Abscisic Acid-Induced Stomatal Closure: An Important Component of Plant Defense Against Abiotic and Biotic Stress

1Plants need to respond quickly to diverse stress conditions, as they cannot move away. Stress can be due to abiotic (e.g., drought, salinity, chilling, and high-temperature) or biotic factors (e.g., pathogens, insects, herbivores). Plants developed various adaptation strategies to cope up with these situations. A typical example is the stomatal closure, limiting the water loss and restricting pathogen entry into the leaves. Plants accumulate hormones [e.g., abscisic acid (ABA) or salicylic acid (SA) or methyl jasmonate (MJ)] under abiotic stress conditions and elicitors [e.g., flagellin 22 (flg22)] under pathogen attack. Among the hormones, ABA is involved in several abiotic and biotic stress conditions and is therefore considered an essential and versatile compound. In contrast, SA, MJ, and ethylene (ET) help in resistance against biotic stress. Under drought, salinity, or cold stress, ABA accumulation causes stomatal closure to conserve water while up-regulating genes to promote osmotic adjustment in leaves. The enhanced ABA levels in plants mediate the cross-adaptation against drought and pathogens besides insect herbivores.

2Several compounds other than ABA also accumulated in plants in response to different stresses (Table 1). These compounds can close stomata and, in many instances, improve plants’ resistance to pathogens. The plant hormones and elicitors can further regulate transcription factors and induce pathogenesis-related (PR) genes. There can also be a cross-talk between the factors involved in abiotic and biotic stress signaling. However, these compounds either require ABA for their action or interact with ABA to activate defense responses.

3Since its discovery, studies on ABA (a sesquiterpene) and its role in plant processes were studied extensively. Plant processes such as seed dormancy, seed development, promotion of desiccation tolerance, abscission, and, most importantly, stomatal closure were all regulated by ABA. Further, ABA can be crucial in also non-stress conditions. The action of ABA was complemented by hormones, such as SA. Similarly, some of the secondary messengers triggered by ABA can also participate in plants’ adaptation to abiotic and biotic stress. Examples are reactive oxygen species (ROS), nitric oxide (NO), and cytosolic free Ca2+. Several compounds like polyamines (PAs), hydrogen sulfide (H2S), and brassinosteroids (BRs) promote drought tolerance by regulating ABA synthesis and vice versa.

4Readers interested in ABA and its role in plants may refer to some recent reviews. Our review emphasizes the role of ABA’s stomatal closure as an adaptive measure against both abiotic and biotic stresses. We have also discussed other compounds that can improve plant adaptations, still involving ABA. The stomatal closure by ABA follows a typical scheme of signal transduction. The interactions of these signaling components with others to synergize the plant’s adaptation against pathogen attacks are described. To limit the length of our article, reviews were cited when available. In some instances, original articles were referred, due to their classic importance.

5When plants were exposed to water stress (drought), an increase in ABA was typical due to either synthesis or degradation of ABA or both. The soil-water deficit could be perceived as a signal by roots to trigger ABA’s de novo synthesis. The increase in ABA of roots in response to drought was correlated with an increase in foliar-ABA concentrations, suggesting drought-induced ABA played a significant role in controlling leaf water potential. ABA accumulated in roots was transported to trigger stomatal closure in leaves and limit transpirational water-loss. An increase in ABA could also occur in response to temperature-stress (high or low) or a newly discovered small peptide, CLE25. found that NCED3 could be the trigger to enhance ABA biosynthesis in Arabidopsis under drought stress. Under these conditions, increased ABA and stomatal closure could limit the water-loss and restrict pathogen entry. This phenomenon was complemented with additional steps of ABA transport from roots to shoots, conversion of bound ABA into free form to mobilize ABA within leaf.

