Hydrogen Peroxide Signaling in Plant Development and Abiotic Responses: Crosstalk with Nitric Oxide and Calcium.

Hydrogen peroxide (H2O2), as a reactive oxygen species, is widely generated in many biological systems. It has been considered as an important signaling molecule that mediates various physiological and biochemical processes in plants. Normal metabolism in plant cells results in H2O2 generation, from a variety of sources. Also, it is now clear that nitric oxide (NO) and calcium (Ca(2+)) function as signaling molecules in plants. Both H2O2 and NO are involved in plant development and abiotic responses. A wide range of evidences suggest that NO could be generated under similar stress conditions and with similar kinetics as H2O2. The interplay between H2O2 and NO has important functional implications to modulate transduction processes in plants. Moreover, close interaction also exists between H2O2 and Ca(2+) in response to development and abiotic stresses in plants. Cellular responses to H2O2 and Ca(2+) signaling systems are complex. There is quite a bit of interaction between H2O2 and Ca(2+) signaling in responses to several stimuli. This review aims to introduce these evidences in our understanding of the crosstalk among H2O2, NO, and Ca(2+) signaling which regulates plant growth and development, and other cellular and physiological responses to abiotic stresses.

Introduction

Hydrogen peroxide (H2O2), a form of reactive oxygen species, is regarded as a common cellular metabolite. H2O2 is continually synthesized through various sources including enzyme and non-enzyme pathways in plants. To date, it has become accepted that H2O2 plays important roles in plant developmental and physiological processes including seed germination (Barba-Espín et al., 2011), programmed cell death (PCD; Cheng et al., 2015; Vavilala et al., 2015), senescence (Liao et al., 2012b), flowering (Liu et al., 2013), root system development (Liao et al., 2009; Ma et al., 2014; Hernández-Barrera et al., 2015), stomatal aperture regulation (Ge et al., 2015) and many others. It is now clear that H2O2 functions as a signaling molecule which may respond to various stimuli in plant cells. These results suggest that H2O2 may be involved in cellular signaling transduction pathways and gene expression modulations in plants.

Nitric oxide (NO), as a small signaling molecule, appears to be involved in plant developmental and physiological processes such as seed germination (Wang et al., 2015), ripening and senescence (Shi Y. et al., 2015) as well as stomatal closure (Shi K. et al., 2015) and pollen tube growth (Wang et al., 2009). Meanwhile, NO signaling may have a vital role in the disease resistance (Kovacs et al., 2015) and response to abiotic stresses such as cold (Fan et al., 2015), salt (Liu W. et al., 2015) and drought (Shan et al., 2015). Calcium ion (Ca2+) signaling is also a core regulator of plant physiological process and stress adaption such as cell polarity regulation (Zhou et al., 2014), leaf de-etiolation (Huang et al., 2012), stomatal closure (Zou et al., 2015). Additionally, Ca2+ signaling is also involved in various responses to abiotic stimuli, including light (Hu et al., 2015) and heavy metal (Li et al., 2016).

A large amount of research show that H2O2, NO and Ca2+ as signaling are involved in plant growth and development as well as response to abiotic stresses. In this review, we focus on H2O2 signaling activities and its cross-talk with Ca2+ and NO in plants.

H2O2 homeostasis

H2O2 generation

H2O2 is a byproduct of aerobic metabolism in plants (Mittler, 2002). Figure 1 shows that H2O2 in plants can be synthesized either enzymatically or non-enzymatically. There are numerous routes of H2O2 production in plant cells, such as photorespiration, electron transport chains (ETC), and redox reaction.

Figure 1

The various routes of hydrogen perioxide (H. Enzymatic production of H2O2 in plants requires several enzymes including cell wall peroxidases (Francoz et al., 2015), oxalate (Hu et al., 2003), amine oxidases and flavin-containing enzymes (Cona et al., 2006), glucose oxidases, glycolate oxidases (Chang and Tang, 2014), and sulfite oxidases (Brychkova et al., 2012). In these enzymes, some of them may convert O to H2O2 and O2. And others may oxidize their each substrates to generate H2O2in biocatalysis processes. Several non-enzymatic reactions are also known to produce H2O2. In peroxisome, H2O2 synthesis is associated with glycolate oxidation during photosynthetic carbon oxidation cycle (Foyer and Noctor, 2003). In chloroplasts, H2O2 production can be produced by the reduction of O by photosynthetic electron transport (PET) chain. H2O2 in chloroplast also may be detected at the manganese-containing, oxygen evolving complex which is the donor site of photosystem II. Moreover, H2O2 could be generated in mitochondria through aerobic respiration because O is produced from complexes I and III in the electron transport chain. H2O2-scavenging enzymes include catalase (CAT; Willekens et al., 1997), peroxidase (POX; Fan and Huang, 2012), ascorbate peroxidase (APX) and glutathione reductase (GR; Jahan and Anis, 2014). In non-enzymatic pathway, Ascorbate (AsA) and glutathione (GSH) are responsible for decreasing H2O2 level (Kapoor et al., 2015).

