J. Pineal Res. 2014; 56:238–245
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Doi:10.1111/jpi.12115
Journal of Pineal Research
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Role of melatonin in alleviating cold stress in Arabidopsis thaliana Abstract: Melatonin (N-acetyl-5-methoxytryptamine) has been implicated in abiotic and biotic stress tolerance in plants. However, information on the effects of melatonin in cold-stress tolerance in vivo is limited. In this study, the effect of melatonin was investigated in the model plant Arabidopsis thaliana challenged with a cold stress at 4⁰C for 72 and 120 hr. Melatonin-treated plants (10 and 30 lM) had significantly higher fresh weight, primary root length, and shoot height compared with the nontreated plants. To aid in the understanding of the role of melatonin in alleviating cold stress, we investigated the effects of melatonin treatment on the expression of coldrelated genes. Melatonin up-regulated the expression of C-repeat-binding factors (CBFs)/Drought Response Element Binding factors (DREBs), a coldresponsive gene, COR15a, a transcription factor involved in freezing and drought-stress tolerance CAMTA1 and transcription activators of reactive oxygen species (ROS)-related antioxidant genes, ZAT10 and ZAT12, following cold stress. The up-regulation of cold signaling genes by melatonin may stimulate the biosynthesis of cold-protecting compounds and contribute to the increased growth of plants treated with exogenous melatonin under cold stress.
Introduction Environmental stresses such as cold temperatures are some of the greatest challenges in crop production. A freezing/ cold temperature adversely impacts crop productivity and is a serious problem to the world’s agriculture. Recent examples of significant reduction in productivity due to frost include loss of fruit crop in many states of USA in 2012 [1], 16% less corn crop in Mexico in 2011 resulting in a threefold increase in food prices [2], and reduced efficiency of flower crop production in Zimbabwe in the same year [3]. Even a modest increase of 1–2°C in the cold tolerance of crop plants can have a dramatic impact on agriculture [4]. Development of cold-tolerant plants via conventional breeding or transgenic approaches is challenging as cold tolerance is a quantitative trait controlled by multiple genes [5, 6]. Cold stress has diverse effects on plant physiology, biochemistry, and molecular biology [7]. At the cellular level, cold affects membrane fluidity and leads to accumulation of low and high molecular weight cryoprotectants, which prevent oxidative damage [4, 5, 8, 9]. The molecular mechanisms of cold tolerance are reasonably well studied in plants, especially in the model plant Arabidopsis where a number of cold-regulated genes are known [10]. In Arabidopsis, cold acclimation involves cold-regulated (COR) genes and transcription activators, C-repeat-binding factor 1 (CBF1), CBF2, and CBF3 also known as Drought Response Element Binding factor 1b (DREB 1b), DREB 1c, and DREB 1a, respectively, that bind to the promoter region of COR genes and regulate their expression [11–13]. The overexpression of CBF1 in Arabidopsis plants increases the expression of COR genes such as COR15a, resulting in increased freezing tolerance [14]. 238
Vikramjit S. Bajwa1,*, Mukund R. Shukla1,*, Sherif M. Sherif1, Susan J. Murch2 and Praveen K. Saxena1 1
Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, Guelph, ON, Canada; 2 Chemistry Department, University of British Columbia, Kelowna, BC, Canada Key words: cold stress, gene expression, growth and development, melatonin Address reprint requests to Praveen K. Saxena, Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: [email protected] *These authors contributed equally to this work. Received October 27, 2013; Accepted December 13, 2013.
