Transcriptional Regulation of Hydrogen Peroxide and Calcium for Signaling Transduction and Stress-defensive Genes Contributing to Improved Drought Tolerance in Creeping Bentgrass

in Journal of the American Society for Horticultural Science

Small molecules, including H2O2 and Ca, mediate stress signaling and drought tolerance in plants. The objective of this study was to determine whether improvement in drought tolerance by H2O2 and Ca were associated with the regulation of transcription factors and stress-protective genes in perennial grass species. Plants of creeping bentgrass (Agrostis stolonifera) were sprayed with water (control), H2O2 (9 mm), or CaCl2 (10 mm) and exposed to drought stress for 20 days in controlled-environment growth chambers. Foliar application of H2O2 or Ca led to significant improvement in drought tolerance of creeping bentgrass, as demonstrated by greater turf quality, leaf relative water content, chlorophyll content, photochemical efficiency, and cell membrane stability, as compared with the untreated control. The application of H2O2 and Ca resulted in significant up-regulation of genes in Ca signaling transduction pathways [Ca-dependent kinase 26 (CDPK26), mitogen-activated protein kinase 1 (MAPK1), and 14-3-3] and transcript factors (WRKY75 and MYB13). For genes encoding antioxidant enzymes, H2O2 mainly enhanced superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) expression, while Ca primarily improved transcript levels of SOD, monodehydroascorbate reductase (MDHAR), and GR. In addition, heat shock protein 70 (HSP70), metallothionein 1 (MT1), and glutamine synthetase 2 (GS2) were also markedly up-regulated by H2O2 and Ca under drought stress. However, the transcript level of lipoxygenase 3 (LOX3) was significantly down-regulated by H2O2 and Ca under well-watered and drought conditions. These results imply that H2O2 and Ca commonly or differentially regulate genes expression in association with drought tolerance through activating Ca signaling pathway and regulating transcription factors and stress-protective genes expression, leading to the alleviation of lipid peroxidation, maintenance of correct protein folding and translocation, and enhancement of nitrogen metabolism under a prolonged period of drought stress in creeping bentgrass.

Abstract

Small molecules, including H2O2 and Ca, mediate stress signaling and drought tolerance in plants. The objective of this study was to determine whether improvement in drought tolerance by H2O2 and Ca were associated with the regulation of transcription factors and stress-protective genes in perennial grass species. Plants of creeping bentgrass (Agrostis stolonifera) were sprayed with water (control), H2O2 (9 mm), or CaCl2 (10 mm) and exposed to drought stress for 20 days in controlled-environment growth chambers. Foliar application of H2O2 or Ca led to significant improvement in drought tolerance of creeping bentgrass, as demonstrated by greater turf quality, leaf relative water content, chlorophyll content, photochemical efficiency, and cell membrane stability, as compared with the untreated control. The application of H2O2 and Ca resulted in significant up-regulation of genes in Ca signaling transduction pathways [Ca-dependent kinase 26 (CDPK26), mitogen-activated protein kinase 1 (MAPK1), and 14-3-3] and transcript factors (WRKY75 and MYB13). For genes encoding antioxidant enzymes, H2O2 mainly enhanced superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) expression, while Ca primarily improved transcript levels of SOD, monodehydroascorbate reductase (MDHAR), and GR. In addition, heat shock protein 70 (HSP70), metallothionein 1 (MT1), and glutamine synthetase 2 (GS2) were also markedly up-regulated by H2O2 and Ca under drought stress. However, the transcript level of lipoxygenase 3 (LOX3) was significantly down-regulated by H2O2 and Ca under well-watered and drought conditions. These results imply that H2O2 and Ca commonly or differentially regulate genes expression in association with drought tolerance through activating Ca signaling pathway and regulating transcription factors and stress-protective genes expression, leading to the alleviation of lipid peroxidation, maintenance of correct protein folding and translocation, and enhancement of nitrogen metabolism under a prolonged period of drought stress in creeping bentgrass.

Plant responses to drought stress involve multiple mechanisms at molecular, biochemical, physiological, and metabolic levels (Bhargava and Sawant, 2013; Shanker et al., 2014; Todaka et al., 2015). Many small molecules, such as Ca and H2O2, play important roles in stress signaling and responses in plants (Apel and Hirt, 2004; Ermak and Davies, 2002; Reddy et al., 2011). Calcium could serve as an inorganic osmolyte for maintaining cell osmotic potential or signaling molecule for stress signal transduction in plants under abiotic stress, such as drought, high temperature, or salt stress (Bush, 1995). The study of Jiang and Huang (2001) found that exogenous Ca application increased Ca concentration in cell saps contributing to improved osmotic adjustment and enhanced antioxidant capacity in two cool-season grass species under heat stress. Stress-triggered change of cytosolic Ca2+ in guard cells was correlated with stomatal closure, indicating the importance of Ca2+ signaling in drought responses (Fu and Lu, 2007). It has been found that exogenously applied Ca altered some antioxidant enzyme activities and enhanced root activity as well as the accumulation of osmolytes, which could improve drought tolerance of white clover (Trifolium repens), wheat (Triticum aestivum), and maize (Zea mays) (Li et al., 2015; Nayyar, 2003; Wang, 2010). Foliar Ca spray also effectively alleviated drought-induced growth inhibition, photoinhibition, and decline in leaf water in maize and sugar beet [Beta vulgaris (Hosseini et al., 2019; Naeem et al., 2018)]. These findings indicate positive function of Ca on improving drought tolerance involved in multiple physiological changes in plants.

