C2H2 Zinc Finger Proteins: Master Regulators of Abiotic Stress Responses in Plants

1As environmental problems become more severe, abiotic stress factors such as high salinity, low temperature, and drought will increasingly limit plant growth and crop yields. During the long process of evolution, plants have evolved protective mechanisms in response to stress involving morphological, physiological, and molecular adaptations. When a plant is subjected to abiotic stress, a series of adaptive changes occur in the plant cells to maintain growth, including the upregulation or downregulation of various genes.

2Regulatory proteins function in stress signal transduction by influencing the expression of downstream target genes (functional genes). These regulatory proteins include protein kinases [including mitogen activated protein kinases (MAPK), calcium dependent protein kinases (CDPK), receptor protein kinases, ribosomal protein kinases, and transcriptional regulatory protein kinases], protein phosphatases, transcription factors (TFs), and proteins involved in inorganic phosphate (Pi) turnover. TFs bind to specific sequences in the promoters of their target genes, thereby regulating gene expression and affecting biological phenotypes. TFs are key regulatory components of abiotic stress signaling pathways.

3Zinc finger proteins form one of the largest TF families in plants and are categorized into subfamilies based on the order of the Cys and His residues in their secondary structures, such as Cys2/His2-type (C2H2), C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6, and C8. Among these, C2H2-type zinc finger protein genes account for ~0.7% of Arabidopsis thaliana genes, 0.8% of yeast genes, and 3% of dipteran and mammalian genes.

4The first C2H2-type zinc finger protein gene discovered in plants was EPF1 from Petunia, encoding a protein with two typical C2H2 zinc finger motifs. Fourteen EPF1-related genes are present in the Petunia genome. Many C2H2-type zinc finger protein genes have since been cloned and studied in model plants such as Arabidopsis, wheat (Triticum aestivum), soybean (Glycine max), and rice (Oryza sativa). These genes encode proteins that play many roles in plant growth, development, and biotic stress resistance.

5In the past two decades, increasing evidence has indicated that C2H2-type zinc finger proteins also play important roles in abiotic stress resistance in plants. In this review, we describe the structures, classifications, and roles of plant C2H2-type zinc proteins in regulating abiotic stress responses in plants.

6Zn2+ binds to several conserved amino acids in C2H2-type zinc finger proteins (generally Cys and His) to form a relatively independent region of the protein. Eukaryotic C2H2-type zinc finger proteins generally contain a specific conserved sequence consisting of 25 to 30 amino acids: C-X2~4-C-X3-P-X5-L-X2-H-X3-H. Two pairs of histidines at the C-terminus of the α-helix and the two cysteines at the end of the β-strand bind to Zn2+ to form a tetrahedral structure. Zn2+ is sandwiched between an α-helix and two antiparallel β-strands; the stability of the ββα structural system is maintained by the interlaced linkage provided by Zn2+ (Figure 1). The presence of Zn2+ ensures that the stability of the entire zinc finger structure and the normal helical structure are maintained.

7Most C2H2 zinc finger proteins in plants contain the highly conserved sequence QALGGH in their zinc finger domains: such proteins are referred to as Q-type zinc finger proteins. Unlike proteins containing the QALGGH sequence, a class of C2H2-type zinc finger proteins do not contain any conserved motifs in the zinc finger region are known as C-type proteins.

8According to the number and distribution of zinc fingers, C2H2-type zinc finger proteins in plants are divided into four categories: (1) proteins containing one C2H2 zinc finger (single-C2H2); (2) proteins containing three C2H2 zinc fingers (triple-C2H2 [tC2H2]); (3) proteins containing more than three adjacent C2H2 zinc fingers (multiple-adjacent-C2H2 [maC2H2]); and (4) proteins containing several C2H2 zinc finger pairs that are widely separated (separated-paired-C2H2 [spC2H2]).