6An increase in endogenous levels of ABA was also observed when plants were infected with pathogens, for e.g., Phaseolus by Colletotrichum, flax by Fusarium, and nced5 mutant of Arabidopsis by Alternaria. Similarly, the clonal variation of chestnut susceptibility or resistance to Fusarium was related to ABA levels under infection. The exact relationship between endogenous ABA levels and disease susceptibility of plants appeared to be complex, as the relationship depended on the duration of infection, other stresses, and the type of pathogen. During the early stages of pathogen infection, the increased ABA levels helped in resistance, while at later stages, high levels of ABA made the plants susceptible to pathogens. The differential effects of ABA on the modulation of pathogen sensitivity need to be examined further, particularly in relation to the predisposition of plant tissue. Readers interested in ABA accumulation mode may refer to relevant articles for further details.

7Stomatal closure is one of the initial responses of plants to stress conditions to retain water status and provide innate immunity against pathogens. The physical barriers on the plant’s outer surface, such as bark, cuticle, and cell wall, could protect against physical and biological factors. However, the microscopic pores on leaf surfaces called stomata are the accessible entrances to several microbes. Stomata form the gateways for transpiration, photosynthetic gas exchange as well as microbial entry into leaves. Stomatal guard cells are quite dynamic in sensing and responding to external microbial pathogens. Stomatal closure can be an essential strategy to defend against abiotic and biotic factors such as drought or pathogens. Several instances of stomatal closure induced by plant pathogens are listed in Table 2. Stomatal closure was triggered by either elicitors or other compounds produced in the leaf in response to pathogens, such as SA, MJ, or PAs. Stomata can sense and respond to microbe-associated molecular patterns, including chitosan, flagellin, and harpin. The sensing of ABA or other compounds and the final response of stomatal closure follows a common signaling pathway involving receptors, protein kinases, secondary messengers, ion channels, ion efflux, and turgor loss in guard cells. Among kinases, OST1 is a primary activating factor NADPH oxidase and raises the ROS levels of guard cells.

8During ABA-induced stomatal closure, an increase in OST1 kinase was followed by the activation of RBOH D/F, and increases in ROS/NO/Ca2+ levels. In turn, Ca2+ dependent CDPKs activated slow anion channel 1 (SLAC1), S-type anion channel 3 (SLAH3) and K+out channels to promote ion efflux from guard cells and forced stomata to close. However, in presence of flg22 or yeast elicitor, the activity of OST1 did not increase. Albeit in a resting stage, OST1 participated in stomatal closure by variety of signals including PAMPs (e.g., flg 22, yeast elicitor, chitosan) or environmental components, such as high CO2 or high humidity. Besides its action through ROS/NO/Ca2+, OST1 could directly modulate ion channels to cause stomatal closure (Figure 1). In a recent study, the events involving OST1/SnRK2s were studied in real-time using FRET sensors. These experiments provided a visual evidence of the interaction of OST1 with signaling components of ABA and elevated CO2. It is obvious that OST1 is an important point of convergence of signals from abiotic and biotic factors. It would be interesting to assess the mechanism by which OST1 keeps up such dual mode of activation and modulating downstream components, all converging to mediate stomatal closure.

9There are reports indicating that stomatal closure can be induced by biotic and abiotic stresses in an “OST1-independent manner”. For e.g., plant elicitor peptides (Peps), a group of damage-associated molecular patterns, can trigger stomatal closure by activating SLAC1 and SLAH3 in an OST1-independent manner. Similarly, elevated CO2 can bypass OST1 kinase and activate SLAC1. SLACs may be activated without the involvement of OST1 but other kinases. For e.g., the signaling events in guard cells can utilize MAPK cascade to up-regulate SLAC1/SLAH3. MAP kinases 3/6 participated upstream of NO during stomatal closure in darkness, while MPK 9/12 activated SLAC1 integrating with Ca2+/CDPK system during the cross-talk of ABA and SA, though the exact mechanism is not known. When exposed to a small elicitor peptide, AtPeps, a two-kinase component of BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1)/BORTRYTIS-INDUCED KINASE 1 (BIK1) was turned on to activate SLAC1 and SLAH3 in guard cells. However, further experiments are necessary to identify the exact components involved in the activation of SLAC1 by MPK 9/12 or BAK1/BIK1.