The higher concentration of H2O2 can cause lipid peroxidation, proteins degradation, accelerated senescence, and even programmed cell death, whereas the lower level and rapidly alteration of H2O2 acts as critical regulatory roles in intermediate signaling transduction for activation of defense mechanisms in plants during early phases of stress responses (Ray et al., 2012; Suzuki et al., 2012; Yu, 1994). Previous studies demonstrated that exogenous H2O2 enhanced heat tolerance through improving cell membrane stability, leaf photosynthesis, and antioxidant enzyme activities in creeping bentgrass [Agrostis stolonifera (Larkindale and Huang, 2004, 2005)]. The appropriate low concentration of foliar H2O2 application alleviated drought damage in soybean (Glycine max) and marigold (Tagetes erecta) associated with carbohydrate accumulation and roots development (Ishibashi et al., 2011; Liao et al., 2012). Abscisic acid (ABA) triggered H2O2 signaling to induce antioxidant defense, thereby alleviating drought-caused oxidative damage in bermudagrass [Cynodon dactylon (Lu et al., 2009)]. It has been widely reported that both of H2O2 and Ca2+ could regulate stress-related downstream genes expression associated with improved drought tolerance in different plant species (Li et al., 2015; Neill et al., 2002; Xu et al., 2015; You et al., 2013). However, H2O2- and Ca-regulated signaling transduction, and key genes controlling drought tolerance were not well documented in plants exposed to severe or a prolonged period of drought stress.

Objectives of this study were to 1) assess effects of H2O2 and Ca signaling molecules on improving drought tolerance through physiological analysis; and 2) examine genes transcript level encoding signaling transduction, transcription factors, antioxidant enzymes, and stress-related proteins that may be commonly or differentially regulated by H2O2 and Ca associated with drought tolerance in creeping bentgrass under a prolonged drought stress.

Materials and Methods

Plant material and treatment.

Creeping bentgrass (‘Penncross’) sod plugs (5 cm diameter) were collected from Rutgers University Horticultural Farm II (North Brunswick, NJ) and planted in plastic containers (40 cm length, 30 cm width, and 35 cm height). A total of eight containers were used, and each container includes three sod plugs. Fritted clay was used as soil matrix, and all containers were placed in a greenhouse [average 23/18 °C of day/night, 790 μmol·m−2·s−1 photosynthetically active radiation (PAR)]. Plants were trimmed to maintain a canopy height of 3 cm and irrigated twice per week with Hoagland’s solution (Hogland and Arnon, 1950) for 2 months in the greenhouse during September–October. Plants were then moved to controlled growth chambers (Environmental Growth Chamber, Chagrin Falls, OH) that provided 21/19 °C (day/night), 70% relative humidity, and 12-h photoperiod at 660 μmol·m−2·s−1 PAR. After acclimation in the growth chamber for 1 week, each sod plug was sprayed with 10 mL of 9 mm H2O2 or 10 mm CaCl2 solution or distilled water (untreated control) once each day for 3 d. After pretreatments, plants were cultivated under the well-watered condition (plants were irrigated every 2 d and soil water content was maintained at the pot capacity) or exposed to drought stress (stopping irrigating) for 20 d during the month of November. The concentration of H2O2 and CaCl2 were selected based on a preliminary experiment. Each treatment has four replications in four different containers that were placed in four growth chambers. Leaf samples were collected from plants at 20 d of drought stress treatment.

Measurements of turf quality and physiological parameters.

Turf quality (TQ) was evaluated by using a scale of 1 to 9 according to color, density, and uniformity of turfgrass (Beard, 2001). Leaf relative water content (RWC) or electrolyte leakage (EL) was detected by using the method of Barrs and Weatherley (1962) or Blum and Ebercon (1981), respectively. Assay methods in details were recorded in our previous study (Li et al., 2016a). For chlorophyll (Chl) content, 0.1 g of fresh leaves were cut from plants and submerged in 10 mL of dimethyl sulphoxide. After being placed in the dark for 48 h, leaves extractions were measured at 663 and 645 nm using a spectrophotometer (Spectronic Instruments, Rochester, NY) (Arnon, 1949). For photochemical efficiency (Fv/Fm), leaves were pretreated into darkness through leaf clips for 20 min. Fv/Fm ratio was then recorded by using a fluorescence meter (Fim 1500; Dynamax, Houston, TX).

Gene expression analysis.

For genes expression analysis, real-time quantitative polymerase chain reaction (qRT-PCR) was used. RNeasy Mini Kit (Qiagen, Duesseldorf, Germany) was used for extracting total RNAs in fresh leaves according to manufacturer’s instructions. The RNA was then reverse-transcribed into cDNA (A revert Aid First Stand cDNA Synthesis Kit; Fermentas, Burlington, ON, Canada). For the PCR protocol, the conditions were set: 5 min at 95 °C, and 40 repeats of denaturation at 95 °C for 15 s, annealing at 60 °C for 45 s, followed by heating the amplicon from 60 to 95 °C to obtain the melting curve. The Eq. The formula 2-ΔΔCt was used for calculating the transcript level of all genes (Xia et al., 2009). Table 1 shows primer sequences of all genes, including reference gene ACT2.

Table 1.

Primer sequences used for detecting transcript levels of genes in real-time quantitative polymerase chain reaction (qRT-PCR) and their corresponding GeneBank accession numbers of the analyzed genes.

Table 1.

Statistical analysis.

Experiment design was a split-plot design with water status as the main plot and small molecules (H2O, Ca, and H2O2) treatments as the sub-plot. Data were analyzed by using the general linear model procedure for the analysis of variance (SAS version 9.1; SAS Institute, Cary, NC). The significance of differences was tested by using Fisher’s protected least significance test with P ≤ 0.05.

Results

Physiological responses to H2O2 and Ca in creeping bentgrass.

