Wei Tang · Anna Y. Tang

Abstract Malonyl-CoA synthetases may modulate cell responses to abiotic stress by regulating stress-related signaling transduction pathways or activating expression of transcription factors. However, the molecular mechanism of cold stress tolerance enhanced by malonyl-CoA synthetase is not fully understood. Here, we report that overexpression of the Arabidopsis thaliana malonyl-CoA synthetase gene AAE13.1 resulted in increased cell viability and growth rate and decreased thiobarbituric acid reactive substances under cold stress in rice ( Oryza sativa L.), tobacco ( Nicotiana tabacum), and slash pine ( Pinus elliottii Engelm.). AAE13.1 was associated with cold stress tolerance by increasing the activity of ascorbate peroxidase, catalase, polyphenol oxidase, and peroxidase and the accumulation of acid phosphatase and alkaline phosphatase. Among six rice mitogenactivated protein kinase ( MAPK) genes examined, AAE13.1 overexpression increased the expression of OsMAPK genes during cold stress. AAE13.1 activated expression of stressresponse genes OsMAPK1, OsMAPK2, and OsMAPK3, indicating that AAE13.1 enhances cold stress tolerance by regulating expression of MAPK genes in plant cells. These results increase our understanding of cold stress tolerance in species of monocotyledons, dicotyledons, and gymnosperms.

Keywords Cold stress · Malonyl-CoA synthetase gene · MAPK transcription factor · Phosphatase · Pinus

Introduction

In response to abiotic stresses such as low temperature, high NaCl, drought, and heavy metals, cells regulate their biological activities at the transcriptional and translational levels. For example, in pea, transcription of DNA helicase 47 was induced in both shoots and roots under cold (4 °C) stress, and helicase activities also changed during distinct cellular processes in cold stressed conditions (Vashisht et al. 2005). Cell and plant growth rate were reduced and APOX capacity increased under cold stress in AtFad transgenic plants (Matos et al. 2007). In rice, the Osmyb4 gene is induced by cold stress and can activate stress responsive pathways in both Arabidopsis thaliana and apple ( Malus pumila Mill.) (Pasquali et al. 2008). A cDNA microarray of Alstroemeria flowers demonstrated that cold stress accelerates many changes in gene expression and activates the expression of 21 transcription factors (Wagstaff et al. 2010). In potato, cold stress also increases expression of transcription factorrelated genes and decreases expression of photosynthesisrelated genes (Evers et al. 2012). In A. thaliana, the cold signaling attenuator HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 negatively regulates cold responses via acting as a chromatin-remodeling factor under cold stress (Jung et al. 2013). In camelina ( Camelina sativa) and rapeseed ( Brassica napus), cold stress can increase H + -ATPase activity and H + -transport (Kim et al. 2013). In A. thaliana, a microarray analysis showed that 1780 genes were differentially regulated in CcCDR-transgenic plants, and CcCDR conferred tolerance to multiple abiotic stresses (Tamirisa et al. 2014). In durum wheat, cold stress increases the level of arabinose, fructose, glucose, raffi nose, sucrose, and hexose phosphates (Shahryar and Maali-Amiri 2016). In apple, different metabolic pathways such as methanol biosynthesis and ethylene biosynthesis pathways are affected by low temperature (Gapper et al. 2017).

Malonyl-CoA is important for the synthesis of fatty acids, phytoalexins, flavonoids, polyketides, anthocyanin pigments, and many malonylated compounds. Malonyl-CoA is derived from acetyl-CoA by acetyl-CoA carboxylase. In A. thaliana, recombinant AAE13, a malonyl-CoA synthetase encoded by the gene Acyl Activating Enzyme13 ( AAE13, AT3G16170), had high activity against malonic acid and is essential for growth and development, likely because it is involved in the detoxification of malonate (Chen et al. 2011). Metabolic engineering analysis demonstrated that malonyl-CoA synthetase increases expression of flavanone and flavone biosynthetic genes (Park et al. 2011). In gerbera ( Gerbera hybrida), expression of both GCHS1 and GCHS4 regulates expression of malonyl-CoA synthetase gene in the epidermal cells (Deng et al. 2014). Overexpression of AtAAE13 in Saccharomyces cerevisiae results in increased level of lipids, suggesting that malonyl-CoA is a critical target for fatty acid (Wang et al. 2014). Expression of a chalcone synthase gene ( PaCHS) in Marchantia paleacea increases the content of flavonoids, suggesting that PaCHS plays a key role in flavonoid biosynthesis under abiotic stress (Yu et al. 2015). Malonyl-CoA synthetase is encoded by AAE13 (AT3G16170) in A. thaliana is localized in the cytosol and the mitochondria, but only mitochondrial AAE13 is essential for plant growth (Guan and Nikolau 2016). In Petunia hybrida, expression of PhAAE13 is highest in corollas, and silencing of PhAAE13 reduces the levels of anthocyanins, fatty acids, and cuticular wax, demonstrating the involvement of PhAAE13 in anthocyanin biosynthesis (Chen et al. 2017).

