Insights on Calcium-Dependent Protein Kinases (CPKs) Signaling for Abiotic Stress Tolerance in Plants

Abstract

Abiotic stresses are the major limiting factors influencing the growth and productivity of plants species. To combat these stresses, plants can modify numerous physiological, biochemical, and molecular processes through cellular and subcellular signaling pathways. Calcium-dependent protein kinases (CDPKs or CPKs) are the unique and key calcium-binding proteins, which act as a sensor for the increase and decrease in the calcium (Ca) concentrations. These Ca flux signals are decrypted and interpreted into the phosphorylation events, which are crucial for signal transduction processes. Several functional and expression studies of different CPKs and their encoding genes validated their versatile role for abiotic stress tolerance in plants. CPKs are indispensable for modulating abiotic stress tolerance through activation and regulation of several genes, transcription factors, enzymes, and ion channels. CPKs have been involved in supporting plant adaptation under drought, salinity, and heat and cold stress environments. Diverse functions of plant CPKs have been reported against various abiotic stresses in numerous research studies. In this review, we have described the evaluated functions of plant CPKs against various abiotic stresses and their role in stress response signaling pathways.

1. Introduction

Plants have several adaptive features to cope with biotic and abiotic stresses under challenging environmental situations. Plants respond to these stresses by inducing the expression of stress-responsive genes through a complex signaling pathway. The expression of these stress-responsive genes is induced upon changes in calcium ion (Ca²⁺) concentrations, due to various biotic and abiotic stimuli [1,2], which enable plant adaptations in a wide range of stressed environments.

Calcium (Ca) as a ubiquitous secondary messenger regulates the stress signaling mechanism in plants. Changes in Ca²⁺ concentration are sensed by several calcium-binding proteins, especially calcium-dependent protein kinases [3]. The calcium-dependent abiotic and biotic stress signaling mechanisms are most commonly dominated by calcium-dependent protein kinases, which play a pivotal role in the regulation of plant responsiveness to salt, drought, and cold and heat stresses as well as other environmental factors. Ca²⁺ is involved in abscisic acid (ABA)-dependent biotic and abiotic stress signals in various plant species [4,5]. The calcium-dependent protein kinases phosphorylate the ABA-responsive element-binding factors (ABFs). ABA regulation by Ca²⁺ is associated with plant defense systems through induction of antioxidants [6], including reactive oxygen species (ROS) [2], and other enzymes like superoxide dismutase (SOD), catalase 3 (CAT3), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) [6,7]. It is also involved in the induction of some nonenzymatic antioxidants like ascorbic acid, α-tocopherol, carotenoids, and glutathione and controls multiple abiotic stress response processes [6,8,9,10]. This review will provide insight into the role of calcium-dependent protein kinases (CPKs) in abiotic stress tolerance in different plant species.

2. CPK Enzymes and Related Kinases

Several calcium-binding protein families have been identified in plants, which are potentially involved in the regulation of calcium-dependent abiotic stress response mechanisms. These Ca²⁺ sensors decode and transmit complex information, present in the form of calcium signal, to the phosphorylation events and regulate stress-responsive genes through protein interactions [11]. These Ca²⁺ signal-decoding groups include calcium-dependent protein kinases (CDPKs or CPKs), calmodulins (CaMs), calmodulin-like protein kinases (CMLs), calcineurin β-like proteins (CBLs), and Ca²⁺/calmodulin-dependent protein kinase (CCaMK) [12,13]. Among all these kinases, CPKs, CMLs, and CBLs have only been discovered in plants and some protozoans, while CaMs are highly conserved among all eukaryotes [11,14]. CaMs, CBLs, and CMLs are small proteins that function as calcium signal communicators through binding to downstream effectors (EFs) [15,16]. CaMs evolved from CMLs, which are considered as the most primitive calcium-binding proteins [13]. Among all these, CPKs were identified in plants as well as green algae, oomycetes, and in some protozoans [17], but they are not present in animals. CPKs, through direct binding with Ca²⁺, have a predominant regulatory role for the Ca-sensing protein families [17].

3. CPK Family in Plants

CPKs are considered as the versatile player for the regulation of abiotic stress management in plants [17]. In 1984, the very first plant CPKs were identified in Pisum sativum [18]. These proteins were initially purified from soybeans in 1987. A CPK encoding gene was cloned from Arabidopsis thaliana in 1991, which opened new ways for CPK gene cloning in several other plant species [11,19,20]. The presence of CPKs in almost all parts of the plant demonstrates that these kinases have a high potential for regulating various signal transduction pathways and have a significant influence on plant growth and development [17,21,22,23].

3.1. CPK Distribution and Localization in Plants

CPKs show a widespread distribution in different plant species. The whole-genome sequencing of plant species (e.g., Arabidopsis [24]) enables researchers to conduct genome-wide identifications of variable CPK encoding genes. These studies identified 34 CPK-encoding genes in the genome of Arabidopsis thaliana, 20 in Triticum aestivum (wheat), and 31 in Oryza sativa (rice) [20,25,26]. Solanum lycopersicum (tomato), which is a model plant of the Solanaceae family, has 29 CPK-encoding genes [27]. Genome-wide exploration of some other plants such as Zea mays (maize), Hordeum vulgare (barley), Cucumis melo (melon), Populus trichocarpa (poplar), Gossypium raimondii (cotton), Manihot esculenta (cassava), and Vitis vinifera (grapevine) revealed the presence of 40, 28, 18, 30, 41, 27, and 19 CPK-encoding genes, respectively [28,29,30,31,32,33,34] (Table 1). Mostly, CPK-encoding genes are expressed in leaves, meristems, roots, and flowers, while some are expressed only in specific tissues [23,35,36].

Table 1.

Genome-wide identification of calcium-dependent protein kinases (CPKs) among various plant species.