H2O2 and Ca application had no significant effects on TQ, RWC, EL, Chl, and Fv/Fm in leaves under well-watered condition. Drought stress significantly decreased TQ, RWC, Chl content, and Fv/Fm ratio, but increased EL of leaves (Figs. 1 and 2). Under drought stress, H2O2- and Ca-treated plants exhibited significantly higher TQ and RWC and lower EL than nontreated plants (Fig. 1). Both H2O2- and Ca-treated plants maintained a 30% increase in Chl content compared with nontreated plants in response to drought stress, and Fv/Fm ratio was also significantly higher in H2O2- and Ca-treated plants than that in nontreated plants under drought stress (Fig. 2).

Fig. 1.
Fig. 1.

Effects of H2O2 and CaCl2 on (A) turf quality, (B) relative water content, and (C) electrolyte leakage in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Fig. 2.
Fig. 2.

Effects of H2O2 and CaCl2 on (A) chlorophyll content and (B) photochemical efficiency in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Expression of genes involved in signaling transduction and transcription factors by H2O2 and Ca.

In response to drought stress, most of genes expression levels were affected by H2O2 and Ca2+ (Fig. 3A). H2O2 and Ca induced significant changes in five genes, while four genes were only regulated by Ca under well-watered conditions (Fig. 3B). Twelve genes were affected by both H2O2 and Ca, and two genes were specifically regulated by H2O2 under drought stress. Only one gene was specifically induced by Ca under drought stress (Fig. 3C). Under well-watered conditions, foliar spraying with Ca upregulated the expression levels of CDPK26, MAPK1, and MYB13, and the application of H2O2 only upregulated MAPK1 expression compared with the well-watered control (Figs. 4 and 5). Drought stress induced significant increases in CDPK26, MAPK1, 14-3-3, ABF3, WRKY75, and MYB13 transcript levels with or without H2O2 and Ca application (Figs. 4 and 5). Under drought stress, both H2O2- and Ca-treated plants showed 4-fold increases in CDPK26 and 14-3-3 as well as 5-fold increase in MAPK1 expression level compared with nontreated plants (Fig. 4). For transcription factors, there was six times as high WRKY75 and MYB13 transcript levels in H2O2-treated plants as in nontreated plants under drought conditions (Fig. 5B and C). Similarly, WRKY75 and MYB13 transcript levels of Ca-treated plants were three times significantly higher than that of nontreated plants under drought stress (Fig. 5B and C). However, H2O2 and Ca had no effect on the transcript level of ABF3 under well-watered and drought conditions (Fig. 5A).

Fig. 3.
Fig. 3.

(A) Heat map of changes in 18 genes expression levels in creeping bentgrass under well-watered and drought condition, (B) differential regulated genes induced by H2O2 and CaCl2 under well-watered condition, and (C) differential regulated genes induced by H2O2 and CaCl2 under drought condition in creeping bentgrass. The log2 fold change ratios are shown in the results. Red indicates an upregulation, and green indicates a downregulation.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Fig. 4.
Fig. 4.

Effects of H2O2 and CaCl2 on genes expression of (A) Ca-dependent kinase 26 (CDPK26), (B) mitogen-activated protein kinase 1 (MAPK1), and (C) 14-3-3 in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Fig. 5.
Fig. 5.

Effects of H2O2 and CaCl2 on genes expression of transcription factors (A) ABRE binding factor 3 (ABF3), (B) WRKY transcription factor 75 (WRKY75), and (C) MYB transcription factor 13 (MYB13) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Expression of genes involved in antioxidant defense affected by H2O2 and Ca.

Exogenous application of H2O2 and Ca significantly upregulated CAT, MDHAR, and GR transcript levels under well-watered conditions (Fig. 6B, D, and F). Under drought conditions, SOD was upregulated by exogenous H2O2 and Ca (Fig. 6A). Exogenous H2O2 application upregulated CAT and DHAR, but Ca had no significant effects on these two genes under drought stress (Fig. 6B and E). Neither H2O2 nor Ca application had significant effects on the expression of ascorbate peroxidase (APX) under well-watered or drought conditions (Fig. 6C). However, exogenous Ca upregulated MDHAR expression under drought stress, and the increased percentage was 114% (Fig. 6D). GR transcript level in H2O2- and Ca-treated plants was significantly higher than that in nontreated plants by two times under drought stress (Fig. 6F).

Fig. 6.
Fig. 6.

Effects of H2O2 and CaCl2 on genes encoding antioxidant enzyme (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) ascorbate peroxidase (APX), (D) monodehydroascorbate reductase (MDHAR), (E) dehydroascorbate reductase (DHAR), and (F) glutathione reductase (GR) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Expression of genes involved in stress-protective proteins and other metabolism affected by H2O2 and Ca.

Under well-watered condition, heat shock protein 90 (HSP90) and MT1 expression were not changed by foliar application of H2O2 and Ca, and exogenous H2O2 upregulated HSP70 and dehydrin 3 (DHN3); but Ca did not show significant effects on HSP70 and DHN3 expression level (Fig. 7). Under drought stress, HSP70 expression level of H2O2- and Ca-treated plants increased by 60% compared with nontreated plants, and H2O2- and Ca-treated plants also had 3-fold increases in MT1 transcript level compared with nontreated plants; but HSP90 expression was unaffected by exogenous application of H2O2 and Ca (Fig. 7A and D). Drought stress induced more than 500 times increase in DHN3 in all treatments relative to well-watered plants, but drought-stressed plants treated with H2O2 and Ca exhibited significantly lower DHN3 expression than drought-stressed plants without chemical treatment (Fig. 7C). Foliar application of H2O2 and Ca had no significant effects on GS2 expression, whereas LOX3 was significantly downregulated by H2O2 under well-watered condition (Fig. 8). Under drought stress, the GS2 transcript level in H2O2- and Ca-treated plants had three times increase compared with drought-stressed control plants (Fig. 8A). On the contrary, LOX3 expression was significantly inhibited by H2O2 and Ca under well-watered and drought conditions (Fig. 8B).