COR15a codes for a major cryoprotective protein, which enhances protoplast and chloroplast freezing tolerance, putatively by changing curvature of the inner membrane and thereby stabilizing the lipid bilayers during freezing [4, 15]. Transgenic Arabidopsis plants overexpressing CBF and COR genes are not only tolerant to low-temperature stress but are also tolerant to other abiotic stresses including drought and salt stresses [14, 16–18]. ZAT10 and ZAT12 are C2H2 zinc-finger transcription activators that also have a role in cold tolerance of plants. ZAT12 regulates the expression of CBFs [19], whereas ZAT10 is a possible subregulator under CBFs affecting CBF-target genes [20]. Other important transcription factors implicated in freezing tolerance in Arabidopsis are calmodulin-binding transcription activators (CAMTAs), which act as positive regulators of CBF2. The impaired freezing tolerance of an Arabidopsis camta1 camta3 double mutant established a positive role for these proteins in cold acclimatization [19]. Mitochondria and chloroplast are hypothesized to be original sites of melatonin synthesis in eukaryotes; therefore, based on this hypothesis, the higher level of melatonin present in plants can be explained as plants contain both mitochondria and chloroplast [21]. Melatonin was first detected in vascular plants in 1995 [22, 23] and was found to be present at much higher levels in medicinal plants, feverfew (Tanacetum parthenium), St John’s wort (Hypericum perforatum), and Huang-qin (Scutellaria baicalensis) [24]. Relatively high levels of melatonin have also been detected in coffee beans, tea, Chinese herbs, fruits such as grapes, and seeds of various edible plant species, and its possible role as a germ tissue protector has been suggested [25–28]. Recently, high concentrations of melatonin (~4 lg/g of tissue) have been detected in photosynthesizing cells of Chara australis, freshwater algae, and
Role of melatonin in cold stress its protective role in preventing chlorophyll degradation and photosynthetic proteins has been described [29]. The discovery of melatonin in plants initiated research efforts to understand its physiological roles [30]. In past few years, several reports have been published that hypothesize a role for melatonin in increasing the plant’s resistance to abiotic and biotic stresses such as light, salt, drought, photoperiod, and apple blotch [31–37]. Dual role of plant melatonin, as a defense compound (phytoalexin) to protect against various environmental stresses, and as a nutraceutical/therapeutic for humans, has been proposed [38, 39]. Melatonin provides necessary protection to cell against oxidative/nitrosative stress by scavenging toxic free radicals such as very reactive hydroxyl radical (•OH), nitric oxide (NO), the peroxynitrite anion (ONOO ), singlet oxygen (1O2), superoxide anion radical (O2 ), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) [40]. Melatonin metabolites, N1-acetyl-5-methoxykynuramine (AMK), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are also powerful free radical scavengers that can scavenge a number of reactive nitrogen species including (OH) and OOCCl3 [41, 42]. The role of melatonin in cold stress is not well studied in plants, and to our knowledge, there are only three reports where melatonin has been used as cold-stress alleviator. Melatonin has been shown to alleviate cold stress in carrot suspension cells [43], cryopreserved Rhodiola crenulata callus, and cryopreserved American elm (Ulmus americana) shoot tips [44, 45]. An increase in melatonin levels was observed in cultured St. John’s wort and Aloe vera plants that were transferred from room temperature to 4°C [43]. However, the physiological and molecular role of melatonin in the induction of tolerance to cold stress in vivo remains undefined. The objectives of the present study were (i) to determine the potential role of melatonin in cold stress in Arabidopsis and (ii) to investigate the putative molecular mechanisms involved. Our data indicate that melatonin enhances cold tolerance in Arabidopsis and up-regulates the expression of cold-inducible transcriptional activators, that is, CBFs, and cold-regulated target genes, that is, COR15a. Melatonin also induces the expression of transcriptional activators such as CAMTA1, ZAT10, and ZAT12, which are implicated for their roles in cold-stress tolerance.
