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Overexpression of the wheat trehalose 6-phosphate synthase 11 gene enhances cold tolerance in Arabidopsis thaliana
Gene 710 (2019) 210–217
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Research paper
Overexpression of the wheat trehalose 6-phosphate synthase 11 gene enhances cold tolerance in Arabidopsis thaliana Xin Liua, Lianshuang Fua, Peng Qina, Yinglu Suna, Jun Liub, Xiaonan Wanga,
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a
Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Ministry of Education, Northeast Agricultural University, Harbin 150030, China b National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
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Keywords: Trehalose 6-phosphate synthase 11 (TaTPS11) Cold tolerance Sucrose Carbohydrate metabolism Triticum aestivum
Low temperature is a key stress factor for the growth and development of wheat (Triticum aestivum L.), and glycometabolism plays an important role in plant cold tolerance. Our previous study identified trehalose 6phosphate synthase 11 gene (TaTPS11), which had a significantly different expression pattern between a high freezing-tolerant wheat cultivar and a low freezing-tolerant wheat cultivar. In this study, TaTPS11 was isolated from a winter-hardy wheat cultivar (D1) and overexpressed in Arabidopsis thaliana to study its effect on cold tolerance in plants. Transgenic plants expressing TaTPS11 had lower sucrose content, higher starch content, and higher activity of key enzyme (sucrose phosphate synthase, sucrose synthase, and invertase) involved in sucrose metabolism. In addition, the expression level of sucrose non-fermenting 1-related kinase 1 (SnRK1), which catalyzes the sucrose in plants, increased in the TaTPS11-overexpressed plants. These results indicated that heterologous expression of TaTPS11 influenced carbohydrate metabolism in Arabidopsis plants. The resultant plants had a significantly higher survival rate after −5 °C treatment for 2 h and exhibited enhanced cold tolerance without unfavorable phenotypes compared to wild-type. Our findings indicated that manipulation of TaTPS11 improved cold tolerance in plants and TaTPS11 had potential values in wheat cold-tolerance breeding.
1. Introduction Low temperature is one of the most important environment factors affecting winter wheat production, especially in high-latitude regions. Low temperature induces a series of complex physiological process, such as glycometabolism, proteometabolism, dehydration and scavenging by reactive oxygen species (Krasensky and Jonak, 2012; Janmohammadi et al., 2015; Kovi et al., 2016). Trehalose (a-D-glucopyranosyl-1, 1-a-D-glucopyranoside) is a non-reducing disaccharide that exists widely in flowering plants. Trehalose biosynthesis in plants occurs via a pathway of hexose-P catabolism followed by the conversion of trehalose 6-phosphate (Tre6P) to trehalose, which is catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Singh et al., 2011; Delorge et al., 2015; Figueroa et al., 2016). Many studies showed that trehalose participates in various aspects of plant growth and development, including seed development, vegetative growth, flowering, and stress response, although only trace
amounts of trehalose are detectable in higher plants (Figueroa et al., 2016). Recent studies indicated that Tre6P rather than trehalose may cause phenotypic changes to leaves, and alter flowering time and branch morphology in Arabidopsis, although the amounts of Tre6P were extremely low (typically pmol g−1 fresh weight) and difficult to measure using existing assays (Schluepmann et al., 2003; Lunn et al., 2014; Figueroa et al., 2016). TPS is a key enzyme to synthesize Tre6P. Arabidopsis has 11 TPS genes, which were classified into Group I (AtTPS14) and Group II (AtTPS5-11) depending on identity with yeast TPS1 or TPS2 (Leyman et al., 2001; Wang et al., 2016). Several studies indicated that regulation of TPS genes could improve abiotic stress tolerance in plants (Pilon-Smits et al., 1998; Garg et al., 2002; Fernandez et al., 2010; Chary et al., 2008; Li et al., 2011; Wang et al., 2016). For example, constitutive expression of yeast ScTPS1 in potato enhanced drought tolerance, but the resulting plants exhibited pleiotropic growth aberrations including dwarfism, chlorotic lancet-shaped leaves, and aberrant root development, indicating that the manipulation of TPS not only improves plant drought tolerance, but also affects plant
Abbreviations: TaTPS11, trehalose 6-phosphate synthase; SuSy, sucrose synthase; SPS, sucrose phosphate synthase; INV, invertase; SnRK1, sucrose non-fermenting 1related kinase 1; TOR, TARGET OF RAPAMYCIN ⁎ Corresponding author. E-mail address: [email protected] (X. Wang). https://doi.org/10.1016/j.gene.2019.06.006 Received 23 January 2019; Received in revised form 26 May 2019; Accepted 5 June 2019 Available online 07 June 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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HYGROMYCIN B PHOSPHOTRANSFERASE (HPT) and TaTPS11. All the gene-specific primers are listed in Table S1.
development (Yeo et al., 2000). Transgenic tomato with constitutive expression of yeast ScTPS1 improved the drought and salt tolerance, but showed pleiotropic changes such as thick shoots, rigid dark-green leaves, erect branches and aberrant root development (Cortina and Culiáñez-Macià, 2005). In tobacco, expression of ScTPS1 with an Arabidopsis stress-induced promoter significantly increased plant drought tolerance without growth aberrations (Karim et al., 2007). Overexpression of OsTPS1 in transgenic rice improved tolerance to cold, high salinity, and drought at the seedling stage, and showed no obvious phenotypic changes comparing to wild-type (WT). Furthermore, the results indicated that OsTPS1 may improve abiotic stress tolerance by increasing the amount of trehalose and regulating expression of stressrelated genes (Li et al., 2011). A similar result was also be described by Jang et al. (2003). The AtTPS11 in Arabidopsis encodes a trehalosesynthesizing enzyme with TPS and TPP activities, and overexpression of homologous TPS11 in Arabidopsis increased starch content and showed an obvious plant defense against aphids, but the study had no reports whether AtTPS11-overexpression influenced the abiotic stress response in Arabidopsis resultant plants (Singh et al., 2011). The cotton GhTPS11 encoded a stress-responsive TPS protein, and its overexpression in Arabidopsis seeds resulted in slower germination than the WT under chilling stress, indicating that GhTPS11 responded to chilling stress during seed germination (Wang et al., 2016). To our knowledge, the manipulation of the Triticum aestivum L. TaTPS genes in plants has not previously been reported. Our previous RNA sequencing analysis showed the transcript level of TaTPS11 was more highly induced in winter wheat cultivars with high freezing-tolerance than in those with low freezing-tolerance. Therefore, in this study, we overexpressed TaTPS11 in transgenic Arabidopsis plants to evaluate its effects on cold tolerance, and assayed the possible TaTPS11 function pathway for improving cold tolerance including sucrose metabolism.