Fig. 7.
Fig. 7.

Effects of H2O2 and CaCl2 on genes encoding stress-related proteins (A) heat shock protein 70 (HSP70), (B) heat shock protein 90 (HSP90), (C) dehydrin 3 (DHN3), and (D) metallothionein 1 (MT1) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Fig. 8.
Fig. 8.

Effects of H2O2 and CaCl2 on genes encoding stress-related proteins (A) glutamine synthetase 2 (GS2) and (B) lipoxygenase 3 (LOX3) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

Discussion

Plant tolerance to drought stress involves the activation of stress signaling transduction pathways, which include multiple transcriptional factors or genes (Dortje et al., 2011). CDPK and MAPK are two critical kinases involved in Ca2+ signaling transduction pathways (Anil and Rao, 2001). CDPK and MAPK signaling transduction regulate transcription factors such as WRKY and MYB families, which play critical roles in drought tolerance in plants via the activation of downstream signaling and stress-defensive genes (Boudsocq and Sheen, 2013; Chen et al., 2012; Danquah et al., 2014; Singh et al., 2002). Previous studies have proved that Ca2+ and reactive oxygen species (ROS) signaling are integrated in cells in most of cases. Ca2+ channels could be activated by H2O2, and ROS signaling production also could be directly regulated by Ca2+ signaling under normal and abiotic stress conditions (Camello-Almaraz et al., 2006; Gilroy et al., 2014; Pei et al., 2000). The research of Li et al. (2015) indicated that H2O2 interacting with Ca2+ signaling were involved in polyamine-regulated drought tolerance in white clover though activating CDPK signaling. The 14-3-3 proteins are important conserved signaling proteins involved in cellular signaling transduction and also have major regulatory function of carbohydrate and nitrogen metabolism in plants (Comparot et al., 2003; Roberts et al., 2002). In addition, 14-3-3 proteins also could regulate multiple signal transduction proteins such as CDPK in response to environmental stresses in higher plants (Roberts et al., 2002). Previous studies indicated that 14-3-3 protein or genes could be differentially mediated by salinity, drought, and cold, which is an important part of stress defense signaling in plants (Chen et al., 1994; Chen et al., 2006; Jarillo et al., 1994). It has been reported that overexpressing a tomato 14-3-3 gene enhanced salt tolerance in Arabidopsis thaliana (Xu and Shi, 2007). A “stay-green” phenotype and better drought tolerance were observed in transgenic cotton overexpressing a 14-3-3 gene (Yan et al., 2004). In this study, the application of H2O2 and Ca up-regulated 14-3-3, CDPK26, MAPK1, WRKY75, and MYB13 in creeping bentgrass exposed to drought stress. Our results, together with reports in previous studies, suggest that H2O2 and Ca could regulate 14-3-3, CDPK26, MAPK1, WRKY75, and MYB13, which may contribute to improved drought tolerance due to the application of those two molecules in creeping bentgrass, as manifested by increased TQ, leaf RWC, Chl content, Fv/Fm, and cell membrane stability under drought stress.

In addition to transcriptional factors in signaling pathways, changes in downstream stress-protective genes also affect plant tolerance to drought stress (Shinozaki and Yamaguchi-Shinozaki, 2007). H2O2 and Ca up-regulated HSP70 and MT1 under drought stress in our study. HSPs are important molecular chaperones that help proteins to fold and assemble correctly related to plant adaption to abiotic stress (Sabehat et al., 1998; Sørensen et al., 2003; Wang et al., 2004). HSPs not only play positive roles in regulating heat tolerance, but also are correlated with the improvement of drought tolerance in plants. For example, the transgenic tobacco (Nicotiana tabacum) constitutively expressing a HSP70 obtained stress tolerance under progressive drought (Cho and Hong, 2006). MT1 gene regulates synthesis of metallothionein, which plays roles in stabilization of cellular membranes, antioxidant, and metal ion homeostasis (Ruttkay-Nedecky et al., 2013). Transgenic rice (Oryza sativa) overexpressing an OsMT1a demonstrated the significant increase in drought tolerance associated with ROS scavenging and ions homeostasis (Yang et al., 2009). The improved accumulation of MT played an important role in spermidine-regulated drought tolerance in white clover (Li et al., 2016b). Current results suggest that H2O2 and Ca-regulated drought tolerance in creeping bentgrass could be associated with activation of HSP70 and MT1 expression. DHNs encoding dedydrins are drought-inducible genes and their up-regulation may protect cells from drought damages and may reflect the level of drought stress that the plant has experienced, depending on stress duration and severity (Hara, 2010). In our study, the transcript level of DHN3 increased by thousands of times in response to drought stress, while H2O2 and Ca application suppressed the up-regulation of DHN3 under drought stress, indicating H2O2- and Ca-treated plants might have suffered less stress damage.