Material and methods Plant material and growth conditions The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in this study. Arabidopsis seeds were surface-sterilized with 70% (v/v) ethanol for 1 min and 15% bleach for 20 min and washed three times with sterile water, 5 min each wash, and then cultured in Petri dishes in a single row on half-strength Murashige and Skoog (MS) salt [46] with 1% sucrose. Media were adjusted to a pH of 5.7 prior to adding 2.2 g/L Phytagel (Sigma Aldrich, St. Louis, MO, USA) and were autoclaved for 20 min at 121°C. For media supplemented with melatonin (0–400 lM), melatonin was filter-sterilized and added to cooled (about 55°C) medium under low light conditions. All petri dishes were kept at an
angle of 65° to allow optimum root and shoot growth. Petri dishes with the seeds were kept at 4°C for two days for stratification and then transferred to standard growth conditions. Standard growth conditions for all experiments were 25°C with a 16-hr photoperiod and a light intensity of 40 lmol/m2/s (LI-250A, LI-CORâ; Biosciences, Lincoln, NE, USA), provided by cool white fluorescent tubes. Cold-stress treatment For cold-stress treatment, five-day-old Arabidopsis seedlings were transferred to 4°C with a 16-hr photoperiod and a light intensity of 15 lmol/m2/s (LI-250A, LI-CORâ; Biosciences), provided by cool white fluorescent tubes. Three levels of melatonin (0, 10, and 30 lM) were used for cold-stress study for 72 and 120 hrs. Growth parameters, including shoot height, primary root length, and fresh weight, were measured after a recovery period of 48 hr by keeping plates in ambient growth conditions. For gene expression analyses, sterilized Arabidopsis seeds were grown in 125-mL Erlenmeyer flasks containing 10 mL half-strength MS liquid medium and kept on an orbital shaker at 75 rpm. The plants were grown in the liquid media to obtain more tissue (more cytoplasmic contents) free from bacteria, fungi, and other contaminating organisms [47], and for better uptake of melatonin by the seedlings. The 11-d-old seedlings were treated with melatonin (0, 30, and 100 lM) for 24 hr and then transferred to 4°C. Tissues were collected from the flasks at different time intervals (0.5 hr, 1.5 hr, 24 hr, and 120 hr) of cold treatment, immediately flash frozen in liquid nitrogen and stored at 80°C until analyzed. RNA extraction and quantitative Real-time RT-PCR Total RNA was extracted from the cold-treated Arabidopsis seedlings using the cetyltrimethylammonium bromide (CTAB) method [48]. To remove any contaminating genomic DNA, RNA samples were treated with DNase using RNase-free DNase set (Qiagen, Toronto, ON, Canada) and then purified using RNAeasyâ Plant Mini Kit (Qiagen). After DNase treatment, total RNA was quantified (A260/280) using a Synergy H1 Hybrid Reader spectrophotometer (BioTek, VT, USA). The quality and integrity of the RNA was also tested by running the RNA samples on denaturing agarose gel, and the samples were mixed with 3X Ambionâ NorthernMaxâ Formaldehyde Load Dye before electrophoresis (Life Technologies Inc., ON, Canada). cDNA was prepared from 2.5 lg of DNase-treated RNA using SuperScriptâ VILOTM cDNA Synthesis Kit (Invitrogen, Burlington, ON, Canada) following manufacturer’s instructions. The qRT-PCR was performed using the BIO-RAD CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada), using gene-specific primers (Table S1) and Bio-Rad SsoFastTM EvaGreenâ Supermix (Bio-Rad). Two sets of synthetic oligonucleotide primers (sets A and B) were used for CBF2 and CBF3, each of the two primer set was designed from different regions of the same gene to get the highest possible confidence in our RT-PCR gene expression data (Table S1). 239
Bajwa et al. Three biological replications were performed for each treatment, and three technical replicates were carried out for each biological replicate. Expression of each gene was normalized to that of Arabidopsis b-actin and was calculated relative to that of nontreated (melatonin) control treatment. Statistical analysis All growth data were analyzed using JMP 10.0.0 (SAS Institute, Cary, NC, USA), and ANOVA was conducted to determine significance of the model followed by means comparison using Student’s t-test with a type II error rate of 0.1. For gene expression study, data were compared for significant differences using two different P-values