9Although many C2H2 transcription factors show transcriptional activation activity, there is no general rule about particular domains that function as transcriptional activators among the various types of domains, such as proline-rich, serine-rich, glutamine-rich, and threonine-rich domains. Many C2H2-type zinc finger proteins function as transcriptional activators during plant growth and abiotic stress, but the activation domain is poorly understood. Several stress-related C2H2-type zinc finger proteins contain a proline-rich region between the NLS and L-box motifs, which is considered to be the activation domain.

10The associated amphiphilic repression (EAR) motif (L/FDLN L/F(x)P) in plants is present in class II AP2/ERF proteins and in the C-termini of C2H2-type zinc finger proteins (TFIIIA class). The EAR motif reduces the underlying transcriptional level of the reporter gene, as well as the transcriptional activation activity of other TFs. The conserved EAR motif in class II ERF TFs plays a key role in their inhibitory activity. For example, the C-terminal inhibitory domain of AtERF4 is DLDLNL; if a mutation occurs in this domain, the protein's inhibitory function is abolished.

11C2H2-type zinc finger proteins with EAR motifs play important roles in regulating abiotic-stress-related gene expression under conditions such as salt, drought, and low temperature. The DLNL sequence (DLN-box) at the C-terminus of STZ, with trans-activation activity, functions in the transcriptional activation of DREB1A under salt stress, while the DLN-box is not the only domain that determines the transcriptional inhibitory activity of C2H2 zinc finger proteins.

12Plant C2H2-type zinc finger proteins have unique structural features: the conserved, unique QALGGH (Q-type C2H2) motif and the long variable spacers between adjacent zinc finger domains. Both features are thought to be important for DNA-binding activity in plants. A mutation of any amino acid in QALGGH sequence except Q will cause the protein to completely lose its ability to bind to DNA, whereas a mutation in Q will greatly reduce its DNA binding ability. Of course, characteristic region of a leucine-rich box (L-box) has also been identified to be related to protein interactions in these proteins. Bioinformatics tools such ZF Models (zinc finger specificity prediction based on the random forest model), ZifRC (zinc finger recognition code), and B1H screening of the C2H2-ZF domain have also been used for binding sequence prediction based on the C2H2-type zinc finger motif.

13In addition to binding to DNA, C2H2-type zinc finger proteins can recognize RNA. Zinc fingers comprehensively recognize specific bases and specific folding backbones in diverse RNA structures. Contact with the phosphoric acid skeleton in RNA is essential for the recognition of this molecule. A phage display assay showed that the amino acids at positions −1 and +2 of the α-helix in C2H2 zinc finger proteins are important for RNA binding.

14Zinc finger proteins interact with other zinc finger proteins or other types of protein to regulate target gene expression. Zinc finger proteins interact with other zinc finger proteins to bind to other DNA sequences or to prevent binding to the corresponding DNA sequences, thereby regulating gene transcription and expression, the potential for mediating protein interactions is much larger compared to DNA binding.

15In vitro experiments such as gel-shift and yeast one-hybrid assays are widely used to study the binding activity of plant C2H2-type zinc finger proteins to cis-elements in the promoter regions of their downstream genes. Several abiotic-stress-related C2H2-type zinc finger proteins, such as AZF1, AZF2, AZF3, and ZAT10/STZ, specifically bind to the A(G/C)T repeat sequences in their target promoters. The DNA sequence A[AG/CT]CNAC, the TGCTANNATTG element, and TACAAT motifs are also possible binding domains of C2H2 TFs in plants. Under abiotic stress conditions, C2H2 zinc finger proteins directly target downstream stress-related genes to activate or inhibit their expression.

16C2H2 zinc finger proteins directly target downstream ion balance-related genes to improve salt resistance in plants. C2H2 zinc finger proteins directly target key genes involved in the biosynthesis of osmotic adjustment substances to improve osmotic stress resistance in plants. C2H2 zinc finger proteins also directly target antioxidant genes associated with reactive oxygen species (ROS) scavenging under abiotic stress conditions. C2H2 zinc finger proteins directly target C-repeat/DRE-binding factor genes (CBFs) to improve cold resistance in plants. These proteins enhance salt tolerance by interacting with the miRNA-transport-related proteins. Finally, C2H2 zinc finger proteins directly target downstream genes involved in hormone signal transduction.