Sr. #	Common Name	Botanical Name	No. of CPKs	Genome Size (Mb)	Reference
1	Algae	Volvox carteri	6	131.2	[37]
2	Apple	Malus domestica	28	881.3	[37]
3	Arabidopsis	Arabidopsis thaliana	34	135	[20]
4	Banana	Musa acuminata	44	523	[38]
5	Barley	Hordeum vulgare	27	667	[39]
6	Barley	Hordeum vulgare	28	667	[31]
7	Barrel clover	Medicago truncatula	11	360	[37]
8	Black cottonwood	Populus trichocarpa	28	422.9	[37]
9	Poplar	Populus trichocarpa	30	500	[34]
10	Butcher	Micromonas pusilla	22	2	[13,37]
11	Cacao tree	Theobroma cacao	17	346	[13,37]
12	Canola	Brassica napus	25	1130	[40]
13	Cassava	Manihot esculenta	26	532.5	[30]
14	Caster bean	Ricinus communis	15	400	[37]
15	Castor bean	Ricinus communis	15	400	[13,37]
16	Chinese liquorice	Glycyrrhiza uralensis	23	379	[41]
17	Chlamydomonas	Chlamydomonas reinhardtii	14	111.1	[13,37]
18	Clementine	Citrus clememtina	26	301.4	[37]
19	Cocoa tree	Theobroma cacao	17	346	[37]
20	Columbine	Aquilegia coerulea	16	306.5	[13,37]
21	Cotton	Gossypium raimondii	41	880	[28]
22	Cotton	Gossypium hirsutum	98	2250–2430	[42]
23	Cucumber	Cucumis sativus	19	323.99	[43]
24	Cucumber	Cucumis sativus	18	203	[37]
25	Finger Millet	Eleusine coracana	4	1593	[44]
26	Flax	Linum usitatissimum	47	318.3	[37]
27	Flooded gum	Eucalyptus grandis	22	691	[37]
28	Foxtail Millet	Setaria italic	27	405.7	[37]
29	Foxtail Millet	Setaria italic	29	405.7	[45]
30	Foxtail millet	Setaria italica	27	405.7	[13,37]
31	Grape	Vitis vinifera	19	500	[29]
32	Grapevine	Vitis amurensis	17	500	[46]
33	Grapevine	Vitis amurensis	13	500	[47]
34	Green algae	Coccomyxa subellipsoidea	2	49	[13,37]
35	Green algae	Ostreococcus lucimarinus	3	13.2	[13,37]
36	Green bean	Phaseolus vulgaris	25	521.1	[37]
37	Linseed	Linum usitatissimum	47	318.3	[13,37]
38	Maize	Zea mays	35	2500	[48]
39	Maize	Zea mays	40	2500	[49]
40	Maize	Zea mays	47	2500	[37]
41	Melon	Cucumis melo	18	375	[32]
42	Monkey flower	Mimulus guttatus	25	321.7	[37]
43	Mustard	Brassica rapa	49	283.8	[37]
44	Norway spruce	Picea abies	11	1960	[37]
45	Oilseed rape	Brassica rapa	49	283.8	[13,37]
46	Orange	Citrus sinensis	24	319	[13,37]
47	Papaya	Carica papaya	15	135	[13,37]
48	Papaya	Carica papaya	15	135	[37]
49	Peach	Prunus persica	17	227.3	[37]
50	Pepper	Capsicum annuum	31	407.5	[50]
51	Pigeon Pea	Cajanus cajan	23	852	[51]
52	Potato	Solanum tubersum	21	800	[37]
53	Potato	Solanum tuberosum	23	800	[52]
54	Purple false brome	Brachypodium distachyon	27	272	[37]
55	Purple false brome	Brachipodium distachyon	27	272	[37]
56	Red Shepherd’s Purse	Capsella rubella	32	134.8	[37]
57	Rice	Oryza sativa	29	430	[53]
58	Rice	Oryza sativa	22	430	[54]
59	Rice	Oryza sativa	30	372	[37]
60	Rubber tree	Hevea brasiliensis	30	1332	[55]
61	Salt cress	Thellungiella halophile	31	238.5	[13,37]
62	Shepherd’s Purse	Capsella rubella	32	134.8	[37]
63	Sorghum	Sorghum bicolor	28	697.5	[37]
64	Soybean	Glycine max	39	1115	[56]
65	Soybean	Glycine max	50	1115	[57]
66	Soybean	Glycine max	39	1115	[58]
67	Soybean	Glycine max	41	978	[13,37]
68	Spikemosses	Selaginella moellendorffii	11	212.5	[13,37]
69	Spreading earthmoss	Physcomitrella patens	25	480	[13,37]
70	Sweet orange	Citrus sinensis	24	319	[37]
71	Switchgrass	Panicum virgatum	53	1358	[37]
72	Tobacco	Nicotiana tabacum	15	323.75	[59]
73	Tomato	Solanum lycopersicum	29	900	[60]
74	Tomato	Solanum lycopersicum	28	900	[37]
75	Tomato	Solanum lycopersicum	29	900	[61]
76	Wheat	Triticum aestivum	20	2125	[26]
77	Wild Strawberry	Fragaria vesca	14	240	[37]

Similarly, CPKs are also found in pollens, embryonic cells, guard cells, xylem, and meristem [36]. These Ca-dependent functional proteins are involved in biological functioning in cellular and subcellular compartments. Numerous CPKs of Arabidopsis are membrane-localized. It is considered that the myristylation causes CPKs to target the membrane [62]. This cellular and subcellular localization indicates a significant role of CPKs in several signaling transduction pathways under stress stimuli.

3.2. CPK Domain Organization and Calcium Ion Signal Decryption

On account of specific abiotic stress stimuli, the plant activates distinct physiological and biochemical response pathways. These stimuli are perceived by some protein and nonprotein elements. Protein elements include enzymes, transcription factors, and disparate receptors, while nonproteins comprise some secondary messengers such as calcium ion cyclic nucleotides, hydrogen ions, lipids, and active oxygen species [17,63]. Among them, Ca is a crucial secondary messenger involved in the signal transduction in all eukaryotes. It regulates the cell polarity and is essential for the regulation of stress-responsive cellular processes, cell morphogenesis, as well as plant growth and development [3,11,64,65]. These calcium signals are recognized by several protein kinases (CPKs), which regulate the response of downstream factors.