Acid phosphatase and alkaline phosphatase are also important in the abiotic stress responses. Acid phosphatase activity significantly increases in tissues under abiotic stress (Malik and Sethi 1975; Erukainure et al. 2017; Zou et al. 2017). Plant-derived alkaline phosphatase also improves abiotic stress tolerance (Leyva et al. 2004). Alkaline phosphatase activity is higher in tomato than in cotton and cabbage under typical condition without stress, indicating that function of alkaline phosphatase is species dependent (Leyva et al. 2004; Sessions 2008; Widyowati et al. 2010; Yan et al. 2011; Sineshchekov et al. 2013). How the AAE13 gene coordinates with acid phosphatase and alkaline phosphatase during a stress response, however, has remained elusive.

Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and guaiacol peroxidase (POX) also increase in cells of many plant species under abiotic stress, suggesting that increased activity of antioxidant enzymes is involved in increased tolerance stress (Gzyl et al. 2009; Arora et al. 2010; Sang and Kim 2011; Sirhindi et al. 2015). In A. thaliana, analysis of cat1, cat2, cat3, cat1 cat2, and cat2 cat3 T-DNA mutants demonstrated that stress has an indirect effect at the leaf level and that cell response to catalase deficiency is independent of the duration ofoxidative stress (Ray et al. 2012; Singh et al. 2012; Spanou et al. 2012; Murota et al. 2017; Yang et al. 2018). In transgenic tobacco and hybrid aspen, overexpression of the horseradish peroxidase gene results in increased stress tolerance (Yoshida et al. 2003). Activation of StPPO (polyphenol oxidase [PPO] derived from Solanum tuberosum) was caused by activation of a latent StPPO by chlorogenic acid quinones in a stress response (Nautiyal et al. 2008; Poiatti et al. 2009; Dirks-Hofmeister et al. 2013; Kuijpers et al. 2014; Molitor et al. 2016; Kampatsikas et al. 2017). How the AAE13 gene co-ordinates with antioxidant enzymes for stress response is not fully understood.

Here, we report that, during cold stress, overexpression of the A. thaliana malonyl-CoA synthetase gene AAE13.1, which encodes a protein of 608 amino acids (Guan and Nikolau 2016), in rice ( Oryza sativa L.), tobacco ( Nicotiana tabacum), and slash pine ( Pinus elliottii Engelm.) increased cell viability and growth rate and decreased the level of thiobarbituric acid reactive substances (TBARS), generated by lipid peroxidation. Measurement of TBARS is a well-established method for monitoring lipid peroxidation caused by cold stress. Lipid peroxidation is a well-defined mechanism of cellular damage in plant cells. Thiobarbituric acid reactive substances (TBARS) assay detects the level of malondialdehyde, the major product of lipid peroxidation and the degradation of unstable lipid peroxides (Tang et al. 2006). Our results showed that overexpression of AAE13.1 enhances cold stress tolerance by increasing the activity of APOX, CAT, PPO, and POD and accumulation of acid phosphatase and alkaline phosphatase and by activating expression of stress-response OsMAPK genes in rice cells.

Materials and methods

Plasmid constructs

The full-length A. thaliana AAE13.1 coding sequence (1827 bp) was cloned into pBI121 as previously described (Chen et al. 2011; Guan and Nikolau 2016). Vector pBI121 and AAE13.1 were digested by restriction enzymes KpnI and XbaI (Promega, Madison, WI, USA) at 37 °C, then purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA, USA) and ligated to generate the expression vector (Tang et al. 2005a, b, 2006). The resulting expression vector, designated pBI- AtAAE13.1, was introduced into Agrobacterium tumefaciens strain LBA4404 using electroporation.

