Integration of Abscisic Acid Signaling with Other Signaling Pathways in Plant Stress Responses and Development

Plants are immobile and, to overcome harsh environmental conditions such as drought, salt, and cold, they have evolved complex signaling pathways. Abscisic acid (ABA), an isoprenoid phytohormone, is a critical signaling mediator that regulates diverse biological processes in various organisms. Significant progress has been made in the determination and characterization of key ABA-mediated molecular factors involved in different stress responses, including stomatal closure and developmental processes, such as seed germination and bud dormancy. Since ABA signaling is a complex signaling network that integrates with other signaling pathways, the dissection of its intricate regulatory network is necessary to understand the function of essential regulatory genes involved in ABA signaling. In the present review, we focus on two aspects of ABA signaling. First, we examine the perception of the stress signal (abiotic and biotic) and the response network of ABA signaling components that transduce the signal to the downstream pathway to respond to stress tolerance, regulation of stomata, and ABA signaling component ubiquitination. Second, ABA signaling in plant development processes, such as lateral root growth regulation, seed germination, and flowering time regulation is investigated. Examining such diverse signal integration dynamics could enhance our understanding of the underlying genetic, biochemical, and molecular mechanisms of ABA signaling networks in plants.


Introduction
Abscisic acid (ABA) signaling (perception, signaling, and tolerance) in plants is a complex response for which there are considerable knowledge gaps at the molecular level. ABA is a plant phytohormone with a small lipophilic sesquiterpenoid (C15) structure [1]. It has a key role in stress adaptation in addition to being critical in numerous biological processes, such as bud dormancy and seed germination [2][3][4][5][6]. In the 1960s, pioneering studies on ABA (initially termed "abscisin" and "dormin") reported that it was accumulated in immature cotton balls that succumbed to ethylene-triggered abscission and over-wintering buds [4,7,8]. Later, it was demonstrated that under such conditions Active SnRKs, CIPKs, and CDPKs (dark blue oval box) play important roles in downstream signal transduction. (C) Stomatal regulation via ABA signaling in response to stress and healthy conditions. Under stress conditions, stomatal regulation (purple arrow →) is carried out by active SnRK2.6/OST1 (blue oval box) through the regulation of downstream ion channel genes (green oval boxes), such as SLAH3, SLAC1, and KAT1. This regulation helps stomata remain closed to avoid loss of excessive water under adverse conditions. Under normal conditions, SnRK2.6/OST1 inactivated by PP2C cannot regulate the downstream genes; thus, stomata remain open. (D) Response to stress tolerance via the ABA signaling pathway. The stress tolerance mechanism (black arrow →) is regulated in Ca 2+ -independent as well as Ca 2+ -dependent manners. The MAP kinase cascade (orange oval box) pathway carries the signal for the response to abiotic stress tolerance. It delays ABA gene expression. Contrarily, signal transduction via only AREB/ABF (yellow oval box) shows early expression of ABA related genes, resulting in an early response to stress tolerance. (E) Involvement of ABA signaling in the plant developmental process. Downstream ABA signaling involved in different developmental processes (red arrow →) such as seed germination (light green oval and square boxes), lateral root growth (light blue oval and square boxes), and regulation of flowering time (yellow oval and square boxes). ABI5 emerges as a critical ABA signaling component in the regulation of the plant developmental process. ABA signaling integrates with light signaling (black dark oval box) to regulate plant development. The brown tack facing up (⊥) indicates the role of ubiquitination in ABA signaling. These E3 ubiquitin ligase elements in ABA signaling guide the inactive protein to undergo degradation. The question mark (?) indicates the unknown pathway. ABD1, DWA1, and DWA2, which are associated with Cul4-based E3 ubiquitin ligases, were reported to be responsible for ABA signaling through the degradation of ABI5 by regulated ubiquitination in the nucleus via the ubiquitin-26S proteasome system [71][72][73]. Single mutants, abd1, dwa1, and dwa2, and a double mutant, dwa1/dwa2, display ABA-hypersensitive phenotypes during seed germination and seedling growth [72,73], which indicates ABI5 acts as a target for ABD1, DWA1, and DWA2, Cul4-based E3 ubiquitin ligases, which leads to the negative regulation of ABA signaling in the nucleus. ABI3 INTERACTING PROTEIN2 is a functional RING-type E3 ligase that interacts with an unstable protein, ABI3, and is degraded via the ubiquitin-26S proteasome system [74]. Different types of E3 ligases with dual roles have been reported participating in the regulation of ABA signaling; however, the knowledge about their substrates and studies related to their association with ABA signaling is an ongoing process.