The CPK-encoding protein commonly has four functional domains, viz., calcium-binding domain (CBD), N terminus variable domain (NTD), protein kinase domain (PKD), and autoinhibitory junction (AJ), but many CPKs also contain an amino-terminal domain with varying sequence lengths, which is a source of functional diversity in the CPK family [62]. Sometimes, the C-terminus variable domain (CTD) also considered as a distinct domain instead of NTD. Different plant species contain varying numbers of CPK genes that are functionally important. The CBD contains four loops where calcium ions directly bind, called EF-hands, and are 20 amino acids in length [20,66,67,68]. The PKD domain has a characteristic serine/threonine phosphorylation site, which responds during regulation of CBD and AJ through Ca signals [68,69]. Among the number of CPK proteins, the majority of them have a myristylation site upstream from their N-terminal variable domain, showing that no CPKs appear in the form of membrane integral proteins [23]. The N-terminus of CPKs has a greater percentage of proline, glutamine, serine, and threonine (PEST) sequences, which carry out swift proteolytic degradation. There is an auto-inhibitory domain adjacent to the conserved domains, having a pseudo-substrate domain activity, and can cause inhibition of the regulatory pathways [68]. The variation in the length of CPK genes is due to the NTD, CT domain, and EF hand of the calcium-binding domain. Ca²⁺ through binding with the EF-hand motif, carries out the phosphorylation of the CPK substrate by removing autoinhibition of kinase activity [22,70]. The highly conserved calmodulin-like domain regulates all the activities of the CPKs by binding the four Ca²⁺ ions to four EF hands at its downstream end. Proteomics of most of the CPKs show that the autophosphorylation of proteins at serine and threonine through a calcium-dependent manner regulate the kinase activity (Figure 1).

Figure 1.

Structure and activation process of plant CPKs. (A) CPK domain structure under the inactive state, (B) activation of CPKs after the binding of Ca²⁺ to the active site of the protein kinase domain (PKD), the autoinhibitory junction (AJ), and calmodulin-like domain (CaM-like domain, CaM-LD).

CPKs are monomolecular Ca-signaling protein kinases that regulate protein phosphorylation. In response to extrinsic and intrinsic cues, the variation in Ca²⁺ concentration, also called “Ca²⁺ signatures”, is recognized, interpreted, and transduced to the downstream toolkit by a group of Ca²⁺-binding proteins. Phosphorylation events cause the activation of CPKs.

3.3. Functional Characterization of Plant CPKs

CPKs are differentially involved in diverse and indispensable functions in various plant species. CPKs show their role against biotic and abiotic stress tolerance upon interaction with specific calcium signals. With respect to abiotic stresses, CPKs are involved in drought [71], salinity [72], and heat [73] and cold [74] stress response signaling by regulating the ABA-responsive transcriptional factors and ion channel regulation [75]. Some Arabidopsis CPKs (e.g., CPK13) are also involved in potassium ion (K⁺) channel regulation and other ion transportation in guard cells [11]. CPKs are also a major participant for providing pathogen-related immunity to plants. In several plant species, CPKs enhance the resistance against fungal elicitors [1,76,77], bacterial invasions [78], and many other pathogen-related diseases [60,79]. Some CPKs are involved in the regulation of the jasmonic acid (JA)-dependent pathway during insect and plant interaction and indirectly regulate plant resistance against insects [80]. The crucial role of CPKs have also been reported in various growth and developmental processes in plants. CPK-encoding genes (AtCPK28) in Arabidopsis play a positive role in stem elongation and contribute to secondary growth by interacting with the gibberellic acid (GA) pathway [81,82]. Similarly, some CPKs regulate pollen tube growth [83], latex biosynthesis [55,84], higher biomass accumulation [85], wounding and herbivory attack [80,86], germination and seedling growth [87], early maturity [88,89], pigmentation and fruit development [90], and several other metabolic and developmental pathways [91]. Still, the role and functionality of various CPK-encoding genes against biotic and abiotic stresses are veiled.

4. Role of CPKs in Abiotic Stress Tolerance

CPKs are recognized as a key Ca sensor group of protein kinase, having a multigene family in the whole plant kingdom [55,92]. The functions of these CPKs are completely dependent on Ca²⁺ signatures. Most of CPK functionality has been identified only in vitro, which is why only specific stress response-associated functions are known [93]. CPKs are not only involved in ion channel regulation but also respond to multiple stress-related pathways through interactions with other distant transcription factors through phosphorylation. Several loss-of-function and gain-of-function studies have confirmed the role of CPKs in abiotic stress tolerance. The cytosolic Ca²⁺ concentration fluxes, induced by various environmental stresses, viz., heat [47], cold [94] light [95], drought [96,97], salt [72,98], and osmotic [99] and pathogen-related factors [100], activate the plant’s transcriptional and metabolic activities [101]. Expression analyses and genome-wide studies have discovered the CPKs transcript activity, protein, and substrate recognition in different plant parts [93]. CPKs are also involved in the ABA-dependent abiotic stress signaling in various plant species. Several CPK genes are involved in the regulation of ABA signaling pathways in plants. Transient gene expression analyses in protoplasts of maize show that CPK11 (closely related to AtCPK4 and AtCPK11) acts upstream of mitogen-activated proteins (MPK5) and is required for the activation of defense functions and antioxidant enzyme activity by regulating the expression of MPK5 genes. Similarly, CPK11 induced by hydrogen peroxide (H₂O₂) regulates and controls the activity of SOD and APX production induced by the ABA signaling pathway [102,103]. CPK activity confirmed by global expression analyses, shows that several CPK members are expressed differentially under varying ABA, salinity, drought, and heat and cold levels [93]. The change in the expression of CPK genes indicates the role of CPKs in plant adaptation against abiotic stress environments.

