*Article* **Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis**

**Edgar Sepulveda-Garcia 1,2, Elena C. Fulton 3, Emily V. Parlan 3, Lily E. O'Connor 3, Anneke A. Fleming 3, Amy J. Replogle 3, Mario Rocha-Sosa 2, Joshua M. Gendron <sup>4</sup> and Bryan Thines 3,\***


**Abstract:** SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

**Keywords:** F-box protein; SCF complex; stress response; WD40 repeat-like protein

### **1. Introduction**

At the onset of environmental stress, the ubiquitin 26S proteasome system (UPS) selectively degrades key cellular proteins to initiate plant responses that promote resilience and survival. Protein targets destined for removal are ubiquitylation substrates for E3 ubiquitin ligases, where one prevalent E3 ligase subtype is the SKP1-CUL1-F-box (SCF) complex [1]. SCF complexes use an interchangeable F-box (FBX) protein subunit as a substrate adaptor to specifically interact with unique protein targets [2–5]. FBX proteins almost universally have a structure with two domains: An N-terminal F-box domain facilitates interaction with a SKP protein and the core SCF complex, and a C-terminal domain interacts specifically with the target(s) [2]. This two-domain structure directly bridges core UPS components to precise protein targets under specific conditions, and it

**Citation:** Sepulveda-Garcia, E.; Fulton, E.C.; Parlan, E.V.; O'Connor, L.E.; Fleming, A.A.; Replogle, A.J.; Rocha-Sosa, M.; Gendron, J.M.; Thines, B. Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis. *Plants* **2021**, *10*, 2228. https:// doi.org/10.3390/plants10102228

Academic Editors: Ewa Muszy ´nska, Kinga Dziurka and Mateusz Labudda

Received: 16 September 2021 Accepted: 11 October 2021 Published: 19 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

places FBX proteins at a dynamic interface that regulates diverse cellular pathways critical for plant life.

A very small number of FBX proteins, however, deviate from this typical two-domain protein structure. Many of these atypical FBX proteins have a centrally located F-box domain, a C-terminal target interaction domain, and an additional protein interaction domain at the N-terminus [6–8]. In humans, N-terminal domains can control subcellular localization [9], bind to an accessory protein that assists with C-terminal targeting events [10] or mediate regulatory interactions with other proteins [6,11,12]. The only plant FBX proteins with established N-terminal interaction dynamics belong to the ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) subfamily, which regulate the circadian clock and flowering time [8,13–16]. In addition to a central F-box domain, the ZTL/FKF1/LKP2 subfamily has a N-terminal blue-light sensing LOV domain and C-terminal kelch repeats [16], which are both used to recruit distinct ubiquitylation substrates [8,15,17,18]. The N-terminal LOV domain has additional roles that regulate the FBX function through an interaction with GIGANTEA (GI), which controls subcellular localization and protein stability [13,14]. Therefore, across kingdoms, a few atypical FBX proteins with a N-terminal protein interaction domain, in addition to a C-terminal targeting domain, achieve an expanded function by having further regulatory capacity and/or coordinating multiple cellular outputs through dual targeting.

F-BOX STRESS INDUCED (FBS) proteins constitute a far less understood subfamily of plant FBX proteins with an atypical structure [19–21]. Arabidopsis FBS1 is the founding member of this subfamily and is noteworthy for its broad biotic and abiotic stress-inducible gene expression profiles [19,21]. In FBS1, a centrally located F-box domain is flanked by two conserved regions present at the N- and C-termini, which do not match any known protein interaction domains or motifs [19]. FBS1 interacts with Arabidopsis SKP1 (ASK1) and can auto-ubiquitylate [19,20], suggesting that it forms a functional SCF-type E3 ligase in vivo. At least five of 13 Arabidopsis 14-3-3 regulatory proteins bind to FBS1 [20]. However, since this interaction requires both the N-terminal region and the F-box domain of FBS1 [20], and ubiquitylation presumably requires an unhindered F-box domain to interact with the SKP subunit of the SCF complex [1], the 14-3-3 proteins are unlikely ubiquitylation targets. Furthermore, an inducible *FBS1* gene construct had no discernable effect on FBS1 interactor 14-3-3λ protein abundance [20]. Importantly though, FBS1-interacting 14-3-3 proteins are negative regulators of Arabidopsis responses to cold and salt stress [22–26], which demonstrates another important link between FBS1 and environmental stress response networks in plant cells.

A more complete understanding of the FBS family protein function in plants has been stymied by two primary limitations. First, not knowing selective targeting relationships between SCFFBS complexes and their putative substrates has left FBS action on cellular output pathways completely enigmatic. Second, functional redundancy within this family has likely thwarted past efforts seeking to establish a biological function. Arabidopsis *fbs1* plants have no obvious phenotype [19,21], however, three additional FBS family members that may be functionally redundant are encoded in the genome. Here, we identify two highly conserved WD40 repeat-like proteins that interact with multiple FBS family members in Arabidopsis, which we have named FBS INTERACTING PROTEINs (FBIPs). Interactions between all four FBS subfamily members and FBIP proteins occur in the nucleus, and interactions occur exclusively via the N-terminal domain of FBS proteins. These findings connect a stress network involving FBS proteins to nuclear processes, and they provide new tools with which to probe unique N-terminal interactions in FBX proteins in the context of plant stress responses.

#### **2. Results**

#### *2.1. FBS Protein Interaction with ASK1*

FBS1 is the founding member of a four-member FBX protein subfamily (FBS1–FBS4) in Arabidopsis. FBS2–FBS4, similar to FBS1, share a non-canonical structure with a centrally

located F-box domain and conserved regions at their N- and C-termini (Figure 1A). The conserved region at the N-termini of FBS proteins spans approximately 20 residues, while the conserved region at the C-terminus encompasses about 35 residues (Figure 1A). FBS1 interacts with ASK1 and auto-ubiquitylates, indicating that FBS1 likely participates in functional SCF complexes [19,20]. However, the ability of other FBS family members to interact with ASK proteins remains unknown, as does the possibility of functional redundancy among family members. To interrogate this possibility, all four FBS family members were tested as bait constructs (DBD, GAL4 DNA-binding domain) for the interaction with ASK1 as prey (AD, GAL4 activation domain) under less stringent (-TLH) and more stringent (-TLHA) nutritional selection. Interactions were apparent between all four FBS family members on -TLH, although only very minimal growth was observed for FBS2 (Figure 1B). Only the interactions between FBS1 and FBS4 with ASK1 were apparent under the most stringent selection (-TLHA) (Figure 1B). Since Arabidopsis has 21 ASK proteins, it is possible that the FBS proteins showing minimal partnering with ASK1 interact more strongly with the other untested ASKs [27]. These interactions show, however, that FBS2–FBS4 are viable candidates for functional SCF complex substrate adapters, similar to FBS1.

**Figure 1.** The Arabidopsis F-BOX STRESS INDUCED (FBS) protein family. (**A**) Full-length protein sequence alignment of the four Arabidopsis FBS family members (FBS1–FBS4) created with the T-COFFEE sequence alignment program. Asterisks are fully conserved residues, colons are strongly conserved residue properties, and periods are weakly conserved residue properties. (**B**) FBS family interactions with ASK1 in yeast two-hybrid assays. Diploid yeast strains with indicated test constructs as bait (DBD) and prey (AD) were grown in liquid culture, diluted (OD600 = 100, 10−1, 10−2, 10−3), and spotted on SD medium minus Trp/Leu (-TL), minus Trp/Leu/His (-TLH), and minus Trp/Leu/His/Ade (-TLHA).

#### *2.2. Identification of a New FBS1 Interactor*

In addition to ASK1, the only established FBS1 interacting proteins belong to the 14-3-3 family [20]. However, since the interaction dynamics are not consistent with ubiquitylation of 14-3-3 proteins by SCFFBS1 [20], we sought additional FBS1 interactors as candidate targets that could connect FBS proteins to biological processes. Two additional related proteins were identified as partners for FBS1, which we have named FBS IN-TERACTING PROTEINs (FBIPs). FBIP1 (At3g54190) was identified in the same yeast two-hybrid screen that found 14-3-3 proteins as FBS1 interactors [20]. FBIP1 is also listed as an FBS1 interactor by the SUBA4 database (http://suba.live/, accessed on 16 September 2021) from high-throughput protein-protein interaction (PPI) screening [28,29]. FBIP1 is 467 residues in length and is a member of the transducin/WD40 repeat-like superfamily of proteins. WD40 repeats typically form a β-propeller domain that acts as a scaffold in mediating protein-protein or protein-DNA interactions [30]. Seven putative WD40 repeat-like sequences were predicted in FBIP1 by the WD40-repeat protein Structures Predictor database version 2.0 (WDSPdb 2.0) [31], although these predictions fall into the low confidence category (Figure 2). A second protein highly similar to FBIP1 was identified in the Arabidopsis genome by BLAST search, which we have named FBIP2 (At2g38630). The protein sequence identity and similarity between FBIP1 and FBIP2 are just over 91% and 96%, respectively (Figure 2).


**Figure 2.** FBS INTERACTING PROTEIN (FBIP) sequence features. Full-length protein sequence alignment of the two Arabidopsis FBIP family members created with the T-COFFEE sequence alignment program. Blue indicates locations of seven WD40-like repeat sequences predicted by the WD40-repeat protein Structure Predictor version 2.0 (WDSPdb 2.0). Asterisks are fully conserved residues, colons are strongly conserved residue properties, and periods are weakly conserved residue properties.