There is evidence for H2O2 production in plants through several enzymes includingcell wall peroxidases (Francoz et al., 2015), oxalate (Hu et al., 2003), amine oxidases and flavin-containing enzymes (Cona et al., 2006; Figure 1). Moreover, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases may also increase H2O2 level through generating superoxide which could be converted to H2O2 by superoxide dismutases (SOD; Grivennikova and Vinogradov, 2013; Brewer et al., 2015). Remans et al. (2010) observed that ROS accumulation, especially H2O2 formation, is mostly related with the stimulation of NADPH oxidase in plants under heavy metal stresses. Moreover, H2O2 produced by NADPH oxidases may significantly increase proline accumulation in Arabidopsis thaliana under salt or mannitol stress (Ben Rejeb et al., 2015). Additionally, some other oxidases such as glucose oxidases, glycolate oxidases (Chang and Tang, 2014), and sulfite oxidases (Brychkova et al., 2012) may oxidize their own substrates to produce H2O2 (Figure 1).

Several non-enzymatic reactions are also known to produce H2O2. For example, many reactions involved in photosynthesis and respiration are responsible for H2O2 production. It is generated continually via electron transport reactions both in mitochondria and chloroplasts (Figure 1).

Peroxisomes

Peroxisome is considered to be the site of photorespiration in plant cell, which needs light-dependent uptake of O2 and releases CO2 accompanying with the generation of H2O2. It is suggested that H2O2 synthesis is associated with the oxidation of glycolate during the photosynthetic carbon oxidation cycle (Foyer and Noctor, 2003; Figure 1).

Chloroplasts

Chloroplast is the source of photosynthesis in plants. Chloroplasts are the crucial sites for H2O2 production during photosynthesis. H2O2 generation is associated with oxygen reduction in chloroplast (Figure 1). Mehler (1951) discovered that reduction of O2 lead to the formation of H2O2 in the presence of light in chloroplast. Moreover, H2O2 production can also be produced by the reduction of O by photosynthetic electron transport (PET) chain components such as Fe–S centers, reduced thioredoxin (TRX), ferredoxin and reduced plastoquinone in the chloroplast (Dat et al., 2000). In addition, non-enzymatic production of H2O2 in chloroplast may be detected at the manganese-containing, oxygen evolving complex which is the donor site of photosystem II (Figure 1). But this process, in most cases, may probably be ignored under physiological conditions.

Mitochondria

One important source of endogenously produced H2O2 in plant cell is mitochondria (Dickinson and Chang, 2011). H2O2 is generated in mitochondria during aerobic respiration when O is produced from complexes I and III in the electron transport chain, which is then rapidly converted to H2O2 by the enzyme superoxide dismutase (Figure 1).

H2O2 removal

The antioxidant systems that regulate H2O2 levels consist of both non-enzymatic and enzymatic H2O2 scavengers (Figure 1). H2O2-scavenging enzymes include catalase (CAT; Willekens et al., 1997), peroxidase (POX; Fan and Huang, 2012), ascorbate peroxidase (APX) and glutathione reductase (GR; Jahan and Anis, 2014). Some studies revealed that APX was found in the cytosol (Begara-Morales et al., 2013), chloroplasts (Asada, 2006), and mitochondria (Navrot et al., 2007). Meanwhile, CAT can decompose H2O2 in peroxisome (Nyathi and Baker, 2006). It is quite clear that these enzymes exist in different organelles and they might decrease H2O2 content efficiently and maintain the stability of membranes.

Ascorbate (AsA) and glutathione (GSH), as non-enzymatic compounds, are constantly participated in regulating ROS level (Kapoor et al., 2015). AsA, a key antioxidant for elimination of H2O2, can react with H2O2 directly. GSH is a crucial antioxidant which may be associated with regenerating AsA, and rapidly oxidizes excess H2O2. Therefore, GSH is also involved in regulating H2O2 level and redox balance in plant cells (Krifka et al., 2012). In fact, H2O2 homeostasis seems to result in some biological effects on plant cells which may be as a signaling sign in signaling transduction pathway.

Responses to H2O2

Growth and development

Table 1 shows that H2O2 mediates various developmental and physiological processes in plants. These findings indicate that H2O2 may affect different parts of plants by increasing endogenous H2O2 level or by regulating relative gene expression. Also, the change of H2O2 level may impact metabolic and antioxidant enzyme activity in favor of plant growth and development (Barba-Espín et al., 2011; Liu et al., 2013). However, the mechanisms that allow different H2O2 function in plants still require examination.

Table 1

The developmental and physiological effects of H.

Developmental and physiological effect	Species	Tissue	H2O2 production	H2O2-mediated effect	References
Seed germination	Pisum sativum L. cv. Alaska	Seed	+	Caused carbonylation of proteins and metabolic enzymeUp-regulated PsMAPK2PsMAPK3 expression	Barba-Espín et al., 2011
PCD	Triticum aestivum L.	Seedling	+	Increased antioxidant enzyme activities and gene expression	Cheng et al., 2015Vavilala et al., 2015
	Chlamydomo-nas reinhardtii		+	Induced cell deathIncreased intracellular H2O2 contentIncreased antioxidant enzyme activities and analyses of transcripts
Senescence	Lilium	Leaf	+	Increased vase life and flower diameterReduced the degradation of RWC, total chlorophyll content and water-soluble carbohydrate	Liao et al., 2012b
Flowering	Monilinia fructicola	Petal	+	Increased H2O2 concentrationEnhanced protein carbonylation (carbonyl content)and lipid peroxidation (MDA content)	Liu et al., 2013
Root system development	Tagetes erecta L.	Root	+	Increased root lengthIncreased root number explant−1	Liao et al., 2009
	Arabidopsis thaliana			Accelerated lateral root formationIncreased endogenous H2O2 productionUp-regulated relative expression levels of HY1	Ma et al., 2014
				Increased sensitivity of the root elongation zone	Hernández-Barrera et al., 2015
Stomatal closure	Arabidopsis thaliana	Leaf	+	Induced stomatal closure	Ge et al., 2015