4.1. CPK-Mediated Drought Response Signaling

Drought stress is a major destructive factor affecting plant growth and development. It decreases water potential in plants as a result, where ABA accumulation controls the opening and closing of stomata, which leads to a lower photosynthetic activity [104]. It decreases the biomass and grain yield in plants. Under drought, plants adopt several conformational changes in the cell. These include ABA-dependent stomatal movement through regulation of guard cells, osmotic adjustments through the accumulation of osmolytes, regulating the oxidative damage by ROS homeostasis, and so on [93,105]. Changes in cytosolic Ca²⁺ concentrations due to water deficiency initiates CPK activity, resulting in the release of ABA in the cell [97]. ABA induces the injection of a calcium chelator (i.e., 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetra acetic acid; BAPTA), into the guard cell, which causes the closing of the stomata and, eventually, control of the transpiration process. Several plant CPKs are involved in drought stress-response mechanisms through an ABA-dependent manner. The CPK-encoding gene (CPK10) of Arabidopsis and an identified interacting heat shock protein (HSP1) lead to a drought-sensitive genotype. CPK10 T-DNA insertional mutants show sensitivity to drought stress as compared to the wild types. AtCPK9 and AtCPK10 are involved in Ca²⁺-dependent ABA-mediated stomatal regulation through interaction with AtCPK33 [106]. The light-induced Arabidopsis encoding gene (CPK13) is involved in inhibiting stomatal opening and contributes to the drought stress responsiveness [11]. Some drought-responsive CPKs also have some associated functions. In rice, for example, OsCPK9 controls both drought stress tolerance and spikelet fertility through an ABA-dependent manner. Results of overexpression of OsCPK9 (OsCPK9-OX) induces stomatal closure through osmotic adjustment and increases the pollen viability and spikelet fertility under polyethylene glycol (PEG-6000)-induced drought stress [71]. Another CPK-encoding gene from the wild grapevine (CPK20) acts as a regulator for drought and its associated with heat/cold responsive pathways. Expression of these genes studied in transgenic Arabidopsis reveals that VaCPK20 overexpression exhibits a high level of tolerance to drought and cold stress through regulation of stress responder genes, viz., ABA-responsive element binding factor 3 (ABF3) or sodium/hydrogen exchanger 1 (NHX1), and cold regulator gene (COR47) [107]. While a CPK-encoding gene of broad bean (VfCPK1) reported being highly expressed in leaf epidermal peels, it is not considered a tissue-specific gene and is only expressed under drought stress [108]. This CPK-encoding gene shows no relationship with both high (37 °C) and low (4 °C) temperatures. The increase in the number of transcripts of VfCPK1 under drought stress only plays a role in the up-regulation of ABA-responsive genes and other kinases that are involved in the signal transduction pathway [108].

Some CPKs are involved in the regulation of antioxidant production and osmolyte homeostasis to combat drought stress. AtCPK8 regulates the movement of the stomatal guard cell and H₂O₂ homeostasis in response to cellular Ca²⁺. An Arabidopsis T-DNA insertion mutant of CPK8 was found to be more sensitive to drought stress as compared to the wild-type plant, which reveals their drought response functionality [97]. CPKs phosphorylate some interactional proteins and perform interactive functioning in plants. Under drought stress, AtCPK8 with an interacting protein CAT3 controls the Ca²⁺-dependent ABA-mediated regulation of stomatal guard cells. The CPK8 mutant was more sensitive to drought stress, while overexpressing CPK8 in transgenic plants exhibited tolerance [97,109]. CaCPK1 activity increases the chickpea responsiveness to drought stress, and its activity is ubiquitous in all tissues of the plant [110]. The activation of drought-responsive CPK-encoding genes is also triggered by various biochemical pathways. A rice CPK-encoding gene (OsCPK1) specifically activated by sucrose starvation was involved in mechanism to prevent drought stress injury during germination by negatively regulating the expression of GA biosynthesis and activating the expression of a 14-3-3 protein ‘GF14c’ [111].

Some closely related CPK-encoding isoforms show functional diversity in response to drought stress. For example, functional divergence is present between two closely homologous (TaCPK7 and TaCPK12) genes of wheat [112]. Functional analysis of TaCPK7 and TaCPK12 reveals that TaCPK7 responded to H₂O₂, drought, salt, and low temperature, while TaCPK12 responded only through the ABA signaling pathway [112]. Several transgenic studies have been conducted to characterize the functions of CPKs in different plant species in relation to drought stress response signaling in plants. The ZoCDPK1 genes from ginger overexpressed in tobacco (Nicotiana tabacum) conferred drought as well as salinity tolerance by improving the photosynthesis and growth of the plant [113]. Enhanced expression of ZoCDPK1 under drought and JA treatment was observed, but no variation was found in expression because of low-temperature stress and abscisic acid treatment. ZoCDPK1 induces the expression of stress-responsive genes (i.e., early responsive to dehydration stress (ERD1) and responsive to dehydration (RD21A)). In ginger, it controls the stress signaling pathway and works in a CTR/DRE-independent manner [113]. Expression of CPK encoding genes of maize studied in Arabidopsis shows that ZmCPK4 is involved in resistance to drought stress through ABA-regulated stomatal regulation. ZmCPK4 induced by H₂O₂ and ABA treatment shows that there might be an association between mitogen-activated protein kinase (MAPKs) members and ZmCPK4 in the upregulation of ABA-regulatory components, especially ABA-insensitive (ABI5), ABF3, and Ras-associated binding protein (RAB18) [87]. The functions of several drought-responsive CPK-encoding genes are summarized in Table 2. (Details of all the genes are given in Table S1)

Table 2.

Various functions of CPKs in biotic and abiotic stresses in different plant species.