We gained no additional insight on the FBIP function using various bioinformatics resources. Other than putative WD repeat-like sequences, no sequence features were identified using various domain or motif prediction programs. BLAST and PSI-BLAST searches with FBIP1 and FBIP2 sequences failed to identify additional significant hits in Arabidopsis. We did, however, find very highly conserved FBIP protein sequences throughout the plant kingdom, including in bryophytes (the top BLAST hit in *Physcomitrella patens* is about 77% identical and 85% similar to *Arabidopsis* FBIP1). By investigating AtGenExpress ATH1 array datasets [32–34], we found that *FBIP1* is constitutively expressed in most tissues and organs of Arabidopsis, and throughout its life cycle, but we found no conditions where *FBIP1* is more highly expressed compared to the other conditions. *FBIP2* is not represented on the ATH1 array.

#### *2.3. FBS Interactions with FBIPs*

We confirmed that the full-length FBS1 and FBIP1 interact with yeast two-hybrid analysis. The interaction between FBS1 and FBIP1 elicited growth in yeast strains on both less stringent (-TLH) and more stringent (-TLHA) nutritional selection, and FBS1 yielded growth with FBIP2 on -TLH (Figure 3A). Family-wide interactions between each FBS protein and the two FBIP proteins were also assessed (Figure S1). Growth was observed for FBS3 and FBIP1, but not with FBS2 or FBS4. No additional interactions were observed with FBIP2. Collectively, the yeast two-hybrid results suggest that FBS1 and FBIP1 might be the primary FBS/FBIP protein interaction pair or possibly bind with the strongest affinity, but that some other family-wide interactions might be possible.

**Figure 3.** Yeast two-hybrid (Y2H) interactions between FBS1 and FBIP proteins. (**A**) Full-length FBS1 interactions with full-length FBIP1 and FBIP2. Diploid yeast strains with indicated test constructs as bait (DBD) and prey (AD) were grown in liquid culture, diluted (OD600 = 100, 10−1, 10−2, 10−3), and spotted on SD medium minus Trp/Leu (-TL), minus Trp/Leu/His (-TLH), and minus Trp/Leu/His/Ade (-TLHA). (**B**) Truncated FBS1 bait (DBD) construct interaction with full length FBIP1 prey (AD). Amino acid deletions are indicated on the left.

FBS proteins have two regions of unknown function outside of the F-box domain and, presumably, at least one of these interacts with a target. In order to determine which parts of FBS1 are important for the FBIP1 interaction, we created truncated versions of FBS1 with the N-terminal (NT), F-box or C-terminal (CT) regions removed in different combinations and tested under stringent (-TLHA) selection (Figure 3B). Removing the N-terminal region (ΔNT-FBS181–185) abolished the ability of FBS1 to interact with FBIP1, while removal of the F-box domain (ΔF-FBS1Δ84–135) or C-terminal region (ΔCT-FBS11–128) did not. The FBS1 N-terminal region (NT-FBS11–80) in combination with the full-length FBIP1 yielded growth on -TLHA, indicating that the FBS1 N-terminal domain alone is sufficient to mediate this interaction.

Near the conserved N-terminal domains of FBS1 and FBS2 we found a LXLXL sequence (Figure 1A), which is the most prominent form of an EAR motif found in many different types of transcriptional regulators [35,36]. The EAR motif mediates the interaction with the WD40 repeat-containing protein TOPLESS (TPL) and TOPLESS RELATED (TPR) co-repressor proteins [37–39]. We considered whether this LXLXL sequence in the N-terminal region of FBS1 might: (1) Function as a canonical EAR motif to interact with TOPLESS, and/or (2) if it could be important for mediating interactions with FBIPs. However, substituting all three leucine residues for alanine in FBS1 did not alter its interaction with FBIP1, and FBS1 did not interact with TPL (both as bait or as prey) in our yeast two-hybrid system.

#### *2.4. FBS Interactions with FBIP Occur in the Nucleus*

Next, we used bimolecular fluorescence complementation (BiFC) to test the FBS interaction with FBIP in plants and determine where the interaction occurs in a cell. The FBS and FBIP family proteins were expressed in *Nicotiana benthamiana* leaves as C-terminal fusions to either N-terminal (nYFP) or C-terminal (cYFP) halves of yellow fluorescent protein (YFP). In multiple independent experiments, the YFP fluorescence was observed for pairings between FBS1 and FBIP1 and FBIP2 (Figure 4). This YFP signal co-localized with that of a co-infiltrated H2B-RFP construct, which localizes exclusively in the nucleus [40], and shows that interactions between FBS1 and FBIP proteins also occur in the nucleus. Similar experiments found that FBS2–FBS4 also interact with FBIP1 in the nucleus (Figure S2). We observed interactions for FBS3 and FBS4 with FBIP2 (Figure S3), although we note that these interactions were more variable in the number of YFP positive nuclei across independent replicates, and with consistently fewer interactions for FBS3 and FBIP2. We did not observe any interactions between FBS2 and FBIP2. All FBS and FBIP fusion protein constructs were tested as pairs with empty nYFP or cYFP vectors, and in all pairings we were unable to detect any fluorescent signal similar to the FBS/FBIP test pairs (Figure S4). These findings show that in plants the FBS proteins participate in family-wide interactions in the nucleus.

**Figure 4.** Bimolecular fluorescence complementation (BiFC) interactions between FBS1 and FBIP proteins. Laser-scanning confocal microscopy of *N. benthamiana* epidermal cells expressing N-terminal nYFP- or cYFP-tagged FBS1 and FBIP proteins. FBS1 interactions with FBIP1 (top row) or FBIP2 (bottom row) are visualized on the BiFC yellow channel (YFP, left column). A co-expressed H2B-RFP (as nuclear marker) is visualized on the red channel (RFP, middle column) and YFP/RFP images are overlaid (Merge, right column). Arrow indicates selected nuclei in the expanded inset image. Scale bar = 100 μm.

#### *2.5. FBS1 Destabilizes FBIP1*

With the interaction established between multiple FBS and FBIP protein pairs, we next asked if the protein abundance relationship between FBS1 and FBIP1 is consistent with FBIP1 being a ubiquitylation target of SCFFBS1. If a protein is ubiquitylated by a particular SCF complex and subsequently degraded by the 26S proteasome, then increasing the abundance of the F-box component typically increases in vivo targeting and decreases substrate abundance [41]. Therefore, we tested the effects of varying FBS1 protein levels on the FBIP1 abundance in our *N. benthamiana* expression system by co-infiltrating *Agrobacterium* harboring these test constructs in different relative concentrations. Increasing the presence of FBS1 protein resulted in a corresponding decrease in the FBIP1 protein abundance by Western blot analysis (Figure 5). In comparison, when the FBS1 abundance was increased relative to the co-infiltrated 14-3-3λ in an identical setup, we did not observe any decrease in 14-3-3λ abundance as the amount of expressed FBS1 was increased (Figure S5). This finding is congruous with previous observations that FBS1 and 14-3-3 interactions are not consistent with targeting [20]. Therefore, since the abundance of FBIP1 decreases in an FBS1-dependent manner, we conclude that FBIPs are viable candidates for SCFFBS1 ubiquitylation targets.

**Figure 5.** FBS1 influence on FBIP1 protein abundance in plants. *N. benthamiana* leaves were infiltrated with *Agrobacterium* (C58C1) strains to express the tagged proteins. *Agrobacterium* mixes contained varying cell densities of strains harboring expression constructs (myc-FBS1 and/or FBIP1-HA), a suppressor protein (p19) or untransformed cells. Total protein was isolated from leaves 3 days after infiltration, separated by SDS-PAGE, transferred, and probed with antibodies against myc (top row, FBS1) or HA (second row, FBIP1). Bottom two rows show Ponceau S staining of the major subunit of Rubisco from the same two blots as a loading control.

#### **3. Discussion**

As substrate adapters for SCF-type E3 ligases, FBX proteins act at the interface between core UPS components and specific cellular outputs, including those that help plant cells mitigate the effects of environmental stress. Previous work with FBS1 strongly alluded to some role in plant stress responses, possibly by regulating the expression of stress genes [19–21], but a more detailed understanding was limited by the unknown identity of ubiquitylation target(s) and by possible redundancy within the *FBS* gene family. Here, we have identified a pair of WD40 repeat-like superfamily proteins, FBIP1 and FBIP2, that both interact with FBS family proteins. These family-wide interactions indicate that functional redundancy within these two families is likely, but at the same time suggest a more robust stress response module. The FBS protein interaction with FBIPs in the nucleus points to a role for these proteins in the regulation of gene expression and/or other chromosomal events. Finally, FBIP proteins are strong candidates for SCFFBS ubiquitylation targets that act in plant stress responses, and they provide new tools with which to investigate unique FBX protein N-terminal events in plants.

The exclusive nuclear localization FBS and FBIP protein interactions under the conditions we tested offer a critical clue as to the molecular functions of both protein families. One hypothesis for the FBIP function stemming from this result is that they regulate gene expression, which is an idea supported by the finding that hundreds of JA/ABA and other stress genes are mis-expressed in the *fbs1-1* background [21]. Some plant nuclear localized WD40 repeat proteins have direct actions in transcription regulation [39,42,43] or chromatin modification [44–46], and in these cases the WD40 repeat proteins are essential components of multi-protein assemblies. For example, TOPLESS (TPL) is a wellstudied WD40 repeat-containing co-repressor protein that acts in diverse developmental and environmental-response pathways [39]. TPL interacts with different DNA-bound transcriptional complexes and it recruits chromatin modifying enzymes and/or Mediator to repress gene expression [40,47,48]. TRANSPARENT TESTA GLABRA 1 (TTG1), another WD40 repeat protein, serves as a scaffold and mediates different combinations of bHLH and R2R3-type MYB DNA-binding transcription factors to regulate flavonoid metabolism and various developmental processes [43,49]. The FBIP proteins may function similarly to TPL or TTG1 and act as scaffolds and/or in recruitment roles for complexes that regulate transcription. Knowing additional FBIP interactors, which may include more recognizable proteins with readily inferred functions, will help address this hypothesis.