Abiotic Stress Signaling Integration with the ABA Signaling Pathway
In plants, ABA signaling is an important tool for robust stress responses to environmental stimuli and developmental processes. Plants encounter numerous abiotic stress factors, such as water scarcity (drought or dehydration), low temperature (cold stress), and salinity (salt stress) [59,127]. The plant utilizes ABA to assess the stress impact and may continuously alter ABA signaling stages based on environmental and physiological conditions to delay processes, such as germination, development, and lateral root formation, as appropriate [128]. Under stress conditions, numerous genes are upregulated in plants via the ABA pathway. Promoter analysis of the ABA-inducible genes has indicated that they must have multiple cis-elements, such as ABREs (PyACGTGG/TC) [129,130]. Plant gene expression analyses have revealed conserved ABREs cis-acting elements in dehydration-inducible promoters [131]. Sequences of ABREs are also present in the genes that are expressed in the seeds ( Figure 1D) [132].
The bZIP subfamily members (AREB1/ABF2, AREB2/ABF4, and ABF3) are induced by ABA, dehydration, and high salinity [133], and the overexpression of the above factors in transgenic plants has led to drought tolerance [133][134][135]. To establish the role of such AREB/ABF TFs in stress-responses in vegetative tissues, Yoshida et al. [136] generated an areb1/areb2/abf3 triple mutant. Microarray analysis revealed impaired stress-responsive gene expression. It also revealed many stress-responsive genes, such as LEA proteins, group A PP2Cs, and various types of TFs that lie downstream of AREB/ABF TFs. Most of such gene promoters contain ABRE sequences. The areb1/areb2/abf3 triple mutant was sensitive to drought-stress and was more resistant to ABA (primary root growth) when compared with other single and double mutants, suggesting that ABF3, AREB1, and AREB2 are the master TFs that regulate the ABRE-dependent gene expression under stress conditions in ABA signaling. HD-ZIP transcription factor (TF), HAT1, a critical regulator in brassinosteroid (BR) signaling, interacts with SnRK2s [137,138]. HAT1 suppresses ABA signaling and is involved in ABA regulation of drought response [138], which also suggests the integration of BR signaling with ABA signaling to regulate the downstream targets of abiotic stress tolerance.

Biotic Stress Signaling Integration with the ABA Signaling Pathway
Plants respond to biotic and abiotic stress via crosstalk signals such as ABA, salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET)-mediated defense signaling [149]. The role of ABA in the crosstalk between biotic and abiotic stress is very broad and is discussed in detail by recently published reviews [150,151]. A restraint function of ABA on the systemic acquired resistance pathway of SA induction has also been reported in tobacco [152]. Elicitors/effectors secreted by Pseudomonas syringae pv. tomato activate ABA biosynthesis along with ABA signaling, which leads to the inhibition of biotic defense responses [23]. However, several reports have shown the positive effect of ABA signaling on biotic and abiotic stress. For example, treatment with ABA and SA resulted in a short-term increase in H 2 O 2 production, which induced tolerance to salinity, heat, and oxidative stress [153]. During infection in plants, stomata can act as passive passage for bacteria. P. syringae pv. tomato pathogen-associated molecular patterns (PAMPs) induce stomatal closure via ABA signaling, NO production, and flagellin receptor (FLS2), indicating the integration of biotic and abiotic signaling with ABA signaling in the regulation of stomata [154]. β-aminobutyric acid (BABA), a non-protein amino acid, has been reported as a link between heat tolerance, biotic stress, and ABA signaling. Plants treated with BABA become resistant to abiotic as well as biotic stress [155][156][157][158]. The ibs3, a BABA-induced sterility mutant, exhibits defected regulation of ABA1, salt resistance, and BABA-induced pathogen [159]. A recent study described BABA as a natural molecule synthesized by plants under stress [160]. Therefore, it may be a new entry into the list of plant hormones. Isolation of an activation-tagged mutant of activated disease resistance 1 (adr1) further consolidates the link between ABA-mediated biotic and abiotic signaling. The adr1 mutant displayed drought tolerance as well as disease resistance. Surprisingly, adr1 plants display sensitive phenotype toward salt and heat stress, suggesting antagonism between biotic stress and abiotic stress [161]. Recently, a study reported that PUB10 acts as a negative regulator of ABA signaling, which could also be intermediatory in JA signaling (Figure 2) [66]. MAPKs are also reportedly involved in plant defense response by regulating the JA-and SA signaling as well as downstreaming transcription factors. This is discussed in detail by a recently published review [162].
In Arabidopsis, the biotic stress-inducible AP2/ERF TF family proteins are associated with different abiotic stresses, such as cold, drought, salinity, heat, and light stress [70,[163][164][165]. Many ROS-inducible genes are also induced by AtERF6 for protection against both biotic and abiotic stress [166]. Most of the ethylene response factors (ERFs) that display abiotic stress tolerance are induced not only by ethylene but also by other biotic stress associated phytohormones, such as JA and SA. Therefore, there is potential crosstalk between abiotic and biotic stress and responses via the ABA signaling pathway [167][168][169][170] (Figure 2).