Sr. #	Specie Name	Gene	Function	Reference
1	Arabidopsis thaliana	AtCPK1	Cellular homeostasis, resistance fungal elicitor.	[76,78,114,115,116]
2		AtCPK3	Salt resistance.	[117,118]
3		AtCPK4	Regulate ABA-regulatory transcription factors (e.g., ABF, ABF4, drought resistance).	[98]
4		AtCPK5	Regulate immunity responses, ROS-dependent cell-to-cell communication.	[78]
5		AtCPK6	Drought tolerance, ABA-dependent osmotic adjustment.	[119]
6		AtCPK8	Drought tolerance through interaction with protein CAT3.	[97,109]
7		AtCPK9	Regulate the ABA-dependent signaling pathway interacting with CPK33.	[75]
8		AtCPK10	Drought responsiveness, ABA-mediated stomatal movements.	[106]
9		AtCPK11	Phosphorylation of AtDi19, ABA signaling.	[120]
10		AtCPK12	Seed germination, activation of ABA regulators.	[72,121]
11		AtCPK16	Root-gravitropism phosphorylate AtACS7.	[122]
12		AtCPK21	Hyperosmotic adjustments.	[123]
13		AtCPK23	Salt stress, drought stress.	[124]
14		AtCPK27	Salinity resistance, H2O2 and ionic homeostasis.	[125]
15		AtCPK28	Vascular development, stem elongation, ethylene synthesis, lignin deposition.	[81,82]
16		AtCPK32	ABA-regulatory gene activation.	[126]
17		AtCPK33	Regulates flowering, biosynthesis of florigen and flowering locus T protein.	[127]
18	Cicer areitinum (Chickpea)	CaCPK1	Salt stress, drought stress, phytohormones, and defense signaling pathways.	[110]
19		CaCPK2
20	Capsicum annuum (Peppers)	CaCPK3	Pathogen resistance, defense functioning (i.e., regulates jasmonic and salicylic acid), ethephon.	[79]
21	Fragaria x ananassa (Garden strawberry)	FaCPK1	low-temperature tolerance, fruit ripening.	[128]
22	Medicago sativa (Alfalfa)	MsCPK3	Heat stress resistance, embryogenesis.	[129]
23	Oryza sativa (Rice)	OsCPK1	Drought stress, seed germination, and GA biosynthesis.	[111]
24		OsCPK4	Microbial-associated immunity, OsRLCK176 degradation.	[130]
25		OsCDPK5	Fungal attacks phosphorylate OsERG1 and OsERG3.	[131]
26		OsCPK9	Drought stress tolerance, ABA sensitivity spikelet fertility.	[71]
27		OsCPK10	Pseudomonas syringae pv resistance, SA and JA regulator.	[132]
28		OsCPK12	Salt tolerance, blast disease resistance, induce ROS production, leaf senescence,	[1,133]
29		OsCDPK13	Regulate cold, salt, dehydration responses.	[134]
30		OsCPK17	Cold stress interacts with sucrose synthase and plasma membrane intrinsic proteins.	[135]
31		OsCPK21	Salt tolerance, ABA pathway activation.	[136]
32		OsCPK24	Cold stress tolerance, inhibition of OsGrx10.	[74]
33		OsCPK31	Starch accumulation, early grain filling.	[137]
34	Nicotiana tabacum (Tobacco)	NtCPK1	Signaling localization for repression of shoot growth, GA biosynthesis.	[138]
35		NtCPK2	Biotic stress immunity.	[139]
36		NtCPK32	Pollen tube growth interacts with CNGC18.	[83]
37	Hevea brasiliensis (Rubber tree)	HbCDPK1	Latex biosynthesis, rubber production.	[84]
38	Panax ginseng (Chinese ginseng)	PgCDPK1a	Regulate ginseng growth.	[85]
39	Phalaenopsis amabilis (Moth orchid)	PaCPK1	Cold stress sensitivity, wounding, pathogen attack.	[86]
40	Triticum aestivum (Wheat)	TaCDPK1	Regulate metabolic and developmental pathways.	[91]
41		TaCPK7	Drought stress, salt stress, ABA signaling pathway.	[112]
42		TaCPK12
43	Zingiber officinale (Ginger)	ZoCDPK1	Salinity and drought stress tolerance.	[113]
44	Zea mays (Maize)	ZmCPK1	Cold stress regulates ZmERF3 expression.	[33]
45		ZmCPK4	Upregulate ABA-regulatory components (i.e., ABI5, ABF3 and RAB18) with MAPKs.	[87]
46		ZmCPK11	Superoxide dismutase and ascorbate peroxidase production, ABA pathway.	[103]
47	Vigna radiata (Mung bean)	VrCPK1	Salt stress tolerance.	[140]
48	Vicia faba (Broad bean)	VfCPK1	Drought stress resistance.	[108]
49	Solanum lycopersicum (Tomato)	SlCDPK2	Flowering.	[141]
50		SlCDPK10	Xanthomonas oryzae pv. oryzae and Pseudomonas syringae resistance.
51		SlCDPK18	Xanthomonas oryzae pv. oryzae and Pseudomonas syringae resistance.	[60]
52	Solanum tuberosum (Potato)	StCPK1	Tuber formation.	[142]
53		StCPK4	Fungal pathogen resistance, ROS production.	[143]
54		StCDPK5	Blight resistance and susceptibility, ROS defense functioning.	[100]
55		StCDPK7	Resistance against Phytophthora infestans.	[77]
56	Nicotiana attenuate (Coyote tobacco)	NaCDPK4	Wound-induced jasmonic acid (JA) accumulation, insect resistance.	[80]
57		NaCDPK5
58	Camellia sinensis (Tea plant)	CsCDPK20	High-temperature stress resistance.	[144]
59		CsCDPK26
60	Hordeum vulgare (Barley)	HvCPK3	Resistance against powdery mildew.	[145]
61		HvCPK4
62	Brassica napus (Oilseed rape)	BnaCPK2	ROS accumulation, cell death.	[2]
63	Musa acuminate (Banana)	MaCDPK7	Heat-induced fruit ripening, chilling, stress tolerance.	[146]
64		MaCDPK2	Sensitive to Foc-TR4 infection, biotic stress tolerance.	[147]
65		MaCDPK4	Sensitive to Foc-TR4 infection, biotic stress tolerance.
66		MaCDPK3	Responsive for drought, cold, and salinity.
67	Vitis amurensis (Grapevine)	VaCPK1	Salt stress, heat-responsiveness, stilbene bio-synthesis.	[89,148]
68		VaCPK26	Salt stress, Stilbene bio-synthesis, through the induced expression of stilbene synthase (STS) genes.	[89,148]
69		VaCPK20	Drought stress, cold stress.	[107]
70		VaCPK21	Salt stress signaling.	[149]
71	Pharbitis nil (Picotee)	PnCPK1	Seed germination, seedling growth, flowering, regulation of light-dependent pathways, embryogenesis.	[90]
72	Populus euphratica (Desert poplar)	PeCPK10	Drought and cold stress tolerance, ABA-responsive genes regulator.	[150]
73	Cucumis melo (Hami melon)	HmCDPK2	Resistance against Penicillium infection.	[151]

4.2. CPKs-Mediated Salt Response Signaling

Salt stress is also a major abiotic factor limiting plant growth and global agricultural productivity. Salinity, mostly due to the accumulation of sodium Na⁺ and chloride Cl⁻ ions, causes an ion imbalance that leads the plants toward oxidative stress [152]. These ions also induce the toxicity of other ions in plants. Salts also increases the production of ROS in plants. Several studies have presented the functioning of CPK-encoding genes in plants against salt stresses. In Arabidopsis, AtCPK27 genes were found in favor of plant adaptation against salt stress [125]. Disruption in the expression of CPK27 in a T-DNA insertional mutant shows salt hypersensitivity at early growth stages in Arabidopsis. CPK27 regulated H₂O₂ and ionic homeostasis. AtCPK3 functions in guard cell movement through osmotic adjustment and ion channel regulation during salt accumulation [11,117,118]. The overexpression of AtCPK3 also increases ABA sensitivity and salt hypersensitivity, affecting the seedling growth and stomatal regulation [98,117]. AtCPK6 belongs to a subclass of the CPK gene family in Arabidopsis whose expression is induced under salt-stressed conditions. AtCPK6 and other kinases are activated because of cytoplasmic Ca²⁺ elevation in the calcium-dependent pathway, which depends on ABA. These kinases combined with AtCPK6 trigger the salt and osmotic stress tolerance. Overexpression of AtCPK6 in Arabidopsis increases the drought and salt tolerance in transgenic plants. RT-PCR analyses showed an increase in the expression of salt-regulated genes in plants, in which the AtCPK6 gene was over-expressed [119].