17Salt stress is a serious problem that limits crop production worldwide. Many C2H2-type TFs act as transcriptional activators or repressors to regulate plant responses to salt stress. Salt stress generates ionic, osmotic, and oxidative stress to plants. C2H2-type zinc finger proteins participate in salt tolerance by influencing salt-regulated genes.

18One way that C2H2-type zinc finger proteins can improve plant salt tolerance is by helping maintain ionic balance. STZ, the first salt-tolerance-related zinc finger protein identified, eliminates the Na+ sensitivity of calcineurin-deficient yeast. STZ appears to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ efflux in yeast. STZ might enhance salt tolerance in plants by regulating the expression of downstream ion-balance-related genes. Arabidopsis AZF1 and AZF3 respond rapidly to salt stress and may improve salt tolerance by regulating downstream ENA1-like genes. In rice, the ZFP182 promoter appears to respond only to monovalent cations such as Na+. Therefore, ZFP182 might regulate downstream ion-transport-related genes, such as Na+ transporter genes, in the salt regulatory signaling pathway. Finally, TaZNF improves salt tolerance in wheat by increasing the excretion of Na+ and reducing stomatal aperture.

19C2H2-type zinc finger proteins also improve salt tolerance by increasing the concentrations of osmotic adjustment substances. ZFP252 and ZFP179 improve salt tolerance in rice by increasing the contents of free proline and soluble sugars and upregulating various genes involved in the biosynthesis of osmotic substances, such as OsDREB2A, OsP5CS, OsProT, and OsLea3. In addition, IbZFP1 in sweet potato (I. batatas (L.) Lam.), ZFP3, and AtSIZ1 in Arabidopsis enhance plant salt tolerance by increasing proline biosynthesis and accumulation.

20C2H2-type zinc finger proteins also improve plant salt tolerance by increasing the ability to scavenge ROS. Arabidopsis plants constitutively expressing ZAT10 show enhanced expression of the ROS scavenging enzymes ascorbate peroxidase1 (APX1), APX2, and Fe-superoxide dismutase1 (FSD1). Transgenic tomato and Arabidopsis plants overexpressing SlZF3 show significantly increased levels of ascorbic acid. Bimolecular fluorescence complementation (BiFC) and Co-Immunoprecipitation (Co-IP) experiments show that SlZF3 directly binds CSN5B, a key component of the COP9 signalosome; CSN5B can promote the degradation of AsA in Arabidopsis by binding to VTC1, so the interaction of SlZF3 and CSN5B inhibited the binding of CSN5B to VTC1, which contributes to AsA biosynthesis.

21Many C2H2-type zinc finger proteins enhance plant salt tolerance through abscisic acid (ABA)-mediated signaling pathways. In addition to high salt, AZF2 and StZFP1 respond rapidly to ABA, suggesting that they improve salt tolerance via an ABA-dependent pathway. In Thellungiella, ThZF1, like AZF2, regulates downstream gene expression under salt stress. Transgenic Arabidopsis plants harboring GhDi19-1 and GhDi19-2 from cotton were more sensitive to salt and ABA than the wild type, suggesting that these genes are involved in plant adaptation to salt stress via the ABA signaling pathway.

22The mitogen-activated protein kinase (MAPK) cascade is involved in various biotic and abiotic stress responses. ZAT6-overexpressing lines in Arabidopsis show improved seed germination under salt stress via ZAT6's effects on the MAPK cascade. ZAT6 interacts with the stress-responsive MAPK MPK6, and the phosphorylation of ZAT6 by MPK6 is required to improve salt tolerance during seed germination. OsZFP213-overexpressing rice lines are more salt tolerant than wild-type and RNAi lines. Yeast two-hybrid, pull-down, and BiFC experiments demonstrate that OsZFP213 interacts with OsMAPK3 to regulate salt tolerance.