Antioxidant defense is another stress-defense pathway including enzymatic and nonenzymatic components. Antioxidant enzymes function as scavengers of ROS in cells, thereby alleviating stress-induced oxidative damage (Hasanuzzaman et al., 2012). H2O2 and Ca act as mediators to participate in regulating antioxidant defense. For example, ABA triggered NADPH oxidase to release H2O2, resulting in the activation of SOD, CAT, and APX against oxidative damage in bermudagrass (Lu et al., 2009). Brassinosteroid-induced H2O2 accumulation was accompanied by increases in SOD, CAT, and key enzymes involved in an ascorbate-glutathione cycle leading to enhanced tolerance to oxidative stress in cucumber (Cucumis sativus) leaves (Xia et al., 2009). Polyamine could activate H2O2 and Ca signaling to regulate antioxidant enzyme activities and genes expression in white clover under water deficit condition (Li et al., 2015). Foliar applied Ca significantly increased CAT, GR, and APX activities associated with significant declines in membrane lipid peroxidation in two cool-season grasses, tall fescue (Festuca arundinacea) and kentucky bluegrass (Poa pratensis), under heat stress (Jiang and Huang, 2001). In this current study, SOD, CAT, DHAR, and GR were up-regulated by H2O2, whereas Ca mainly induced SOD, MDHAR, and GR expression under drought stress, which may contribute to improved drought tolerance; however, H2O2 and Ca regulated antioxidant defense through different enzymatic components in creeping bentgrass. In addition to ROS-induced oxidative damages, dioxygenation of polyunsaturated fatty acids catalyzed by lipoxygenase (LOX) also causes membrane lipid peroxidation in plants (Ángel-Coronel et al., 2017; Fukuchi-Mizutani et al., 2000; Siedow, 1991). The suppression of LOX activity by silicon has been associated with mitigation of drought-induced oxidative damages in eight chickpea (Cicer arietinum) cultivars (Gunes et al., 2007). In this study, the transcript level of LOX3 was significantly down-regulated by H2O2 and Ca in creeping bentgrass under drought stress, indicating that H2O2 and Ca could also alleviate oxidative damages involving LOX.

Abiotic stress causes excessive NH3-NH4+ accumulation that is toxic to plants (Rare, 1990; Yu et al., 2005). Glutamine synthetase (GS) is responsible for amine assimilation and catalyzing the synthesis of glutamine under environmental stress. It helps plants to detoxify the excess free ammonia in cells in response to abiotic stress, leading to effective alleviation of stress-caused excessive NH3-NH4+ accumulation (Guan et al., 2016; Yu et al., 2005). In this study, both H2O2 and Ca significantly up-regulated GS2 expression under drought stress, which implies that H2O2 and Ca could regulate amine assimilation in plants suffering from drought stress.

Conclusions

In summary, the application of H2O2 and Ca significantly enhanced drought tolerance of creeping bentgrass, as demonstrated by improved turf quality, leaf RWC, Chl content, photochemical efficiency, and cell membrane stability under drought stress. The positive effects of H2O2 and Ca on drought tolerance could be associated with the activation of Ca signaling pathways and transcript factors (14-3-3, CDPK26, MAPK1, WRKY75, and MYB13), antioxidant defense (SOD, CAT, GR, MDHAR, and DHAR) alleviating oxidative damage, and stress protection genes (HSP70, MT1, and GS2) assisting protein folding and translocation, and maintaining nitrogen metabolism under a prolonged drought stress (Fig. 9).

Fig. 9.
Fig. 9.

Proposed pathways regulated by H2O2 and CaCl2 contributing to drought tolerance in creeping bentgrass.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 4; 10.21273/JASHS04901-19

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  • DanquahA.de ZelicourtA.ColcombetJ.HirtH.2014The role of ABA and MAPK signaling pathways in plant abiotic stress responsesBiotechnol. Adv.324052

    • Search Google Scholar
    • Export Citation
  • DortjeG.InesL.OksoonY.2011Plant tolerance to drought and salinity: Stress regulating transcription factors and their functional significance in the cellular transcriptional networkPlant Cell Rep.3013831391

    • Search Google Scholar
    • Export Citation
  • ErmakG.DaviesK.J.2002Calcium and oxidative stress: From cell signaling to cell deathMol. Immunol.38713721

  • FuD.LuM.2007The structural basis of water permeation and proton exclusion in aquaporinsMol. Membr. Biol.24366374

  • Fukuchi-MizutaniM.IshiguroK.NakayamaT.UtsunomiyaY.TanakaY.KusumiT.UedaT.2000Molecular and functional characterization of a rose lipoxygenase cDNA related to flower senescencePlant Sci.160129137

    • Search Google Scholar
    • Export Citation
  • GilroyS.SuzukiN.MillerG.ChoiW.G.ToyotaM.DevireddyA.R.MittlerR.2014A tidal wave of signals: Calcium and ROS at the forefront of rapid systemic signalingTrends Plant Sci.19623630

    • Search Google Scholar
    • Export Citation
  • GuanM.de BangT.PedersenC.SchjoerringJ.K.2016Cytosolic glutamine synthetase Gln1; 2 is the main isozyme contributing to GS1 activity in arabidopsis shoots and can be up-regulated to relieve ammonium toxicityPlant Physiol.17119211933

    • Search Google Scholar
    • Export Citation
  • GunesA.PilbeamD.J.InalA.BagciE.G.CobanS.2007Influence of silicon on antioxidant mechanisms and lipid peroxidation in chickpea (Cicer arietinum L.) cultivars under drought stressJ. Plant Interact.2105113

    • Search Google Scholar
    • Export Citation
  • HaraM.2010The multifunctionality of dehydrins: An overviewPlant Signal. Behav.5503508

  • HasanuzzamanM.HossainM.A.da SilvaJ.A.T.FujitaM.2012Plant response and tolerance to abiotic oxidative stress: Antioxidant defense is a key factor p. 261–315. In: B. Venkateswarlu A.K. Shanker C. Shanker and M. Maheswari (eds.). Crop stress and its management: Perspectives and strategies. Springer Berlin Germany