OsCPK12 positively modulates salt stress tolerance, and it is associated with decreases in the resistance against blast disease by increasing the sensitivity to ABA and inducing the accumulation of ROS in rice [1]. In Arabidopsis, AtCPK27 was found to be favorable for plant adaptation against salt stress. Disruption in the expression of CPK27 in T-DNA insertional mutant shows salt hypersensitivity at early growth stages. Under salt stress, CPK27 regulates H₂O₂ and ionic homeostasis and makes plants resistant to salt stress (Figure 2) [125].

Figure 2.

Role of different CPKs under various abiotic stresses; (A) Ca²⁺-dependent ABA-mediated drought and salt stress signal recognition by CPKs; (B) Ca²⁺ binding at the active site of protein kinase domain (PKD); (C) some drought-responsive genes involved in metabolite regulation and signal transduction pathways; (D) some salt-responsive genes and their role in antioxidant production (i.e., H₂O₂), as well as ROS detoxification; (E) some cold stress-responsive genes and their interaction genes activation; and (F) phosphorylation events controlling the anion channel regulation, K⁺-inward channel regulation, Ca²⁺-concentration, and channel regulation in the cell, and ABA-mediated CATALASE 3 regulation in plant cells.

OsCPK21 genes regulate the ABA-dependent salt stress signaling pathway. The high survival rate of transgenic rice seedlings developed by a mini scale, full-length cDNA over-expresser (FOX) gene hunting system was found due to the overexpression of OsCPK21-FOX under salt stress. In these plants, many salt-induced and ABA-regulating genes were expressed more as compared to wild-type plants. Overexpression of OsCPK21 increases exogenous ABA and enhances salt tolerance by regulating and inducing the salt tolerance genes [136].

VaCPK21 gene up-regulation is positively involved in salt stress-response signaling mechanisms in grapevines. Overexpression of this gene in transgenic Arabidopsis and V. amurensis callus cell lines shows that under the salt stress, VaCPK21 acts as a regulator for genes that respond to salt stress (i.e., AtRD26, kinase-like protein (AtKIN1), AtRD29B, AtNHX1, catalase (AtCAT1), copper superoxide dismutase (AtCSD1), cold regulator (AtCOR15 and AtCOR15)), and are found functionally important for salt stress tolerance [149]. Similarly, CaCPK1 and CaCPK2 activities are enhanced during high salt stress in leaves of chickpea plants. These isoforms play a role in the regulation of phytohormones and defense signaling pathways [110].

4.3. CPK-Dependent Cold and Heat Stress Signaling

Several CPK-encoding genes are differentially expressed under cold and heat treatments, but their exact molecular response mechanism is still unknown. OsCPK17 was reported to be important for the cold stress response by targeting the sucrose synthase and plasma membrane intrinsic proteins in rice [135]. OsCPK24 causes inhibition of glutaredoxin (OsGrx10) to sustain higher glutathione levels and phosphorylation, through the Ca²⁺ signaling pathway, and responds positively to cold stress tolerance in rice [74]. MaCDPK7 was found as a positive regulator of heat-induced fruit ripening and chilling stress tolerance in bananas [146].

PeCPK10 provides cold and drought stress tolerance through ABA-induced stomatal closing in P. euphratica. Its constitutive expression regulates ABA-responsive genes (i.e., RD29B and COR15A) that regulate the cellular functioning. Transgenic Arabidopsis with over-expressed PeCPK10 showed lower water loss under drought stress and tolerance against freezing. Expression analyses reveal that PeCPK10 localizes in cytoplasm quickly in response to changes in Ca²⁺ concentrations and regulates the stomata guard cells, while nuclear-localized PeCPK10 only regulates the transcriptional factors [150]. CPK16 and CPK32 in grapevine plants positively regulate stilbene (a phenolic secondary metabolite) biosynthesis and CPK30 individually involved in both cold and drought tolerance [153]. In maize, ZmCPK1 and ZmCPK25 gene expressions were increased or decreased, respectively, upon exposure to cold stress. ZmCPK1 is negatively related with the regulation of the cold stress signaling mechanism. Studies of transgenic Arabidopsis also show that ZmCPK1 inversely regulates the expression of ethylene response factor (ZmERF3) genes and impairs cold stress tolerance [33]. CsCDPK20 and CsCDPK26 act as regulatory factors for heat stress-responsive genes and control positive heat stress signaling in the tea plant [144].

4.4. Role of CPKs in ROS Detoxification

Drought, salt, and heat stress triggers ROS production in plants, which must be detoxified by the plant to prevent itself from oxidative stress. Mitochondria, chloroplasts, and peroxisomes are the central organelles for ROS accumulation [105,154]. ABA-induced ROS production in plants is reported to be dependent on nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase [105], which plays a vital role in oxidative bursting and activating plant defense responses [155,156]. Plant CPKs have been reported to regulate ROS production [2]. For instance, StCPK4 functions in the phosphorylation of NADPH oxidase and indirectly regulates ROS accumulation [143]. In B. napus, BnaCPK2 controls the activity of the respiratory burst oxidase homolog protein D (RbohD) during cell death and ROS production [2]. Arabidopsis CPK32 interacts with ABF4 in the ABA signaling pathway [126]. AtCPK6 from Arabidopsis decreases ROS production by reducing lipid peroxidation and confers drought stress [119]. Likewise, OsCPK12 promotes salt stress tolerance in rice through decreasing ROS accumulation [1]. The other CPKs and ROS responses are summarized in Table 2.