Stress condition

Recent studies have demonstrated that H2O2 is a key signaling molecule in the signaling pathway, which associated with abiotic stress response. A number of discussions showed that H2O2 could respond to abiotic stresses such as drought (Hameed and Iqbal, 2014; Ashraf et al., 2015), salinity (Sathiyaraj et al., 2014; Mohamed et al., 2015), cold (Orabi et al., 2015), high temperatures (Wang Y. et al., 2014; Wu et al., 2015), UV radiation (He et al., 2005), ozone (Oksanen et al., 2004), and heavy metal (Wen et al., 2013; Table 2). It is clear from these studies that H2O2 could enhance abiotic stress resistance through protecting organelle structure under abiotic stress conditions. For instance, H2O2 may protect chloroplast ultrastructure to preserve photosynthesis under abiotic stress. Similarly, to improve plant abiotic stress tolerance, H2O2 may modulate the expression of resistance genes and antioxidant enzyme activities during abiotic stress response.

Table 2

Report on H.

Stress	Plant species	Tissue	H2O2-mediated effect	References
Drought	Triticum aestivum L.	Leaf	Increased SOD, POD, CAT activitiesRaised total phenolic and reducing sugars content	Hameed and Iqbal, 2014
	Zea mays L.	Leaf	Reduced degradation of chlorophyll increased endogenous H2O2, MDA contentsIncreased antioxidant enzymes activitiesIncreased ascorbic acid content and ion contents	Ashraf et al., 2015
Salt	Panax ginseng	Leaf	Increased chlorophyll and carotenoid contentIncreased Relative water contentIncreased growth height and dry-weightIncreased antioxidant activityUp-regulated relative gene expression of defense related genes	Sathiyaraj et al., 2014
	Lycopersicon esculentum L.		Decreased electrolyte leakageIncreased endogenous H2O2 and MDA contentIncreased antioxidant enzymes activitiesAffect protein pattern and peroxidase enzymes	Mohamed et al., 2015
Cold	Lycopersicon esculentum L.	Seedling	Increased antioxidant enzymes activitiesIncreased MDA content Decreased electrolyte leakageIncreased total soluble solids	Orabi et al., 2015
Heat	Festuca arundinacea Lolium perenne	Leaf	Decreased the GSH/GSSG ratioIncreased POD, CAT, APC, GR, and GPX activities	Wang Y. et al., 2014
	Arabidopsis thaliana	Seedling	Increased thermotoleranceEnhanced antioxidant enzyme activitiesIncreased endogenous NO contentIncreased HSFs activity and HSP21 accumulation	Wu et al., 2015
UV-B	Vicia faba L.	Leaf	Increased endogenous H2O2 productionInduced Stomatal closure	He et al., 2005
Ozone	Betula papyrifera	Leaf	Induced proliferation of peroxisomesIncreased Level of gene expression for catalase (Cat)	Oksanen et al., 2004
Heavy metal	Zea mays var. rugosa Bonaf	Seedling	Decreased the activities of proline dehydrogenaseIncreased the activities of Arginase and OAT, P5CS and GDHUp-regulated the expression levels of P5CS, GDH, Arginase, OAT and ProDH genes	Wen et al., 2013

H2O2 as a signaling molecule in plant

Among ROS, H2O2 has comparatively long life span and small size, which permit it to traverse through cellular membranes to different cellular compartments. García-Mata and Lamattina (2013) found that H2O2 may move between cells through aquaporin channels for signaling transduction. Increasing evidences point out that H2O2 signaling may regulate various plant physiological processes. For example, H2O2 as signaling molecule may participate in nitrosative stress-triggered cell death in kimchi cabbage (Brassica rapa var. glabra Regel) seedlings (Kim et al., 2015). Also, Li et al. (2015) suggested that H2O2 is involved in signaling crosstalk between NO and hydrogen sulfide (H2S) to induce thermotolerance in maize seedlings. Moreover, the interaction among H2O2, NO and Ca2+ could relieve copper stress in Ulva compressa (González et al., 2012). H2O2 signaling was also demonstrated to play a salient role in brassinosteroid-regulated stomatal movement (Shi C. et al., 2015). As stated above, H2O2 as an important signaling molecule may play a significant role at every stage of plant life and under various abiotic stress conditions. H2O2 signaling appears to crosstalk with many different signaling molecules such as hormones (Shi C. et al., 2015), protein kinase (González et al., 2012) and many other small signaling molecules (Li et al., 2015). H2O2 and these signaling molecules may influence each other through various positive and negative feedback loops. Thus, they co-regulate cell division and differentiation, antioxidant system as well as gene expression involved in plant development and defense.