2.3. RNA isolation and qRT-PCR analysis Total RNA was isolated from leaves using TRIzol reagent (Invitrogen, Carlsbad, USA), and each sample was a mixture of RNA extracted from eight plants. First-strand cDNA was synthesized using EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, China), according to the manufacturer protocol. The relative expression analysis was performed using a Roche LightCycler® 480 system with TransStart Top Green qPCR SuperMix (Transgen, China), as described by Xin et al. (2016). The Arabidopsis thaliana actin 2 (ACT2) was used as an internal control in quantitative RT-PCR. Data were calculated using 2(−ΔΔCt) method (Livak and Schmittgen, 2001). The gene-specific primers for qRT-PCR are listed in Table S1. 2.4. Determination of carbohydrate and enzyme activities Approximately 0.1 g of Arabidopsis shoot was mixed with 0.5 mL of 80% ethanol and boiled for 40 min. Then the extracts were centrifuged at 14,000g for 5 min. The residue was re-extracted again, as above. The supernatant was used for sucrose and fructose measurements (Qi et al., 2007). Debris was used for starch determinations using glucose equivalents, and the detailed procedure was in accordance with previous method (Baud et al., 2002). For assays of enzyme activity, 1 g fresh sample was placed in liquid nitrogen and ground into powder. The powder was suspended in 3 mL extraction buffer (100 mM HEPES pH 7.5, 2 mM MEDTA, 2 mM dithiothreitol, 1 mM PMSF and 10 mL/L protease inhibitor cocktail), and centrifuged at 14,000g with 4 °C for 10 min. The supernatant was used for determination of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) activities (Bahaji et al., 2015). For invertase (INV) assays, 1 g fresh sample was ground into powder using liquid nitrogen and suspended in 3 mL extraction buffer (50 mM Tris-acetate, pH 7.5; 10 mM EDTA; 5 mM DTT), and the homogenates were directly used for INV assays, as previously described (Qi et al., 2007). One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 μmol of product per minute.
2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis plants were routinely grown in a growth chamber at 22 °C with 150 μM m−2·s−1 light intensity, a photoperiod of 16 h-light/ 8 h-dark, and 60%–70% humidity. After 15 d, seedlings were subjected to −5 °C for 2 h, and then transferred back to the previous growth conditions for 2 d recovery. The living plants after treatment were counted for surviving rate. The high freezing-tolerance wheat cultivar (D1), which was the only winter wheat variety planted in high-latitude regions of China, was selected for the freezing treatment, and seedling plants were grown in artificial climatic chamber condition at 25 °C under photoperiod conditions of 14 h light/10 h dark, 50%–60% humidity, and 150 μM m−2·s−1 light intensity for 15 d, then transferred to a low temperature conditions, as follows: W0, control; W1, 4 °C cold acclimation for 30 d; W2, treatment as W1, then −10 °C/2 h; W3, treatment as W2, then −12 °C/2 h; W4, treatment as W3, then −14 °C/ 2 h; W5, treatment as W4, then −16 °C/2 h; W6, treatment as W5, then −18 °C/2 h.
2.5. Statistical analysis All numerical data were presented as mean ± SE calculated from three biological replicates. All P values were based on a two-tailed ttest. P < 0.05 and P < 0.01 were considered as statistically significant. 3. Results 3.1. Identification and sequence analysis of wheat cDNA encoding TPS Previous RNA sequencing analysis had revealed seven TPS-related genes that showed differential expression between high freezing-tolerant winter wheat (D1) and low freezing-tolerant winter wheat (J22) under low temperatures (Xie and Li, 2015; Xie et al., 2015). Quantitative RT-PCR analysis showed that one gene was induced under cold stress in both high freezing-tolerant winter wheat (D1) and low freezing-tolerant winter wheat (J22), but the expression level of the gene in D1 was significantly higher than in J22 (Fig. S1). Then, we compared the gene sequence in the coding region and found 7 SNPs and 5 InDels between D1 and J22 (Fig. S2), which resulted in a frameshift mutation and the changes of 207 encoded amino acids in J22. Herein, we aligned the encoding protein sequence of the gene in D1 with Arabidopsis using TAIR BLAST (https://www.arabidopsis.org/). The candidate gene shared the greatest identity with Arabidopsis trehalose6-phosphate synthase 11 (AtTPS11, GenBank accession: AT2G18700).
2.2. Plant transformation For the production of transgenic Arabidopsis plants, TaTPS11 cDNA was isolated by reverse-transcription PCR using gene-specific primers with total mRNA from the leaves of wheat D1, and inserted into pCAMBIA1305 vector with NcoI and SpeI restriction enzymes. The TaTPS11 fragment was placed under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter. The resulting construct was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis thaliana with a Columbia (Col-0) background by the floral dip method (Clough and Bent, 1998). The transformants were then screened by PCR amplification using specific primers for the 211
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Fig. 1. The phylogenetic relationships of TaTPS11 and other plant TPSs. (A) Amino acid sequence alignment of TaTPS11 with other plant species comprising Arabidopsis AtTPS11, barley HvTPS, rice OsTPS11, and maize ZmTPS11. Green underline domain (1-386 aa) indicates conserved trehalose-6phosphate synthase domain, and blue underlined domain (436-670 aa) indicates conserved trehalose phosphatase domain. (B) A phylogenetic tree derived from 11 Arabidopsis TPSs (AtTPS1-11), maize TPS (ZmTPS), barley TPS (HvTPS), and wheat TPS11 (TaTPS11). From the GenBank database: AtTPS1 (Q9SYM4); AtTPS2 (Q9FZ57); AtTPS3 (Q9SHG0); AtTPS4 (Q9T079); AtTPS5 (O23617); AtTPS6 (Q94AH8); AtTPS7 (Q9LMI0); AtTPS8 (Q0WUI9); AtTPS9 (Q9LRA7); AtTPS10 (O80738); AtTPS11 (Q9ZV48); HvTPS11 (BAJ97294); ZmTPS11 (AQK75662); OsTPS11 (AEB53186). The scale bar indicates distance value of 0.2 substitutions per site. The alignment was done using CLUSTALW in MEGA7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
aligned sequences from different organisms, and TaTPS11 shares conserved function domains (1-386 aa, trehalose-6-phosphate synthase domain; 436-670 aa, trehalose phosphatase domain), which were predicted by InterPro (http://www.ebi.ac.uk/interpro/search/sequencesearch) (Fig. 1B). In Arabidopsis, 11 TPS genes were classified into Group I (AtTPS1-4) and Group II (AtTPS5-11) through the presence or absence of the two conserved motifs (LDYDGTLM and GDDRSD) in the TPP domain (Singh et al., 2011). This indicated that TaTPS11 may encode a bifunctional enzyme with trehalose-6-phosphate synthase and trehalose phosphatase activities, and TaTPS11 belonged to the Class II TPS. 3.2. Expression of TaTPS11 in wheat plants under abiotic stress Previous transcriptome analysis showed that TaTPS11 was induced by low temperatures (Xie et al., 2015). To verify the result, TaTPS11 transcripts in the wheat were monitored by quantitative real time-PCR (qRT-PCR). During the whole treated process, we gradually decreased the treated temperature and increased the treated time to imitate the outside freezing stress. The results showed that the expression of TaTPS11 through 4 °C cold acclimation (W1) was obviously induced, and transcript abundance was almost 4-fold as no cold acclimation control (W0) (Fig. 2A). With treated temperature decrease and time increase, the transcript abundance of TaTPS11 presented a downward trend during the initial W2, W3, and W4 low-temperature treatment stages, however dramatically increased under long time freezing W5 and W6 treatment stages, indicating that TaTPS11 may involve in plant freezing tolerance (Fig. 2A). In addition, the detailed transcript abundance of TaTPS11 in 15-day-old wheat seedlings treated with different abiotic stresses for different times was determined. The result showed that the expression of TaTPS11 was significantly induced under 25% PEG (simulation of osmotic stress), 300 mM NaCl (simulation of salt stress) at 12 h and 24 h (Fig. 2B, C). Previous reports showed that the metabolism of trehalose and sucrose had close relationship (Figueroa et al., 2016; Wang et al., 2016). We also examined the transcript abundance of TaTPS11 under sucrose stress, and found that TaTPS11 was obviously induced under 6% sucrose at 12 h rather than at 24 h (Fig. 2D). Above results indicated TaTPS11 participated in plant stress response. 3.3. Overexpression of TaTPS11 increases cold tolerance in Arabidopsis Therefore, the gene was named TaTPS11 (Accession: MK330685). The TaTPS11 contains a 2121-bp open reading frame and encodes 706 amino acids, which shared 63.57% identity with AtTPS11 at protein level. Then Amino acid sequence of TaTPS11 was compared with those of Arabidopsis (AtTPS11, accession: NP_179460), barley (HvTPS, accession: BAJ97294), maize (ZmTPS, accession: AQK55931) and rice (OsTPS, accession: XP_015625782) (Fig. 1A). The deduced amino acid sequence of TPS11 has high sequence identity of 86.36% among the
To survey the potential function of TaTPS11 in plant cold stress responses, a vector carrying TaTPS11 under the control of constitutive the cauliflower mosaic virus (CaMV) 35S promoter and HPT selectable marker gene was introduced into Arabidopsis (Fig. 3A). Fourteen homozygous transgenic lines were obtained, and the transcript abundance of TaTPS11 was detected in TaTPSOE-01, -04, -05, -07 and -09 lines (referred to as TaTPSOE-01-14. OE = overexpression). The expression levels of TaTPS11 in TaTPS11OE-01, TaTPS11OE-07 and 212
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Fig. 2. Expression analysis of TaTPS11 under abiotic stress in wheat. (A) qRT-PCR analysis of TaTPS11 under cold stress. W0, normal culture; W1, 4 °C cold acclimation for 30 d; W2, treatment as W1 then −10 °C/2 h; W3, treatment as W2 then −12 °C/2 h; W4, treatment as W3 then −14 °C/2 h; W5, treatment as W4 then −16 °C/2 h; W6, treatment as W5 then −18 °C/2 h. (B) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated with 30% PEG stress over different time periods. (C) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated with 300 mM NaCl stress for different time periods. (D) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated by 6% sucrose stress for different time periods. Error bars indicate the SE calculated from three replicates.