  • HoglandC.R.ArnonD.I.1950The solution culture method for growing plants without soil. Calif. Agr. Exp. Circ. 347

  • HosseiniS.A.RéthoréE.PluchonS.AliN.BilliotB.YvinJ.C.2019Calcium application enhances drought stress tolerance in sugar beet and promotes plant biomass and beetroot sucrose concentrationIntl. J. Mol. Sci.203777

    • Search Google Scholar
    • Export Citation
  • IshibashiY.YamaguchiH.YuasaT.Iwaya-InoueM.ArimaS.ZhengS.H.2011Hydrogen peroxide spraying alleviates drought stress in soybean plantsJ. Plant Physiol.16815621567

    • Search Google Scholar
    • Export Citation
  • JarilloJ.A.CapelJ.LeyvaA.Martínez-ZapaterJ.M.SalinasJ.1994Two related low-temperature-inducible genes of arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulatorsPlant Mol. Biol.25693704

    • Search Google Scholar
    • Export Citation
  • JiangY.HuangB.2001Effects of calcium on antioxidant activities and water relations associated with heat tolerance in two cool-season grassesJ. Exp. Bot.52341349

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HuangB.2004Thermotolerance and antioxidant systems in Agrostis stolonifera: Involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethyleneJ. Plant Physiol.161405413

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HuangB.2005Effects of abscisic acid, salicylic acid, ethylene and hydrogen peroxide in thermotolerance and recovery for creeping bentgrassPlant Growth Regulat.471728

    • Search Google Scholar
    • Export Citation
  • LiZ.ZhangY.PengD.WangX.PengY.HeX.ZhangX.MaX.HuangL.YanY.2015Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signalingFront. Physiol.6280

    • Search Google Scholar
    • Export Citation
  • LiZ.PengY.HuangB.2016aPhysiological effects of γ-aminobutyric acid application on improving heat and drought tolerance in creeping bentgrassJ. Amer. Soc. Hort. Sci.1417684

    • Search Google Scholar
    • Export Citation
  • LiZ.ZhangY.ZhangX.PengY.MerewitzE.MaX.HuangL.YanY.2016bThe alterations of endogenous polyamines and phytohormones induced by exogenous application of spermidine regulate antioxidant metabolism, metallothionein and relevant genes conferring drought tolerance in white cloverEnviron. Exp. Bot.1242238

    • Search Google Scholar
    • Export Citation
  • LiaoW.B.HuangG.B.YuJ.H.ZhangM.L.2012Nitric oxide and hydrogen peroxide alleviate drought stress in marigold explants and promote its adventitious root developmentPlant Physiol. Biochem.58615

    • Search Google Scholar
    • Export Citation
  • LuS.SuW.LiH.GuoZ.2009Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activitiesPlant Physiol. Biochem.47132138

    • Search Google Scholar
    • Export Citation
  • NaeemM.NaeemM.S.AhmadR.IhsanM.Z.AshrafM.Y.HussainY.FahadS.2018Foliar calcium spray confers drought stress tolerance in maize via modulation of plant growth, water relations, proline content and hydrogen peroxide activityArch. Agron. Soil Sci.64116131

    • Search Google Scholar
    • Export Citation
  • NayyarH.2003Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonistsEnviron. Exp. Bot.50253264

    • Search Google Scholar
    • Export Citation
  • NeillS.J.DesikanR.ClarkeA.HurstR.D.HancockJ.T.2002Hydrogen peroxide and nitric oxide as signalling molecules in plantsJ. Exp. Bot.5312371247

    • Search Google Scholar
    • Export Citation
  • PeiZ.M.MurataY.BenningG.ThomineS.KlüsenerB.AllenG.J.GrillE.SchroederJ.I.2000Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cellsNature406731734

    • Search Google Scholar
    • Export Citation
  • RareE.1990Stress physiology: The functional significance of the accumulation of nitrogen-containing compoundsJ. Hort. Sci.65231243

  • RayP.D.HuangB.W.TsujiY.2012Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalingCell. Signal.24981990

  • ReddyA.S.AliG.S.CelesnikH.DayI.S.2011Coping with stresses: Roles of calcium- and calcium/calmodulin-regulated gene expressionPlant Cell2320102032

    • Search Google Scholar
    • Export Citation
  • RobertsM.R.SalinasJ.CollingeD.B.200214-3-3 proteins and the response to abiotic and biotic stressPlant Mol. Biol.5010311039

  • Ruttkay-NedeckyB.NejdlL.GumulecJ.ZitkaO.MasarikM.EckschlagerT.StiborovaM.AdamV.KizekR.2013The role of metallothionein in oxidative stressIntl. J. Mol. Sci.1460446066

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • TodakaD.ShinozakiK.Yamaguchi-ShinozakiK.2015Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plantsFront. Plant Sci.684

    • Search Google Scholar
    • Export Citation
  • WangC.Q.2010Exogenous calcium alters activities of antioxidant enzymes in Trifolium repens L. leaves under peg-induced water deficitJ. Plant Nutr.3318741885

    • Search Google Scholar
    • Export Citation
  • WangW.VinocurB.ShoseyovO.AltmanA.2004Role of plant heat-shock proteins and molecular chaperones in the abiotic stress responseTrends Plant Sci.9244252

    • Search Google Scholar
    • Export Citation
  • XiaX.J.WangY.J.ZhouY.H.TaoY.MaoW.H.ShiK.AsamiT.ChenZ.YuJ.Q.2009Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumberPlant Physiol.150801814

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Contributor Notes

This research was supported by Sichuan Science and Technology Program (Grant No. 2017HH0060) and the Center for Turfgrass Science at Rutgers University.B.H. is the corresponding author. E-mail: huang@sebs.rutgers.edu.
  • View in gallery

    Effects of H2O2 and CaCl2 on (A) turf quality, (B) relative water content, and (C) electrolyte leakage in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Effects of H2O2 and CaCl2 on (A) chlorophyll content and (B) photochemical efficiency in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    (A) Heat map of changes in 18 genes expression levels in creeping bentgrass under well-watered and drought condition, (B) differential regulated genes induced by H2O2 and CaCl2 under well-watered condition, and (C) differential regulated genes induced by H2O2 and CaCl2 under drought condition in creeping bentgrass. The log2 fold change ratios are shown in the results. Red indicates an upregulation, and green indicates a downregulation.