Crosstalk between H2O2 and No

NO is a diatomic free radical gas. Previous studies suggested that NO could take part in a wide range of physiological processes such as vasorelaxation, nervous system, defense against pathogens in animals (Mayer and Hemmens, 1998). In mammals, NO is synthesized via three different isoforms of NO synthase (NOS) including inducible NOS (iNOS; Nathan and Hibbs, 1991), endothelial NOS (eNOS) and neuronal NOS (nNOS; Förstermann et al., 1994). In plants, NO could be synthesized through enzymatic and non-enzymatic pathways (Figure 2). The enzymatic pathway includes nitrate reductase (NR; Rockel et al., 2002), nitric oxide-like (NOS-like) synthase (Guo et al., 2003), Nitrite-NO reductase (Ni-NOR; Stöhr et al., 2001) and xanthine oxidase (XOR; Corpas et al., 2004) pathways. The non-enzymatic generation of NO includes nitrification or de-nitrification processes (Skiba et al., 1993, Figure 2).

Figure 2

Summary of the main NO systhetic pathways and NO functions in plant growth, development and defense processes. NO may be synthesized by enzymatically and non-enzymatically pathways. In enzymatic pathway, nitrate reductase (NR; Rockel et al., 2002), Nitrite-NO reductase (Ni-NOR; Stöhr et al., 2001) and xanthine oxidase (XOR; Corpas et al., 2004) could convert NO and NO to NO. Meanwhile, because of NOS-like enzyme (Guo et al., 2003), L-Arginine may be catalyzed to NO. In non-enzymatic pathway, N could be transformed to NO through nitrification and denitrification (Skiba et al., 1993). NO plays an important signaling molecule in plant. It could regulate developmental and physiological processes such as seed germination (Wang et al., 2015), root development (Liao et al., 2011) and stomatal closure (Shi C. et al., 2015). Also, it may be involved in response to abiotic stresses such as cold (Fan et al., 2015), salt (Liu W. et al., 2015) and drought (Shan et al., 2015).

A plethora of evidences suggest that NO, as a versatile signaling molecule, is involved in regulating every aspect of plant growth and developmental processes such as seed germination (Fan et al., 2013; Wang et al., 2015), flowering (Liu W. W. et al., 2015), root growth and development (Liao et al., 2011; Wu et al., 2014; Xiang et al., 2015), ripening and senescence (Liao et al., 2013; Shi Y. et al., 2015). Meanwhile, as a physiological regulator, NO signaling is involved in mediating stomatal closure (Noelia et al., 2015; Shi K. et al., 2015; Chen et al., 2016), pollen tube growth (Wang et al., 2009). Also, NO plays an essential role in plant disease resistance (Rasul et al., 2012; Kovacs et al., 2015) and responses to various abiotic stresses such as cold (Fan et al., 2015), heat (Yu et al., 2015), salt (Liu W. et al., 2015), drought (Shan et al., 2015), UV-B (Esringu et al., 2015) and heavy metal (Alemayehu et al., 2015; Chen et al., 2015; Kaur et al., 2015). These studies have paved the way to understand the signaling roles of NO which may affect cell metabolism, cellular redox balance and gene expression in plants. The relative target receptor may receive signaling activated by various stimuli. As a result, NO may activate regulatory mechanism to promote developmental and physiological processes and regulate abiotic stress response in plants.

Interaction in growth and development

To date, the interaction between H2O2 and NO has been demonstrated clearly in plants. The signaling crosstalk between H2O2 and NO has been considered to be an essential factor to influence plant developmental and physiological processes such as leaf cell death (Lin et al., 2012), delay senescence (Iakimova and Woltering, 2015), root growth and development (Liao et al., 2010, 2011), stomatal closure (Huang et al., 2015; Shi K. et al., 2015), and pollen tube growth (Serrano et al., 2012). Table 3 shows the interaction of H2O2 and NO at different levels in a great number of developmental and physiological processes in plants. On the one side, H2O2 may act as a cofactor to promote endogenous NO synthesis. For example, Lin et al. (2012) implied that H2O2 may stimulate NO production through increasing NR activity in leaves of noe1 plants under high light. Shi C. et al. (2015) reported that Gα-activated H2O2 production may induce NO synthesis. The research found that NO could modulate stomatal closure in H2O2 mutants AtrbohF and AtrbohD AtrbohF and in the wild type treated with H2O2 scavenger and inhibitor. However, H2O2 did not close or reduce the stomatal closure in mutants Nia1-2 and Nia2-5 Nia1-2, and in the wild type treated c-PTIO or tungstate (Shi C. et al., 2015). These results clearly show that H2O2 might induce NO synthesis in stomatal closure. On the other side, NO may induce H2O2 generation in plants. Liao et al. (2011) reported cPTIO or L-NAME could inhibit the endogenous H2O2 generation implying that NO was required for the production of H2O2 during adventitious rooting. Meanwhile, NO could mediate antioxidant enzyme activities to influence the H2O2 level (Zhang et al., 2007). Thus, the interaction of H2O2 and NO may trigger a serious of physiological and biological response in plant cells.

Table 3

The developmental and physiological effects of crosstalk between H.