TaTPS11OE-09 lines were significantly higher than in other lines (Fig. 3B). These three homozygous lines screened by HPT and TaTPS11 exhibited no visible morphological differences with WT at the seedling stage, and were selected for further studies (Fig. S3). To study the cold response of TaTPS11, two-week-old Arabidopsis plants were subjected to −5 °C for 2 h, and recovered for 2 d. All resultant lines had an increased cold tolerance over the WT. The survival rates of TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines were 65%, 60% and 63%, respectively, but only 17% in the WT after 2 d recovery (Fig. 4). The above results indicated that TaTPS11 played a positive role in cold tolerance in plants.
that heterologous expression of TaTPS11 influenced sucrose metabolism in Arabidopsis. Previous studies have shown that expression of TPSs could affect starch metabolism (Gomez et al., 2010; Singh et al., 2011). Therefore, we also measured starch content, which was found to be significantly higher in the OE-lines than in the WT (Fig. 5F). These results indicated that heterologous expression of TaTPS11 influenced carbohydrate metabolism in Arabidopsis plants.
4. Discussion Glycometabolism has been confirmed to play a key role in overwintering response to cold stress in plants (Krasensky and Jonak, 2012; Nägele and Heyer, 2013; Kovi et al., 2016). Previous studies showed that sucrose metabolism had a close correlation with plant cold tolerance, which was coordinated with the sucrose increases (Livingston and Henson, 1998; Janska et al., 2011). In rice plants, increased sucrose content occurred when plants underwent cold stress (McKown et al., 1996; Wanner and Junttila, 1999; Reyes-Díaz et al., 2006). Overexpressing of OsDREB1 in rice increased cold tolerance and was accompanied by increases in sucrose (Ito et al., 2006). However, in our study, constitutive expression of TaTPS11 in Arabidopsis increased cold tolerance, but decreased sucrose content comparing to WT (Fig. 5A), indicating no direct correlation between cold tolerance and increase of sucrose content in TaTPS11OE Arabidopsis plants. The increase of fructose content was also deemed as an important component of plant cold response process (Livingston and Henson, 1998; Janska et al., 2011). Synthetic wheat lines with high freezing tolerances had higher fructan
3.4. Overexpression of TaTPS11 influenced the carbohydrate metabolism in Arabidopsis Previous studies reported that Tre6P catalyzed by TPS could modulate sucrose levels by affecting sucrose metabolism in plants (Wingler et al., 2012; Figueroa et al., 2016). To confirm the results, the sucrose content was compared between TaTPS11OE lines and WT. The sucrose contents in TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines were 1.74 mg/g, 1.91 mg/g and 1.82 mg/g, respectively, and were significantly < 2.10 mg/g measured in WT (Fig. 5A). Similar results were also observed for the fructose contents in TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines, which were significantly lower than in the WT (Fig. 5B). The key enzyme activities in sucrose metabolism (SPS, SuSy, and INV) were also examined. Intriguingly, all detected enzyme activities were obviously increased comparing to WT (Fig. 5C-E), indicating 213
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Fig. 3. Construction of TaTPS11 overexpression vector and molecular identification of transgenic Arabidopsis plants. (A) Structure of TaTPS11 overexpression (OE) vector. HPT, hygromycin phosphotransferase gene; TaTPS11, cDNA of trehalose-6-phosphate synthase 11 from wheat; CaMV35S, promoter sequence of cauliflower mosaic virus (CaMV) 35S. (B) Analysis of five independent transgenic Arabidopsis lines: OE-1, OE-4, OE-5, OE-7, OE-9. Values are means ± SEM with three replicates.
accumulation compared to the low freezing tolerance lines (Kovi et al., 2016). Hisano et al. (2004) reported that overexpression of wheat fructosyltransferase genes increased accumulation of fructans and improved freezing tolerance at the cellular level (Hisano et al., 2004). In the present study, the fructose content in transgenic Arabidopsis plants also decreased comparing to WT (Fig. 5B). Our results suggested that complex interactions existed between the content of soluble sugars (such as sucrose and fructose) and cold tolerance in plants, which needs further detailed studies. Previous study showed that the starch content in tps1 mutant of Arabidopsis was significantly increased (Gomez et al., 2010). Overexpression of AtTPS11 constitutively elevated the starch content comparing to WT in Arabidopsis (Singh et al., 2011). In our study, we found that the starch content in TaTPS11-OE lines was also elevated, indicating that TaTPS11 possibly has functions similar to AtTPS11 in Arabidopsis. SPS, SuSy and INV are key numbers of sucrose-metabolizing enzymes that regulate sucrose synthesis and catabolism (Winter and Huber, 2000; Kulshrestha et al., 2013; Volkert et al., 2014). All three enzyme activities (SPS, SuSy and INV) in TaTPS11-OE Arabidopsis plants increased comparing to WT (Fig. 5C–E). These results suggested that TaTPS11-overexpression influenced the sucrose metabolism in Arabidopsis plants possibly by positive regulation of SPS, SuSy and INV activities. The expression of SuSy and INV genes was reported to be regulated under abiotic and biotic stresses (Cabello et al., 2014). INV genes responded to high salinity, drought and low temperature (Zeng et al., 1999; Cabello et al., 2014). In the present study, we observed significant increases of SPS, SuSy, and INV activities accompanied with cold tolerance in TaTPS11-OE Arabidopsis plants, indicating a positive relationship between sucrose-metabolizing enzymes and cold tolerance. However, more efforts are worth further studying the detailed correlations. In higher plants, TPS catalyzes Tre6P biosynthesis (Chary et al., 2008; Figueroa et al., 2016). Recent findings have shown that Tre6P could regulate Suc consumption and growth, probably acting via multiple mechanisms, including inhibition of one conserved protein kinase,
Fig. 4. Cold stress analysis of TaTPS11 overexpression (OE) Arabidopsis plants. 15-day-old seedlings were incubated under growth conditions of −5 °C for 2 h, before being transferred back to normal growth conditions for 2 d recovery, and then the living plants after treatment were counted for surviving rate. Error bars indicate SE calculated from three biological replicates. All P values are based on a two-tailed t-test: **, P < 0.01 statistical significance.