  • View in gallery

    Effects of H2O2 and CaCl2 on genes expression of (A) Ca-dependent kinase 26 (CDPK26), (B) mitogen-activated protein kinase 1 (MAPK1), and (C) 14-3-3 in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Effects of H2O2 and CaCl2 on genes expression of transcription factors (A) ABRE binding factor 3 (ABF3), (B) WRKY transcription factor 75 (WRKY75), and (C) MYB transcription factor 13 (MYB13) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Effects of H2O2 and CaCl2 on genes encoding antioxidant enzyme (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) ascorbate peroxidase (APX), (D) monodehydroascorbate reductase (MDHAR), (E) dehydroascorbate reductase (DHAR), and (F) glutathione reductase (GR) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Effects of H2O2 and CaCl2 on genes encoding stress-related proteins (A) heat shock protein 70 (HSP70), (B) heat shock protein 90 (HSP90), (C) dehydrin 3 (DHN3), and (D) metallothionein 1 (MT1) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Effects of H2O2 and CaCl2 on genes encoding stress-related proteins (A) glutamine synthetase 2 (GS2) and (B) lipoxygenase 3 (LOX3) in creeping bentgrass under well-watered and drought condition for 20 d. Vertical bars indicate ±se (n = 4). Different letters above columns indicate significant differences among control, H2O2, and Ca treatment under a given condition (well-watered or drought); asterisk (*) indicates significant difference for one particular treatment (control, H2O2, or Ca) between well-watered and drought condition.

  • View in gallery

    Proposed pathways regulated by H2O2 and CaCl2 contributing to drought tolerance in creeping bentgrass.

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    • Search Google Scholar
    • Export Citation
  • DortjeG.InesL.OksoonY.2011Plant tolerance to drought and salinity: Stress regulating transcription factors and their functional significance in the cellular transcriptional networkPlant Cell Rep.3013831391

    • Search Google Scholar
    • Export Citation
  • ErmakG.DaviesK.J.2002Calcium and oxidative stress: From cell signaling to cell deathMol. Immunol.38713721

  • FuD.LuM.2007The structural basis of water permeation and proton exclusion in aquaporinsMol. Membr. Biol.24366374

  • Fukuchi-MizutaniM.IshiguroK.NakayamaT.UtsunomiyaY.TanakaY.KusumiT.UedaT.2000Molecular and functional characterization of a rose lipoxygenase cDNA related to flower senescencePlant Sci.160129137

    • Search Google Scholar
    • Export Citation
  • GilroyS.SuzukiN.MillerG.ChoiW.G.ToyotaM.DevireddyA.R.MittlerR.2014A tidal wave of signals: Calcium and ROS at the forefront of rapid systemic signalingTrends Plant Sci.19623630

    • Search Google Scholar
    • Export Citation
  • GuanM.de BangT.PedersenC.SchjoerringJ.K.2016Cytosolic glutamine synthetase Gln1; 2 is the main isozyme contributing to GS1 activity in arabidopsis shoots and can be up-regulated to relieve ammonium toxicityPlant Physiol.17119211933

    • Search Google Scholar
    • Export Citation
  • GunesA.PilbeamD.J.InalA.BagciE.G.CobanS.2007Influence of silicon on antioxidant mechanisms and lipid peroxidation in chickpea (Cicer arietinum L.) cultivars under drought stressJ. Plant Interact.2105113

    • Search Google Scholar
    • Export Citation
  • HaraM.2010The multifunctionality of dehydrins: An overviewPlant Signal. Behav.5503508

  • HasanuzzamanM.HossainM.A.da SilvaJ.A.T.FujitaM.2012Plant response and tolerance to abiotic oxidative stress: Antioxidant defense is a key factor p. 261–315. In: B. Venkateswarlu A.K. Shanker C. Shanker and M. Maheswari (eds.). Crop stress and its management: Perspectives and strategies. Springer Berlin Germany

  • HoglandC.R.ArnonD.I.1950The solution culture method for growing plants without soil. Calif. Agr. Exp. Circ. 347

  • HosseiniS.A.RéthoréE.PluchonS.AliN.BilliotB.YvinJ.C.2019Calcium application enhances drought stress tolerance in sugar beet and promotes plant biomass and beetroot sucrose concentrationIntl. J. Mol. Sci.203777

    • Search Google Scholar
    • Export Citation
  • IshibashiY.YamaguchiH.YuasaT.Iwaya-InoueM.ArimaS.ZhengS.H.2011Hydrogen peroxide spraying alleviates drought stress in soybean plantsJ. Plant Physiol.16815621567

    • Search Google Scholar
    • Export Citation
  • JarilloJ.A.CapelJ.LeyvaA.Martínez-ZapaterJ.M.SalinasJ.1994Two related low-temperature-inducible genes of arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulatorsPlant Mol. Biol.25693704

    • Search Google Scholar
    • Export Citation
  • JiangY.HuangB.2001Effects of calcium on antioxidant activities and water relations associated with heat tolerance in two cool-season grassesJ. Exp. Bot.52341349