23C2H2-type zinc finger proteins can improve plant salt tolerance via several mechanisms simultaneously, such as promoting ionic balance, scavenging ROS, and increasing the levels of osmotic adjustment substances. GsZFP1 from soybean enhances salt tolerance in transgenic alfalfa by maintaining ionic balance, removing peroxides, and increasing the levels of osmotic adjustment substances. TaZFP1 overexpression lines show improved salt tolerance due to increased photosynthesis, osmolyte accumulation, and ROS scavenging. RNA-seq analysis show that TaZFP1 regulates photosynthesis, osmolyte metabolism, and ROS-homeostasis-related genes involved in salt stress. AtRZFP enhances salt tolerance by increasing ROS scavenging and maintaining Na+ and K+ homeostasis.

24C2H2-type zinc finger proteins can also improve salt tolerance via other mechanisms. ZAT7 may enhance salt tolerance by interacting with the miRNA-transport-related proteins WRKY70 and HASTY. A DST loss-of-function rice mutant shows enhanced salt tolerance due to increased stomatal closure and reduced stomatal density. ZjZFN1 from Zoysia japonica increases salt tolerance in transgenic Arabidopsis. RNA-seq analysis of these plants suggests that ZjZFN1 regulates phenylalanine metabolism, α-linolenic acid metabolism, and phenylpropanoid biosynthesis during salt stress.

25Osmotic stress is induced by many abiotic stresses such as salinity, cold, and drought stress. Osmotic stress causes physiological drought, ion imbalance, oxidative damage, and growth inhibition in plants. The ability of plants to resist osmotic stress is related to their ability to alter osmotic pressure and increase the biosynthesis of osmotic regulators.

26The C2H2 zinc finger proteins ZAT12 and ZAT10 are involved in regulating the osmotic stress pathway in Arabidopsis. Both ZAT10-overexpressing lines and zat10 mutant showed enhanced tolerance to osmotic stress. ZAT10 is phosphorylated by MPKs that are thought to function in abiotic stress tolerance. ZAT10 is a positive regulator of osmotic stress responses that is regulated by MAP kinases in Arabidopsis. In rice, RZF71 was strongly induced by 20% PEG6000 treatment, suggesting that RZF71 plays an important role in the response to osmotic stress. Moreover, six C2H2-type zinc finger proteins were reported to be involved in osmotic stress responses in poplar. Finally, heterologously expressing GmZAT4 from soybean enhanced the tolerance of Arabidopsis to 20% PEG treatment. Expression analysis of marker genes indicated that GmZAT4 enhances osmotic stress tolerance via an ABA pathway in both soybean and Arabidopsis.

27Cold snap often cause serious damage to plants and can kill plants when severe. A comprehensive understanding of the mechanism underlying cold damage in plants would help resolve the problem of cold stress injury.

28C2H2 zinc finger proteins enhance cold resistance by directly regulating downstream cold-related genes in plants. For instance, ZAT12 regulates cold acclimation by controlling the expression of 15 cold-suppressed genes and 9 cold-inducible genes. ZAT12 also downregulates the expression of CBF genes, suggesting it plays a negative role in plant adaptation to cold stress. Transgenic Arabidopsis and tobacco (Nicotiana tabacum) plants overexpressing SCOF-1 show increased expression of COR (cold-regulated) genes and enhanced cold tolerance. The SCOF-1 transgenic plants recover from chilling stress more rapidly than the control, and the T2 generation of SCOF-1 transgenic Arabidopsis still expresses cold regulatory genes, such as COR15a, COR47, and RD29B, at higher levels than the wild type at normal growth temperatures. SCOF-1 does not directly interact with the CTR/DRE, ABRE, or cis-acting elements in the promoter regions of COR genes in vitro, but yeast two-hybrid experiments demonstrated that SCOF-1 interacts with SGBF-1. Interestingly, SCOF-1 significantly enhances the activity of SGBF-1 bound to ABRE sequences in vitro, thereby promoting COR genes' expression and enhancing cold tolerance. The interaction of these two proteins is required for the participation of ABRE in the expression of cold-regulated genes to enhance cold tolerance. In addition, SCOF-1 increases cold tolerance in transgenic sweet potato and transgenic potato. SlCZFP1 enhances cold tolerance in transgenic Arabidopsis and rice by inducing the constitutive expression of COR or cold-responsive genes. The cold-stress-related gene COR6.6 was significantly upregulated in GmZF1 transgenic plants, suggesting that GmZF1 regulates cold stress resistance in transgenic Arabidopsis by binding to the COR6.6 promoter region. In banana, the overexpression of MaC2H2-2 and MaC2H2-3 significantly represses the transcription of MaICE1, a key component of the cold signaling pathway. Therefore, MaC2H2s might enhance cold resistance in banana by suppressing the transcription of MaICE1.