Transformation of rice, tobacco, and pine cells

AtAAE13.1-transgenic cell lines of three plant species rice ( O. sativa), tobacco ( N. tabacum ), and pine ( P. elliottii) were generated as described before (Tang and Page 2013), using A. tumefaciens strain LBA4404 carrying pBI- AtAAE13.1 (Tang et al. 2006, 2007a, b). Cell cultures of rice, tobacco, and pine were then grown for 7 weeks before further analysis.

Polymerase chain reaction analyses of transgenic cells

Genomic DNA was extracted from 7 g of transgenic rice, tobacco, and pine cells using a Genomic DNA Isolation Kit (Sigma). For the PCR using the method of Tang and Page ( 2013), neomycin phosphotransferase II gene ( NPTII) was amplified using forward primer nfp and reverse primer nrp; AtAAE13.1 was amplified using forward primer zaf and reverse primer zar. The PCR mixture, the PCR conditions, and gel electrophoresis have been described previously (Tang et al. 2006, 2007a, b). The PCR was run in a PTC-100TM machine (MJ Research, San Francisco, CA, USA) using 300 ng of genomic DNA as a template.

Southern blot analysis of transgenic cells

Southern blots were done as previously described (Tang et al. 2006, 2007a, b). Genomic DNA was extracted from 9 g of control cells or transgenic cells of rice, tobacco, and pine using a Genomic DNA Isolation Kit (Sigma). Twenty-eight micrograms of DNA for each was digested with the restriction enzyme XbaI (Boehringer Mannheim) for 16 h at 37 °C. The molecular probe (1827 bp fragment of AtAAE13.1) were labeled with digoxigenin (DIG) (Roche Diagnostics, Indianapolis, IN, USA).

RNA isolation and northern blot analysis

Total RNA was extracted from transgenic and control cells using an RNeasy Mini Plant Kit (Qiagen, Germantown, MD, USA) and the instructions, and 9 μg of total RNA was used for northern blotting as described (Tang et al. 2007a, b). The DIG-labelled AtAAE13.1 DNA fragment (1827 pb) (Roche Diagnostics) was used as the hybridization probe. Tobacco rRNA was used as the loading control for RNA samples.

Cold treatment

The AtAAE13.1-transgenic cell lines of O. sativa, N. tabacum, and P. elliottii and wild-type control cells were incubated in growth incubators at ? 10, ? 4, 10, or 24 °C in the dark for 24 h. Cells were then moved to typical growth conditions incubated at 24 °C with 16 h light/8 h dark.

Determination of cell viability and growth rate

After the 24-h temperature treatment and 7 days in typical growth conditions incubated at 24 °C with 16 h light/8 h dark, the influence of cold stress on cell growth and viability was analyzed as previously described (Tang et al. 2006, 2007a, b). The average growth rate was expressed as mg/g FW/day. Cell samples from both chilled and control cell cultures were collected with 3 biological replicates. Cells were imaged using a confocal microscope. In a preliminary experiment, cell viability of nontransgenic cells of O. sativa, N. tabacum, and P. elliottii was evaluated at 0, 1, 3, 5, 7, and 9 days after the cold treatment to select an appropriate assay time (7 days). The effect of cold stress at a shorter time (2 days after cold treatment) was also verified.

Measurement of thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS) were quantified using the thiobarbituric acid (TBA) reaction as described previously (Tang et al. 2005a, b; Tang and Page 2013) using 3 g of transgenic or control cells from each species. The sample was heated at 95 °C for 30 min and centrifuged at 10,000 × g for 15 min before absorbance was measured at 532 nm. A preliminary time-course study was done as described for cell viability to determine the assay time (7 days).

Activity of APOX, CAT, PPO, and POD

Activity of ascorbate peroxidase (APOX), catalase (CAT), polyphenol oxidase (PPO), and peroxidase (POD) was measured as previously described (Tang et al. 2005a, b, 2006; Tang and Page 2013) 7 days after the temperature treatment using 5 g of control or transgenic cells. Three biological replicates were assayed for each treatment.

Activity of acid phosphatase and alkaline phosphatase

Activity of acid phosphatase and alkaline phosphatase in a methanolic extract of cells (Ma et al. 2013; Selin-Rani et al. 2016) was measured as previously described (Kaida et al. 2009; Johnson et al. 2015) 7 days after the temperature treatment of control and transgenic cells of the three species and quantified by the method of Sakthivel and Guruvayoorappan ( 2013) was subjected to the measurement of acid phosphatase and alkaline phosphatase enzymatic activities (Ma et al. 2013; Selin-Rani et al. 2016). Three biological replicates were used for each treatment.