Role of ABA Signaling in Seed Germination and Lateral Root Formation
ABA accumulates during seed development and seed germination. In mature seeds, ABA promotes the synthesis of LEA (Late embryogenesis abundant) proteins for desiccation tolerance. ABA also inhibits germination and stimulates dormancy in mature seeds [55]. ABI3 and ABI4 control seed sensitivity and embryonic gene expression in plants [171]. abi3 mutant seeds display reduced dormancy and vivipary, caused by the strongest alleles. To control seed maturation, ABI3/VP1 binds directly to the promoters of Sph/RY. FUSCA3 (FUS3) and LEAFY COTYLEDON 2 (LEC2) genes encode TFs that are structurally related to VP1/ABI3 [172,173], and the genes interact with ABI5 [174], although VP1/ABI3 is involved directly in ABA signaling. A bZIP protein, ABA-INSENSITIVE5 (AB15), was identified via ABA insensitive germination screening [171]. In addition to ABI5, three AREB/ABF-type bZIP proteins, namely EEL, AREB3, and AtbZIP67/AtDPBF2, are expressed in the nuclei of developing seeds and play vital roles in seed germination [175,176]. During early germination and seed maturation under stress conditions, ABI5 regulates the direct expression of AtEm1 and AtEm6 (LEA class genes) [130,175,177]. A seed expressed gene, DELAY OF GERMINATION 1 (DOG1), is critical for dormancy induction. During Arabidopsis seed development, DOG1 interacts with ABI3 and influences ABI5 expression [178] ( Figure 1E). PGIP1 and PGIP2 are associated with the process of seed germination, and they are direct targets of ABI5 [179,180]. Overall, all the above studies highlight the key role of ABI5 as a master regulator of seed development through the ABA signaling pathway. A negative regulator of lateral root formation, MYB96, activates the expression of ABI5 and is involved in plant responses to salt and drought stress [181]. MYB7 also negatively regulates ABI5 expression in seeds [182] ( Figure 1E). The above studies support the functional role of ABI5 in the ABA signaling pathway-dependent inhibition of lateral root growth under stress conditions [183].

ABA and Light Signaling Convergence
ABA and light are the endogenous hormonal and the external environmental cues that play vital roles in the regulation of seed germination and seed development. The ability of plants to integrate external signals with internal regulatory pathways is crucial for their survival [184,185]. However, the crosstalk between ABA signaling and light signaling and its underlying molecular mechanisms remain largely unclear. The involvement of TF HY5 in promoting seedling photomorphogenesis, root development, and early seedling growth has been studied extensively. It mediates ABA signaling responses in seed germination by binding directly to the ABI5 promoter and regulating its expression [186]. Two major TFs in the phytochrome A pathway, FAR-RED IMPAIRED RESPONSE1 (FAR1) and FAR-RED ELONGATED HYPOCOTYL3 (FHY3), positively regulate ABA signaling by inducing ABI5 expression directly [187]. PIL5 (also known as PIF1), a phytochrome-interacting bHLH TF, also targets ABI5 [188]. Conversely, BBX21, a transcriptional regulator that is involved in the regulation of seedling photomorphogenesis, negatively regulates ABI5 expression by intervening in the binding of HY5 to the ABI5 promoter [189]. In addition, ABI5 can regulate its own expression while BBX21 inhibits ABI5 activation ( Figure 1E). BBX21 represses ABI5 activity by regulating the binding activities of both ABI5 and HY5 to the ABI5 promoter [189]. The findings suggest that, in the light signaling pathway, multiple TFs regulate ABI5 expression in the ABA signaling responses.