10There can be additional components of a leaf that can provide resistance to microbes as well as environmental stresses, e.g., callose or silicon deposition, cuticular waxes and trichomes. ABA was involved in some of these responses. Cuticular wax biosynthesis was ABA-dependent and mediated by MYB94 and MYB96 transcription factors. A light-induced increase in trichome density and thick leaves was due to high ABA levels in the leaves. Furthermore, ABA promoted callose synthesis and deposition by negatively regulating callose degrading pathogenesis-related protein 2 (PR2). Similarly, ABA up-regulated callose deposition and antiviral RNA silencing mechanism to evade virus attack.

11Besides ABA, several other compounds increase when plants are exposed to stress, which close stomata and help plant defense responses. These compounds can be grouped into three categories: hormones, elicitors, and metabolites (Table 3). The hormones include MJ, SA, ET, and BRs. MJ was the most effective one and induced stomatal closure by elevating pH, ROS, NO, and Ca2+ leading to activation of anion channels, similar to ABA action. Further studies in detail are needed to understand the effects of ET as well as BRs on closure.

12Salicylic acid (SA) is considered a plant defense hormone with overlapping functions as an elicitor. SA-induced stomatal closure was mediated by ROS produced primarily through peroxidase (not NADPH oxidase, as in ABA). The other downstream events of NO production and ion channel modulation in guard cells were similar to ABA’s action. Thus, there is an overlapping of signaling pathways mediated by SA and ABA to cause stomatal closure in Arabidopsis. Several microbial elicitors (chitosan, flg22, harpin, and cryptogein) promoted stomatal closure and prevented pathogens’ entry. These elicitors produce NO and ROS via nitric oxide synthase (NOS) and NADPH oxidase, respectively. The combined action of ROS and NO could be imparting pathogen resistance.

13Allyl isothiocyanate (AITC), proline, and PAs are examples of metabolites that accumulate under stress. Despite accumulation in large quantities, proline, a compatible osmolyte, caused only a partial closure. PAs (including putrescine, spermidine, and spermine) accumulated during water stress and pathogen attack. The oxidation of PAs by polyamine oxidase raised ROS levels, followed by NO to cause stomatal closure similar to ABA. Similarly, AITC promoted stomatal closure and defense responses against biotic components.

14The hormones, elicitors, and metabolites described above interact markedly with ABA and act in tandem to promote abiotic stress tolerance (Table 4). ABA’s interactions with SA or MJ to work together during stomatal closure and pathogen resistance are well-known. For e. g., MJ promoted ABA biosynthesis by inducing the AtNCED3 gene expression in Arabidopsis. ABA was needed during SA-action on stomata. Conversely, elevated ABA triggered SA biosynthesis by activating SID2 and promoted stomatal closure. These reports confirm the synergy between MJ, SA, and ABA during stomatal closure.

15An SA-receptor, NPR1, mediated chitosan signaling in guard cells. SA, chitosan, and ABA interacted during stomatal closure by activating MAP kinases (MPK9 and MPK12). Elevated levels of PAs stimulated biosynthesis of ABA. In turn, ABA stimulated oxidation of Pas to elevate H2O2 and NO, and stomatal closure. Such interactions could fine-tune ABA’s effects to strengthen the plant defense reactions against both abiotic and biotic stresses. The direct role of PAs and proline in pathogen resistance is not clear.

16The stomatal closure by ABA cannot be a permanent strategy to prevent microbial entry, as pathogens, such as Puccinia, can enter leaves through places other than stomata. Therefore, we do not mean to overemphasize the role of ABA-induced stomatal closure as the sole mode of adaptation. Also, stomata need to open subsequently to keep up the gas exchange and normal plant function. At the same time, microbial pathogens initiate counteractive measures to reopen stomata, by either effectors, (such as coronatine or fusicoccin,;; ), locking the open-stomata or even killing guard cells to prevent their closure.