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HuangB.2004Thermotolerance and antioxidant systems in Agrostis stolonifera: Involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethyleneJ. Plant Physiol.161405413

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HuangB.2005Effects of abscisic acid, salicylic acid, ethylene and hydrogen peroxide in thermotolerance and recovery for creeping bentgrassPlant Growth Regulat.471728

    • Search Google Scholar
    • Export Citation
  • LiZ.ZhangY.PengD.WangX.PengY.HeX.ZhangX.MaX.HuangL.YanY.2015Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signalingFront. Physiol.6280

    • Search Google Scholar
    • Export Citation
  • LiZ.PengY.HuangB.2016aPhysiological effects of γ-aminobutyric acid application on improving heat and drought tolerance in creeping bentgrassJ. Amer. Soc. Hort. Sci.1417684

    • Search Google Scholar
    • Export Citation
  • LiZ.ZhangY.ZhangX.PengY.MerewitzE.MaX.HuangL.YanY.2016bThe alterations of endogenous polyamines and phytohormones induced by exogenous application of spermidine regulate antioxidant metabolism, metallothionein and relevant genes conferring drought tolerance in white cloverEnviron. Exp. Bot.1242238

    • Search Google Scholar
    • Export Citation
  • LiaoW.B.HuangG.B.YuJ.H.ZhangM.L.2012Nitric oxide and hydrogen peroxide alleviate drought stress in marigold explants and promote its adventitious root developmentPlant Physiol. Biochem.58615

    • Search Google Scholar
    • Export Citation
  • LuS.SuW.LiH.GuoZ.2009Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activitiesPlant Physiol. Biochem.47132138

    • Search Google Scholar
    • Export Citation
  • NaeemM.NaeemM.S.AhmadR.IhsanM.Z.AshrafM.Y.HussainY.FahadS.2018Foliar calcium spray confers drought stress tolerance in maize via modulation of plant growth, water relations, proline content and hydrogen peroxide activityArch. Agron. Soil Sci.64116131

    • Search Google Scholar
    • Export Citation
  • NayyarH.2003Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonistsEnviron. Exp. Bot.50253264

    • Search Google Scholar
    • Export Citation
  • NeillS.J.DesikanR.ClarkeA.HurstR.D.HancockJ.T.2002Hydrogen peroxide and nitric oxide as signalling molecules in plantsJ. Exp. Bot.5312371247

    • Search Google Scholar
    • Export Citation
  • PeiZ.M.MurataY.BenningG.ThomineS.KlüsenerB.AllenG.J.GrillE.SchroederJ.I.2000Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cellsNature406731734

    • Search Google Scholar
    • Export Citation
  • RareE.1990Stress physiology: The functional significance of the accumulation of nitrogen-containing compoundsJ. Hort. Sci.65231243

  • RayP.D.HuangB.W.TsujiY.2012Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalingCell. Signal.24981990

  • ReddyA.S.AliG.S.CelesnikH.DayI.S.2011Coping with stresses: Roles of calcium- and calcium/calmodulin-regulated gene expressionPlant Cell2320102032

    • Search Google Scholar
    • Export Citation
  • RobertsM.R.SalinasJ.CollingeD.B.200214-3-3 proteins and the response to abiotic and biotic stressPlant Mol. Biol.5010311039

  • Ruttkay-NedeckyB.NejdlL.GumulecJ.ZitkaO.MasarikM.EckschlagerT.StiborovaM.AdamV.KizekR.2013The role of metallothionein in oxidative stressIntl. J. Mol. Sci.1460446066

    • Search Google Scholar
    • Export Citation
  • SabehatA.WeissD.LurieS.1998Heat-shock proteins and cross-tolerance in plantsPhysiol. Plant.103437441

  • ShankerA.K.MaheswariM.YadavS.DesaiS.BhanuD.AttalN.B.VenkateswarluB.2014Drought stress responses in cropsFunct. Integr. Genomics141122

    • Search Google Scholar
    • Export Citation
  • ShinozakiK.Yamaguchi-ShinozakiK.2007Gene networks involved in drought stress response and toleranceJ. Exp. Bot.58221227

  • SiedowJ.N.1991Plant lipoxygenase: Structure and functionAnnu. Rev. Plant Physiol.42145188

  • SinghK.FoleyR.C.OñatesánchezL.2002Transcription factors in plant defense and stress responsesCurr. Opin. Plant Biol.5430436

  • SørensenJ.G.KristensenT.N.LoeschckeV.2003The evolutionary and ecological role of heat shock proteinsEcol. Lett.610251037

  • SuzukiN.KoussevitzkyS.MittlerR.MillerG.2012ROS and redox signalling in the response of plants to abiotic stressPlant Cell Environ.35259270

    • Search Google Scholar
    • Export Citation
  • TodakaD.ShinozakiK.Yamaguchi-ShinozakiK.2015Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plantsFront. Plant Sci.684

    • Search Google Scholar
    • Export Citation
  • WangC.Q.2010Exogenous calcium alters activities of antioxidant enzymes in Trifolium repens L. leaves under peg-induced water deficitJ. Plant Nutr.3318741885

    • Search Google Scholar
    • Export Citation
  • WangW.VinocurB.ShoseyovO.AltmanA.2004Role of plant heat-shock proteins and molecular chaperones in the abiotic stress responseTrends Plant Sci.9244252

    • Search Google Scholar
    • Export Citation
  • XiaX.J.WangY.J.ZhouY.H.TaoY.MaoW.H.ShiK.AsamiT.ChenZ.YuJ.Q.2009Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumberPlant Physiol.150801814

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