Expression of mitogen-activated protein kinase genes

Expression of mitogen-activated protein kinase genes in control and transgenic cells of the three species 7 days after treatment was quantified using qPCR as previously described (Wan et al. 2007). Total RNA was extracted from frozen cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). First-strand cDNA was synthesized in a 50-μL reaction containing 2.5 mM oligo (dT) primers, 2.5 mM random hexamer, and 2.5 μg of total RNA. The PrimeScript RT reagent kit (TaKaRa, Ohtsu, Japan) was used according to the instructions. Samples were analyzed in triplicate on the Applied Biosystems 7900HT System as described in the manufacturer’s manual. Primers for qPCR are listed in Table 1. Small noncoding RNA U6 was used as an internal control for normalizing data. The delta–delta Ct method was used to obtain expression value. Three biological replicates were used for each treatment.

Statistical analyses

The General Linear Model procedure and ANOVA in SAS 9.4m4 (SAS Institute, Cary, NC, USA) were used to analyze significant differences among means between the control and transgenic cell lines for each species at 5% level of probability.

Results

Molecular analysis of transgenic cells

Among the 27 AAE13.1-transgenic cell lines of O. sativa, 33 of N. tabacum, and 38 transgenic cell lines of P. elliottii obtained, transgenic cell lines Os1, Os2, and Os3 of O. sativa, Nt1, Nt2, and Nt3 of N. tabacum, and Pe1, Pe2, and Pe3 of P. elliottii were selected for PCR analysis of NPTII (Fig.1 b) and AAE13.1 (Fig.1 c) and found to be positive for the genes. The lines were then examined by Southern blot analysis using the DNA fragment of AAE13.1 as probe (Fig.1 d) and by Northern blot analysis for AAE13.1 (Fig.1 e). These cell lines were then functionally analyzed after a cold stress.

Fig.1 Expression vector and molecular analyses of transgenic cells. a Expression vector showing the T-DNA region that includes right border (RB), left border (LB), the nos promoter (nos Pro), the NPTII gene ( NPTII), the nos terminator (nos Ter), the 35 S promoter (35S Pro), and the AAE13.1 gene ( AAE13.1). PCR of b NPTII gene (717 bp) and c AAE13.1 (1827 bp) in O. sativa, N. tabacum, and P. elliottii. d Southern blots and northern blots of AAE13.1 gene in O. sativa, N. tabacum, and P. elliottii. Tobacco 25S rRNA was used as the loading control. Lane P: plasmid positive control; C negative control

Overexpression of AAE13.1 increases growth rate and viability and decreases TBARS during cold stress

Cell viability and TBARS were evaluated through a time course (0, 1, 3, 5, 7, 9 days after cold treatment) using nontransgenic cells of O. sativa, N. Tabacum, and P. elliottii to determine the assay time point (Fig.2 a-f). Compared tothe growth rate of the control cells, cell growth rate was 76–91% higher in the three transgenic lines of O. sativa (Fig.2 g), 28–39% higher in three transgenic lines of N. tabacum (Fig.2 h), and 35–43% higher in the three transgenic lines of P. elliottii (Fig.2 i) after the treatment with ? 10 °C and ? 4 °C. Compared to viability of the control cells, cell viability of O. sativa transgenic cell lines was 31–43% higher (Fig.2 j), 32–41% higher for the N. tabacum transgenic cell lines (Fig.2 k), and 21–46% higher for the three P. elliottii transgenic cell lines (Fig.2 l) after treatment with ? 10 °C and ? 4 °C. Overexpression of AAE13.1 also decreased TBARS in transgenic cells of O. sativa by 12–19% (Fig.2 m), N. tabacum by 13–18% (Fig.2 n), P. elliottii by 13–17% (Fig.2 o) after treatment with ? 10 °C and 4 °C.