5. Functional Interaction of CPKs with Other Kinases in Abiotic Stress Signaling

CPK crosstalk and several interactions have been revealed in molecular regulatory pathways by functional studies. CPKs are not only involved in specific stress responses but also in multiple stress-related pathways by interacting with other distant proteins and regulating phosphorylation events. In Arabidopsis, CPK28 supports the turnover and phosphorylation of plasma membrane-related receptor-like cytoplasmic kinase (botrytis-induced kinase 1, BIK1), an important convergent substrate of multiple pattern recognition receptor (PRR) complexes for plant immunity [36]. AtCPK8 regulates and phosphorylates CAT3. It is involved in Ca²⁺-dependent ABA and H₂O₂-induced guard cell regulation and provides drought resistance [97,109]. Molecular responses of AtCPK1 studied by using real-time PCR (RT-PCR) show that the investigated gene expressions, viz., pyrroline-5-carboxylate synthetase 1(P5CS1), galactinol synthase 1(GOLS1), RD22 (dehydration-responsive protein), RD29A, C-repeat binding factor (CBF4), and KIN2 (kinases), were upregulated by ATCPK1 and conferred salinity stress tolerance [157]. Further, AtCPK1 in loss-of-function and gain-of-function mutants were studied. It provides salt and drought stress resistance by up and down-regulation of stress responder genes, viz., zinc finger protein (ZAT10), APX2, COR15A, and RD29A [157]. AtCPK12 phosphorylates several salt stress response-related proteins during regulatory functioning [72]. Another grapevine gene (VaCPK21) transgenically expressed in Arabidopsis interacts with several salt stress-related genes (i.e., AtRD29, AtRD26, AtKIN1, AtNHX1, AtCSD1, AtCAT1, AtCOR15, and AtCOR47). Likewise, VaCPK20 responds to cold and drought stress tolerance by regulating COR47, NHX1, KIN1, or ABF3 in transgenic Arabidopsis [107,149].

In vivo interaction validated by co-immunoprecipitation assays (Co-IP) revealed that OsCPK4, a dual-face protein, was involved in the regulation of the stability of cytoplasmic kinase (CPK176) in rice. OsCPK4 plays a vital role in the negative regulation of receptor-like OsCPK176 accumulation. OsCPK4 and OsCPK176 phosphorylation events provide pattern-triggered immunity [130]. OsCPK17 phosphorylates the sucrose-phosphate synthase (OsSPS4) and plasma membrane intrinsic proteins (OsPIP2;1 and OsPIP2;6) (aquaporin), which are essential in sugar metabolism and membrane channel activity against cold stress responses in rice [135]. Moreover, OsCPK24 is involved in the phosphorylation of glutathione-dependent thioltransferase and inhibition of OsGRX10 to maintain a higher level of glutathione. This regulatory pathway induces the overall cold stress responsiveness in rice [74]. The plant CPK-encoding genes also induce the regulation of other stress-responsive genes, viz., AtRBOHF, AtRBOHD, AtABI1, AtRAB18, AtRD29B, AtHSP101, AtHSP70, Arabidopsis heat stress transcription factor A2 (AtHSFA2), AtP5CS2, proline transporter (AtProT1), AtPOD, and AtAPX1 for drought, salt, heat and cold stresses [11]. In tea plants, CsCDPK20 and CsCDPK26 have an interactive function for thermo-tolerance [144]. BnaCPK2 interacts with NADPH oxidase-like RbohD and controls ROS accumulation and cell death in oilseed rape [2]. In Arabidopsis, CPK9 controls the ABA ion channel regulation through a Ca²⁺-dependent manner. Overexpression studies revealed that CPK9 and CPK33 mutually controlled the regulation of guard cells and stomatal movement [75]. CPK16 and CPK32 in grapevine plants positively regulate stilbene (a phenolic secondary metabolite) biosynthesis and CPK30 individually involved in both drought and cold tolerance [153]. Moreover, VaCPK1 and VaCPK26 genes are also involved in the same regulatory pathway ([89]. The overexpression of VaCPK29 up-regulates stress-responsive genes (i.e., dehydration elements (DREs) AtABF3, AtDREB1A, AtDREB2A, AtRD29A, and AtRD29B), which provide resistance to heat as well as osmotic stress [73]. Under in vitro conditions, post-transcriptionally miR390-regulated StCDPK1 controls the downstream auxin efflux carrier of PIN-proteins (StPIN4), which are involved in potato tuber development [142].

Arabidopsis CPKs interact and phosphorylate the basic leucine zipper domain (bZIP) transcription factor FD and have a crucial role in florigen complex formation, which induces late flowering in plants [127]. Biochemical analyses show that the cold-induced marker gene (Zmerf3), which is a type II ethylene response factor, is suppressed by ZmCPK1 in maize. It is supposed that the ZmCPK1 directly phosphorylates the ERF3 protein and, as a result, inactivates ERF and has a negative role in the cold stress response [33]. ZmCPK11 controls the upstream ZmMPK5, which is involved in ABA-dependent defense-related signaling in maize. CPK-encoding genes also have several interactive functions concerning plant growth and development. In Xenopus oocytes, AtCPK32 potentially regulates the cyclic nucleotide-gated ion channel regulating gene (CNGC18). AtCPK32 stimulation of CNGC18 regulates pollen tube depolarization in Arabidopsis [83]. Constitutively active OsCDPK1 in gain and loss-of-function transgenic rice targets the G-box factor 14-3-3c protein (GF14c). The expression of this protein causes the biosynthesis of GA and improves drought tolerance in rice seedlings [111]. AtCPK28 seems to be a regulatory component for the control of stem length and vascular development in Arabidopsis. The mutant of CPK28 (i.e., cpk28) was involved in the altered expression of NAC transcriptional regulators, such as NST1 and NST3, as well as gibberellin-3-beta-oxigenase 1 (GA3ox1), a regulator of gibberellic acid homeostasis [81]. After ABA treatment, the dual functioning OsCPK9-OX in rice increases the transcript levels of drought and spikelet fertility-responsive genes, viz., OsRSUS, Rab21, Osbzip66, and OsNAC45. The results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrate that OsCPK9 in interacting with these genes switches on the molecular regularization of ABA and stress-associated pathways [71]. The ZoCDPK1 gene from ginger promotes the expression of drought and salinity stress associated genes, viz., RD2A (dehydration responsive protein 2A) and ERD1 (early responsive to dehydration stress 1) in tobacco. This DRE/CRT-independent regulatory pathway improves photosynthesis and plant growth as well [113]. Constitutive expression of calcium-dependent protein kinase of Populus euphratica (PeCPK10) regulates (RD29B and COR15A) cold and drought genes [150]. This cross-talk between CPK isoforms and the interactive partners increases the complexities among the signaling pathways.