17Some of the pathogens secrete a cocktail of cell wall digesting enzymes to facilitate the entry through the epidermis into the leaves. The pathogens can also restrict the biosynthesis/actions of ABA and related hormones. Further, pathogens too can trigger programmed cell death (PCD) of host tissue facilitating the spread of infection. Despite the counteractive efforts by pathogens, ABA can still contribute to plant defense. It is known that ABA could initiate multifaceted measures involving hypersensitive response (HR) and long-term adaptation on its own or by synergetic interaction with other hormones, such as SA or MJ, to ensure improved resistance (described below).

18The role of gasotransmitters in stomatal regulation requires special mention. In addition to NO, two more gaseous signaling molecules (gasotransmitters), hydrogen sulfide (H2S), and carbon monoxide (CO) produced within plant cells are an integral part of ABA-dependent stomatal closure as well as other stress conditions. These three gasotransmitters interacted with ABA-signaling during drought. Under abiotic stress, ABA could elevate the levels of NO as well as CO or H2S. For e.g., ABA activated heme oxygenase (HO), thereby increased CO levels and caused stomatal closure. In turn, NO elevated the levels of H2S by regulating H2S producing enzymes (L/D-cysteine desulfhydrases). Similarly, CO promoted both NO and ROS synthesis, facilitating stomatal closure during abiotic stress. Thus, a triangular interaction appears to be operating in guard cells. These interactions and synergistic actions need to be examined further. We, however, feel that among the three gasotransmitters, NO could be the significant signaling molecule. Like in the case of ROS, the production of NO can also be triggered by microbial pathogens to activate defense-related genes (e.g., phenylalanine ammonia-lyase and pathogenesis-related protein-1) that play a significant role in acquired pathogen resistance. NO produced in response to lipopolysaccharide contributed towards resistance against Pst DC3000. The upregulation of H2S production suggested a strong association between H2S and plant defense. Further studies on these protective abilities of gasotransmitters to improve pathogen resistance could help achieve plants’ resilience.

19Gasotransmitters exert their actions by mediating post-translational modifications (PTMs) such as S-nitrosylation, nitridation, and persulfidation of target proteins. These PTMs seem to exert different effects. Accumulation of H2S by ABA mediates the persulfidation of SnRK2.6 to promote stomatal closure by ABA. S-nitrosylation, mediated by NO, inhibited OST1/SnRK2.6 kinase activity and limited stomatal closure. Detailed experiments on such contrasting effects of NO and H2S would unravel the mechanism of interaction between gasotransmitters and ABA during stomatal closure and plant defense against pathogens.

20Stomatal closure is the result of turgor loss in guard cells because of increased cation/anion efflux. A well-defined transduction pathway mediates the events during stomatal closure by ABA or other compounds, as illustrated in Figure 1. Binding of ABA to its receptor inactivates protein phosphatase 2C resulting in the activation of OST1 kinase, which stimulates NADPH oxidase (due to phosphorylation) enzyme to generate ROS and then the production of NO. Both ROS and NO can elevate levels of cytosolic Ca2+. The high levels of ROS, NO, and Ca2+ act either directly or together to activate anion/cation efflux channels while inhibiting the influx channels. The final result is the loss of cations/anions from guard cells, resulting in turgor loss and stomatal closure. These three secondary messengers (ROS, NO and Ca2+) can also stimulate the production of other signaling components such as phospholipase C, phospholipase D, phosphatidic acid, and inositol 1,4,5-triphosphate besides raising cytosolic pH, all contributing to stomatal closure. Apart from well-known NO, other gasotransmitters, i.e., CO and H2S, are also involved in ABA-induced stomatal closure.

21In recent years, another signaling component, reactive carbonyl species (RCS) was found to play a significant role in stomatal closure. These RCS are products of lipid oxidation, produced and scavenged during various developmental processes, including PCD. suggested that RCS (also called oxylipins) played a dominant role during stomatal closure by biotic factors (e.g., elicitors from pathogens), compared to ROS during the action of ABA (typical of abiotic stress factor). Soon, detailed reports appeared that RCS could function downstream of ROS production during closure by ABA and MJ in Nicotiana tabacum and Arabidopsis thaliana. Recently, RCS was found to activate CPK6, promote the elevation of Ca2+ and activate SLAC1, leading to stomatal closure. These observations imply that RCS and ABA could enable guard cells to respond to both biotic and abiotic stress conditions. Several authors had reviewed the details of the ABA-induced signal transduction pathway.