Future work will also be guided by questions that address interaction dynamics between FBIPs and the N-terminal region of FBS proteins, and the consequences of these associations. There are 13 residue positions in the FBS N-terminal region, ranging from moderately to absolutely conserved, that could be critical for the interaction with FBIPs. Future work will include identification of the exact residue or residues in FBS proteins mediating this interaction. Given numerous FBS connections to stress, but that *FBIP1* appears to be constitutively expressed across different plant organs and environmental conditions, it could be the case that FBIP proteins are components of a stress-response system working at the post-translational level. Next steps include a rigorous assessment of conditions under which SCFFBS complexes form and interact with FBIP proteins in vivo. Furthermore, whether some additional factor (i.e., post-translational modification) stimulates SCFFBS association with FBIP proteins, as in the case of some other SCF targeting events [50], is well worth investigating. The idea that additional in vivo factors or modification mediates the FBS/FBIP interaction is consistent with the finding that we observed more family-wide interactions in plant BiFC experiments compared to yeast two-hybrid. Knowing that SCF complexes in some atypical contexts ubiquitylate targets via the FBX protein N-terminal interactions [8,17], and that FBS1 appears to destabilize FBIP1, a leading hypothesis for future work is that FBIP proteins are bona fide ubiquitylation substrates for SCFFBS. Considering our work here and the general knowledge surrounding SCF action, our current model is that stress stimulates increased SCFFBS-dependent ubiquitylation of FBIP proteins, which are then degraded in response to this environmental trigger, resulting in cellular changes.

The atypical structure of FBS proteins, along with the identification of FBIPs as FBS Nterminal interactors, leads to a few intriguing hypotheses regarding how this SCF complex may impact cellular pathways in plant stress. If FBIP is a bona fide target with a biological function distinct from a more typical C-terminal target, then SCFFBS complexes provide an exciting opportunity to study how plants coordinate more than one cellular pathway

related to stress. N- and C-terminal targeting events might be simultaneous under a given condition, and in this situation SCFFBS may integrate a response by ubiquitylating two distinct protein types, each interacting with a different region of the FBS substrate adapter. Alternatively, N- and C-terminal targeting may be asynchronous and condition dependent, in which case SCFFBS may entail a switch that works in or leads to two different cellular states. At this point, however, we cannot completely exclude the possibility that FBIPs are not targets (see above), but instead serve in an alternative capacity that enables or inhibits the FBS action. One idea then is that FBIPs are accessories that help recruit other proteins as ubiquitylation targets. In humans, Cks1 directly associates with the N-terminus of FBX protein Skp2 to direct SCFSkp2 interaction with ubiquitylation target p27 in human cell cycle regulation [10,50]. In Arabidopsis, KAI2 and D14 interact with FBX protein MAX2 in SCFMAX2 complex to mediate ubiquitylation of SMXL transcription factors [51], though in these cases KAI2 and D14 are also FBX C-terminal interactors. To address these scenarios or others a critical piece of information to learn is the identity of a FBS C-terminal region-interacting protein that we presume to exist. Future work can then investigate higher order SCFFBS complex assembly and action.

The 14-3-3 proteins directly regulate a range of cellular processes in plant cells [52], including core signaling pathways and transcriptional reprogramming events in cold and salt stress responses [22–26]. FBS protein interactions with FBIPs will almost certainly be a vital tool used to fully understand the connections between FBS proteins and the 14-3-3 protein regulatory network. Five of 13 Arabidopsis 14-3-3 proteins interact with FBS1. However, 14-3-3 proteins are unlikely ubiquitylation targets of SCFFBS1 and the consequences of these interactions are unknown [20]. One hypothesis regarding this interaction is that 14-3-3 proteins promote dimerization of SCFFBS ligases [20], which in other situations enhances ubiquitylation targeting by SCF complexes [53,54]. As our understanding further develops regarding FBIPs as putative targets, their cellular abundance will be an essential readout in studies that investigate 14-3-3 effects on SCFFBS activity. The 14-3-3 proteins exert regulatory effects through other mechanisms, however, through controlling the subcellular localization of client proteins or by shifting the location themselves [52]. In salt stress, FBS1 interactors 14-3-3λ and 14-3-3κ act at the plasma membrane and release signaling component SOS2 to activate salt stress tolerance [26,55]. Cold temperature triggers the FBS1 interactor 14-3-3λ to translocate from the cytosol into the nucleus where it interacts with and adjusts cold-responsive C-repeat-binding factor (CBF) action [25]. Considering that the FBS1 interaction with FBIPs was exclusively nuclear under the conditions tested here, an investigation of temporal and spatial aspects of 14-3-3/FBS interactions relative to FBS/FBIP interactions in plant cells before and during environmental stress will add more broadly to our understanding of the 14-3-3 stress response network in plant cells.

#### **4. Materials and Methods**

Bioinformatics: Gene and protein sequences were obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org, accessed on 16 September 2021). Protein sequences were aligned using T-COFFEE (http://www.ebi.ac.uk/Tools/msa/tcoffee, accessed on 16 September 2021) accessed through the European Bioinformatics Institute (EBI) website (http://www.ebi.ac.uk, accessed on 16 September 2021) [56]. WD40 repeat-like sequences were identified in FBIP1 and FBIP2 using the WD40-repeat protein Structures Predictor database version 2.0 (WDSPdb 2.0; http://www.wdspdb.com/wdsp/, accessed on 16 September 2021) [31]. Basic Local Alignment Search Tool (BLAST) and Position-Specific Iterative (PSI)-BLAST were accessed through the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov, accessed on 16 September 2021) and used to search the RefSeq database. Candidate protein interactors were identified by searching the SUBA4 database (http://suba.live/, accessed on 16 September 2021) [29].

Gateway cloning: Gene-specific primers (Supplementary Table S1) were used with PCR to amplify coding sequences from pooled *Arabidopsis thaliana* (accession Col-0) cDNA. Amplicons were inserted into the pENTR/D-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. Then, the genes were transferred with the LR Clonase II enzyme mix (Thermo Fisher Scientific, Waltham, MA, USA) into pCL112 or pCL113 [57] destination vectors for BiFC experiments, and into pGBKT7-GW (Addgene plasmid #61703) or pGADT7-GW (Addgene plasmid #61702) destination vectors for yeast two-hybrid experiments. Alternatively (Figure 3B), *FBS1* and *FBIP1* sequences were cloned into pBI770/pBI771 and tested for the interaction, as done previously [20]. Primers used to create *FBS1* truncation constructs are indicated in Supplementary Table S1.

Yeast two-hybrid assays: *Saccharomyces cerevisiae* cells were grown, transformed, mated, and selected by standard yeast protocols. Bait constructs (GAL4 DNA-binding domain, DBD) were transformed into Y2H Gold and prey constructs (GAL4 activation domain, AD) and Y187 strains by the LiAc method (Takara Bio; San Jose, CA, USA). Haploid strains were mated to produce diploid strains to test for the interactions. Diploid strains were grown for 24 h at 30 ◦C in the liquid synthetic defined (SD) medium minus Trp/Leu (-TL) medium with shaking. Thereafter, cells were washed in sterile water, cell concentrations were adjusted to OD600 = 100, 10−1, 10−2, 10−3, and 10 μL was spotted on SD -TL (control), SD minus Trp/Leu/His (-TLH), and SD minus Trp/Leu/His (-TLHA) selective plates. The plates were incubated for 2 days at 30 ◦C and then scanned to produce images.

Bimolecular fluorescence complementation (BiFC): Recombinant plasmids were transformed into the *Agrobacterium tumefaciens* strain GV3101 (pMP90) by electroporation and selected under appropriate antibiotics. *A. tumefaciens* seed cultures were grown in LB with the appropriate antibiotic selection for 2 days with shaking at 30 ◦C. Then, they were used to inoculate 50 mL LB containing the appropriate antibiotics plus 10 μM acetosyringone and grown for an additional 24 h. The cells were pelleted and resuspended in the infiltration medium (10 mM MES, 10 mM MgCl2, 100 μM acetosyringone) and incubated for 5 h with rocking at room temperature. The cells were pelleted a second time, resuspended in the infiltration medium, and the appropriate nYFP/cYFP, H2B-RFP constructs were combined at a final OD600 of 1.0 for each test/control construct with suppressor strains (p19, γβ, PtoHA, HcPro) at a final OD600 of 0.5. *Nicotiana benthamiana* leaves from 4-week-old plants were infiltrated by a syringe with the *A. tumefaciens* mixes. The underside of whole leaf mounts was visualized using laser-scanning confocal microscopy 3 days after infiltration with a Nikon D-Eclipse C1 Confocal laser scanning microscope (Nikon Instruments) with either: (1) Excitation at 488 nm with an emission band pass filter of 515/30 or (2) excitation at 561 nm with an emission band pass filter of 650 LP.

Co-infiltration: *FBS1*, *FBIP1*, and *14-3-3λ* were cloned into pN-TAPa (9X myc tag), pGWB14 (3X HA tag) or pGWB12 (VSVG tag) vectors [58], respectively, using a Gateway strategy as above. Recombinant plasmids were transformed by electroporation into the *A. tumefaciens* strain C58C1Rif/pGV2260. *A. tumefaciens* was grown to a stationary phase in the LB medium containing the appropriate antibiotics plus 50 μg/mL acetosyringone. Bacteria were pelleted and washed with 10 mM MgCl2, and then resuspended in 10 mM MgCl2 and 150 μg/mL acetosyringone. Cell densities were adjusted to OD600 of 0.5. After 3 h of incubation, *A. tumefaciens* strains containing each construct were adjusted to varying concentrations and mixed with the same volume of an *A. tumefaciens* strain containing the viral suppressor p19, treated in the same way, but adjusted to OD600 of 1.0. The abaxial side of leaves from 3–4 week-old *N. benthamiana* were infiltrated with this bacterial suspension. After 3 days, the leaf material was collected and immediately frozen in liquid N2 for protein extraction.