6. Conclusions

The multifaceted role of CPKs in plants is consequential for abiotic stress tolerance in plants. Regardless of the reported functional detail on CPK-encoding genes, there are many other important isoforms identified whose expression profiles and involvement in abiotic stress signal transduction pathways in plants are still not clearly known. Future research is required to extend and identify the remaining CPK-encoding genes, their interactional regulators, and their functional exploration with respect to abiotic stress responses. These research studies are helpful to improve the plant’s adaptation under unpredictable environments and to minimize threats to the world’s food security.

Abbreviations

ABA	Abscisic acid
AJ	Autoinhibitory junction
APX	Ascorbate peroxidase
AtABI1	Arabidopsis thaliana ABA-insenstive-1
AtCDPK/AtCPK	Arabidopsis thaliana-calcium dependent protein
AtCSD1	Arabidopsis thaliana copper superoxide dismutase 1
AtHSP101 and 70	Arabidopsis thaliana heat shock protein 101 and 70
AtProT1	Arabidopsis thaliana proline transporter 1
AtRBOHD	Arabidopsis thaliana respiratory burst oxidase protein D
AtRBOHF	Arabidopsis thaliana respiratory burst oxidase protein F
BaCDPK/BaCPK	Brassica napus calcium dependent protein kinase
BIK1	Botrytis-induced kinase 1
bZIP	Basic leucine zipper domain
Ca	Calcium
Ca2+	Calcium ion
CaCDPK/CaCPK	Cicer arietinum calcium dependent protein kinase
CaMs	Calmodulins
CAT	Catalase
CAT3	Catalase-3
CBD	Calcium binding domain
CBF4	C-repeat binding factor 4
CBLs	Calcineurin β-like proteins
CCaMK	Calcium/Calmodulin-dependent protein kinase
CDPKs/CPKs	Calcium dependent protein kinases
CMLs	Calmodulin-like protein Kinase
CNGC18	Cyclic nucleotide-gated ion channel 18
Co-IP	Co-immunoprecipitation assay
COR	Cold regulator
CT	C-terminus
CTR	C-repeat
DRE	Dehydration elements
EF	Elongation Factor
ERD1	Early responsive to dehydration stress 1
ERF3	Ethylene response factor 3
FaCDPK/FaCPK	Fragaria x ananassa calcium dependent protein kinase
GA	Gibberellic acid
GA3ox1	Gibberellin-3-betaoxigenase 1
GOLS1	Galactinol synthase 1
GPX	Glutathione peroxidase
GR	Glutathione reductase
H2O2	Hydrogen peroxide
HSF	Heat stress transcription factor
HSP	Heat shock protein
HvCDPK/HvCPK	Hordeum vulgare calcium dependent protein kinase
JA	Jasmonic acid
K+	Potassium ion
LeCDPK/LeCPK	Solanum lycopersicum calcium dependent protein kinase
MaCDPK/MaCPK	Musa acuminate calcium dependent protein kinase
MPK5	Mitogen-activated protein kinase 5
MsCPK	Medicago sativa calcium dependent protein kinase
NaCDPK/NaCPK	Nicotiana attenuate calcium dependent protein kinase
NADPH	Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
NHX	Sodium/Hydrogen exchanger
NST	NAC-transcription factors
NtCDPK/NtCPK	Nicotiana tabacum calcium dependent protein kinase
N-VD	N-terminus variable domain
OsCPK/OsCDPK	Oryza sativa calcium dependent protein kinase
OsGrx10	Oryza sativa glutaredoxin 10
OX	Overexpression
P5CS1	Pyrroline-5-carboxylate synthetase 1
PaCDPK/PaCPK	Phalaenopsis amabilis calcium dependent protein kinase
PeCDPK/PeCPK	Populus euphratica calcium dependent protein kinase
PEG	Polyethylene glycol
PEST	Proline, glutamine, serine and threonine
PgCDPK/PgCPK	Panax ginseng calcium dependent protein kinase
PIP	Plasma membrane intrinsic protein
PKD	Protein kinase domain
PnCDPK/PnCPK	Populus euphratica calcium dependent protein kinase
PRR	Pattern recognition receptor
qRT-PCR	Quantitative reverse transcription Polymerase chain reaction
RAB18	Ras-associated binding protein 18
RbohD	Respiratory burst oxidase homolog protein D
RD2A	Dehydration responsive protein 2A
RD29A	Dehydration responsive protein 29A
ROS	Reactive oxygen species
RT-PCR	Real-time PCR
SiCDPK/SiCPK	Setaria italic calcium dependent protein kinase
SOD	Superoxide dismutase
StCDPK/StCPK	Solanum tuberosum calcium dependent protein kinase
TaCDPK/TaCPK	Triticum aestivum calcium dependent protein kinase
T-DNA	Transfer DNA
VaCDPK/VaCPK	Vitis amurensis calcium dependent protein kinase
VaCPK/VaCDPK	Vitis amurenssis calcium dependent protein kinase
VfCPK/VfCDPK	Vicia faba calcium dependent protein kinase
VrCDPK/VrCPK	Vigna radiata calcium dependent protein kinase
ZmCDPK1/ZmCPK1	Zea mays calcium dependent protein kinase 1
ZoCDPK/ZoCPK	Zingiber officinale calcium dependent protein kinase

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/20/21/5298/s1.

Author Contributions

Conceptualization, R.M.A. and L.S.; Validation, G.C., M.W., M.A.R.R., F.A., and S.H.W.; Investigation, L.S., M.W., and F.A.; Resources, M.W., B.A., M.A.R.R., M.A.N., and S.H.W.; Writing—original draft preparation, R.M.A. and L.S.; Writing—review and editing, M.A.R.R., L.S. and M.A.N.; Visualization, G.C., M.W., M.A.R.R.; Supervision, L.S.; Project administration, R.M.A.; Funding acquisition, R.M.A.

Funding

This research was funded by the Punjab Agricultural Research Board (PARB) of Pakistan under the project grant # PARB-938.

Conflicts of Interest

The authors declare no conflicts of interest.