*2.2.1. ChlH/ABAR (H subunit of the chloroplast magnesium chelatase/ ABA receptor)*

The primary function of ChlH is chlorophyll synthesis. ChIH was initially identified as an ABA-binding protein in broad bean (*Vicia faba*) and the Arabidopsis protein was named as ABAR. Later it was found that binding of ABA to ChIH/ABAR depends on the stereochemistry and it specifically binds to only (+) ABA to mediate ABA responses. Although ChIH/ABAR is localized in the chloroplast envelope, it functions as a negative regulator of ABA signaling in the nucleus. The cytosolic C-terminus of the ABAR interacts with WRKY transcription factors (WRKY 18, 40, 60) which act as transcriptional repressors, repressing ABA-responsive gene expression in the nucleus. Binding of ABA with ChlH/ABAR promotes interaction with WRKYs, preventing them from repressing downstream genes such as *ABI5* and *DREB2* [28-31]. Thus, it has been proposed that ChlH mediates nuclear-chloroplast signaling. However, another research group has not been able to reproduce these results using *wrky* loss of function mutants. In addition, barley ChIH does not bind to ABA and ChIH loss of function mutants do not show any impaired ABA responses. Despite its ABA-binding properties in Arabidopsis, it is not confirmed whether ChIH functions as an ABA receptor (reviewed in [5,27]). However, ChlH/ABAR mediates ABA-induced stomatal closure and ABA inhibition of blue-lightmediated stomatal opening. In addition, ChIH/ABAR has a role in ABA-mediated fruit ripening in peach and strawberry (reviewed in [31]).

#### *2.2.2. GTG1/GTG2 (G protein coupled receptor type G protein 1 and 2)*

G protein coupled ABA receptors are plasma membrane localized cell surface receptors that are widely expressed in plants. Both GTG1 and GTG2 showed specific and saturable ABA- binding activity in direct ABA-binding assays. GTGs have GTPase activity and GDP bound GTGs have enhanced ABA-binding ability, which in turn initiates ABA signaling. GTG1/2 bind with GPA1 (G-PROTEIN α SUBUNIT 1), which abolishes its GTPase activity and represses ABA binding. GTP bound GPA1 represses ABA signalling. However, the downstream components of this pathway are not characterized yet [31,32].

#### **2.3. Recent studies on ABA perception and signaling mechanisms**

Recent findings that several ABA receptors exist in different parts of the cell provide evidence that ABA is active in a variety of subcellular compartments. ABA synthesis enzymes are present in different compartments, suggesting that ABA synthesis occurs in different parts of the cell and that these ABA levels contribute to overall ABA homeostasis. For example, the ABA biosynthesis enzyme AtABA1 is localized in the chloroplast, whereas AtABA2 is in the cytosol [33,34]. It has also been proposed that ABA produced in cytoplasm, plastids, vacuole, and other subcellular organelles may have different physiological roles initiated by signaling networks via different ABA receptors in each specific compartment [35].

Takeuchi *et al*. [36] identified a potential ABA analog AS6 that can inhibit the activity of PYLs. X-ray crystallography studies showed the structure of ABA facilitates the binding of ABA to PYR/PYL/RCAR receptors and thereby inhibits interaction with PP2Cs. The AS6 ABA analog was able to block PYL-PP2C interaction, indicating that binding of ABA to PYL receptors initiates ABA responses by repressing PP2Cs.

Inhibition of PP2Cs results in autoactivation of SnRK2 kinases and thereby positive regulation of ABA signaling. Recently, the crystal structures of SnRK2.3 and SnRK2.6 were elucidated, providing evidence that kinase activation is a two-step mechanism as well as details of how the ABA signal is transmitted to downstream components [37]. This study also showed that autophosphorylation of SnRK2.6 is more efficient than that of SnRK2.3.

Lumba *et al*. [20] did a comprehensive transcriptomic data analysis in order to generate a mesoscale ABA signaling network. They showed that there are 3 main kinase hubs, MAP3K∂4, SnRK3.15, and SnRK3.22, that interact with PP2Cs and these kinases act as negative regulators of ABA response, in contrast to the SnRK2s involved in ABA signaling. SnRK3.15 and SnRK3.22 also interact with a large number of transcription factors and may have a role in overall ABA responses in the plant [20].