sucrose non-fermenting 1-related kinase 1 (SnRK1), in Arabidopsis plants (Zhang et al., 2009; Delatte et al., 2011). Figueroa proposed that the relationship between Tre6P and SnRK1 in developing tissues was complex and not yet fully resolved (Figueroa et al., 2016). In our study, we also examined the expression level of AtSnRK1 in TaTPS11-overexpressing Arabidopsis. Contrary to expectation, the resultant plants promoted the expression of SnRK1 rather than inhibit it (Fig. 5G), 214
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Fig. 5. Carbohydrate metabolism changes in TaTPS11 overexpression (OE) Arabidopsis plants. (A) Sucrose content. (B) Fructose content. (C) Sucrose phosphate synthase (SPS) activity assay. (D) Sucrose synthase (SuSy) activity assay. (E) Invertase activity assay. (F) Starch content. (G) The relative expression level of AtSnRK. (H) The relative expression level of AtTOR. Error bars indicate SE calculated from three biological replicates. All P values were based on a two-tailed t-test: **, P < 0.01 statistical significance.
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indicating that the effect of heterologous expression of TaTPS11 was different from endogenous expression of AtTPSs in Arabidopsis, but this hypothesis needs further study. We examined the transcript abundance of the other conserved protein kinase, TARGET OF RAPAMYCIN (TOR), in the TaTPS11-overexpressing Arabidopsis. The result showed that the expression level of TOR was roughly the same between OE lines and WT (Fig. 5H), indicating that expression of Tre6P and TOR was not correlated, and these also corroborated the previous opinion of the relationship between Tre6P and TOR (Figueroa et al., 2016). Particular focus has recently been directed towards understanding the regulation role of TPSs in enhancing cold tolerance (Pilon-Smits et al., 1998; Garg et al., 2002; Karim et al., 2007; Chary et al., 2008; Li et al., 2011; Wang et al., 2016). The OsTPS1-overexpression transgenic rice improved the tolerance of cold, and no obvious phenotypic changes were apparent (Li et al., 2011). Overexpression of homologous AtTPS11 in Arabidopsis increased starch content and showed increased plant defense against aphids but without reports related to abiotic stress response (Singh et al., 2011). Resent research showed that cotton GhTPS11 that encoded a stress-responsive TPS protein functioned in chilling stress during seed germination in Arabidopsis (Wang et al., 2016). To our knowledge, few TaTPSs were identified and characterized in wheat, and no detailed functional studies of TaTPSs in abiotic stress have been reported. The present study showed that TaTPS11 was obviously induced not only by cold, salt and osmotic stress (simulation by PEG), but also by sucrose in wheat (Fig. 2). In conclusion, the wheat TaTPS11 was obviously induced by multiple abiotic stresses in wheat, and TaTPS11-overexpression increased the cold tolerance in Arabidopsis plants via influencing the carbohydrate metabolism such as sucrose, fructose and starch, indicating that TaTPS11 had potential values in wheat cold-tolerance breeding.
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Karim, S., Aronsson, H., Ericson, H., Pirhonen, M., Leyman, B., Welin, B., Mantyla, E., Palva, E.T., Van Dijck, P., Holmstrom, K.O., 2007. Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol. Biol. 64, 371–386. Kovi, M.R., Ergon, A., Rognli, O.A., 2016. Freezing tolerance revisited-effects of variable temperatures on gene regulation in temperate grasses and legumes. Curr. Opin. Plant Biol. 33, 140–146. Krasensky, J., Jonak, C., 2012. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J. Exp. Bot. 63, 1593–1608. Kulshrestha, S., Tyagi, P., Sindhi, V., Yadavilli, K.S., 2013. Invertase and its applications – a brief review. J. Pharm. Res. 7, 792–797. Leyman, B., Van Dijck, P., Thevelein, J.M., 2001. An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana. Trends Plant Sci. 6, 510–513. Li, H.W., Zang, B.S., Deng, X.W., Wang, X.P., 2011. Overexpression of the trehalose-6phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234, 1007–1018. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Method 25, 402–408. Livingston, D.P., Henson, C.A., 1998. Apoplastic sugars, fructans, fructan exohydrolase, and invertase in winter oat: responses to second-phase cold hardening. Plant Physiol. 116, 403–408. Lunn, J.E., Delorge, I., Figueroa, C.M., Van Dijck, P., Stitt, M., 2014. Trehalose metabolism in plants. Plant J. 79, 544–567. McKown, R., Kuroki, G., Warren, G., 1996. Cold responses of Arabidopsis mutants impaired in freezing tolerance. J. Exp. Bot. 47, 1919–1925. Nägele, T., Heyer, A.G., 2013. Approximating subcellular organisation of carbohydrate metabolism during cold acclimation in different natural accessions of Arabidopsis thaliana. New Phytol. 198, 777–787. Pilon-Smits, E.A.H., Terry, N., Sears, T., Kim, H., Zayed, A., Hwang, S., van Dun, K., Voogd, E., Verwoerd, T.C., Krutwagen, R.W.H.H., Goddijn, O.J.M., 1998. Trehaloseproducing transgenic tobacco plants show improved growth performance under drought stress. J. Plant Physiol. 152, 525–532. Qi, X., Wu, Z., Li, J., Mo, X., Wu, S., Chu, J., Wu, P., 2007. AtCYT-INV1, a neutral invertase, is involved in osmotic stress-induced inhibition on lateral root growth in
Author contributions XL designed the research, performed most of the experiments, analyzed the data and wrote the manuscript; LSF and YLS provided technical assistance; PQ participated in carbohydrate and enzyme activity assays; JL helped to correct the manuscript; XNW designed the research and corrected the manuscript. All authors approved the final manuscript. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgements This research was financially supported by National Key Research and Development Program of China (grant number: 2016YFD0100502), Natural Science Foundation of Heilongjiang Province of China (grant numbers: QC2018020, QC2016026), Young Talents Program of Northeast Agricultural University (grant number: 17QC04), and Applied Technology Research and Development Project of Harbin (grant number: 2016RAQXJ034). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.06.006. References Bahaji, A., Baroja-Fernandez, E., Ricarte-Bermejo, A., Sanchez-Lopez, A.M., Munoz, F.J., Romero, J.M., Ruiz, M.T., Baslam, M., Almagro, G., Sesma, M.T., Pozueta-Romero, J., 2015. Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis. Plant Sci. 238, 135–147.
