly improved the concentration of cellular osmoprotectants, including glycine betaine and proline, which enhanced chilling resistance and delayed leaf cell death in cotton. More importantly, the transgenic cotton showed encouraging results in agronomic traits because of a broad spectrum of resistance to abiotic stresses, including low temperature, drought, and salinity 371

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

tolerance. Our findings demonstrate that AhCMO is an ideal candidate for genetic engineering, which may greatly contribute to enhance cotton low temperature resistance. Thus, the introduction of AhCMO cotton varieties could have rational applications in actual agricultural practice.

Groot, G.J.J.B.D., Hundt, A., Murphy, A.B., Bange, M.P., Mai-Prochnow, A., 2018. Cold plasma treatment for cotton seed germination improvement. Sci. Rep. 8, 14372. Gwathmey, C.O., Bange, M.P., Brodrick, R., 2016. Cotton crop maturity: a compendium of measures and predictors. Field Crop Res. 191, 41–53. Hildebrandt, T.M., 2018. Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol. Biol. 98, 121–135. Holmstrom, K.O., Somersalo, S., Mandal, A., Palva, T.E., Welin, B., 2000. Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J. Exp. Bot. 51, 177–185. Jorge, T.F., Rodrigues, J.A., Caldana, C., Schmidt, R., van Dongen, J.T., Thomas-Oates, J., António, C., 2016. Mass spectrometry-based plant metabolomics: metabolite responses to abiotic stress. Mass Spectrom. Rev. 35, 620–649. Kenji, M., Tsuyoshi, F., 2013. Cold signaling and cold response in plants. Int. J. Mol. Sci. 14, 5312–5337. Kishitani, S., Watanabe, K., Yasuda, S., Arakawa, K., Takabe, T., 2010. Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants. Plant Cell Environ. 17, 89–95. Kreps, J.A., Wu, Y., Chang, H.-S., Zhu, T., Wang, X., Harper, J.F., 2002. Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol. 130, 2129–2141. Liu, Y., He, C., 2017. A review of redox signaling and the control of MAP kinase pathway in plants. Redox Biol. 11, 192–204. Liu, J., Shi, Y., Yang, S., 2017. CBF: a key factor balancing plant cold stress responses and growth. Chin Bull Bot. 52, 689–698. Liu, C., Ou, S., Mao, B., Tang, J., Wei, W., Wang, H., Cao, S., Schläppi, M.R., Zhao, B., Xiao, G., 2018. Early selection of bZIP73 facilitated adaptation of Japonica rice to cold climates. Nat. Commun. 9, 3302. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using RealTime quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408. Luo, D., Niu, X., Wang, Y., Zheng, W., Chang, L., Wang, Q., Wei, X., Yu, G., Lu, B.-R., Liu, Y., 2007. Functional defect at the rice choline monooxygenase locus from an unusual post-transcriptional processing is associated with the sequence elements of short-direct repeats. New Phytol. 175, 439–447. Luo, D., Niu, X., Yu, J., Yan, J., Gou, X., Lu, B.R., 2012. Rice choline monooxygenase (OsCMO) protein functions in enhancing glycine betaine biosynthesis in transgenic tobacco but does not accumulate in rice (Oryza sativa L. ssp. japonica). Plant Cell Rep. 31, 1625–1635. Lv, S., Yang, A., Zhang, K., Lei, W., Zhang, J., 2007. Increase of glycinebetaine synthesis improves drought tolerance in cotton. Mol Breeding. 20, 233–248. Ma, Y., Dai, X., Xu, Y., Luo, W., Zheng, X., Zeng, D., Pan, Y., Lin, X., Huanhuan, L., Zhang, D., Xiao, J., Guo, X., Xu, S., Niu, Y., Jin, J.B., Zhang, H., Xu, X., Li, L., Wang, W., Chong, K., 2015. COLD1 confers chilling tolerance in rice. Cell 160, 1209–1221. Mansour, M.M.F., 1998. Protection of plasma membrane of onion epidermal cells by glycinebetaine and proline against NaCl stress. Plant Physiol. Biochem. 36, 767–772. Mcneil, S.D., Rhodes, D., Russell, B.L., Nuccio, M.L., Shachar-Hill, Y., Hanson, A.D., 2000. Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol. 124, 153–162. Meng, X., Zhao, Q., Jin, Y., Yu, J., Yin, Z., Chen, S., Dai, S., 2016. Chilling-responsive mechanisms in halophyte Puccinellia tenuiflora seedlings revealed from proteomics analysis. J. Proteomics 143, 365–381. Møller, I.M., Jensen, P.E., Hansson, A., 2007. Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 58, 459–481. Morran, S., Eini, O., Pyvovarenko, T., Parent, B., Singh, R., Ismagul, A., Eliby, S., Shirley, N., Langridge, P., Lopato, S., 2011. Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors. Plant Biotechnol. J. 9, 230–249. Nuccio, M.L., Rhodest, D., Mcneil, S.D., Hanson, A.D., 1999. Metabolic engineering of plants for osmotic stress resistance. Curr. Opin. Plant Biol. 2, 128–134. Park, E.-J., Jeknić, Z., Sakamoto, A., DeNoma, J., Yuwansiri, R., Murata, N., Chen, T.H.H., 2004. Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J. 40, 474–487. Park, E.-J., Chen, T.H.H., Jeknic, Z., 2006. Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol. 47, 706–714. Pham, H.M., Kebede, H., Ritchie, G., Trolinder, N., Wright, R.J., 2018. Alternative oxidase (AOX) over-expression improves cell expansion and elongation in cotton seedling exposed to cool temperatures. Theor. Appl. Genet. 131, 2287–2298. Rathinasabapathi, B., Burnet, M., Russell, B.L., Gage, D.A., Liao, P.C., Nye, G.J., Scott, P., Golbeck, J.H., Hanson, A.D., 1997. Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. Proc. Natl. Acad. Sci. U. S. A. 94, 3454–3458. Rivero, R.M., Mestre, T.C., Mittler, R., Rubio, F., Garcia-Sanchez, F., Martinez, V., 2014. The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell Environ. 37, 1059–1073. Russell, B.L., Rathinasabapathi, B., Hanson, A.D., 1998. Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth. Plant Physiol. 116, 859–865. Saneoka, H., Nagasaka, C., Hahn, D.T., Yang, W.J., Premachandra, G.S., Joly, R.J., Rhodes, D., 1995. Salt tolerance of glycinebetaine-deficient and -containing maize lines. Plant Physiol. 107, 631–638. Shen, Y.G., Du, B.X., Zhang, W.K., Zhang, J.S., Chen, S.Y., 2002. AhCMO, regulated by stresses in Atriplex hortensis, can improve drought tolerance in transgenic tobacco. Theor. Appl. Genet. 105, 815–821. Shi, Y., Ding, Y., Yang, S., 2018. Molecular regulation of CBF signaling in cold acclimation. Trends Plant Sci. 23, 623–637. Shirasawa, K., Takabe, T., Takabe, T., Kishitani, S., 2006. Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation

Competing financial interests The authors declare no competing financial interests. CRediT authorship contribution statement Yanan Wang: Investigation, Data curation, Formal analysis, Writing - original draft. Chengzhen Liang: Project administration, Conceptualization, Formal analysis, Funding acquisition, Writing - review & editing. Yanyan Li: Investigation. Muhammad Ali Abid: Investigation. Muhammad Askari: Data curation. Peilin Wang: Investigation. Yuan Wang: Data curation. Guoqing Sun: Data curation. Yongping Cai: Data curation. Shou-Yi Chen: Data curation. Yi Lin: Data curation. Rui Zhang: Project administration, Conceptualization, Formal analysis, Funding acquisition, Writing - review & editing. Sandui Guo: Data curation, Project administration, Conceptualization, Formal analysis, Funding acquisition, Writing - review & editing. Acknowledgments This work was supported by grants from the Ministry of Agriculture (Grant No. 2016ZX08009003-003-004 to Chengzhen Liang and Grant No. 2016ZX08005004 to Rui Zhang), the National Natural Science Foundation of China (Grant No. 31601349 to Chengzhen Liang), and the Innovation Program of the Chinese Academy of Agricultural Sciences. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.envexpbot.2019.03. 012. References Agarwal, P.K., Gupta, K., Lopato, S., Agarwal, P., 2017. Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance. J. Exp. Bot. 68, 2135. Ahuja, I., Vos, R.C.H.D., Bones, A.M., Hall, R.D., 2010. Plant molecular stress responses face climate change. Trends Plant Sci. 15, 664–674. Aihong, L., Yiqin, W., Jiuyou, T., Peng, X., Chunlai, L., Linchuan, L., Bin, H., Fuquan, Y., Loake, G.J., Chengcai, C., 2012. Nitric oxide and protein s-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice. Plant Physiol. 158, 451–464. Artur, C., M Manuela, C., Hernani, G., 2011. Membrane transport, sensing and signaling in plant adaptation to environmental stress. Plant Cell Physiol. 52, 1583–1602. Bartels, D., Sunkar, R., 2005. Drought and salt tolerance in plants. Crit. Rev. Plant Sci. 24, 23–58. Bhuiyan, N.H., Hamada, A., Yamada, N., Rai, V., Hibino, T., Takabe, T., 2007. Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor. J. Exp. Bot. 58, 4203–4212. Burnet, M., Lafontaine, P.J., Hanson, A.D., 1995. Assay, purification, and partial characterization of choline monooxygenase from Spinach. Plant Physiol. 108, 581–588. Chen, T.H., Murata, N., 2010. Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ. 34, 1–20. Couée, I., Sulmon, C., Gouesbet, G., El Amrani, A., 2006. Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J. Exp. Bot. 57, 449–459. Delauney, A.J., Hu, C.A., Kishor, P.B., Verma, D.P., 1993. Cloning of ornithine δ-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. J. Biol. Chem. 268, 18673. Forlani, G., Makarova, K.S., Ruszkowski, M., Bertazzini, M., Nocek, B., 2015. Evolution of plant δ1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives. Front. Plant Sci. 6, 567. Foyer, C., Buchanan, B., Pfannschmidt, T., 2009. Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid. Redox Signal. 11, 861–905.