ABA Signaling and Control of Flowering Time
A variety of ABA signaling activities are involved in controlling meristem function or flowering time [171,190]. In addition, the ABA inhibitory effect in floral transition was described very well in a study on an ABA-deficient mutant [191]. Such an inhibitory effect could be due to the modulation of DELLA protein activity [184]. Therefore, ABA is also considered a floral repressor. FLOWERING LOCUS C (FLC) is a key repressor integrator that tightly controls flowering signals [192]. FLC also mediates seed germination via genes, such as SOC1, APETALA1, and FT, making FLC an effective regulator in temperature-dependent seed germination [193]. ABFs are the bZIP TFs that are involved in ABA signaling during seed germination in plants [194,195]. Another bZIP protein, FD, mediates signals from FT at the shoot apex [196]. Overexpression of another bZIP TF, ABI5, upregulates FLC expression and delays flowering initiation. Phosphorylation of ABI5/SnRK2 during ABA signaling directly affects floral transition, and the inhibitory effect of ABI5 on floral transition disappears without phosphorylation. Transactivation of FLC expression could occur by direct binding of ABI5 to FLC promoter regions [197]. AtU2AF65b, a putative U2AF65 spliceosome, participates in ABA-mediated flowering via the regulation of the pre-mRNA splicing of ABI5 and FLC [198], which indicates the positive regulation of FLC activity by ABI5 during ABA signaling. Furthermore, AtU2AF65b-mediated mRNA splicing is critical for ABA-regulated flowering transition for the control of floral transition in plants ( Figure 1E).

Other Aspects of ABA Signaling
ABA transporters are also a significant part of ABA signaling, as it is important to transport ABA from its sites of synthesis to its multiple sites of action within plants. In Arabidopsis, four ABA transporters have been identified (AtABCG25, AtABCG30, AtABCG31, and AtABCG40) all of which are ATP-binding cassette transporter G subfamily members [199][200][201][202]. AtABCG25 is involved in exporting ABA from the vasculature [201], while AtABCG40 is a plasma-membrane ABA-uptake transporter in guard cells, and is necessary for timely closure of stomata in response to drought stress and seed germination [199,200]. AtABCG30 mediates ABA uptake into the embryo, while AtABCG31 brings about ABA secretion from the endosperm [200]. A recent study reported ABA transporter-like 1 (AhATL1) gene from peanut (Arachis hypogaea L.) whose cognate protein, AhATL1, is a member of the ATP-binding cassette transporter G subfamily and localizes to the plasma membrane [203]. The expression of both the AhATL1 transcript and the corresponding protein was upregulated by water stress and treatment with exogenous ABA. Another report suggested that in Medicago truncatula, MtABCG20 acts as an ABA exporter that influences root morphology and seed germination [204]. These data indicate that the ABA transport system plays a significant role in water deficit tolerance and growth regulation [203].
ABA signaling crosstalk occurs with other hormones that are involved in plant growth and stress response. These hormones include strigolactone, cytokinin, and karrikin. Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching [205]. ABA signaling may regulate SL biosynthesis [206]. The antagonistic action of ABA and cytokinin signaling mediates drought stress response in Arabidopsis [207]. Karrikin signaling pathway seems to be upstream of ABA signaling pathway and karrikin mediates changes in ABA-related gene expression [208]. DELLA protein is important for seed germination [209]. ABA also interacts with DELLA protein when DELLA/ABI3/ABI5 complex is involved in seed germination [210].

Conclusions
It is evident that ABA is an important signaling compound. In stress and developmental responses in plants, ABA signaling largely depends on the SnRK family of protein kinases. ABA signaling integrates other signaling components, such as Ca 2+ , light, MAP kinase, SA, JA, and ET signaling, in response to environmental cues, developmental activities, and biotic stress (Figure 3). Such integration is vital for response stress and plant development; however, there are still gaps regarding to what extent and how often such integrations occur. In addition, it is important to reveal the complex ABA signaling network by adopting more integrated and more detailed genome-wide studies to identify the critical components of stress responses and developmental processes and to develop scientific tools for the genetic engineering of stress-tolerant and robust plants. Furthermore, it is critical to determine the role of all ABA signaling-related genes to fill any knowledge gaps about ABA signaling. In the future, studying the function of ABA signaling-related genes under different combined stress conditions and the regulation of developmental processes would offer detailed insights into the underlying mechanism of ABA signaling.

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