Table 1 Primers used in qRTPCR in this study

Fig.2 Time course of cell viability ( a– c) and TBARS ( d– f) of O. sativa, N. tabacum, and P. elliottii cells under cold treatment. Overexpression of AAE 13.1 increases cell growth rate and viability, and decreases TBARS levels after cold treatments in transgenic rice ( g, j, m), tobacco ( h, k, n), and pine ( i, l, o)

Fig.3 Effect of AAE13.1 on activity of APOX, CAT, PPO, and POD in transgenic cells of rice ( a, d, g, j), tobacco ( b, e, h, k), and pine ( c, f, i, l). Data are means of three independent experiments; error bars represent standard deviations. One-way ANOVA was used to test for significant differences among treatments. * P < 0.05, significant relative to control as assessed by a t test

Activity of ascorbate peroxidase (APOX), catalase (CAT), polyphenol oxidase (PPO), and peroxidase (POD)

Overexpression of AAE13.1 increased the activity of APOX, CAT, PPO, and POD in transgenic cell lines of O. sativa (Fig.3 a, d, g, j), N. tabacum (Fig.3 b, e, h, k), and P. elliottii (Fig.3 c, f, i, d) after treatment at ? 10 °C and ? 4 °C but activity did not change at 10 °C or 24 °C compared to controls.

Acid phosphatase and alkaline phosphatase of transgenic cell lines

Compared to the activity in the controls, activity of acid phosphatase and alkaline phosphatase increased significantly in the transgenic cell lines of O. sativa (Fig.4 a, d), N. tabacum (Fig.4 b, e), and P. elliottii (Fig.4 c, f) after treatment with ? 10 °C and ? 4 °C but not after treatment with 10 °C and 24 °C.

Effect of AAE13.1 gene on the morphology of transgenic cells

Overexpression of the AAE13.1 gene decreased the size of transgenic cells in rice (Fig.5 a–d), tobacco (Fig.5 e–h), and pine (Fig.5 i–l), after treatment with ? 10 °C and ? 4 °C compared to the transgenic cells of O. sativa, N. tabacum, and P. elliottii treated at 10 °C, and 24 °C.

Expression of MAPK genes in transgenic cells

Compared to expression of the respective MAPK genes in the controls, the expression of OsMAPK1 (Fig.6 a), OsMAPK2 (Fig.6 b), OsMAPK3 (Fig.6 c), OsMAPK4 (Fig.6 d), OsMAPK5 (Fig.6 e), and OsMAPK6 (Fig.6 f) was significantly higher in transgenic rice cells after treatment with ? 10 °C and ? 4 °C, but there was no difference after treatment with 10 °C and 24 °C. At 2 days after the cold treatment, results were similar (Fig.7).

Discussion

Fig.4 Effect ofoverexpression of AAE13.1 on activity of acid phosphatase and alkaline phosphatase in transgenic cells of rice ( a, d), tobacco ( b, e), and pine ( c, f). Data are means of three independent experiments; error bars represent standard deviations. One-way ANOVA was used to test for significant differences among treatments. * P < 0.05, significant relative to control as assessed by a t test

Fig.5 Effect ofoverexpression of AAE13.1 on morphology of transgenic cells of rice ( a– h), tobacco ( i– p), and pine ( q- x) and cell size ( y– za) after treatment with different temperature (? 10 °C, ? 4 °C, 10 °C, and 24 °C)

In response to signal transduction during abiotic stress, cells regulate their biological activities at the transcriptomic and proteomic levels. Increased activity of antioxidant enzymes is involved in tolerance to stress. In our genetic and biochemical assays to explore the effect ofoverexpression of AAE13.1 after cold stress (? 10, ? 4, and 10 °C) in different plant species, overexpression increased cell viability and growth rate, and decreased TBARS in transgenic cells of rice, tobacco, and pine. Thus, overexpression of the AAE13.1 gene has the potential to enhance cold stress tolerance by decreasing TBARS and might be achieved in different plant species.

The activity of APOX, CAT, PPO, and POD is related to cold stress tolerance in many plant species (Yang et al. 2018; Faltin et al. 2010; Correa-Aragunde et al. 2015). In a study of PPO activity in 60 plant or fungal species including S. tuberosum and Agaricus bisporus, RP-UHPLC-MS analysis showed that the extracts of O. sativa, N. tabacum, and P. elliottii cells contained a mixture of phenolic compounds, and the phenolic fraction was mainly responsible for inhibiting PPO (Nautiyal et al. 2008; Poiatti et al. 2009; Dirks-Hofmeister et al. 2013; Kuijpers et al. 2014; Molitor et al. 2016; Kampatsikas et al. 2017). In our present study, overexpression of AAE13.1 during cold stress was associated with increased activity of APOX, CAT, PPO, and POD. Among the six MAPK genes analyzed in O. sativa, only OsMAPK1 (Fig.6 a), OsMAPK2 (Fig.6 b), OsMAPK3 (Fig.6 c) increased significantly; the other did not change after the treatment with ? 10 °C and ? 4 °C; thus, OsMAPK1 (Fig.6 a), OsMAPK2 (Fig.6 b), OsMAPK3 (Fig.6 c) apparently are involved in AAE13.1-enhanced cold stress tolerance.