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Doi:10.1111/jpi.12115
Journal of Pineal Research
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Role of melatonin in alleviating cold stress in Arabidopsis thaliana Abstract: Melatonin (N-acetyl-5-methoxytryptamine) has been implicated in abiotic and biotic stress tolerance in plants. However, information on the effects of melatonin in cold-stress tolerance in vivo is limited. In this study, the effect of melatonin was investigated in the model plant Arabidopsis thaliana challenged with a cold stress at 4⁰C for 72 and 120 hr. Melatonin-treated plants (10 and 30 lM) had significantly higher fresh weight, primary root length, and shoot height compared with the nontreated plants. To aid in the understanding of the role of melatonin in alleviating cold stress, we investigated the effects of melatonin treatment on the expression of coldrelated genes. Melatonin up-regulated the expression of C-repeat-binding factors (CBFs)/Drought Response Element Binding factors (DREBs), a coldresponsive gene, COR15a, a transcription factor involved in freezing and drought-stress tolerance CAMTA1 and transcription activators of reactive oxygen species (ROS)-related antioxidant genes, ZAT10 and ZAT12, following cold stress. The up-regulation of cold signaling genes by melatonin may stimulate the biosynthesis of cold-protecting compounds and contribute to the increased growth of plants treated with exogenous melatonin under cold stress.
Introduction Environmental stresses such as cold temperatures are some of the greatest challenges in crop production. A freezing/ cold temperature adversely impacts crop productivity and is a serious problem to the world’s agriculture. Recent examples of significant reduction in productivity due to frost include loss of fruit crop in many states of USA in 2012 [1], 16% less corn crop in Mexico in 2011 resulting in a threefold increase in food prices [2], and reduced efficiency of flower crop production in Zimbabwe in the same year [3]. Even a modest increase of 1–2°C in the cold tolerance of crop plants can have a dramatic impact on agriculture [4]. Development of cold-tolerant plants via conventional breeding or transgenic approaches is challenging as cold tolerance is a quantitative trait controlled by multiple genes [5, 6]. Cold stress has diverse effects on plant physiology, biochemistry, and molecular biology [7]. At the cellular level, cold affects membrane fluidity and leads to accumulation of low and high molecular weight cryoprotectants, which prevent oxidative damage [4, 5, 8, 9]. The molecular mechanisms of cold tolerance are reasonably well studied in plants, especially in the model plant Arabidopsis where a number of cold-regulated genes are known [10]. In Arabidopsis, cold acclimation involves cold-regulated (COR) genes and transcription activators, C-repeat-binding factor 1 (CBF1), CBF2, and CBF3 also known as Drought Response Element Binding factor 1b (DREB 1b), DREB 1c, and DREB 1a, respectively, that bind to the promoter region of COR genes and regulate their expression [11–13]. The overexpression of CBF1 in Arabidopsis plants increases the expression of COR genes such as COR15a, resulting in increased freezing tolerance [14]. 238
Vikramjit S. Bajwa1,*, Mukund R. Shukla1,*, Sherif M. Sherif1, Susan J. Murch2 and Praveen K. Saxena1 1
Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, Guelph, ON, Canada; 2 Chemistry Department, University of British Columbia, Kelowna, BC, Canada Key words: cold stress, gene expression, growth and development, melatonin Address reprint requests to Praveen K. Saxena, Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: [email protected] *These authors contributed equally to this work. Received October 27, 2013; Accepted December 13, 2013.