Protein extraction and Western blotting: Approximately 100 μg of frozen tissue was homogenized in 200 μL of 1× Laemmli loading buffer plus 4 M urea, boiled for 5 min, and centrifuged at 10,000× *g* for 5 min. Then, 10 μL of the supernatant were loaded onto 8%, 10% or 15% polyacrylamide gels and subjected to SDS-PAGE using the standard protocols. The separated proteins were blotted onto a Hybond-P+ membrane (Amersham Pharmacia Biotech, Amersham, UK) using the standard protocols, and then the membranes were probed with anti-c-Myc, anti-HA antibody or anti-VSVG antibodies (all from SigmaAldrich, St. Louis, MO, USA). The blots were developed using an alkaline phosphatase kit (BCIP/NBT kit; Invitrogen; Waltham, MA, USA).

AGI numbers: FBS1 (At1g61340), FBS2 (At4g21510), FBS3 (At4g05010), FBS4 (At4g35930), FBIP1 (At3g54190), and FBIP2 (At2g38630).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants10102228/s1, Figure S1: Yeast two-hybrid FBS1–FBS4 interactions with FBIP1 and FBIP2. Figure S2: Bimolecular fluorescence complementation (BiFC) interactions between FBS1–FBS4 and FBIP1. Figure S3: Bimolecular fluorescence complementation (BiFC) interactions between FBS1– FBS4 and FBIP2. Figure S4: YFP channel positive and negative controls. Figure S5: FBS1 influence on 14-3-3 λ protein abundance in plants. Table S1: Primer sequences used for cloning.

**Author Contributions:** E.S.-G., E.C.F., E.V.P., L.E.O., A.A.F., A.J.R., M.R.-S., J.M.G. and B.T. designed the experiments. E.S.-G., E.C.F., E.V.P., L.E.O., A.A.F., A.J.R. and B.T. conducted the experiments and analyzed the data. B.T. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from the M.J. Murdock Charitable Trust (NS-2016262 and 20141205:MNL:11/20/14) for materials and student summer research stipends, and funds from the University Enrichment Committee (UEC) at the University of Puget Sound for materials and student summer research stipends.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank David Somers (The Ohio State University) for the H2B-RFP construct, Frank Harmon (University of California at Berkeley/USDA Plant Gene Expression Center) for the yeast two-hybrid vectors, Faride Unda (University of British Columbia) for the BiFC vectors, and Ruirui Huang and Vivian Irish (Yale University) for the yeast two-hybrid TOPLESS constructs. Moreover, we thank Andreas Madlung (University of Puget Sound) for critical reading of the manuscript and other helpful discussions. Finally, we would like to thank Michal Morrison-Kerr (University of Puget Sound) for her indispensable help in supporting Puget Sound undergraduate research students.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


### *Review* **Crosstalk between Ca2+ and Other Regulators Assists Plants in Responding to Abiotic Stress**

**Yaoqi Li †, Yinai Liu †, Libo Jin \* and Renyi Peng \***

Biomedicine Collaborative Innovation Center of Zhejiang Province, Institute of Life Sciences, College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China; 20461337004@stu.wzu.edu.cn (Y.L.); 21461338012@stu.wzu.edu.cn (Y.L.)

**\*** Correspondence: libo9518@126.com (L.J.); 20170032@wzu.edu.cn (R.P.)

† These authors contributed equally to this work.

**Abstract:** Plants have evolved many strategies for adaptation to extreme environments. Ca2+, acting as an important secondary messenger in plant cells, is a signaling molecule involved in plants' response and adaptation to external stress. In plant cells, almost all kinds of abiotic stresses are able to raise cytosolic Ca2+ levels, and the spatiotemporal distribution of this molecule in distant cells suggests that Ca2+ may be a universal signal regulating different kinds of abiotic stress. Ca2+ is used to sense and transduce various stress signals through its downstream calcium-binding proteins, thereby inducing a series of biochemical reactions to adapt to or resist various stresses. This review summarizes the roles and molecular mechanisms of cytosolic Ca2+ in response to abiotic stresses such as drought, high salinity, ultraviolet light, heavy metals, waterlogging, extreme temperature and wounding. Furthermore, we focused on the crosstalk between Ca2+ and other signaling molecules in plants suffering from extreme environmental stress.

**Keywords:** Ca2+; abiotic stress response; Ca2+ sensors; signal transduction; abiotic stress tolerance calcium; heat stress; cold stress

### **1. Introduction**

Calcium ions (Ca2+) are important ions that maintain the normal physiological functions of plant cells and are involved in physiological metabolism in plants [1]. Ca2+ also functions as a ubiquitous secondary messenger involved in plant responses to various stresses [2]. Usually, there is a significant increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) in plant cells that is caused by low temperature [3], salt [4], drought [5] and other abiotic stresses. Ca2+ spikes are triggered by Ca2+ influx through channels or Ca2+ efflux through pumps. This increase is recognized, amplified and transmitted downstream by Ca2+-binding proteins, also known as calmodulin or Ca2+ sensors, which regulate plant cell division, cell elongation, stomatal movement, various stress responses and growth and development through a series of conduction cascades [6].

The main function of Ca2+ in plant stress resistance is to stabilize plant cell walls and membranes. It can activate or inhibit various ion channels on the membrane to achieve a balance of ion concentrations inside or outside the cell. The activities of specific enzymes are activated or inhibited by Ca2+ in cells to regulate biochemical reactions in plants [7,8]. Moreover, the transcriptional expression of multiple anti-stress genes is regulated by changes in calcium signaling to enhance the adaptability of plants exposed to extreme environments [9]. Under abiotic stress conditions, changes in the calcium ion concentration in the plant cytoplasm can be generally recognized as a cellular secondary messenger to distinguish different original signals; it also continues to transmit the signal downstream by interacting with calcium-binding proteins, causing a series of biochemical reactions in the cells to adapt or resist various stresses [10].

**Citation:** Li, Y.; Liu, Y.; Jin, L.; Peng, R. Crosstalk between Ca2+ and Other Regulators Assists Plants in Responding to Abiotic Stress. *Plants* **2022**, *11*, 1351. https://doi.org/ 10.3390/plants11101351

Academic Editors: Ewa Muszy ´nska, Petronia Carillo, Kinga Dziurka and Mateusz Labudda

Received: 25 February 2022 Accepted: 18 May 2022 Published: 19 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Ca2+, considered a secondary messenger for plant signal transduction, transmits extracellular information and regulates many physiological and biochemical responses to primary signals, such as light, hormones, and gravity [11]. Cytosolic Ca2+ cannot be maintained at a high level for a long time. If the concentration is too high, the Ca2+ will react with phosphoric acid, which is necessary for the metabolism of energy substances, and produce a precipitate that inhibits the normal physiological growth of cells or even causes cell death. In normal plant cells, most Ca2+ exists in a bound form, collectively known as the calcium pool, and calcium-storing proteins with high capacity and low affinity for Ca2+ can enhance Ca2+-buffering capacity. Due to this low affinity, when Ca2+ channels in calcium banks open, Ca2+-binding proteins can be rapidly dissociated from Ca2+, releasing it into the cytoplasm so that Ca2+ signals can be accurately and rapidly transmitted [12]. Ca2+ enters the cell through the Ca2+ channel, which is actually a protein on the plasma membrane that is maintained in the on or off state according to changes in its conformation. This channel rapidly stimulates and induces Ca2+ release from the vacuole. There are two major vacuolar uptake mechanisms, including P-type Ca2+ pumps and a family of cation/H<sup>+</sup> exchangers, which are responsible for high-affinity Ca2+ uptake and low-affinity with high-capacity Ca2+ uptake, respectively. Although research on the Ca2+ transport pathway mainly focuses on the regulation of [Ca2+]cyt by calmodulin (CaM) on the cell membrane, Ca2+ flow through internal membrane systems, such as the endoplasmic reticulum and mitochondrial membrane, is also critical when studying the transport patterns of Ca2+ signals [13,14].

Because the distribution and transfer of intracellular Ca2+ are the basis for the formation of Ca2+ signals, the increase or decrease in intracellular Ca2+ concentrations directly affect the generation and termination of Ca2+ signals. When there is no external stimulation, cytosolic Ca2+ is insufficient to activate CaM, which lacks its own catalytic activity. However, under extreme environmental conditions, [Ca2+]cyt increases rapidly, producing Ca2+ signals, and the reaction with CaM transmits the signal downward to allow subsequent physiological and biochemical reactions to occur [15]. Finally, restoration of the normal [Ca2+]cyt levels occurs by reloading calcium stores after completing Ca2+ signalling, and through the calcium efflux system, which consists of Ca2+-ATPase pumps and Ca2+/H+ exchangers, to remove excess Ca2+(Figure 1) [14,16].

Calcium sensors in plants are composed of Ca2+-binding proteins, such as CaMs, calmodulin-like-proteins (CMLs), calcineurin-B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs). CBLs interact with CBL-interacting protein kinases (CIPKs) to form a CBL/CIPK signaling network, which plays a key role in the plant response to abiotic stress. These networks may contain many interactions, with CBLs activating CIPKs and CIPKs phosphorylating CBLs. Phosphorylation is the major mechanism affecting downstream proteins [17].