Environmental stresses can alter cell cycle regulation by shortening the G2 period and thus altering growth (Matia et al. 2010; Herranz and Medina 2014; de Buanafina et al. 2017; Lee et al. 2017; Majda et al. 2017). In embryogenic cell suspensions of wheat ( Triticum aestivum L.), cold stress led to an abnormal chromosome number and low cell viability (Ahmed and Sagi 1993). Cell viability and cell size are important for increased cold stress tolerance (Tang et al. 2006). In the present study, growth rate was significantly increased in cultures with high viability. Increased sensitivity of the cells to stresses was associated with a 35% increase in cell size of Medicago sativa plants (Steward et al. 1999). In our study, overexpression of the AAE13.1 gene increased the size of transgenic cells in rice (Fig.5 a–d), tobacco (Fig.5 e–h), and pine (Fig.5 i–l) after low temperature (? 10 °C and ? 4 °C) treatment, but not increased cell size under growth at 24 °C.

Fig.6 MAPK expression is regulated by overexpression of AAE13.1 gene in transgenic rice cells. OsMAPK1 ( a), OsMAPK2 ( b), OsMAPK3 ( c), OsMAPK4 ( d), OsMAPK5 ( e), and OsMAPK6 ( f). Data are means of three independent experiments; error bars represent standard deviations. One-way ANOVA was used to test for significant differences among treatments. * P < 0.05, significant relative to control as assessed by a t test

Fig.7 Verification ofeffect of cold stress in transgenic rice cells. Effect of a shorter cold stress (2 days) on growth rate ( a), cell viability ( b), TBARS ( c), APOX ( d), CAT ( e), PPO ( f), POD ( g), acid phosphatase ( h), alkaline phosphatase ( i), OsMAPK1 ( j), OsMAPK2 ( k), OsMAPK3 ( l), OsMAPK4 ( m), OsMAPK5 ( n), and OsMAPK6 ( o) in control and transgenic rice cell lines. Data are means of three independent experiments; error bars represent standard deviations. One-way ANOVA was used to test for significant differences among treatments. * P < 0.05, significant relative to control as assessed by a t test

The effects of chilling can also affect newly formed cells that were not stressed. On the basis of previous reports and our own experimental results here, we propose a mechanism by which previous chilling can affect new cells: (1) Cold stress affects cell wall formation and microtubules organization when the new cell wall is formed during the division of cold-stress-treated cells (Lee et al. 2017; Majda et al. 2017). The effect on this molecular machinery may last a few generations. (2) Cold stress affects cell metabolism and the biosynthesis of stress-related proteins that are associated with the growth of cells and their internal structures, such as the formation of the mitochondria envelope (Deng et al. 2014 ). (3) Cold stress may lead to alterations in the ultrastructure of nuclei, plasmalemma, and chromatin (Johnson et al. 2015). (4) Cold stress causes DNA damage and affects the formation of secondary structures of RNA that are related to the process of cell death (Johnson et al. 2015; Majda et al. 2017).

Conclusion

Overexpression of the AAE13.1 gene increased cold stress tolerance in association with increased cell viability and growth rate and lower levels of TBARS in rice, tobacco, and slash pine. Activity of APOX, CAT, PPO, and POD and accumulation of acid phosphatase and alkaline phosphatase also increased. Among six MAPK genes in rice, AAE13.1 overexpression changed expression of all examined MAPK genes after cold stress. AAE13.1 counteracts cold stress by activating expression of stress-response genes OsMAPKs, indicating that AAE13.1 enhances cold stress tolerance by regulating expression of transcription factor genes in plant cells. These findings may provide new information for our understanding of the AAE13.1 gene-related cold stress tolerance in different plant species including monocotyledonous, dicotyledonous, and gymnosperm plants.

AcknowledgementsThe authors are grateful to Dr. T. Bradshaw, Dr. R. Lischewski, and Dr. D. Thompson for their critical reading and suggestions during the preparation of this manuscript.

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