Developmental and physiological effect	Species	Tissue	Crosstalk between H2O2 and NO mediated effects	References
Cell death	Oryza sativa	Leaf	H2O2 induced NR-dependent NO generationNO Is required for H2O2-induced leaf cell death increased NR enzyme	Lin et al., 2012
Senescence	Lactuca sativa L.	Leaf	NO decreased endogenous H2O2 contentDelay senescence	Iakimova and Woltering, 2015
Root growth	Dendranthema morifolium	Root	Increase the activities of PPO, IAAO and the content of WSC and total nitrogenDecrease the total polyphenol contentNO and H2O2 may act synergistically to mediate adventitious root generation and development	Liao et al., 2010Liao et al., 2011
	Tagetes erecta L.		NO may be involved as an upstream signaling molecule for H2O2 production
Pollen tube growth	Olea europaea L.	Flower	Decreased cell deathIncreased nitrated proteins	Serrano et al., 2012
Stomatal movement	Arabidopsis	Leaf	H2O2 production was required for NO synthesisRegulated stomatal closure	Shi C. et al., 2015 Huang et al., 2015
	Vicia faba		Regulated stomatal closureH2O2 induced NO production

Interaction during abiotic stress

Recently, the roles of H2O2and NO signaling and their crosstalk in mediating plant response to abiotic stresses have been largely established (Table 4).

Table 4

Reports on interaction between H.

Stress	Plant species	Tissue	Crosstalk between H2O2 and NO mediated effects	Reference
Salt	Citrus aurantium L.	Leaf	Alleviated salinity-induced protein carbonylationShifted the accumulation levels of leaf S-nitrosylated proteins acclimation to salinityIdentified a number of proteins which were modulated by both H2O2 and NO treatments	Tanou et al., 2009Tanou et al., 2010
	Populus euphratica	shoot	Increased K/Na ratio	Zhang et al., 2007
	Medicago falcata	Seedling	Stimulated expression of PM H+-ATPase Induced MfMIPSI transcriptIncreased the level of myo-inositol	Tan et al., 2013
Drought	Tagetes erecta L.	Root	Alleviated the destruction of mesophyll cell ultrastructureIncreased leaf chlorophyll contentMediated chlorophyll fluorescence parametersEnhanced carbohydrate accumulationDecreased starch contentH2O2 generation may be affected by NO	Liao et al., 2012a
	Tagetes erecta L.	Leaf	Increased RWCDecrease ion leakageIncreased antioxidant enzyme, PEPCase, HK activities and MDA contentNO acted downstream of H2O2	Lu et al., 2009
UV-B	Arabidopsis	Leaf	NO production depends on H2O2Mediated stomatal closure	He et al., 2013
			The UV-B Photoreceptor UVR8 was mediated by H2O2 and NO	Tossi et al., 2014
Heat	Zea may L.	seedling	Improved survival percentage of maize seedlingsH2O2 increased endogenous NO contentH2O2 may be involved in downstream signal of NO	Li et al., 2015
	Arabidopsis		NO is involved in H2O2 signaling as a downstream factor.Increased HS factor activity and HS protein accumulation.	Wang L. et al., 2014
	Triticum aestivum L.		Increased seedling resistanceIncreased H2O2 and NO contentIncreased survival percentage of seedlings	Karpets et al., 2015
Cold	Medicago sativa subsp. falcata	Leaf	Mediated cold-induced MfSAMS1 expression	Guo et al., 2014
	Medicago falcate	Seedling	Up-regulated MfMIPSI expression	Tan et al., 2013
	Medicago sativa		Increased myo-inositol content
Heavy metal	Ulva compressa	Cell	Increased PDH,IDH,OGDH activity and increased relative transcript levels	González et al., 2012
	Triticum aestivum	Root	Decreased lipid peroxidationIncreased NOS activityIncreased antioxidative enzyme activities	Duan et al., 2015

Drought

Drought stress is a major environmental factor that affects plant growth and development. As reported by Liao et al. (2012a), both H2O2 and NO could protect mesophyll cells ultrastructure and improve the photosynthetic level of leaves under drought stress during adventitious rooting in marigold explants. Similarly, the interplay between H2O2 and NO signaling may increase the activity of myo-inositol phosphate synthase to alleviate drought stress (Tan et al., 2013). Additionally, Lu et al. (2009) suggested that endogenous NO and H2O2 may be involved in ABA-induced drought tolerance of bermudagrass by increasing antioxidant enzyme activities. NO may be considered to be upstream or downstream signaling molecule of H2O2 (Lu et al., 2009; Liao et al., 2012a). Thus, the interaction between H2O2 and NO may alleviate drought stress through up-regulating antioxidant defense system to protect cell membrane and maintain ion homeostasis in plants.

Salt

The interaction between H2O2 and NO plays an important role in plant tolerance to salt stress (Zhang et al., 2007; Tan et al., 2013). Tanou et al. (2009) suggested that H2O2 and NO pre-treatments could alleviate salinity-induced protein carbonylation in citrus. The authors suggested an interaction between H2O2 and NO during salt stress response. Furthermore, H2O2- and NO-responsive proteins have been identified which may further reveal a protein interaction network between H2O2 and NO signaling under salt stress (Tanou et al., 2010).