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Winter, H., Huber, S.C., 2000. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit. Rev. Biochem. Mol. Biol. 35, 253–289. Xie, D., Li, Z., 2015. Transcriptome profile and differentially expressed genes analysis in winter wheat under cold stress conditions. Res. J. Biotechnol. 10, 73–88. Xie, D.W., Wang, X.N., Fu, L.S., Sun, J., Zheng, W., Li, Z.F., 2015. Identification of the trehalose-6-phosphate synthase gene family in winter wheat and expression analysis under conditions of freezing stress. J. Genet. 94, 55–65. Xin, L., Zhang, C., Wang, X., Liu, Q., Yuan, D., Gang, P., Sun, S.S.M., Tu, J., 2016. Development of high-lysine rice via endosperm-specific expression of a foreignLYSINE RICH PROTEINgene. BMC Plant Biol. 16, 1–13. Yeo, E.T., Kwon, H.B., Han, S.E., Lee, J.T., Ryu, J.C., Byu, M.O., 2000. Genetic engineering of drought resistant potato plants by introduction of the trehalose-6phosphate synthase (TPS1) gene from Saccharomyces cerevisiae. Mol. Cell 10, 263–268. Zeng, Y., Wu, Y., Avigne, W.T., Koch, K.E., 1999. Rapid repression of maize invertases by low oxygen. Invertase/sucrose synthase balance, sugar signaling potential, and seedling survival. Plant Physiol. 121, 599–608. Zhang, Y., Primavesi, L.F., Jhurreea, D., Andralojc, P.J., Mitchell, R.A., Powers, S.J., Schluepmann, H., Delatte, T., Wingler, A., Paul, M.J., 2009. Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6phosphate. Plant Physiol. 149, 1860–1871.
Arabidopsis. Plant Mol. Biol. 64, 575–587. Reyes-Díaz, M., Ulloa, N., Zúñiga-Feest, A., Gutiérrez, A., Gidekel, M., Alberdi, M., Corcuera, L.J., Bravo, L.A., 2006. Arabidopsis thaliana avoids freezing by supercooling. J. Exp. Bot. 57, 3687–3696. Schluepmann, H., Pellny, T., van Dijken, A., Smeekens, S., Paul, M., 2003. Trehalose 6phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 100, 6849–6854. Singh, V., Louis, J., Ayre, B.G., Reese, J.C., Pegadaraju, V., Shah, J., 2011. TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae. Plant J. 67, 94–104. Volkert, K., Debast, S., Voll, L.M., Voll, H., Schiessl, I., Hofmann, J., Schneider, S., Bornke, F., 2014. Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis. J. Exp. Bot. 65, 5217–5229. Wang, C.L., Zhang, S.C., Qi, S.D., Zheng, C.C., Wu, C.A., 2016. Delayed germination of Arabidopsis seeds under chilling stress by overexpressing an abiotic stress inducible GhTPS11. Gene 575, 206–212. Wanner, L.A., Junttila, O., 1999. Cold-induced freezing tolerance in Arabidopsis. Plant Physiol. 120, 391–400. Wingler, A., Delatte, T.L., O'Hara, L.E., Primavesi, L.F., Jhurreea, D., Paul, M.J., Schluepmann, H., 2012. Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability. Plant Physiol. 158, 1241–1251.
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Research paper
Overexpression of the wheat trehalose 6-phosphate synthase 11 gene enhances cold tolerance in Arabidopsis thaliana Xin Liua, Lianshuang Fua, Peng Qina, Yinglu Suna, Jun Liub, Xiaonan Wanga,
T
⁎
a
Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Ministry of Education, Northeast Agricultural University, Harbin 150030, China b National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Trehalose 6-phosphate synthase 11 (TaTPS11) Cold tolerance Sucrose Carbohydrate metabolism Triticum aestivum
Low temperature is a key stress factor for the growth and development of wheat (Triticum aestivum L.), and glycometabolism plays an important role in plant cold tolerance. Our previous study identified trehalose 6phosphate synthase 11 gene (TaTPS11), which had a significantly different expression pattern between a high freezing-tolerant wheat cultivar and a low freezing-tolerant wheat cultivar. In this study, TaTPS11 was isolated from a winter-hardy wheat cultivar (D1) and overexpressed in Arabidopsis thaliana to study its effect on cold tolerance in plants. Transgenic plants expressing TaTPS11 had lower sucrose content, higher starch content, and higher activity of key enzyme (sucrose phosphate synthase, sucrose synthase, and invertase) involved in sucrose metabolism. In addition, the expression level of sucrose non-fermenting 1-related kinase 1 (SnRK1), which catalyzes the sucrose in plants, increased in the TaTPS11-overexpressed plants. These results indicated that heterologous expression of TaTPS11 influenced carbohydrate metabolism in Arabidopsis plants. The resultant plants had a significantly higher survival rate after −5 °C treatment for 2 h and exhibited enhanced cold tolerance without unfavorable phenotypes compared to wild-type. Our findings indicated that manipulation of TaTPS11 improved cold tolerance in plants and TaTPS11 had potential values in wheat cold-tolerance breeding.
1. Introduction Low temperature is one of the most important environment factors affecting winter wheat production, especially in high-latitude regions. Low temperature induces a series of complex physiological process, such as glycometabolism, proteometabolism, dehydration and scavenging by reactive oxygen species (Krasensky and Jonak, 2012; Janmohammadi et al., 2015; Kovi et al., 2016). Trehalose (a-D-glucopyranosyl-1, 1-a-D-glucopyranoside) is a non-reducing disaccharide that exists widely in flowering plants. Trehalose biosynthesis in plants occurs via a pathway of hexose-P catabolism followed by the conversion of trehalose 6-phosphate (Tre6P) to trehalose, which is catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Singh et al., 2011; Delorge et al., 2015; Figueroa et al., 2016). Many studies showed that trehalose participates in various aspects of plant growth and development, including seed development, vegetative growth, flowering, and stress response, although only trace
amounts of trehalose are detectable in higher plants (Figueroa et al., 2016). Recent studies indicated that Tre6P rather than trehalose may cause phenotypic changes to leaves, and alter flowering time and branch morphology in Arabidopsis, although the amounts of Tre6P were extremely low (typically pmol g−1 fresh weight) and difficult to measure using existing assays (Schluepmann et al., 2003; Lunn et al., 2014; Figueroa et al., 2016). TPS is a key enzyme to synthesize Tre6P. Arabidopsis has 11 TPS genes, which were classified into Group I (AtTPS14) and Group II (AtTPS5-11) depending on identity with yeast TPS1 or TPS2 (Leyman et al., 2001; Wang et al., 2016). Several studies indicated that regulation of TPS genes could improve abiotic stress tolerance in plants (Pilon-Smits et al., 1998; Garg et al., 2002; Fernandez et al., 2010; Chary et al., 2008; Li et al., 2011; Wang et al., 2016). For example, constitutive expression of yeast ScTPS1 in potato enhanced drought tolerance, but the resulting plants exhibited pleiotropic growth aberrations including dwarfism, chlorotic lancet-shaped leaves, and aberrant root development, indicating that the manipulation of TPS not only improves plant drought tolerance, but also affects plant
Abbreviations: TaTPS11, trehalose 6-phosphate synthase; SuSy, sucrose synthase; SPS, sucrose phosphate synthase; INV, invertase; SnRK1, sucrose non-fermenting 1related kinase 1; TOR, TARGET OF RAPAMYCIN ⁎ Corresponding author. E-mail address: [email protected] (X. Wang). https://doi.org/10.1016/j.gene.2019.06.006 Received 23 January 2019; Received in revised form 26 May 2019; Accepted 5 June 2019 Available online 07 June 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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HYGROMYCIN B PHOSPHOTRANSFERASE (HPT) and TaTPS11. All the gene-specific primers are listed in Table S1.