29C2H2 zinc finger proteins can enhance plant cold resistance by increasing the levels of osmotic substances. ZFP182 significantly enhances cold tolerance in rice overexpression lines by increasing the expression of OsP5CS and OsLEA3 and the accumulation of osmoprotectants. GmZF1 improved cold resistance in transgenic Arabidopsis by increasing proline and soluble contents and reducing membrane lipid peroxidation under cold stress.

30Finally, several C2H2-type zinc finger proteins regulate low-temperature stress through the ABA signaling pathway. SCOF-1 enhances plant cold tolerance through ABA-dependent signaling pathways. GmZF1 is highly induced by ABA treatment, suggesting that GmZF1 might participate in an ABA-dependent signaling pathway.

31Drought is an important factor limiting plant growth worldwide, inducing adverse reactions such as osmotic imbalance, membrane system damage, and decreased respiratory and photosynthetic rates. Drought not only hinders plant growth and metabolism at various stages but also affects crop quality and yields.

32C2H2 zinc finger proteins enhance plant drought resistance by increasing the levels of osmotic adjustment substances. For example, rice plants overexpressing ZFP252 have a significantly higher survival rate than wild-type and antisense-ZFP252 plants under drought stress. The overexpression lines exhibit enhanced expression of stress-related genes, such as Oslea3, OsP5CS, and OsProT, which contribute to the accumulation of osmotic substances. Overexpressing OsMSR15 and ZFP3 in transgenic Arabidopsis confers increase in drought tolerance by maintaining higher proline contents, reducing electrolyte leakage, and increasing stress-responsive gene expression.

33C2H2 zinc finger proteins also enhance drought resistance by improving the ability to scavenge ROS. ZFP245 improves drought resistance in rice by enhancing the activities of the ROS scavenging enzymes superoxide dismutase (SOD) and peroxide (POD) and increasing resistance to H2O2. When ZxZF from the highly drought-tolerant plant Zygophyllum xanthoxylum is expressed under the control of the drought-inducible promoter rd29A in Arabidopsis and poplar, the resulting transgenic plants have significantly increased photosynthetic efficiency, ROS, and scavenging ability. ZAT18 knock down mutant exhibits reduced drought tolerance, while the overexpression lines show higher leaf water content and higher antioxidant enzyme activity than wild-type plants under drought stress.

34C2H2 zinc finger proteins also enhance drought resistance via the ABA and other signaling pathways. Under dehydration stress, AZF2 expression is lower in the aba1 and abi1 mutants than in the wild type, indicating that AZF2 confers drought stress tolerance through an ABA-dependent pathway. GmZFP3 plays a negative role in plant tolerance to drought stress. Transgenic Arabidopsis plants of GmZFP3 show enhanced expression of ABA-related marker genes, including DREB2A, LHY1, MYB2, RCI3, PAD3, CCA1, and UGT71B6, suggesting that GmZFP3 regulates the drought stress response through an ABA-dependent signaling pathway. Transgenic plants overexpressing OsMSR15 show hypersensitivity to exogenous ABA. Transgenic plants also showed increased expressions of a number of stress-responsive genes, including RD29A and DREB1A under drought stress, which suggested that OsMSR15 may play important roles in response to drought stress both in ABA-dependent and -independent pathways. In the ZAT18 overexpression lines, RNA-seq analysis revealed the enrichment of pathways, such as hormone metabolism, stress response, and signaling pathways; stress-related genes, such as COR47, ERD7, LEA6, RAS1, and hormone-signaling-transduction-related genes, such as JAZ7 and PYL5, appear to be the downstream target genes of ZAT18.