COR15a codes for a major cryoprotective protein, which enhances protoplast and chloroplast freezing tolerance, putatively by changing curvature of the inner membrane and thereby stabilizing the lipid bilayers during freezing [4, 15]. Transgenic Arabidopsis plants overexpressing CBF and COR genes are not only tolerant to low-temperature stress but are also tolerant to other abiotic stresses including drought and salt stresses [14, 16–18]. ZAT10 and ZAT12 are C2H2 zinc-finger transcription activators that also have a role in cold tolerance of plants. ZAT12 regulates the expression of CBFs [19], whereas ZAT10 is a possible subregulator under CBFs affecting CBF-target genes [20]. Other important transcription factors implicated in freezing tolerance in Arabidopsis are calmodulin-binding transcription activators (CAMTAs), which act as positive regulators of CBF2. The impaired freezing tolerance of an Arabidopsis camta1 camta3 double mutant established a positive role for these proteins in cold acclimatization [19]. Mitochondria and chloroplast are hypothesized to be original sites of melatonin synthesis in eukaryotes; therefore, based on this hypothesis, the higher level of melatonin present in plants can be explained as plants contain both mitochondria and chloroplast [21]. Melatonin was first detected in vascular plants in 1995 [22, 23] and was found to be present at much higher levels in medicinal plants, feverfew (Tanacetum parthenium), St John’s wort (Hypericum perforatum), and Huang-qin (Scutellaria baicalensis) [24]. Relatively high levels of melatonin have also been detected in coffee beans, tea, Chinese herbs, fruits such as grapes, and seeds of various edible plant species, and its possible role as a germ tissue protector has been suggested [25–28]. Recently, high concentrations of melatonin (~4 lg/g of tissue) have been detected in photosynthesizing cells of Chara australis, freshwater algae, and
Role of melatonin in cold stress its protective role in preventing chlorophyll degradation and photosynthetic proteins has been described [29]. The discovery of melatonin in plants initiated research efforts to understand its physiological roles [30]. In past few years, several reports have been published that hypothesize a role for melatonin in increasing the plant’s resistance to abiotic and biotic stresses such as light, salt, drought, photoperiod, and apple blotch [31–37]. Dual role of plant melatonin, as a defense compound (phytoalexin) to protect against various environmental stresses, and as a nutraceutical/therapeutic for humans, has been proposed [38, 39]. Melatonin provides necessary protection to cell against oxidative/nitrosative stress by scavenging toxic free radicals such as very reactive hydroxyl radical (•OH), nitric oxide (NO), the peroxynitrite anion (ONOO ), singlet oxygen (1O2), superoxide anion radical (O2 ), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) [40]. Melatonin metabolites, N1-acetyl-5-methoxykynuramine (AMK), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are also powerful free radical scavengers that can scavenge a number of reactive nitrogen species including (OH) and OOCCl3 [41, 42]. The role of melatonin in cold stress is not well studied in plants, and to our knowledge, there are only three reports where melatonin has been used as cold-stress alleviator. Melatonin has been shown to alleviate cold stress in carrot suspension cells [43], cryopreserved Rhodiola crenulata callus, and cryopreserved American elm (Ulmus americana) shoot tips [44, 45]. An increase in melatonin levels was observed in cultured St. John’s wort and Aloe vera plants that were transferred from room temperature to 4°C [43]. However, the physiological and molecular role of melatonin in the induction of tolerance to cold stress in vivo remains undefined. The objectives of the present study were (i) to determine the potential role of melatonin in cold stress in Arabidopsis and (ii) to investigate the putative molecular mechanisms involved. Our data indicate that melatonin enhances cold tolerance in Arabidopsis and up-regulates the expression of cold-inducible transcriptional activators, that is, CBFs, and cold-regulated target genes, that is, COR15a. Melatonin also induces the expression of transcriptional activators such as CAMTA1, ZAT10, and ZAT12, which are implicated for their roles in cold-stress tolerance.