There are three major elements, influx, efflux and decoding, that affect Ca2+-signal translation. Ca2+ influx is mediated by depolarization-activated, hyperpolarization-activated and voltage-independent Ca2+-permeable channels, which are encoded by genes, including *cyclic nucleotide-gated channels* (*CNGCs*), *glutamate receptor-like channels* (*GLRs*), *mechanosensitive channels of small* (*MscS*) and *conductance-like channels* (*MSLs*), *annexins*, *mid1-complementing activity channels* (*MCAs*), *Piezo channels* and *channel 1* (*OSCA1*) [18]. The Ca2+-efflux system, the calcium-dependent protein kinase ZmCDPK7 consisting of autoinhibited Ca2+-ATPases (ACAs), ER-type Ca2+-ATPases (ECAs), and P1-ATPases (HMA1), enables Ca2+ efflux to form an informative signature. Specificity in Ca2+-based signaling is achieved via Ca2+ signatures with cognate Ca2+-binding proteins. The decoding step is carried out by protein families such as CDPKs, CBL, CIPKs, CaM and CMLs (Figure 2) [19,20].

Plants constantly suffer from various abiotic stresses during their growth and development. Ca2+, acting as a secondary messenger, plays an essential role in the plant response to abiotic stresses; it can not only transmit and recognize various regulatory signals but also participate in gene expression and normal protein functions [21,22]. This review summarizes the biological process of cytosolic Ca2+ in response to abiotic stresses, such

as drought, high temperature, high salinity, heavy metals, waterlogging, and mechanical damage. Furthermore, we focus on both the crosstalk of cytosolic Ca2+ with other signaling molecules and biomacromolecules in plants suffering from extreme environmental stresses.

**Figure 1.** The Ca2+ signaling network in plant cells. Abiotic stress, including high-temperature stress (heat), low-temperature stress (cold), salt stress (salt), waterlogging stress (water), drought stress (drought), heavy-metal stress (metal), ultraviolet-B radiation stress (UV-B) and wound stress (wound), gives rise to an increase in [Ca2+], which is subsequently decoded by Ca2+ sensors such as Ca2+-dependent protein kinases (CDPKs), calmodulin-like-proteins (CMLs), calmodulins (CaMs), and calcineurin-B like proteins (CBLs) and their interacting protein kinases (CIPKs). These sensors activate various downstream responses that in turn result in an overall response precisely according to the original stimulus.

**Figure 2.** The generation and translation of Ca2+ signals in plant cells. Three major processes, including influx, efflux and decoding, can alter the effects of Ca2+-signal translation. GLRs: glutamate receptor-like channels, CNGCs: cyclic nucleotide-gated channels, OSCAs: hyperosmolality-induced Ca2+ increase channels, ACAs: Ca2+-ATPases, ECAs: Ca2+-ATPases, HMA1: P1-ATPases, MCUC: mitochondrial calcium uniporter complex, CAX: Ca2+ exchangers, CDPKs: calcium-dependent protein kinases, CBL: calcineurin B-like, and CIPKs: protein kinases. Reproduced with permission from [20], copyright 2017 Elsevier.

### **2. Molecular Mechanisms of Crosstalk between Ca2+ and Other Regulators in Response to Abiotic Stresses in Plants**

#### *2.1. Drought Stress*

Drought is a common adverse factor inhibiting plant growth and development; high levels of drought lead to an increase in the content of reactive oxygen species (ROS) that promote membrane peroxidation and damage membrane structure [23]. Ca2+ plays an important regulatory role in the signaling related to the plant drought stress response, reflecting its ability to regulate the activity of some enzymes and improve the ROS-scavenging ability. In addition, damage caused by drought can be reduced with Ca2+ channel activation mediating stomatal closure, which reduces transpiration flux to control water loss, thus improving plant water use efficiency [24].

By monitoring the water potential of the root vascular system, plants can transmit stress signals from roots to leaves, regulate stomatal closure and induce the expression of related genes to avoid dehydration [25]. Ca2+ efflux was observed in epidermal cells and mesophyll cells of barley roots under drought stress conditions. Extracellular pH affects K+ absorption, Ca2+ outflow and H<sup>+</sup> influx/alkalization in the leaves, which may be a chemical signal in the barley response to drought stress [26]. The application of molybdenum to wheat decreased the transpiration of wheat leaves but increased the Ca2+ concentration and other osmotic substances in wheat roots, which increased the osmotic pressure and further enhanced the water absorption capacity of wheat roots [27].

The abscisic acid (ABA)-dependent Ca2+ signaling pathway is the main response to drought stress in plants. ABA activates plasma membrane calcium channels in various ways to stimulate the release of Ca2+ from intracellular calcium stores, and several secondary messengers, including ROS, nitric oxide (NO), inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR), are involved in this process. When water deficit occurs, ABA accumulates in the leaves. On the one hand, it activates phospholipase C and decomposes IP3, which can activate the intracellular calcium pool in guard cells to allow stomatal closure. On the other hand, intracellular Ca2+ can also be increased by cADPR, but no receptors for IP3 and cADPR have been identified until now in plants [28,29]. ABA can also rapidly induce an intracellular Ca2+ increase through hydrogen peroxide (H2O2), leading to plasma membrane hyperpolarization and direct activation of plasma membrane hyperpolarizationactivated calcium channels (HACCs) and vacuolar membrane Ca2+ channels to achieve stomatal closure regulation [30]. At present, the pathway of NO modulating the crosstalk between ABA and H2O2 and activating the calcium signaling pathway has also been further revealed [31]. Wang et al. mentioned that extracellular Ca2+ and ABA promote stomatal closure by promoting H2O2 to produce calcium signals dependent on NO synthesis [32]. In *Arabidopsis* ABI mutants, H2O2 and NO activate calcium signals depending on cyclic guanosine 3 ,5 -monophosphate (cGMP), which likely acts upstream of calcium signals. After exogenous calcium treatment, ion channels can be activated by intracellular calcium signaling, and calcium signal production processes mediated by ABA and H2O2 may be performed in the following sequence: ABA→H2O2→NO→cGMP→Ca2+. The upstream calcium-sensing signal is converted to a calcium-receiving signal, and then the downstream calcium signal can produce biological reactions promoting stomatal closure [33].

Ca2+ not only acts as a secondary messenger in the rapid response to upstream stimulation, but more importantly, the Ca2+ signaling system also contains a large number of different types of calcium signal receptors, such as CDPKs, CaM, CBL, and CIPK, which receive exogenous calcium signals and convert them into endogenous calcium signals. These signals are then phosphorylated and dephosphorylated or eventually interact with other proteins to regulate stomatal movement [34–36]. The interaction between CBL9 and CIPK3 negatively regulates Ca2+-dependent ABA signaling in *Arabidopsis* [37]. It was found that VvK1.1 in grapevine corresponds to the AKT1 channel in *Arabidopsis*, and dominates K+ uptake in root periphery cells. VvK1.1 and AKT1 have common functions, such as regulation by CIPK23, which occurs independently in grapevine under drought stress; this process is essential for stomatal movement regulated by K<sup>+</sup> flow [38]. During stomatal

closure, the relationship between ABA and Ca2+ is not a simple upstream and downstream regulatory process. In the early stage of drought stress, Ca2+ can rapidly induce ABA biosynthesis and activate Ca2+ channels on the plasma membrane by utilizing turgor pressure or pH change to increase the intracellular Ca2+ concentration instantly. Then, the expression of related transcription factors and genes, including zeaxanthin epoxidase, ninecis-epoxy carotenoid dioxygenase, abscisic aldehyde oxidase and molybdenum cofactor sulfurase, is increased by the protein kinase cascade reaction. ABA inhibition of type 2C protein phosphatase leads to phosphorylation and activation of sucrose-nonfermenting-1-related protein kinase 2, which in turn stimulates the expression of ABA-responsive genes, thereby promoting ABA biosynthesis; then, the generated ABA in turn promotes an increase in Ca2+ concentration [39]. Another hypothesis is that ABA induces the activation of calcium decoding signal elements, including calcium-permeable ion channels, Ca2+/H+ antiporters and Ca2+-ATPases, and transduces calcium signals to alter stomatal aperture and transpiration efficiency to regulate water use efficiency in plants. Moreover, calcium channel proteins, such as *Arabidopsis thaliana two-pore channel 1* (*AtTPC1*) and *TaTPC1* from wheat, also regulate stomatal closure [40]. This hypothesis may explain the role of these genes in plant responses to drought and cold stress.

In addition to stomatal closure, plants can increase their water retention capacity by regulating stomatal density and other developmental processes to respond to drought. GT-2like 1 (GTL), a trihelix transcription family member, regulates stomatal motility by regulating the expression of *stomatal density and distribution1* (*SDD1*) genes. When PtaGTL1 identified in *Populus tremula* × *P. alba* was transferred to *Arabidopsis thaliana*, GTL increased *SDD1* gene expression by binding to Ca2+-CaM, thus reducing stomatal density and the transpiration rate and improving water use efficiency under drought stress [41]. Therefore, to adapt to different degrees of water deficit, plants adjust the stomatal number and leaf area through growth and development and balance the relationship between water use efficiency and photosynthesis to achieve the optimal adaptation point, which may be an effective strategy for plants to cope with long-term drought stress [42].