372

Environmental and Experimental Botany 162 (2019) 364–373

Y. Wang, et al.

biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells. Front. Plant Sci. 9, 1469. Yamada, M., Morishita, H., Urano, K., Shiozaki, N., Yamaguchi-Shinozaki, K., Shinozaki, K., Yoshiba, Y., 2005. Effects of free proline accumulation in petunias under drought stress. J. Exp. Bot. 56, 1975–1981. Yamaguchi-Shinozaki, K., Shinozaki, K., 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 57, 781–803. Yu, H.X., 2007. The analysis of the reasons causing premature senenscence in Xinjiang cotton. CN.Cotton. 34, 41–42. Zhao, L., Chanon, A.M., Chattopadhyay, N., Dami, I.E., Blakeslee, J.J., 2016. Quantification of carbohydrates in grape tissues using capillary zone electrophoresis. Front. Plant Sci. 7, 818. Zhou, Q., Luo, D., Chai, X., Wu, Y., Liu, Z., 2018. Multiple regulatory networks are activated during cold stress in Medicago sativa L. Int. J. Mol. Sci. 19, 3169. Zhu, T., Liang, C., Meng, Z., Sun, G., Meng, Z., Guo, S., Zhang, R., 2017. CottonFGD: an integrated functional genomics database for cotton. BMC Plant Biol. 17, 101.

of their tolerance to abiotic stress. Ann. Bot. 98, 565–571. Taïbi, K., Del Campo, A.D., Vilagrosa, A., Bellés, J.M., López-Gresa, M.P., López-Nicolás, J.M., Mulet, J.M., 2018. Distinctive physiological and molecular responses to cold stress among cold-tolerant and cold-sensitive Pinus halepensis seed sources. BMC Plant Biol. 18, 236. Tao, J.J., Wei, W., Pan, W.J., Lu, L., Li, Q.T., Ma, J.B., Zhang, W.K., Ma, B., Chen, S.Y., Zhang, J.S., 2018. An Alfin-like gene from Atriplex hortensis enhances salt and drought tolerance and abscisic acid response in transgenic Arabidopsis. Sci. Rep. 8, 2707. Tiryaki, D., Aydın, İ., Atıcı, Ö., 2018. Psychrotolerant bacteria isolated from the leaf apoplast of cold-adapted wild plants improve the cold resistance of bean (Phaseolus vulgaris L.) under low temperature. Cryobiology 86, 111–119. Wang, L., Shan, T., Xie, B., Ling, C., Shao, S., Jin, P., Zheng, Y., 2019. Glycine betaine reduces chilling injury in peach fruit by enhancing phenolic and sugar metabolisms. Food Chem. 272, 530–538. Wankun, D., Yongbo, W., Zexian, L., Han, C., Yu, X., 2014. HemI: a toolkit for illustrating heatmaps. PLoS One 9, e111988. Xu, Z., Sun, M., Jiang, X., Sun, H., Dang, X., Cong, H., Qiao, F., 2018. Glycinebetaine

373