UV-B

UV-B, a key environmental signal, initiates diverse responses in plants (Jansen and Bornman, 2012). UV-B radiation can also influence plant growth, development, and productivity. It has been shown that the crosstalk between H2O2 and NO could be involved in the response to UV-B stress. There was an interrelationship among Gα protein, H2O2, and NO during UV-B-induced stomatal closure in Arabidopsis leaves (He et al., 2013). This study found that there was a significant increase in H2O2 or NO levels which associated with stomatal closure in the wild type by UV-B stress. However, these effects were abolished by double mutants of AtrbohD and AtrbohF or Nia1 mutants. These results strongly suggested that the crosstalk between H2O2 and NO signaling might play an essential role during UV-B-induced stomatal closure in guard cells. Recently, Tossi et al. (2014) also showed a mechanism involving both H2O2 and NO generation in response to UV-B exposure. Therefore, the crosstalk between H2O2 and NO can regulate stomatal movement to reduce UV-B stress damage to plant cells.

Cold

Cold stress adversely influences plant growth and development. Guo et al. (2014) reported that the interaction of H2O2 and NO may affect cold-induced S-adenosylmethionine synthetase and increase cold tolerance through up-regulating polyamine oxidation in Medicago sativa subsp. falcate. Moreover, signaling interplay of H2O2 and NO was essential for cold-induced gene expression of falcata myo-inositol phosphate synthase (MfMIPS), which improved tolerance to cold stress (Tan et al., 2013). Thus, the interaction between H2O2 and NO may initiate different mechanisms to response to cold stresses.

Heat

Recently, many studies have been conducted to investigate the relationship between H2O2 and NO under heat stress. Li et al. (2015) reported that a signaling crosstalk between H2O2 and NO may be involved in inducing thermotolerance in maize seedlings. Moreover, H2O2 may be upstream signaling of NO in the heat shock pathway in Arabidopsis seedlings (Wang L. et al., 2014). In addition, treatment with low level of H2O2 or NO could increase seedling viability under heat resistance (Karpets et al., 2015). These studies support the existence of crosstalk between H2O2 and NO in heat responses in plants.

Heavy metal stress

Alberto et al. (2012) suggested that the signaling interaction between H2O2 and NO was involved in alleviating copper stress of Ulva compressa through mediating antioxidant enzyme activities and activating relative gene expression. Besides, the interplay of NO and H2O2 in wheat seedlings participated in regulating root growth under zinc stress and alleviated zinc stress through increasing antioxidant system, decreasing lipid peroxidation as well as up-regulating resistance gene expression (Duan et al., 2015). Obviously, the crosstalk of H2O2 and NO has been found under heavy metal stress condition, which may trigger a variety of antioxidant responses in plants.

As stated above, the physiological effect of H2O2 and NO is similar and synergetic. In different cases, these forms of interaction are various. However, the form of H2O2 and NO crosstalk depend on plant species and environmental stresses. H2O2 and NO could modulate each other through regulating antioxidant enzymes activities and relative gene expression in plants. Meanwhile, H2O2 and NO may synergistically regulate many common target genes which were related to signaling transduction, defense reaction, plant hormone interactions, protein transport and metabolism. Therefore, it has a significant meaning to elaborate the mechanism of the interaction between H2O2 and NO in plant developmental processes and response to abiotic stresses.

Crosstalk between H2O2 and Ca2+

Ca2+ is a widespread signaling molecule in plants. When plants receive stimuli, the change of intracellular Ca2+ concentration may transfer signaling to regulate a series of cellular processes in plants (Kong et al., 2015; Tang et al., 2015). There are various types of Ca2+ receptors and channels in plants such as Ca2+-ATPases (Pászty et al., 2015), Ca2+-binding sensor protein (Wagner et al., 2015), inositol-1,4,5-trisphosphate (IP3; Serrano et al., 2015) and cyclic ADP-ribose (cADPR, Gerasimenko et al., 2015). It is well known that Ca2+ is involved in plant growth and development such as seed germination (Kong et al., 2015), pollen tube growth (Zhou et al., 2014), leaf de-etiolation (Huang et al., 2012), root growth and development (Liao et al., 2012a; Han et al., 2015) and other physiological processes including cell polarity regulation (Zhou et al., 2014; Himschoot et al., 2015), stomatal closure (Zou et al., 2015) and immune response (Seybold et al., 2014). Furthermore, variations in cytosolic free Ca2+ concentration have been demonstrated to response to a wide range of environmental stresses such as heat shock (Urao et al., 1994), drought (Zou et al., 2015), light (Hu et al., 2015), salt (Tepe and Aydemir, 2015), and heavy metal (Li et al., 2016). Because of Ca2+ has various receptors and channels in plants, it may receive different upstream signaling molecules quickly and then respond to abiotic stress.