#### *2.2. Salt Stress*

Salt stress usually causes ion toxicity, osmotic imbalance and oxidative stress, resulting in limited plant growth and thereby affecting the sustainability of crop yields. In the external environment, hypersaline stress occurs when a high enough salt content significantly changes the water potential, thus affecting the plant [43]. Ca2+ also plays a significant regulatory role in plant resistance to salt stress. For example, Ca2+ inhibits Na+ influx by regulating Na+ entry into the main cell channel nonselective cation channels (NSCCs). Moreover, Ca2+ prevents the outflow of K+ by inhibiting K<sup>+</sup> permeable outwardly rectifying conductance (KORC) channel and initiates the salt overly sensitive (SOS) signal transduction pathway, which regulates the development of plasticity in roots during salt stress adaptation; for example, SOS3 is required for auxin biosynthesis, root polar movement and the formation and maintenance of auxin gradients [44–46].

Usually, Ca2+ influx is related to hydroxyl radicals (OH·) and Ca2+ influx channels on the plasma membrane in wheat roots. Salt-stress-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the plasma membrane produces a large number of superoxide anion radicals (O2 −) extracellularly during electron transfer to O2, which are then rapidly converted into H2O2 and OH·. Notably, both OH· and H2O2 can activate the Ca2+ channels to induce extracellular Ca2+ flow into the cells [19]. Overall, ROS have been identified as key regulators of Ca2+ influx.

When suffering from salt stress, roots are the sensory part of plants that initiate the response and adaptive behavior to defend against stress damage as part of first-line defense. The SOS signaling pathway is activated by the increase in Ca2+ in the root cytoplasm caused by salt stress, which mediates cell signal transduction by SOS3/SCABp8-SOS2-SOS1 at the cellular level [47]. In this process, SOS3 functions as a Ca2+-binding protein, interacts with SOS2 to form a complex and then activates downstream SOS1 through phosphorylation, thus maintaining K<sup>+</sup> and Na<sup>+</sup> homeostasis inside and outside the cell. Furthermore, SOS3 has also been shown to play a key role in mediating the recombination of Ca2+-dependent actin filaments during salt stress [48].

CDPKs are a large polygenic family whose members contain a serine/threonine protein kinase catalytic domain as an effector region and a calmodulin-like domain for binding to Ca2+. These proteins can directly activate and regulate target proteins when sensing Ca2+ signals, thus playing an essential role in a variety of physiological processes in plants. OsCPK12 has been shown to be a crucial factor in salt stress tolerance, acting as a positive regulator of stress tolerance by regulating ABA signaling and reducing ROS accumulation in rice [49]. For example, rice overexpressing OsTPC1 show enhanced tolerance to stress through positive regulation of ABA signaling and salt signaling pathways. Some researchers suggest that ABA receptors may be upstream factors that regulate intracellular Ca2+ levels in plants under salt stress conditions [50]. Other experiments have shown that ABA receptors may exist inside cells or outside the plasma membrane. On the surface of the plasma membrane, when ABA acts on its receptor, the activated part interacts with G protein, which binds to the plasma membrane to activate phospholipase C and stimulate the release of Ca2+ from the calcium pool [51].

Ca2+-ATPase (PCA1) has been identified as essential for the adjustment of salt tolerance in the moss *Physcomitrella patens*. PCA1 encodes a PIIB-type Ca2+-ATPase, which is a plantspecific Ca2+ pump with an N-terminal autoinhibitory calmodulin-binding domain that has been confirmed with in vivo complementation analysis of Ca2+ transport-deficient yeast strains. This class of Ca2+ pumps may trigger the initiation of stress adaptation mechanisms in Ca2+ signaling pathways. In contrast to the transient [Ca2+]cyt increase caused by NaCl in the wild-type, hyperaccumulation of cytosolic Ca2+ in PCA1 mutants remained high and did not return to prestimulus [Ca2+]cyt levels. Therefore, Ca2+ pumps contribute to the production of stress-induced Ca2+ signatures [52]. In addition, based on the isolation of *monocation-induced [Ca2+]i increases 1 Arabidopsis* mutant, which affects Ca2+ influx under salt stress, an association between salt sensing and GIPC-gated Ca+2+ influx has been inferred. It has been demonstrated that Ca2+ channels are gated by GIPCs in plants [53].

#### *2.3. Extreme Temperature Stress*

#### 2.3.1. Low-Temperature Stress

A large number of free radicals are produced in plants exposed to low-temperature stress, thereby damaging the membrane system. When plants are subjected to lowtemperature stress, Ca2+ channels are opened, and intracellular [Ca2+]cyt increases rapidly to induce calcium signaling [54]. Finally, the process is completed after signal transfer from the extramembrane into the membrane. On the one hand, the results of Ca2+ treatment of tobacco seedlings subjected to low-temperature stress showed that Ca2+ could increase the content of intracellular bound calcium and improve the activities of catalase, superoxide dismutase (SOD), peroxidase (POD) and other antioxidant enzymes, but reduce the content of malondialdehyde [55,56]. Furthermore, the decrease in enzyme activity after Ca2+ treatment was lower than that after Ca2+-free treatment, and the membrane permeability of tobacco seedlings also recovered quickly after growth had stopped. Therefore, it is speculated that Ca2+ can improve plant cold resistance and maintain the stability of the membrane system [57,58]. Another study demonstrated that Ca2+ and CaM could regulate the freezing resistance of citrus protoplasts, while treatment with the exogenous CaM blocker TFP or the Ca2+-chelating agent ethylene glycol diethyl ether diamine tetra-acetic acid (EGTA) could also inhibit the freezing resistance of citrus [59]. CBLs are a special class of Ca2+ receptors that specifically interact with CIPK protein kinases to activate downstream target proteins and decode Ca2+ signals. The expression of CIPK7 is induced by low temperature, can interact with the CBL1 protein in vitro and may be associated with CBL1 protein in vivo. Compared with wild-type plants, CBL1 mutant plants showed CIPK7

expression is affected by CBL1, suggesting that CIPK7 may bind to the calcium receptor CBL and participate in plants' cold response [60,61].

In contrast to CaM and CBL, which have to couple with Ca2+ to change their conformation and be activated, CDPKs, which are constitutively activated and directly phosphorylated, transduce calcium signals by interacting with the site of the calcium receptor or forming a peptide chain [62]. CDPKs are involved in the intermediate process instead of participating in the initial response to low temperature in rice. Moreover, several Ca2+ related genes, such as CDPK13, are regulated by low-temperature stress in plants [63]. In rice, the CDPK13 gene is expressed in leaf sheaths and calli during the initial 2 weeks of growth, and CDPK13 is phosphorylated in response to low temperature and gibberellin (GA) signaling. Simultaneously, low temperature or exogenous GA3 treatment resulted in the elevation of CDPK13 gene expression and protein accumulation. Compared to wild-type and cold-sensitive rice, CDPK13-overexpressing-line rice showed stronger cold tolerance and a higher rate of plant recovery from cold injury, implying that CDPK13 might be a key protein in the rice signaling network responding to low-temperature stress [64,65].

Calcium channels are not only the key to the generation of calcium signals but also the rapid transport pathway and regulatory element for Ca2+ across the membrane [66]. At present, *Arabidopsis thaliana* two-pore channel 1 (AtTPC1) is the most studied calcium channel protein. Stomatal closure of *attpc1-2* functional deficient mutants treated with ABA, methyl jasmonate (MeJA) and Ca2+ was detected, and the results demonstrated that both ABA and MeJA can induce the accumulation of ROS and NO to cause an increase in [Ca2+]cyt and cytoplasmic alkalization and activate anion channels in both wild-type and mutant plants, thus causing the stomata to be closed. However, compared with that in wild-type *Arabidopsis*, exogenous Ca2+ could not induce stomatal closure or activate anion channels on the plasma membrane in *attpc1-2* mutants. Taken together, we can conclude that AtTPC1 protein is involved in both stomatal closure and plasma membrane anion channel activation and is regulated by exogenous calcium signals in guard cells; however, it is not regulated by ABA and MeJA [67]. Stomatal closure is a common adaptive response of plants to low temperature. Stomatal guard cells respond quickly to abiotic stress stimuli, such as low temperature and drought [68].

Studies in eukaryotic cells suggest the overall translation rate can be regulated by an increased AMP/ATP ratio, which leads to activation of 5 -AMP-activated protein kinase and the release of Ca2+ from the endoplasmic reticulum, which triggers the phosphorylation of eukaryotic extension factor 2 by its activated specific kinase eukaryotic elongation factor 2 kinase [69].

#### 2.3.2. High-Temperature Stress

High-temperature stress also gives rise to plant cell membrane damage, osmotic regulation imbalance, an accumulation of ROS, an inhibition of photosynthesis, cell aging and death, thus limiting plant distribution, growth and productivity [70]. Exogenous application of Ca2+ effectively improves high-temperature stress resistance in laver and tomato [71,72] and alleviates the damage caused by high-temperature stress in ornamental plants such as chrysanthemum [73]. In tomato, spraying calcium chloride on the leaf surface can increase the activities of protective enzymes and soluble protein contents in leaf intima and reduce the malonic acid content, thus enhancing high-temperature-stress adaptability [71]. Further research showed that Ca2+ treatment can significantly improve the net photosynthetic rate, transpiration rate and stomatal conductance of tomato leaves suffering from high-temperature stress [74]. On the other hand, significant upregulation of PhCAM1 and PhCAM2 expression is related to the change in [Ca2+]cyt when high-temperature stress occurs, while the expression of PhCAM1 and PhCAM2 is not obviously changed after EGTA is added, implying that the Ca2+ signaling system and CAM play a major role in the regulation of resistance to high-temperature stress in *Pyropia haitanensis* [58,75]. Based on the above descriptions, it can be clearly seen that Ca2+ can not only stabilize the cell membrane structure but also prevent damage to photosynthetic organs from ROS under high-temperature stress by regulating osmotic balance and the antioxidant system. Additionally, Ca2+, acting as an essential signaling substance, participates in signal transduction when high-temperature stress occurs and enhances high-temperature resistance in plants [76,77].