22Several of the signaling components during ABA-induced stomatal closure can protect against pathogens (Table 2). The three major secondary messengers, triggered by ABA (namely ROS, NO, and Ca2+) can initiate defense processes such as stomatal closure and PCD. ABA-induced NO can act as a signaling molecule to initiate adaptive responses against abiotic (UV, drought, and salinity) or biotic factors (pathogens or elicitors). The reaction products of ROS and NO (like peroxynitrite) and NO-mediated post-translational modifications can all act together to initiate defense responses. The rise in cytosolic Ca2+ was often required to induce HR as a plant immunity response (e.g., against microbial pathogens). Other compounds involved in ABA signaling, like phospholipase D and phosphatidic acid, were also associated with plants’ defense against pathogens. The ability of H2S, a gasotransmitter, to impart resistance against typical plant pathogen (Pseudomonas syringae) suggests a link between stomatal closure and adaptation to plant pathogens. The promotion or inhibition of ROS and NO production by gasotransmitters can be a significant factor during plant defense.

23Arabidopsis thaliana had been an excellent model to study and validate the components/mechanisms of plant function. Several mutants of A. thaliana were employed to establish ABA’s signaling components (Table 5). These mutants fall under three groups: those with altered ABA biosynthesis/reception or deficient in signaling compounds or those with altered stomatal response independent of ABA. The mutants who cannot close their stomata also lose their ability to resist pathogens, becoming hypersensitive to pathogens. These observations emphasize the strong association of stomatal closure by ABA or related compounds with altered pathogen resistance.

24Evidence is emerging that ABA may not always impart resistance but increase plants’ susceptibility to abiotic or biotic factors by compromising defense responses. For e.g., ABA can act differently depending on the pathogen status: pre-entry or post-entry phases. During the early stages, induction of stomatal closure along with stimulation of wax and callose synthesis could reinforce the plant’s defense. However, in the post-invasion stage, ABA can be antagonistic and increase susceptibility to microbes. Accumulation of ABA at infection site repressed events involving disease resistance against Cercospora in beetroot. Elevated ABA levels promoted sugar transport to fungi and enhanced the infection of wheat by Puccinia striiformis sp. tritici (pst). Furthermore, increased ABA levels antagonized plant’s defense responses by suppressing SA or MJ induced defense gene expression, callose deposition, and basal resistance against Fusarium oxysporum or Magnaporthe grisea.

25We believe that ABA could play a significant role in restricting or at least delaying the pathogen entry and subsequent infections, at least in the case of bacteria (Table 5 and description below). We acknowledge that the role of stomata should not be generalized, as the experiments done on pathogen infection with Arabidopsis (non-host) may not be all applicable with typical host species, such as wheat or barley.

26Pathogen resistance cannot be entirely due to stomatal closure, and the action of ABA needs to continue beyond stomata. Often during infection, an increase in ABA levels led to multiple events that help against abiotic stress and disease resistance. Even if pathogens manage to enter the intercellular spaces, elevated ABA can initiate a spectrum of events that restrict the multiplication and spread of plant pathogens inside leaves. For e.g., ABA can raise the levels of ROS, NO, and Ca2+. An important consequence of elevated ROS, NO, and Ca2+ is HR response, leading to PCD in several crop species while protecting against pathogens. The HR may also include callose deposition in cell walls, increased cuticular biosynthesis, and blocking of plasmodesmata. Further, the modulation by ABA of miRNA can restrict viral replication and the viral movement due to blocked plasmodesmata, thus causing antiviral silencing.