Crosstalk between H2O2 and Ca2+ occurs in plant cells (Table 5). For example, exogenous H2O2 caused transiently dose-dependent increase in Ca2+ influx in Arabidopsis thaliana root epidermis (Demidchik et al., 2007). Two Ca2+ channels could be regulated by H2O2 level in root elongation zone. Han et al. (2015) demonstrated that H2O2 signaling could induce root elongation by mediating Ca2+ influx in the plasma membrane of root cells in Arabidopsis seedlings. Richards et al. (2014) also suggested that Annexin 1, a Ca2+ transport protein, may regulate H2O2-induced Ca2+ signature in Arabidopsis thaliana roots to promote root growth and development. Additionally, Ca2+ signaling was involved in H2O2-induced adventitious rooting in marigold because removal of Ca2+ could inhibit H2O2-induced adventitious root development (Liao et al., 2012a). Interestingly, Wu et al. (2010)'s findings strongly suggested that spermidine oxidase (Spd)-derived H2O2 signaling may mediate Ca2+ influx. Spd was probably related to downstream induction of H2O2 signaling and then H2O2 activated Ca2+-permeable channels during pollen tube growth (Wu et al., 2010). Cross talk between Ca2+–Calmodulin (CaM) and H2O2 also played a significant role in antioxidant defense in ABA signaling in maize leaves (Hu et al., 2007; Table 5). Thus, the signaling crosstalk between H2O2 and Ca2+ may affect every stage of plant development by modulating cell elongation and division, antioxidant enzyme activity and gene expression. H2O2 may activate Ca2+ receptors and target proteins to increase [Ca2+]cyt level and Ca2+ may induce endogenous H2O2 generation during plant growth and development.

Table 5

The developmental and physiological effects of crosstalk between H.

Developmental and physiological effect	Species	Tissue	Crosstalk between H2O2 and Ca2+ mediated effects	References
Root growth and elongation	Arabidopsis	Root	H2O2 induce Ca2+ influxIncreased root elongationEndogenous H2O2 resulted in Ca2+ fluxEnhanced root growth	Han et al., 2015Demidchik et al., 2007
Adventitious root development	Arabidopsis	Root	Extracellular H2O2 induced a sustained increase in cytosolic free Ca2+Exogenous H2O2 induced expression of AtANN1	Richards et al., 2014Liao et al., 2012a
	Tagetes erecta L.		Endogenous H2O2 increased Cytosolic free Ca2+ and CaM contentInduced adventitious root development
Pollen growth	P.Dyrifolia Nakai cv.Hosui Imamuraaki	Flower	H2O2 activates Ca2+ currentsInduced pollen tube growth	Wu et al., 2010
Antioxidant defense	Zea may L.	Leaf	H2O2 increased the concentration of cytosolic Ca2+ in the protoplasts of mesophyll cells and the expression of the calmodulin 1 (CaM1) gene and CaM content in leavesEnhanced the expression of the antioxidant genes	Hu et al., 2007

Interaction in abiotic stress

Clearly, correlations also exist between H2O2 and Ca2+ in response to abiotic stresses in plants (Table 6). Shoresh et al. (2011) investigated that supplemental Ca2+ had a significant effect on H2O2 metabolism and regulating leaves and roots growth in maize under salt stress. The authors indicated that extracellular Ca2+ may modulate endogenous H2O2 levels through activating polyamine oxidase activity. Also, salt stress may induce H2O2 accumulation in Ca2+-dependent salt resistance pathway in Arabidopsis thaliana roots (Li et al., 2011). Moreover, Lu et al. (2013) suggested that exogenous H2O2 and Ca2+ may mediate root ion fluxes in mangrove species under NaCl stress. Obviously, H2O2 may interact with Ca2+ under salt stress in plants through mediating root ion balance, increasing antioxidant enzymatic activity and up-regulating the expression of related genes. Moreover, H2O2 and Ca2+ signaling were also involved in ABA responses to drought stress in Arabidopsis thaliana through Ca2+-dependent protein kinase8 (CPK8) which could regulate catalase3 (CAT3) activity mediating stomatal movement (Zou et al., 2015). In addition, Qiao et al. (2015) reported that a Ca2+-binding protein (rice annexin OsANN1) could enhance heat stress tolerance by modulating H2O2 production. Over production of H2O2 induced by heat stress increased OsANN1 expression and up-regulated the level of SOD and CAT expression, which constructed a signaling mechanism for stress defense in plants (Qiao et al., 2015). Until now, the signaling crosstalk between H2O2 and Ca2+ may regulate various responses to abiotic stresses in plants. It may be connected with the regulation of antioxidant system. Thus, the interaction between H2O2 and Ca2+ may increase antioxidant enzyme activities such as APX, SOD, and GR. These antioxidant enzymes may alleviate stress damages in plants. In addition, the crosstalk between H2O2 and Ca2+ could regulate gene expression level and induce protein interactions.

Table 6

Reports on interaction between H.

Stress	Plant species	Tissue	Crosstalk between H2O2 and Ca2+ mediated effects	References
Salt	Bruguiera gymnorrhiza L.Kandelia candel L.	Root Leaf	Mediated root ion fluxIncreased K+ flux and Na+/H+ antiport	Lu et al., 2013
	Arabidopsis	Root	Increased NADPH/NADP+,G6PDH activityUp-regulated expression of PM H+-ATPase gene	Li et al., 2011
Drought	Zea may L.	Root	Increased root viabilityDecreased membrane leakageIncreased chlorophyll contentIncreased peroxidase activity	Shoresh et al., 2011
	Arabidopsis	Seedling	Induced stomatal closureMediated protein interaction between CPK8 and CAT3	Zou et al., 2015
Heat	Oryza sativa subsp. japonica	Seedling	Up-regulated OsANN1 expressionEnhanced the level of SOD, CAT expression	Qiao et al., 2015

It appears that the interrelationship between H2O2 and Ca2+ may be involved in various aspects of plant growth and development processes and abiotic stress responses. In fact, the change of Ca2+ concentration is closely related to H2O2 burst in plant cells. The combination of H2O2 and Ca2+ may play crucial roles in plants. Different plants even different parts of the same plant may have different modulation mechanisms. Thus, relationship between H2O2 and Ca2+ signaling in plants is very complex. The interplay of H2O2, Ca2+ and its mechanism need to be illustrated clearly in the future.