development (Yeo et al., 2000). Transgenic tomato with constitutive expression of yeast ScTPS1 improved the drought and salt tolerance, but showed pleiotropic changes such as thick shoots, rigid dark-green leaves, erect branches and aberrant root development (Cortina and Culiáñez-Macià, 2005). In tobacco, expression of ScTPS1 with an Arabidopsis stress-induced promoter significantly increased plant drought tolerance without growth aberrations (Karim et al., 2007). Overexpression of OsTPS1 in transgenic rice improved tolerance to cold, high salinity, and drought at the seedling stage, and showed no obvious phenotypic changes comparing to wild-type (WT). Furthermore, the results indicated that OsTPS1 may improve abiotic stress tolerance by increasing the amount of trehalose and regulating expression of stressrelated genes (Li et al., 2011). A similar result was also be described by Jang et al. (2003). The AtTPS11 in Arabidopsis encodes a trehalosesynthesizing enzyme with TPS and TPP activities, and overexpression of homologous TPS11 in Arabidopsis increased starch content and showed an obvious plant defense against aphids, but the study had no reports whether AtTPS11-overexpression influenced the abiotic stress response in Arabidopsis resultant plants (Singh et al., 2011). The cotton GhTPS11 encoded a stress-responsive TPS protein, and its overexpression in Arabidopsis seeds resulted in slower germination than the WT under chilling stress, indicating that GhTPS11 responded to chilling stress during seed germination (Wang et al., 2016). To our knowledge, the manipulation of the Triticum aestivum L. TaTPS genes in plants has not previously been reported. Our previous RNA sequencing analysis showed the transcript level of TaTPS11 was more highly induced in winter wheat cultivars with high freezing-tolerance than in those with low freezing-tolerance. Therefore, in this study, we overexpressed TaTPS11 in transgenic Arabidopsis plants to evaluate its effects on cold tolerance, and assayed the possible TaTPS11 function pathway for improving cold tolerance including sucrose metabolism.
2.3. RNA isolation and qRT-PCR analysis Total RNA was isolated from leaves using TRIzol reagent (Invitrogen, Carlsbad, USA), and each sample was a mixture of RNA extracted from eight plants. First-strand cDNA was synthesized using EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, China), according to the manufacturer protocol. The relative expression analysis was performed using a Roche LightCycler® 480 system with TransStart Top Green qPCR SuperMix (Transgen, China), as described by Xin et al. (2016). The Arabidopsis thaliana actin 2 (ACT2) was used as an internal control in quantitative RT-PCR. Data were calculated using 2(−ΔΔCt) method (Livak and Schmittgen, 2001). The gene-specific primers for qRT-PCR are listed in Table S1. 2.4. Determination of carbohydrate and enzyme activities Approximately 0.1 g of Arabidopsis shoot was mixed with 0.5 mL of 80% ethanol and boiled for 40 min. Then the extracts were centrifuged at 14,000g for 5 min. The residue was re-extracted again, as above. The supernatant was used for sucrose and fructose measurements (Qi et al., 2007). Debris was used for starch determinations using glucose equivalents, and the detailed procedure was in accordance with previous method (Baud et al., 2002). For assays of enzyme activity, 1 g fresh sample was placed in liquid nitrogen and ground into powder. The powder was suspended in 3 mL extraction buffer (100 mM HEPES pH 7.5, 2 mM MEDTA, 2 mM dithiothreitol, 1 mM PMSF and 10 mL/L protease inhibitor cocktail), and centrifuged at 14,000g with 4 °C for 10 min. The supernatant was used for determination of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) activities (Bahaji et al., 2015). For invertase (INV) assays, 1 g fresh sample was ground into powder using liquid nitrogen and suspended in 3 mL extraction buffer (50 mM Tris-acetate, pH 7.5; 10 mM EDTA; 5 mM DTT), and the homogenates were directly used for INV assays, as previously described (Qi et al., 2007). One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 μmol of product per minute.
2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis plants were routinely grown in a growth chamber at 22 °C with 150 μM m−2·s−1 light intensity, a photoperiod of 16 h-light/ 8 h-dark, and 60%–70% humidity. After 15 d, seedlings were subjected to −5 °C for 2 h, and then transferred back to the previous growth conditions for 2 d recovery. The living plants after treatment were counted for surviving rate. The high freezing-tolerance wheat cultivar (D1), which was the only winter wheat variety planted in high-latitude regions of China, was selected for the freezing treatment, and seedling plants were grown in artificial climatic chamber condition at 25 °C under photoperiod conditions of 14 h light/10 h dark, 50%–60% humidity, and 150 μM m−2·s−1 light intensity for 15 d, then transferred to a low temperature conditions, as follows: W0, control; W1, 4 °C cold acclimation for 30 d; W2, treatment as W1, then −10 °C/2 h; W3, treatment as W2, then −12 °C/2 h; W4, treatment as W3, then −14 °C/ 2 h; W5, treatment as W4, then −16 °C/2 h; W6, treatment as W5, then −18 °C/2 h.
2.5. Statistical analysis All numerical data were presented as mean ± SE calculated from three biological replicates. All P values were based on a two-tailed ttest. P < 0.05 and P < 0.01 were considered as statistically significant. 3. Results 3.1. Identification and sequence analysis of wheat cDNA encoding TPS Previous RNA sequencing analysis had revealed seven TPS-related genes that showed differential expression between high freezing-tolerant winter wheat (D1) and low freezing-tolerant winter wheat (J22) under low temperatures (Xie and Li, 2015; Xie et al., 2015). Quantitative RT-PCR analysis showed that one gene was induced under cold stress in both high freezing-tolerant winter wheat (D1) and low freezing-tolerant winter wheat (J22), but the expression level of the gene in D1 was significantly higher than in J22 (Fig. S1). Then, we compared the gene sequence in the coding region and found 7 SNPs and 5 InDels between D1 and J22 (Fig. S2), which resulted in a frameshift mutation and the changes of 207 encoded amino acids in J22. Herein, we aligned the encoding protein sequence of the gene in D1 with Arabidopsis using TAIR BLAST (https://www.arabidopsis.org/). The candidate gene shared the greatest identity with Arabidopsis trehalose6-phosphate synthase 11 (AtTPS11, GenBank accession: AT2G18700).