High-temperature stress also induces heat stress transcription factor (HSP) expression, and many of these factors act as molecular chaperones to prevent protein denaturation and maintain protein homeostasis [78]. Similarly to mammalian heat shock transcription factors (HSFs), plant HSFs are released from the binding and inhibition of HSP70 and HSP90 and combine with misfolded proteins under high-temperature stress. Therefore, HSFs can be used to activate the high-temperature stress response. In contrast, high-temperature stress also activates mitogen-activated protein kinases (MAPKs) and regulates the expression of HSP genes. This may be closely related not only to changes in membrane fluidity but also to calcium signaling induced by high-temperature stress, which is especially required for HSP gene expression and high-temperature stress tolerance acquisition [79,80]. The common features between signals of low- and high-temperature stress are not limited to membrane fluidity changes, calcium signaling and MAPK activation, as they also include ROS, NO, and phospholipid signaling [81,82].

The calcium-dependent protein kinase ZmCDPK7 positively regulates heat stress tolerance in maize. ABA regulates ZmCDPK7 expression by phosphorylation of the respiratory burst oxidase homologue RBOHB in a Ca2+-dependent manner, thus triggering ROS accumulation, which further promotes ZmCDPK7 expression. Moreover, ZmCDPK7 plays a crucial role in maintaining protein quality and reducing heat stress damage by activating the chaperone function of sHSP17.4 through Ca2+-dependent phosphorylation [83].

The Ca2+/calmodulin-dependent phosphatase calcineurin plays a role in morphogenesis and calcium homeostasis during temperature-induced mycelium-to-yeast dimorphism of *Paracoccidioides brasiliensis*. Intracellular Ca2+ levels increased immediately after the onset of dimorphism. The extracellular or intracellular chelation of Ca2+ inhibits dimorphism, while extracellular Ca2+ addition accelerates dimorphism. In addition, the calcineurin inhibitor cyclosporine A disrupts intracellular Ca2+ homeostasis and reduces mRNA transcription of the *CCH1* gene in the Ca2+ channel of the yeast cell plasma membrane, effectively reducing cell growth or resulting in abnormal growth morphology *P. brasiliensis* [84].

#### *2.4. Heavy-Metal Stress*

Increasing the Ca2+ content in soil can enhance the heavy-metal tolerance of plants. The accumulation of active Al3+ and Mn2+, as well as the lack of nutrients in acidic soil, are important limiting factors for crop growth [85]. Earlier studies showed that Al3+ could induce Ca2+ loss in plants and inhibit Ca2+ absorption and root growth, thereby suppressing plant growth and development. However, salicylic acid (SA) can alleviate Al3+-induced inhibition of soybean root elongation and reduce the Al3+ content in plants. The plant response to Al3+ stress requires endogenous SA and Ca2+ for the transmission and amplification of the Al3+ stress signal, which strengthens the subsequent physiological response [86]. In addition, citric acid (CA) secreted from soybean roots can alleviate Al3+ toxicity. Both CA secretion and SA content changes are affected by Ca2+, and it has been speculated that SA and Ca2+ might be linked to the Al3+ tolerance mechanism of soybean. Moreover, both Ca2+ and SA can alleviate the physiological reaction of root growth inhibition caused by aluminum, promote the secretion of citric acid, improve the enzyme activities of SOD, POD, ascorbate peroxidase and other antioxidant systems, reduce the accumulation of ROS, and alleviate oxidative stress damage to improve the Al3+ tolerance of soybean. Additionally, SA may participate in the Al3+ tolerance mechanism by increasing the endogenous Ca2+ level [87,88]. Exogenous Ca2+ can increase the relative expression levels of PLC and PLD genes, indicating that Ca2+ has some effects on the changes in phospholipase in soybean root tip cells, which may be related to changes in microtubule structure [89].

The addition of exogenous calcium can reduce the content of heavy metal ions in plants growing in soils with excessive amounts of heavy metals, such as Cu2+, Cr6+ and Pb2+, and improve their ability to resist heavy metal stress [90,91]. According to research findings, when the Cu2+ concentration increased, the Ca2+ content in plant roots increased, which may be significant for improving plant resistance to Cu2+ stress [92]. In addition, Cr6+ stress activates plant endogenous hydrogen sulfide (H2S) synthesis and Ca2+ signal transduction. H2S and Ca2+ alone or in combination can significantly reduce the injury caused by Cr6+ stress; however, the effect is better when they are used in combination. In contrast, treatment with H2S synthesis inhibitors or Ca2+ chelating agents enhances environment-induced stress. This result suggests the synergistic effects of H2S and Ca2+ in response to Cr6+ stress in *Setaria italica* [93].

#### *2.5. Wound Stress*

Usually, wounds from mechanical damage caused by harsh weather conditions, such as wind and rain, or by geological disasters, including debris flows and landslides, induce the release of calcium signals to regulate the overall response to stress and further improve the survival ability of plants [94,95]. Wound signaling is required for initiating plant regeneration. Plants promote changes in downstream cell fate due to signal transduction cascades induced by wounds [96]. Wounds also promote changes in cell membrane potential (Vm), fluctuations in Ca2+ concentration, ROS bursts, and drastic increases in the concentrations of jasmine, ethylene, SA and other plant hormones [97]. Therefore, Ca2+, as a vital part of wound signaling, may regulate the transcription of downstream genes accompanied by signal transduction and trigger some physiological and biochemical reactions locally or systemically. Studies have shown that the loss of cell membrane integrity at the site of injury may allow cytoplasmic inclusions of damaged cells to enter the intercellular space, thus changing the original ion concentration and composition, which further affects the state of various ion channels on the cell membrane and leads to fluctuations in transmembrane potential and calcium concentration [98,99]. Furthermore, GdCl3, a calcium channel inhibitor, has been shown to inhibit plasma membrane depolarization induced by single-cell injury [100].

Ca2+ signals respond to wounds rapidly (often within just 2 s) in plants suffering from mechanical damage and then propagate to specific undamaged distal tissues after 2 min. The ethylene synthesis-related genes ACS2, ACS6, ACS7 and ACS8 were rapidly upregulated within 30 min after leaf injury in *Arabidopsis.* At the same time, wounding rapidly activated the expression of mitogen-activated protein kinase (MPK) along with calcium-dependent protein kinase (CPK) [101]. To some extent, its transmission depends on glutamate receptor-like 3.3/3.6 (glR3.3/3.6) proteins, which are regulated by glutamate concentration. Mutation of both glr3.3 and glr3.6 leads to the long-distance transport of Ca2+ being blocked, and the expression of defense genes is subsequently reduced in undamaged areas, while glutamate contents are reduced concurrently [102]. Moreover, Ca2+ also functions as an intracellular secondary messenger to regulate the biochemical state of cells near wounds, and Ca2+-dependent MC4 in the cytoplasm has catalytic activity due to the wound-induced [Ca2+]cyt increase. The defense response occurs by catalyzing the elicitor peptide precursors into mature peptides located on the cytoplasmic side of the vacuole membrane; in turn, these peptides are recognized by the cytoplasmic vacuolar membrane-targeted receptor-elicitor peptide receptors [103,104]. Although Ca2+ transfer over long distances depends on ROS produced by NADPH oxidase, inhibition of calcium ion signaling can weaken the wound response to jasmonic acid (JA) and ethylene production [97,105]. Therefore, these results indicate that there is a closely linked interaction among various substances related to wounding signals.

Ca2+ is directly involved in the generation and propagation of long-distance signals in plants. Under strong local stress, variation potential (VP), a long-distance intercellular electrical signal, is the potential mechanism for coordinating functional responses to different plant cells, which can cause functional changes in unstimulated organs and tissues, namely, systematic responses of plants. Specifically, ligand-dependent or mechanically sensitive Ca2+ channels are activated by the propagation of chemical or hydraulic signals or a combination of these potentially distant signals. Subsequent Ca2+ influx can trigger VP production, thus inducing H+-ATPase inactivation and possibly Cl<sup>−</sup> channel activation [106].

In long-distance ROS signal transduction, RESPIRATORY BURST HOMOLOG D (RBOHD) is a ferric oxidoreductase that can be activated directly by calcium ions binding to its EF-Hand motif and phosphorylated by various protein kinases, such as CPK5 and CIPK. *Botrytis*-induced kinase 1 (BIK1) is also under Ca2+-dependent regulation by CPK28 and phosphorylates RBOHD. ROS-activated Ca2+-permeable channels on the plasma membrane provide a mechanism for RBOHD to trigger its further activation [107]. The crosstalk between Ca2+ and ROS to transmit these signals among cells across long distances, namely, that of RBOH, is activated by Ca2+-dependent protein kinases in the presence of Ca2+. This leads to the accumulation of nonprotoplast ROS, leading to induced Ca2+ release from adjacent cells. Then, another Ca2+-dependent protein kinase is activated circularly [99,108]. In this way, signals are transmitted over long distances within plants (Figure 3) [109].

**Figure 3.** Schematic model of Ca2+- and ROS-mediated cell-to-cell signal propagation over long distances in plants. Stimulating the production of cytosolic Ca2+ signals results in the activation of RBOHD by Ca2+-regulated kinases, which produce ROS and then propagate the signal by activating Ca2+ channels in neighboring cells.