Crosstalk among H2O2, No and Ca2+

It has been suggested that there is a connection among H2O2, NO, and Ca2+ in plants. H2O2, NO, and Ca2+ may act as essential signaling molecules which may form a complex signaling network to regulate different developmental and physiological processes in plants (Figure 3). For instance, during adventitious rooting of mung bean, Ca2+ signaling played a pivotal role and functioned as a downstream molecule of H2O2 and NO signal pathway (Li and Xue, 2010; Figure 3). Similarly, there is a possible relationship among H2O2,NO and Ca2+/CaM during adventitious rooting in marigold explants (Liao et al., 2012a). The authors found that exogenous NO and H2O2 promoted adventitious root development in marigold explants through increasing endogenous Ca2+ and CaM levels. Moreover, H2O2, NO and Ca2+ were also involved in oligochitosan-induced programmed cell death in tobacco suspension cells (Zhang et al., 2012). Pharmacological experiments revealed that Ca2+ signaling induced NO accumulation through inducing H2O2 generation during stomatal closure in Arabidopsis guard cells (Li et al., 2009). Furthermore, Wang et al. (2011) suggested a functional correlationship among H2O2, calcium-sensing receptor (CAS) and NO in Ca2+-dependent guard cell signaling. It was shown that CAS may transduce Ca2+ signaling through activating its downstream target NO and H2O2 signaling pathway (Wang et al., 2011). Therefore, it is thus clear that the interplay of H2O2, NO, and Ca2+ may have an significant effect on plant growth and physiological processes through promoting cell proliferation, controlling cell metabolism, meanwhile, regulating modes of cell death. Moreover, Vandelle et al. (2006) has reported that NO and H2O2 synthesis could also act upstream to increase cytosolic Ca2+ concentration during hypersensitive response (HR) through activating plasma membrane- and intracellular membrane-associated Ca2+ channels. Besides, the interaction among H2O2, NO, and Ca2+ signaling may regulate ABA-induced antioxidant defense in maize (Ma et al., 2012). Obviously, the mutual effect among H2O2, NO and Ca2+ may increase antioxidant system and induce disease defense in plants.

Figure 3

Schematic model of the interaction among H. H2O2, NO and Ca2+ may receive various stimuli through signaling sensors. They might interact via cross-regulation and transduce signaling to downstream molecules by activating phosphokinase like MAPKs, or relative enzyme activity in order to regulate plant development and growth and abiotic stress responses.

Furthermore, the interplay among H2O2, NO, and Ca2+ also have an effect on abiotic stress response in plants. For example, Lang et al. (2014) reported that NO likely interacted with Ca2+ and H2O2 in Aegiceras corniculatum to up-regulate Na+/H+ antiport system of plasma membrane under salt stress. There were species-specific interactions between H2O2, Ca2+, NO, and ATP in salt-induced reduction of K+ efflux (Lang et al., 2014). Moreover, there was a crosstalk among H2O2, NO, and Ca2+ when Ulva compressa exposed to copper excess and the interaction had a significant effect on transcriptional activation of target genes (Alberto et al., 2012). The H2O2-induced NO generation could be inhibited by Ca2+ channel blockers, implicating that Ca2+ may mediate the effect of H2O2 on NO production. Furthermore, Ca2+ release through different type of Ca2+ channels was also shown to be activated by NO and H2O2 (Alberto et al., 2012; Figure 3). The interrelationship between H2O2, NO and Ca2+ may provide additional layers of responses to abiotic stresses through controlling ion transport, increasing antioxidant enzyme activities and affecting expression of resistance genes, indicating a feedback mechanism between H2O2, NO and Ca2+ under abiotic stresses. In a word, the combination of these findings strongly supports the view that there has an interaction among H2O2, NO, and Ca2+ signaling pathway in plant growth, development and abiotic stress responses. During signaling transduction, Ca2+ signaling could be activated by H2O2 and NO; it could also regulate H2O2 and NO signaling. Ca2+ may act as a point of signaling convergence between H2O2 and NO signaling pathways in plants. However, the network of H2O2, NO, and Ca2+ seems to be intricate and multidimensional. Therefore, considerably more work will need to be done to determine the interaction among H2O2, NO and Ca2+ signaling in plants.

Conclusion

H2O2 was once considered as a poisonous molecule in plants. Based on current studies, H2O2 may be a vital signaling molecule which controls plant growth and development. Interestingly, NO and Ca2+ which also act as the key component of signaling transduction in plants seem to be as upstream or downstream signaling molecules of H2O2. Meanwhile, H2O2 modulates NO and Ca2+ signaling pathways. There is a complex interactive network among H2O2, NO, and Ca2+ in plants. Moreover, the interplay among them has functional implications for regulating developmental and physiological processes which may increase the possibility of signal reception and transduction in plants. Future work will need to focus on the molecular mechanism of the interplay among H2O2, NO, and Ca2+ during signaling transduction in plants.

Author contributions

LN wrote the paper. WL provided the idea and revised the paper.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.