2.2. Plant transformation For the production of transgenic Arabidopsis plants, TaTPS11 cDNA was isolated by reverse-transcription PCR using gene-specific primers with total mRNA from the leaves of wheat D1, and inserted into pCAMBIA1305 vector with NcoI and SpeI restriction enzymes. The TaTPS11 fragment was placed under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter. The resulting construct was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis thaliana with a Columbia (Col-0) background by the floral dip method (Clough and Bent, 1998). The transformants were then screened by PCR amplification using specific primers for the 211
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Fig. 1. The phylogenetic relationships of TaTPS11 and other plant TPSs. (A) Amino acid sequence alignment of TaTPS11 with other plant species comprising Arabidopsis AtTPS11, barley HvTPS, rice OsTPS11, and maize ZmTPS11. Green underline domain (1-386 aa) indicates conserved trehalose-6phosphate synthase domain, and blue underlined domain (436-670 aa) indicates conserved trehalose phosphatase domain. (B) A phylogenetic tree derived from 11 Arabidopsis TPSs (AtTPS1-11), maize TPS (ZmTPS), barley TPS (HvTPS), and wheat TPS11 (TaTPS11). From the GenBank database: AtTPS1 (Q9SYM4); AtTPS2 (Q9FZ57); AtTPS3 (Q9SHG0); AtTPS4 (Q9T079); AtTPS5 (O23617); AtTPS6 (Q94AH8); AtTPS7 (Q9LMI0); AtTPS8 (Q0WUI9); AtTPS9 (Q9LRA7); AtTPS10 (O80738); AtTPS11 (Q9ZV48); HvTPS11 (BAJ97294); ZmTPS11 (AQK75662); OsTPS11 (AEB53186). The scale bar indicates distance value of 0.2 substitutions per site. The alignment was done using CLUSTALW in MEGA7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
aligned sequences from different organisms, and TaTPS11 shares conserved function domains (1-386 aa, trehalose-6-phosphate synthase domain; 436-670 aa, trehalose phosphatase domain), which were predicted by InterPro (http://www.ebi.ac.uk/interpro/search/sequencesearch) (Fig. 1B). In Arabidopsis, 11 TPS genes were classified into Group I (AtTPS1-4) and Group II (AtTPS5-11) through the presence or absence of the two conserved motifs (LDYDGTLM and GDDRSD) in the TPP domain (Singh et al., 2011). This indicated that TaTPS11 may encode a bifunctional enzyme with trehalose-6-phosphate synthase and trehalose phosphatase activities, and TaTPS11 belonged to the Class II TPS. 3.2. Expression of TaTPS11 in wheat plants under abiotic stress Previous transcriptome analysis showed that TaTPS11 was induced by low temperatures (Xie et al., 2015). To verify the result, TaTPS11 transcripts in the wheat were monitored by quantitative real time-PCR (qRT-PCR). During the whole treated process, we gradually decreased the treated temperature and increased the treated time to imitate the outside freezing stress. The results showed that the expression of TaTPS11 through 4 °C cold acclimation (W1) was obviously induced, and transcript abundance was almost 4-fold as no cold acclimation control (W0) (Fig. 2A). With treated temperature decrease and time increase, the transcript abundance of TaTPS11 presented a downward trend during the initial W2, W3, and W4 low-temperature treatment stages, however dramatically increased under long time freezing W5 and W6 treatment stages, indicating that TaTPS11 may involve in plant freezing tolerance (Fig. 2A). In addition, the detailed transcript abundance of TaTPS11 in 15-day-old wheat seedlings treated with different abiotic stresses for different times was determined. The result showed that the expression of TaTPS11 was significantly induced under 25% PEG (simulation of osmotic stress), 300 mM NaCl (simulation of salt stress) at 12 h and 24 h (Fig. 2B, C). Previous reports showed that the metabolism of trehalose and sucrose had close relationship (Figueroa et al., 2016; Wang et al., 2016). We also examined the transcript abundance of TaTPS11 under sucrose stress, and found that TaTPS11 was obviously induced under 6% sucrose at 12 h rather than at 24 h (Fig. 2D). Above results indicated TaTPS11 participated in plant stress response. 3.3. Overexpression of TaTPS11 increases cold tolerance in Arabidopsis Therefore, the gene was named TaTPS11 (Accession: MK330685). The TaTPS11 contains a 2121-bp open reading frame and encodes 706 amino acids, which shared 63.57% identity with AtTPS11 at protein level. Then Amino acid sequence of TaTPS11 was compared with those of Arabidopsis (AtTPS11, accession: NP_179460), barley (HvTPS, accession: BAJ97294), maize (ZmTPS, accession: AQK55931) and rice (OsTPS, accession: XP_015625782) (Fig. 1A). The deduced amino acid sequence of TPS11 has high sequence identity of 86.36% among the
To survey the potential function of TaTPS11 in plant cold stress responses, a vector carrying TaTPS11 under the control of constitutive the cauliflower mosaic virus (CaMV) 35S promoter and HPT selectable marker gene was introduced into Arabidopsis (Fig. 3A). Fourteen homozygous transgenic lines were obtained, and the transcript abundance of TaTPS11 was detected in TaTPSOE-01, -04, -05, -07 and -09 lines (referred to as TaTPSOE-01-14. OE = overexpression). The expression levels of TaTPS11 in TaTPS11OE-01, TaTPS11OE-07 and 212
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Fig. 2. Expression analysis of TaTPS11 under abiotic stress in wheat. (A) qRT-PCR analysis of TaTPS11 under cold stress. W0, normal culture; W1, 4 °C cold acclimation for 30 d; W2, treatment as W1 then −10 °C/2 h; W3, treatment as W2 then −12 °C/2 h; W4, treatment as W3 then −14 °C/2 h; W5, treatment as W4 then −16 °C/2 h; W6, treatment as W5 then −18 °C/2 h. (B) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated with 30% PEG stress over different time periods. (C) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated with 300 mM NaCl stress for different time periods. (D) qRT-PCR analysis of TaTPS11 in 15-day-old wheat seedlings treated by 6% sucrose stress for different time periods. Error bars indicate the SE calculated from three replicates.
TaTPS11OE-09 lines were significantly higher than in other lines (Fig. 3B). These three homozygous lines screened by HPT and TaTPS11 exhibited no visible morphological differences with WT at the seedling stage, and were selected for further studies (Fig. S3). To study the cold response of TaTPS11, two-week-old Arabidopsis plants were subjected to −5 °C for 2 h, and recovered for 2 d. All resultant lines had an increased cold tolerance over the WT. The survival rates of TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines were 65%, 60% and 63%, respectively, but only 17% in the WT after 2 d recovery (Fig. 4). The above results indicated that TaTPS11 played a positive role in cold tolerance in plants.
that heterologous expression of TaTPS11 influenced sucrose metabolism in Arabidopsis. Previous studies have shown that expression of TPSs could affect starch metabolism (Gomez et al., 2010; Singh et al., 2011). Therefore, we also measured starch content, which was found to be significantly higher in the OE-lines than in the WT (Fig. 5F). These results indicated that heterologous expression of TaTPS11 influenced carbohydrate metabolism in Arabidopsis plants.
4. Discussion Glycometabolism has been confirmed to play a key role in overwintering response to cold stress in plants (Krasensky and Jonak, 2012; Nägele and Heyer, 2013; Kovi et al., 2016). Previous studies showed that sucrose metabolism had a close correlation with plant cold tolerance, which was coordinated with the sucrose increases (Livingston and Henson, 1998; Janska et al., 2011). In rice plants, increased sucrose content occurred when plants underwent cold stress (McKown et al., 1996; Wanner and Junttila, 1999; Reyes-Díaz et al., 2006). Overexpressing of OsDREB1 in rice increased cold tolerance and was accompanied by increases in sucrose (Ito et al., 2006). However, in our study, constitutive expression of TaTPS11 in Arabidopsis increased cold tolerance, but decreased sucrose content comparing to WT (Fig. 5A), indicating no direct correlation between cold tolerance and increase of sucrose content in TaTPS11OE Arabidopsis plants. The increase of fructose content was also deemed as an important component of plant cold response process (Livingston and Henson, 1998; Janska et al., 2011). Synthetic wheat lines with high freezing tolerances had higher fructan
3.4. Overexpression of TaTPS11 influenced the carbohydrate metabolism in Arabidopsis Previous studies reported that Tre6P catalyzed by TPS could modulate sucrose levels by affecting sucrose metabolism in plants (Wingler et al., 2012; Figueroa et al., 2016). To confirm the results, the sucrose content was compared between TaTPS11OE lines and WT. The sucrose contents in TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines were 1.74 mg/g, 1.91 mg/g and 1.82 mg/g, respectively, and were significantly < 2.10 mg/g measured in WT (Fig. 5A). Similar results were also observed for the fructose contents in TaTPS11OE-01, TaTPS11OE-07 and TaTPS11OE-09 lines, which were significantly lower than in the WT (Fig. 5B). The key enzyme activities in sucrose metabolism (SPS, SuSy, and INV) were also examined. Intriguingly, all detected enzyme activities were obviously increased comparing to WT (Fig. 5C-E), indicating 213
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Fig. 3. Construction of TaTPS11 overexpression vector and molecular identification of transgenic Arabidopsis plants. (A) Structure of TaTPS11 overexpression (OE) vector. HPT, hygromycin phosphotransferase gene; TaTPS11, cDNA of trehalose-6-phosphate synthase 11 from wheat; CaMV35S, promoter sequence of cauliflower mosaic virus (CaMV) 35S. (B) Analysis of five independent transgenic Arabidopsis lines: OE-1, OE-4, OE-5, OE-7, OE-9. Values are means ± SEM with three replicates.