35C2H2-type zinc finger proteins can also improve plant drought resistance through various mechanisms at the same time. Transformation with CgZFP1 and DgZFP3 from chrysanthemum, BcZAT12 from tomato, and IbZFP1 from sweet potato improve drought resistance in transgenic plants by increasing the levels of osmotic adjustment substances, improving ROS scavenging ability, and regulating downstream stress response genes. The Arabidopsis azf2 mutant expressing the Thellungiella halophila gene ThZF1 driven by the 35S promoter displays a flowering time phenotype similar to that of the wild type under drought stress. ThZF1 and AZF2 can both activate the transcription of AtEPSP. GsZFP1 overexpression enhances drought tolerance in transgenic alfalfa. The expression levels of stress-responsive marker genes, including MtCOR47, MtRAB18, MtP5CS, and MtRD2, were much higher in these plants than in the wild type under drought stress.

36Abiotic stress usually leads to the production of ROS. Low levels of ROS benefit plant growth and development by functioning as signals and might play other important roles as well, while the excessive accumulation of ROS causes secondary damage to plants. In the presence of excessive ROS, C2H2 zinc finger proteins are expressed at higher levels to help maintain stable ROS levels in plants.

37The expression of antioxidase genes that are associated with ROS scavenging under abiotic stress can be regulated by C2H2 zinc finger proteins. Ascorbate peroxidase 1 (Apx1) is an important scavenger of H2O2 in plants. The expression levels of TF genes ZAT12 and ZAT7 increase with increasing H2O2 levels in the loss-of-function mutant apx1. Plants lacking ZAT12 are more sensitive to high H2O2 contents than wild-type plants. APX1, APX2, and FeSOD1 are significantly upregulated in ZAT10 and ZAT12 transgenic lines, but are significantly downregulated in zat10 loss-of-function mutants under high-light stress. DST directly binds the promoter region of peroxidase24 precursor to regulate its expression and reduces the accumulation of H2O2 in plants. SOD and POD activity is significantly enhanced in ZFP245 and ZFP179 overexpression rice plants, thereby increasing resistance to abiotic stress and ROS at the seedling stage. Under water and oxidative stress, overexpressing ZFP36 reduces the damage from ROS by increasing SOD and APX activity, whereas ZFP36-RNAi lines exhibit severe ROS damage due to reduced SOD and APX activity.

38C2H2-type zinc finger proteins participate in antioxidative stress responses through ABA and MAPK signaling pathways. ZFP36 is significantly induced by ABA-triggered increases in H2O2 and OsMPK activity. ZFP36 also increases the expression levels of NADPH oxidase and MAPK genes in the ABA signaling pathway. These findings indicate that ZFP36 is involved in regulating the crosstalk among NADPH oxidase, H2O2, and MAPK in the ABA signaling pathway.

39Plants are highly sensitive to strong light. The ability of plants to withstand strong light depends on the duration of light exposure and light quality. Overexpressing the C2H2-type zinc finger protein gene RHL41, which is identical to the ZAT12 gene in Arabidopsis, significantly improved resistance to high-light conditions, as manifested by dramatic changes in plant morphology, such as increased production of thin-walled tissues of the barrier and increased anthocyanin and chlorophyll contents. The expression of ZAT12 increases in response to light stress. Perhaps ZAT12 is part of the active oxygen scavenging signal transduction pathway, which responds to H2O2 produced during light stress. ZAT10 expression is induced by light stress, and ZAT10-overexpressing transgenic lines showed enhanced tolerance to photo-inhibitory light stress. Under high-light stress, the levels of Apx1, Apx2, and iron superoxide dismutase 1 (FSD1) significantly increased in ZAT10-overexpressing transgenic plants, whereas suppressed expression was observed in ZAT10 knockout plants, suggesting that the increased tolerance of ZAT10-overexpressing plants to high light results from the specific expression of ROS clearance-related genes. However, to date, few studies have focused on the roles of zinc finger proteins in plant responses to high-light stress; more work needs to be done in this area.