27Other ABA-promoted events include the activation of genes involved in either accumulation of compatible solutes for osmotic adaptation or PCD to restrict the spread of pathogens within leaves or enhanced secondary metabolites production (Figure 2). For example, ABRE-binding proteins (AREBs) and ABRE-binding factors (ABFs) up-regulate stress-responsive genes, involved in short-term and long term adaptations to abiotic stresses, including drought, cold, and heat. The accumulation of proline on exposure to water stress or ABA can serve the dual purpose of providing compatible solute for osmotic adjustments in leaves and causing partial stomatal closure in the epidermis. Elevated proline levels due to ABA can further offer the plant defense against pathogens. There have been claims that proline accumulation is not all ABA-dependent. It is not clear if ABA is the master regulator of proline accumulation or a consequence of stomatal closure. ABA and proline’s combined action can be beneficial under hypoxic stress. Similarly, ABA, proline, and PAs can help together during plant adaptation to osmotic stress.

28Several ABA responses are beneficial during biotic stress, as well. ABA and SA promoted the accumulation of anti-microbial flavan-3-ols, enhancing the plant defense against rust infection in two popular trees, Poplar and Malus. Similarly, the ABA-activated MYC2 transcription factor was essential for defense against Meloidogyne incognita. PCD is a controlled process and is a consequence of high ROS and NO levels typically up-regulated by ABA. In plants, PCD is often an adaptive response during abiotic/biotic stress. However, PCD can also aggravate plant disease. ABA was involved in the induction of cell death around the wounded site. Further experiments are needed to establish if the process of PCD is incidentally associated with ABA or if ABA is the causal factor.

29Enhanced production of secondary metabolites is another defense mechanism of plants against stress. Abscisic acid itself is a secondary metabolite produced during stress and can play a significant role in secondary metabolite production when plants encounter abiotic or biotic stress factors. ABA-induced increase in flavonoids and other metabolites served as a defensive measure against UV-B radiation. Trichoderma harzianum infection increased ABA levels, which helped in osmotic adaptation under drought by restricting water loss and increasing osmolytes, like proline. Such interactions between ABA and secondary metabolite production are quite exciting and need to be examined in detail.

30Based on the extensive literature available, we tried to emphasize the role of ABA-induced stomatal closure as an essential component of plant defense against both limited water and pathogens (Figure 2). ABA’s role is complemented further by the cross-talk and interaction of ABA with other hormones, microbial elicitors, and metabolites. We, therefore, emphasize ABA can play either a direct role or an indirect role as well. The stomatal closure by ABA can be considered a quick short-term response. However, the three vital secondary messengers involved in ABA-signaling, namely ROS, NO, and cytosolic free Ca2+, can promote events, such as osmolyte accumulation, up-regulation of adaptive genes, HR and PCD. These events facilitate the long-term adaptation of plants against abiotic stress as well as pathogens. ABA’s ability to induce stomatal closure may not always be due to changes occurring in guard cells’ secondary messengers. For e.g., chitosan, a microbial elicitor, can cause the death of guard cells, likely to make them non-functional. This aspect is thought-provoking and needs to be examined further using ABA or other hormones, such as SA or MJ.

31There are emerging areas that are related to the role of ABA and stomatal closure in plant defense. ABA’s ability to induce priming could help plants tolerate heat or drought stress occurring later. Similarly, plants could have a transcriptional memory of ABA and MJ that can be useful for long-term adaptations. Because of the well-documented importance, there had been recurring attempts to discover ABA-analogs or ABA-agonists. Stomatal closure and signaling components in guard cells can be excellent model systems to monitor such compounds. A few ABA-analogs were found, which mimic ABA to induce stomatal closure. An extension of such work would open up an exciting possibility of exploiting ABA-analogs to improve plants/crops’ water-use efficiency. There were reports that ABA and or stomatal closure may not be crucial, particularly during fungal pathogen infection of crops, e.g., barley and wheat. A few experiments on mutants of such crop plants deficient in ABA or ability to close stomata may help us clarify the exact situation. Plants are indeed known to employ more than one strategy to overcome stress conditions or to optimize metabolism. ABA-induced stomatal closure is one of the approaches, while plants/pathogens could evoke other strategies, as well.

32AR conceived the idea and developed the outline. All authors reviewed the literature and wrote the article. AR edited the final draft of the review. All authors approved the final version.

33The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.