accumulation compared to the low freezing tolerance lines (Kovi et al., 2016). Hisano et al. (2004) reported that overexpression of wheat fructosyltransferase genes increased accumulation of fructans and improved freezing tolerance at the cellular level (Hisano et al., 2004). In the present study, the fructose content in transgenic Arabidopsis plants also decreased comparing to WT (Fig. 5B). Our results suggested that complex interactions existed between the content of soluble sugars (such as sucrose and fructose) and cold tolerance in plants, which needs further detailed studies. Previous study showed that the starch content in tps1 mutant of Arabidopsis was significantly increased (Gomez et al., 2010). Overexpression of AtTPS11 constitutively elevated the starch content comparing to WT in Arabidopsis (Singh et al., 2011). In our study, we found that the starch content in TaTPS11-OE lines was also elevated, indicating that TaTPS11 possibly has functions similar to AtTPS11 in Arabidopsis. SPS, SuSy and INV are key numbers of sucrose-metabolizing enzymes that regulate sucrose synthesis and catabolism (Winter and Huber, 2000; Kulshrestha et al., 2013; Volkert et al., 2014). All three enzyme activities (SPS, SuSy and INV) in TaTPS11-OE Arabidopsis plants increased comparing to WT (Fig. 5C–E). These results suggested that TaTPS11-overexpression influenced the sucrose metabolism in Arabidopsis plants possibly by positive regulation of SPS, SuSy and INV activities. The expression of SuSy and INV genes was reported to be regulated under abiotic and biotic stresses (Cabello et al., 2014). INV genes responded to high salinity, drought and low temperature (Zeng et al., 1999; Cabello et al., 2014). In the present study, we observed significant increases of SPS, SuSy, and INV activities accompanied with cold tolerance in TaTPS11-OE Arabidopsis plants, indicating a positive relationship between sucrose-metabolizing enzymes and cold tolerance. However, more efforts are worth further studying the detailed correlations. In higher plants, TPS catalyzes Tre6P biosynthesis (Chary et al., 2008; Figueroa et al., 2016). Recent findings have shown that Tre6P could regulate Suc consumption and growth, probably acting via multiple mechanisms, including inhibition of one conserved protein kinase,
Fig. 4. Cold stress analysis of TaTPS11 overexpression (OE) Arabidopsis plants. 15-day-old seedlings were incubated under growth conditions of −5 °C for 2 h, before being transferred back to normal growth conditions for 2 d recovery, and then the living plants after treatment were counted for surviving rate. Error bars indicate SE calculated from three biological replicates. All P values are based on a two-tailed t-test: **, P < 0.01 statistical significance.
sucrose non-fermenting 1-related kinase 1 (SnRK1), in Arabidopsis plants (Zhang et al., 2009; Delatte et al., 2011). Figueroa proposed that the relationship between Tre6P and SnRK1 in developing tissues was complex and not yet fully resolved (Figueroa et al., 2016). In our study, we also examined the expression level of AtSnRK1 in TaTPS11-overexpressing Arabidopsis. Contrary to expectation, the resultant plants promoted the expression of SnRK1 rather than inhibit it (Fig. 5G), 214
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Fig. 5. Carbohydrate metabolism changes in TaTPS11 overexpression (OE) Arabidopsis plants. (A) Sucrose content. (B) Fructose content. (C) Sucrose phosphate synthase (SPS) activity assay. (D) Sucrose synthase (SuSy) activity assay. (E) Invertase activity assay. (F) Starch content. (G) The relative expression level of AtSnRK. (H) The relative expression level of AtTOR. Error bars indicate SE calculated from three biological replicates. All P values were based on a two-tailed t-test: **, P < 0.01 statistical significance.
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indicating that the effect of heterologous expression of TaTPS11 was different from endogenous expression of AtTPSs in Arabidopsis, but this hypothesis needs further study. We examined the transcript abundance of the other conserved protein kinase, TARGET OF RAPAMYCIN (TOR), in the TaTPS11-overexpressing Arabidopsis. The result showed that the expression level of TOR was roughly the same between OE lines and WT (Fig. 5H), indicating that expression of Tre6P and TOR was not correlated, and these also corroborated the previous opinion of the relationship between Tre6P and TOR (Figueroa et al., 2016). Particular focus has recently been directed towards understanding the regulation role of TPSs in enhancing cold tolerance (Pilon-Smits et al., 1998; Garg et al., 2002; Karim et al., 2007; Chary et al., 2008; Li et al., 2011; Wang et al., 2016). The OsTPS1-overexpression transgenic rice improved the tolerance of cold, and no obvious phenotypic changes were apparent (Li et al., 2011). Overexpression of homologous AtTPS11 in Arabidopsis increased starch content and showed increased plant defense against aphids but without reports related to abiotic stress response (Singh et al., 2011). Resent research showed that cotton GhTPS11 that encoded a stress-responsive TPS protein functioned in chilling stress during seed germination in Arabidopsis (Wang et al., 2016). To our knowledge, few TaTPSs were identified and characterized in wheat, and no detailed functional studies of TaTPSs in abiotic stress have been reported. The present study showed that TaTPS11 was obviously induced not only by cold, salt and osmotic stress (simulation by PEG), but also by sucrose in wheat (Fig. 2). In conclusion, the wheat TaTPS11 was obviously induced by multiple abiotic stresses in wheat, and TaTPS11-overexpression increased the cold tolerance in Arabidopsis plants via influencing the carbohydrate metabolism such as sucrose, fructose and starch, indicating that TaTPS11 had potential values in wheat cold-tolerance breeding.
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Author contributions XL designed the research, performed most of the experiments, analyzed the data and wrote the manuscript; LSF and YLS provided technical assistance; PQ participated in carbohydrate and enzyme activity assays; JL helped to correct the manuscript; XNW designed the research and corrected the manuscript. All authors approved the final manuscript. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgements This research was financially supported by National Key Research and Development Program of China (grant number: 2016YFD0100502), Natural Science Foundation of Heilongjiang Province of China (grant numbers: QC2018020, QC2016026), Young Talents Program of Northeast Agricultural University (grant number: 17QC04), and Applied Technology Research and Development Project of Harbin (grant number: 2016RAQXJ034). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.06.006. References Bahaji, A., Baroja-Fernandez, E., Ricarte-Bermejo, A., Sanchez-Lopez, A.M., Munoz, F.J., Romero, J.M., Ruiz, M.T., Baslam, M., Almagro, G., Sesma, M.T., Pozueta-Romero, J., 2015. Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis. Plant Sci. 238, 135–147.
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