Material and methods Plant material and growth conditions The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in this study. Arabidopsis seeds were surface-sterilized with 70% (v/v) ethanol for 1 min and 15% bleach for 20 min and washed three times with sterile water, 5 min each wash, and then cultured in Petri dishes in a single row on half-strength Murashige and Skoog (MS) salt [46] with 1% sucrose. Media were adjusted to a pH of 5.7 prior to adding 2.2 g/L Phytagel (Sigma Aldrich, St. Louis, MO, USA) and were autoclaved for 20 min at 121°C. For media supplemented with melatonin (0–400 lM), melatonin was filter-sterilized and added to cooled (about 55°C) medium under low light conditions. All petri dishes were kept at an
angle of 65° to allow optimum root and shoot growth. Petri dishes with the seeds were kept at 4°C for two days for stratification and then transferred to standard growth conditions. Standard growth conditions for all experiments were 25°C with a 16-hr photoperiod and a light intensity of 40 lmol/m2/s (LI-250A, LI-CORâ; Biosciences, Lincoln, NE, USA), provided by cool white fluorescent tubes. Cold-stress treatment For cold-stress treatment, five-day-old Arabidopsis seedlings were transferred to 4°C with a 16-hr photoperiod and a light intensity of 15 lmol/m2/s (LI-250A, LI-CORâ; Biosciences), provided by cool white fluorescent tubes. Three levels of melatonin (0, 10, and 30 lM) were used for cold-stress study for 72 and 120 hrs. Growth parameters, including shoot height, primary root length, and fresh weight, were measured after a recovery period of 48 hr by keeping plates in ambient growth conditions. For gene expression analyses, sterilized Arabidopsis seeds were grown in 125-mL Erlenmeyer flasks containing 10 mL half-strength MS liquid medium and kept on an orbital shaker at 75 rpm. The plants were grown in the liquid media to obtain more tissue (more cytoplasmic contents) free from bacteria, fungi, and other contaminating organisms [47], and for better uptake of melatonin by the seedlings. The 11-d-old seedlings were treated with melatonin (0, 30, and 100 lM) for 24 hr and then transferred to 4°C. Tissues were collected from the flasks at different time intervals (0.5 hr, 1.5 hr, 24 hr, and 120 hr) of cold treatment, immediately flash frozen in liquid nitrogen and stored at 80°C until analyzed. RNA extraction and quantitative Real-time RT-PCR Total RNA was extracted from the cold-treated Arabidopsis seedlings using the cetyltrimethylammonium bromide (CTAB) method [48]. To remove any contaminating genomic DNA, RNA samples were treated with DNase using RNase-free DNase set (Qiagen, Toronto, ON, Canada) and then purified using RNAeasyâ Plant Mini Kit (Qiagen). After DNase treatment, total RNA was quantified (A260/280) using a Synergy H1 Hybrid Reader spectrophotometer (BioTek, VT, USA). The quality and integrity of the RNA was also tested by running the RNA samples on denaturing agarose gel, and the samples were mixed with 3X Ambionâ NorthernMaxâ Formaldehyde Load Dye before electrophoresis (Life Technologies Inc., ON, Canada). cDNA was prepared from 2.5 lg of DNase-treated RNA using SuperScriptâ VILOTM cDNA Synthesis Kit (Invitrogen, Burlington, ON, Canada) following manufacturer’s instructions. The qRT-PCR was performed using the BIO-RAD CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada), using gene-specific primers (Table S1) and Bio-Rad SsoFastTM EvaGreenâ Supermix (Bio-Rad). Two sets of synthetic oligonucleotide primers (sets A and B) were used for CBF2 and CBF3, each of the two primer set was designed from different regions of the same gene to get the highest possible confidence in our RT-PCR gene expression data (Table S1). 239
Bajwa et al. Three biological replications were performed for each treatment, and three technical replicates were carried out for each biological replicate. Expression of each gene was normalized to that of Arabidopsis b-actin and was calculated relative to that of nontreated (melatonin) control treatment. Statistical analysis All growth data were analyzed using JMP 10.0.0 (SAS Institute, Cary, NC, USA), and ANOVA was conducted to determine significance of the model followed by means comparison using Student’s t-test with a type II error rate of 0.1. For gene expression study, data were compared for significant differences using two different P-values