Generally, Jasmonate-associated VQ domain protein 1 (JAV1) associates with JAS-MONATE ZIM domain protein 8 (JAZ8) and WRKY51 to form the JAV1-JAZ8-WRKY51 (JJW) complex, which inhibits the expression of jasmonate (JA) synthesis genes. Once the plant sustains an injury, the sudden increase in the concentration of Ca2+ causes calmodulin to sense Ca2+ and combine with JAV1, thus phosphorylating JAV1, depolymerizing the JJW complex, and releasing the transcriptional inhibition of the JA synthesis gene lipoxygenase 2 (LOX2), which finally results in the accumulation of large quantities of jasmine in response to wound stress [110,111].

#### *2.6. Waterlogging Stress*

Plants will be damaged by a lack of sufficient oxygen (O2) for respiration when they are exposed to waterlogging or submergence stress [112]. Under flooding conditions, when O2 is lacking, it will likely cause a massive buildup of CO2 as respiration and metabolism proceed, and when this occurs, intracellular Ca2+ in plants is required for the response to waterlogging-induced hypoxia stress in nonphotosynthetic organs [113,114]. Hypoxia promotes a real-time [Ca2+]cyt increase and ROS accumulation, which may be interdependent [115]. For example, ROS in guard cells and root cells can activate Ca2+ channels, and Ca2+ can also promote ROS accumulation. Furthermore, in mutants with

a loss of function of the PM-NAD(P)H oxidase subunits, ROS produced by the defective enzyme can activate Ca2+ channels on the cell membrane to achieve Ca2+ flow, thus contributing to the promotion of root tip growth. It has been demonstrated that ROS accumulation is coupled with Ca2+ dynamics in pollen tubes and root tips; however, relevant and reliable biochemical evidence about whether ROS directly activate NADPH oxidase is necessary [116].

Previous research showed that [Ca2+]cyt acts as a key transducer of hypoxic signals in rice and wheat protoplasm exposed to hypoxia stress [117], and alcohol dehydrogenases (ADH), whose activity is involved in resistance to waterlogging, displayed significant improvement in maize [118]. In corn cells, hypoxic signaling rapidly elevates [Ca2+]cyt by the release of intracellular stores of Ca2+; however, Ca2+ is not only involved in hypoxic signal transduction but also affects the activity of related Ca2+-dependent enzymes, such as alcohol dehydrogenase, reflecting tolerance to hypoxia [119]. Studies indicate that Ca2+ influx can promote the reduction in H2S in plants suffering from waterlogginginduced hypoxia stress [120]. H2S production by CBS is 3.5 times higher in the presence of Ca2+/CaM than in the absence of Ca2+, but it is inhibited by treatment with CaM inhibitors. The application of exogenous Ca2+ and its ion carrier A23187 markedly increased H2S-induced antioxidant activity, while the calcium-chelating agent EGTA, the plasma membrane channel blocker La3+, and calmodulin antagonists attenuated this resistance [58]. During waterlogging, hypoxia stress causes the accumulation of H2O2, activates the ROSinduced Ca2+ channel and triggers the self-amplifying "ROS-Ca2+ hub", which further increases K<sup>+</sup> loss and cell inactivation. The increased content of gamma-aminobutyric acid (GABA) induced by hypoxia is beneficial to the recovery of membrane potential and the maintenance of homeostasis between cytosolic K+ and Ca2+ signaling. In addition, the ROS-Ca2+ hub can be better regulated by elevated GABA through transcriptional control of RBOH gene expression, thus preventing the excessive accumulation of H2O2 and allowing plants to more easily survive waterlogging [121].

#### *2.7. UV-B Radiation Stress*

UV-B radiation stress not only has adverse effects on plant morphology, such as plant dwarfing and leaf thickening, but also harms plant physiological processes, including chloroplast structure damage, photosynthetic rate decreases, and transpiration weakening [122–124]. Studies suggest that there are at least two pathways involved in the cytoplasmic Ca2+ response to UV-B radiation stress in plants. On the one hand, enhanced UV-B radiation triggers a significant increase in the free Ca2+ concentration in the cytoplasm of wheat mesophyll cells, which may release Ca2+ from the intracellular calcium pool or increase intracellular Ca2+ influx. UV-B radiation inhibits CaM, leading to it dissociating from the inhibitory region and in turn binding to the active site, which leaves the Ca2+ pump in a resting state [125]. On the other hand, UV-B radiation possibly promotes phosphatase dephosphorylation in the inhibitory region and combines with the active site to play an inhibitory role [126]. In addition, the calcium pump is directly activated to change the transport of intracellular Ca2+ under UV-B radiation conditions, thereby increasing [Ca2+]cyt. Furthermore, a slightly increased [Ca2+]cyt can not only act on the membrane skeleton and significantly reduce the deformability of cells but is also involved in the lipid redistribution of the membrane and the decline in membrane stability [127].

The total phenol content of wheat under UV-B+CaCl2 treatment increased by 10.3% compared with UV-B treatment alone. Most of the genes related to phenolic biosynthesis were upregulated during wheat germination, suggesting that exogenous Ca2+ promotes the accumulation of free phenols and bound phenols in germinal wheat exposed to UV-B radiation. In addition, treatment with Ca2+ can significantly alleviate membrane lipid peroxidation, activate antioxidant enzymes and regulate plant hormone levels. However, the Ca2+ channel blocker LaCl3 significantly reduced TPC and APX activity [128]. These contrasting results suggested that Ca2+ was involved in the regulation of phenolic metabolism, antioxidant enzyme activity and endogenous plant hormone levels of germinal wheat in response to UV-B radiation stress [129].

SA is considered to be a synergist of H2O2, which may contribute to the generation or maintenance of ROS signaling levels and participate in many signaling responses to abiotic stresses, such as UV-B [130] and heavy metals [86]. Ca2+ is essential for H2O2- and SAmediated signal transduction. *Arabidopsis thaliana* BTB and TAZ domain proteins (AtBTs) are Ca2+-dependent CaM-binding proteins. The AtBT family may be a signal transduction center, and the signal transduction chain includes Ca2+, H2O2 and SA. These signals may regulate transcription by altering AtBT expression and conformation [131].

#### **3. Calcium Ion Downstream Signaling Response**

Under abiotic stress conditions, plants transmit information through a second messenger, allowing cells to transmit external information into the cell interior. The cells then respond by triggering downstream reactions, consisting of transcriptional regulation and protein modification, to influence appropriate adaptive responses [132]. For example, in response to heat stress, altered membrane fluidity is sensed through Ca2+ channels and receptor-like kinases. Heat stress transcription factor A1 (HsfA1) transcription factors are the main heat-stress-resistance regulatory factors in plants. When activated by heat, they target downstream transcription factors, microRNAs and *ONSEN* (a copia-like retrotransposon) to induce the expression of heat stress-responsive genes that are critical for ROS clearance, protein homeostasis and heat stress memory [133]. Downstream events of Ca2+ signal transduction are mainly mediated by Ca2+-binding proteins. In *Arabidopsis*, membrane hyperpolarization and ROS-activated Ca2+-permeable channels under K+ deficiency result in an increase in cytoplasmic Ca2+, and Ca2+ signals are sensed by specific sensors and transmitted downstream. CBL1/CBL9 recruits the cytoplasmic kinase CIPK23 to the plasma membrane, where CIPK23 activates AKT1-mediated uptake of K+ through phosphorylation. [134,135].

Calcium regulates the actin cytoskeleton either directly by binding to actin-binding proteins (ABPs) and regulating their activity or indirectly through calcium-stimulated protein kinases, such as CDPKs. The oscillation of the Ca2+ concentration gradient in the tip region of the pollen tube affects actin dynamics, and the remodeling of the actin cytoskeleton is associated with pollen tube elongation, showing that the Ca2+ concentration gradient may precisely regulate actin dynamics and promote pollen tube growth [136].

#### **4. Conclusions and Perspectives**

As one of the most important signaling molecules in cells, the Ca2+ signal transduction pathway is widely involved in the regulation of growth and development, abiotic stress response and many other physiological processes. Various studies have confirmed that abiotic stresses such as drought, high salt, ultraviolet light, heavy metal, waterlogging and extreme temperature can lead to a rapid increase in intracellular Ca2+ via the regulation of a variety of Ca2+ channels and trigger the Ca2+ signaling process. Then, the signals are decoded by Ca2+ sensors, following a series of physiological reactions through appropriate transduction pathways. Ca2+ is involved in crosstalk between other signaling molecules and phytohormone interactions when plants suffer from abiotic stress. In general, calcium, as the central node of the regulatory network, assists other regulators in adapting to adverse abiotic stresses.

Although the many molecular mechanisms behind Ca2+ involvement in abiotic stress responses have been elucidated, it remains unclear how plants can accurately distinguish the types and intensities of external stimuli and thus regulate [Ca2+]cyt in a precise and complex way so that they can respond to a series of complex upstream signals accurately and exclusively and ensure signal transduction sensitivity and specificity concurrently. Furthermore, because crosstalk between Ca2+ and other signaling molecules is vital for the stress response, the mechanism of stress perception and the system of signal transduction at the biological level should be investigated. Therefore, the next important task for Ca2+ signaling research is to determine which physiological reactions are involved in the various Ca2+-targeted proteins downstream of calcium signaling and which downstream molecules are regulated to affect gene expression. Moreover, with recent advances in techniques and the development of molecular biology, cell biology, genetics and other disciplines, the role of Ca2+ signaling will certainly be elucidated more thoroughly.

**Author Contributions:** R.P. and L.J. contributed to the conception of this review. Y.L. (Yaoqi Li), Y.L. (Yinai Liu) and R.P. (Renyi Peng) designed and produced the figures. R.P. (Renyi Peng) and Y.L. (Yaoqi Li) wrote the manuscript. Y.L. (Yaoqi Li), Y.L. (Yinai Liu), L.J. and R.P. (Renyi Peng) revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support from the Natural Science Foundation of Zhejiang Province (Grant No. LQ20C020003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**