40As is well known, many plant hormones participate in physiological adaptations to abiotic stress, and many stress related plant hormones that play an important role in resistance to many stresses mediated by C2H2 type zinc finger proteins have been studied. Abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are important plant hormones in pathogens and abiotic stresses signaling pathways.

41Typically, ABA is responsible for plant defense against abiotic stresses such as salinity, cold, osmotic, drought, active oxygen, and heat. Many studies found that the application of exogenous ABA can induce the expression of many stress related marker genes. There are at least two ABA-mediated signal pathways existing in plants to respond to abiotic stresses: ABA-dependent and ABA-independent pathways; the two pathways also have some degree of crosstalk in response to abiotic stresses. C2H2 zinc finger proteins can confer abiotic stress tolerance by increasing the contents of abscisic acid (ABA), proline, soluble sugars or chlorophyll, and by reducing the water loss rate. Zinc finger proteins are also involved in high temperature and H+ tolerance mediated by ABA signaling pathways. C2H2 zinc finger protein genes improve plant tolerance to stresses by ABA-dependent pathway or ABA-dependent pathway, whereas some genes improve plant tolerance to stresses through both ABA-dependent and ABA-independent pathways. Abiotic stresses, as well as ABA signaling pathways, together with C2H2 zinc finger proteins constitute a complex network and there is many overlaps between different signaling pathways.

42Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play major roles in biotic stress responses, as their levels increase in response to pathogen infection and herbivorous insect attack. Of course, the stress response of hormones to stress is not completely strict. SA is a phenolic hormone that functions as an important signaling molecule in plant responses to biotic and abiotic stress. C2H2 TFs are involved in the early response to SA in Carica papaya. AtZAT6 plays important roles in stress responses by activating the expression of SA-related genes in Arabidopsis. JAs are important class of lipid phytohormones. ZPT2-3 expression is induced by mechanical wounding and abiotic stress, a process mediated by a JA-dependent, ET-independent pathway. AtZFP11-overexpressing lines exhibited changes in the expression of a number of genes involved in JA and stress responses. ETHYLENE RESPONSE FACTOR1 (ERF1), a downstream component of the ET signaling pathway, is involved in plant resistance to several necrotrophic fungi. High salt levels and drought stress significantly induced the expression of ERF1 in Arabidopsis. ERF1 specifically regulates gene expression by integrating the JA, ET, and ABA signaling pathways and plays a positive regulatory role in plant resistance to salt, drought, and heat stress.

43It is now clear that C2H2-type zinc finger proteins play central roles in plant responses to high salt, cold, osmotic, drought, and oxidative stresses. Identifying abiotic stress sensors is crucial for understanding the molecular mechanisms underlying abiotic stress resistance in plants. For example, Glycosyl inositol phosphorylceramide (GIPC) was recently shown to be a salt receptor in plants. REDUCED HYPEROSMOLALITY-INDUCED CALCIUM INCREASE 1 (OSCA1) is considered to be an osmotic stress sensor, but how it perceives osmotic stress is not clear. Chilling-Tolerance Divergence 1 (COLD1), which regulates cold stress perception in rice, is another potential stress receptor. However, much more effort will be needed to identify abiotic stress sensors and to unravel the detailed roles of C2H2-type zinc finger proteins in abiotic stress resistance in plants.

44After stress signals are perceived by the plant, second messengers (such as Ca2+ and ROS) are generated in the cytosol, where they mediate the phosphorylation of downstream proteins (by protein kinases such as MAPKs) as well as ABA-dependent or -independent pathways. Various cis-elements are present in the promoter regions of high salt-, drought-, and ABA-induced genes. For example, C2H2-type zinc finger protein genes AZF2, AtSIZ1, and STZ contain cis-elements such as DRE (CCGAC), MYCRS (CANNTG), and MYBRS (RAACYR) elements in their promoter regions, conferring salt tolerance (Han et al., 2019). Protein kinases and ABA-dependent or -independent pathways directly or indirectly regulate C2H2 transcription factors, which in turn specifically activate or inhibit a group of target genes containing C2H2 zinc finger protein-binding sequences such as A [AG/CT]CNAC in their promoter regions, thereby enhancing plant stress tolerance (Figure 2).

45Although abiotic stress responses are regulated by multiple pathways controlled by C2H2 zinc finger proteins, there is a certain degree of overlap among these signaling pathways and regulatory networks. The roles of plant C2H2-type zinc finger proteins in different abiotic stress responses and signaling pathways are summarized in Supplementary Table 1. As shown in the table, the same C2H2 zinc finger protein gene can be induced by multiple abiotic stresses at the same time. In addition, different C2H2 zinc finger proteins increase plant stress resistance through similar regulatory mechanisms, and different C2H2 zinc finger proteins improve plant stress resistance through similar ABA or protein kinase signaling pathways. Therefore, the stress response network of plants is very complex, and the many unknown proteins and related pathways must be further elucidated.

46C2H2-type zinc finger proteins play important roles in plant development and growth as well as abiotic stress resistance. In-depth research on these proteins is needed to better understand their functions and mechanisms.

47The roles of C2H2 zinc finger proteins in plant stress-resistance mechanisms must be analyzed in more detail, such as how these transcription factors respond to various signaling molecules, how they regulate downstream gene expression, and how they interact with other transcription factors. Whether (and how) C2H2 zinc finger proteins mediate abiotic stress responses in plants by interacting with noncoding RNAs is currently unclear. The continuous development of biotechnology methods such as CRISPR-Cas should greatly facilitate the study of abiotic-stress-related C2H2-type zinc finger proteins in plants.

48In addition to the model plant Arabidopsis, more studies should focus on the roles of C2H2-type zinc finger proteins in stress-resistant plants, such as halophytes and xerophytes. During the course of evolution, stress-resistant plants have evolved various physiological and molecular mechanisms to adapt to stress. Halophytes can grow and finish their life cycles in saline soils with a salt concentration equivalent to ≥200mM NaCl, and euhalophytes grow vigorously in sea water. C2H2 zinc finger proteins might be associated with these morphological features or play distinct roles in these plants. For instance, overexpressing the C2H2 zinc finger gene ZjZFN1 from the halophyte Z. japonica improved salt tolerance in Arabidopsis by altering phenylalanine metabolism, α-linolenic acid metabolism, and phenylpropanoid biosynthesis. The expression of a homolog of the C2H2 zinc finger gene AtSIZ1 from the halophyte Limonium bicolor improved salt tolerance in Arabidopsis by maintaining ionic homeostasis and osmotic balance.

49These issues have been clarified in succession, which will be of great benefit to make full use of plant C2H2 zinc finger protein gene resources to improve crop varieties and increase plant stress resistance.

50GH and BW conceived and designed this study. GH and BW wrote the manuscript. CL, JG, ZQ, NS, and NQ proposed related theories and assisted with the interpretation of some references. All authors have read, edited, and approved the current version of the manuscript.

51This work was supported by the NSFC (National Natural Science Research Foundation of China, project No. 31570251; 31600200; 31770288), the Shandong Province Key Research and Development Plan (2017CXGC0313; 2016GNC113012), the Natural Science Research Foundation of Shandong Province (ZR2014CZ002; ZR2017MC003; ZR2019MC065), the Higher Educational Science and Technology Program of Shandong Province (J15LE08; J17KA136), and the Open Fund of Shandong Provincial Key Laboratory of Plant Stress (KLPS2018-01).

52The 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.