HOS15: A missing link that fine-tunes ABA signaling and drought tolerance in Arabidopsis

ABSTRACT

Among the phytohormones, abscisic acid (ABA) specifically regulates plant adaptation to osmotic stresses, such as drought and high salinity, by controlling the internal water status in plants. A significant accumulation of ABA occurs in response to conditions of water deficit; this is followed by a sophisticated signaling relay, known as the ABA signaling pathway, which decreases the rate of transpiration through stomatal closure, thereby suppressing photosynthetic activity. Snf1-related kinases (SnRK2s) are the major components regulating the ABA signaling pathway. Of these, SnRK2.6 (OST1) and SnRK2.3 are negatively regulated by HOS15 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE15), in an ABA-dependent manner, to cease the signaling relay. HOS15 is a WD40-repeat protein that regulates several physiological processes, including plant growth and development, freezing stress responses, and ABA signaling. Here, we provide a brief overview of the functional importance of HOS15 in the regulation of ABA signaling and drought stress.

Introduction

Phytohormones play a central role in environmental adaptation by inducing many biochemical and physiological changes to protect against abiotic stresses, including high salinity, dehydration, and temperature changes.^1-3 In addition to abiotic stresses, plant hormones are involved in plant responses to biotic stresses, such as pathogen and insect attacks.^4,5 Among the plant hormones, abscisic acid (ABA) is one of the most important regulators of plant growth and development and plays a central role in both biotic and abiotic stress responses.^1,4,6,7 The phenotypes of ABA-deficient mutants, exhibiting loss of dormancy, a reduction in size, and wilting, support the importance of ABA in developmental and physiological responses.^6,8,9 ABA signaling is regulated by the combined activities of several proteins, known as ABA signaling core proteins. Of these, the ABA receptors PYR/PYL/RCAR (pyrabactine resistance/PYR-like/regulatory components of ABA response) and co-receptors clade A protein phosphatase 2 Cs (PP2 Cs) function at the top of the signaling relay. On receiving the ABA signal, the PYR/PYL/RCAR receptors interact with and inhibit the phosphatase activity of PP2 Cs, resulting in the release of subgroup III Snf1-related kinase 2 s (SnRK2s) sequestered to PP2 Cs.^10,11 Following their release, SnRK2s first undergo autophosphorylation and then transphosphorylate the downstream transcription factors, such as the ABA response element binding factors (ABFs).¹⁰ ABFs include members of the bZIP transcription factors (TFs) that regulate ABA-mediated gene expression under stress conditions.^10,12 Transcription factors in this family recognize the ABA response elements (ABREs) involved in ABA-regulated gene expression and upregulate the expression of ABA-responsive genes, such as RD29A and RD29B.^12-14

The Arabidopsis subgroup III SnRK2 family includes three members, SnRK2.2, SnRK2.3, and SnRK2.6,^12,15 and their triple mutants show greater insensitivity to ABA than their single or double mutants, demonstrating that these subgroup III SnRK2s are highly redundant.

BRASSINOSTEROID INSENSITIVE2 (BIN2), a GSK3-like kinase that functions as a negative regulator in brassinosteroid signaling, interacts directly with and phosphorylates SnRK2s (SnRK2.2/2.3/2.6).¹⁶ Phosphorylation by BIN2 positively regulates the kinase activity of SnRK2s, indicating that BIN2 positively regulates SnRK2s.¹⁶ In line with this a recent study shows ARK, an Arabidopsis Raf-like kinase phosphorylate and activate SnRK2E (OST1), highlighting the involvement of Raf-like kinases in activation of SnRK2s.¹⁷ However, the manner in which SnRK2s are deactivated at the protein level (such as proteasomal degradation) is poorly understood. In contrast, other components of the ABA signaling pathway, such as the ABA receptors, PP2Cs and TFs, have been extensively studied.

More recently, Cheng et al. (2017) found that AtPP2-B11, a component of the SCF ubiquitin E3 ligase complex, interacts with and promotes the ABA-dependent ubiquitination and degradation of SnRK2.3.¹⁸ Interestingly, SnRK2.2 and SnRK2.6 (OST1) were also ubiquitinated and degraded; however, the E3 ligases responsible for the degradation of these two SnRK2s were not known.¹⁸ In this regard, the identification of HOS15 as a promoter of OST1 degradation suggests that it could be one of the possible E3 ligases mentioned previously.^18,19 Loss-of-function hos15-2 mutant plants are hypersensitive to exogenous ABA during seed germination and stomatal movement, while being extremely tolerant to drought stress, indicating the importance of HOS15 as a negative regulator (Figure 1).¹⁹ Moreover, ABA responsive genes as well as genes responsible for drought tolerance exhibit hyper induction in hos15-2 plants under stress, compared to the wild type (Figure 2), suggesting that HOS15 negatively regulates the ABA signaling pathway.

Figure 1.

Loss-of-function HOS15-mutant plants are ABA sensitive and drought tolerant. (a) hos15-2 plants are sensitive to exogenous ABA during germination. Seeds of Col-0, hos15-2, and two complementation lines, Comp #1 and Comp #2, were germinated on 1/2 MS medium in the presence of the indicated concentrations of ABA (μM) in a long-day chamber at 22ºC. Photographs were taken 7 days after germination. (b) Green cotyledons were counted on day 4. Error bars indicate SE. (c) HOS15 loss-of-function mutant plants tolerate dehydration stress. Seeds of Col-0, hos15-2, and two complementation lines, Comp #1 and Comp #2, were germinated on 1/2 MS medium for 1 week and then transplanted into soil. Drought tolerance assay of 3-week-old Col-0, hos15-2, Comp #1, and Comp #2 plants was performed by withholding water for 14 days followed by re-watering. Photographs were taken 2 days after re-watering. Each test was replicated three times.

Figure 2.

ABA- and dehydration-stress responsive genes are highly induced in hos15-2 under stress. (a) Expression of ABA-related genes in Col-0 and hos15-2. Seeds of wild type (Col-0) and hos15-2 were cultured on 1/2 MS medium for 7 days and then treated with 100 μM ABA for the indicated time intervals. Total RNA was extracted from these seedlings, and RT-qPCR analysis was performed. UBQ5 was used as the internal control. Error bars indicate SD. (b) Expression of dehydration-related genes in Col-0 and hos15-2. Seeds of Col-0 and hos15-2 were cultured on 1/2 MS medium for 2 weeks and then dehydrated at room temperature for 1 hour. Total RNA was extracted from these seedlings, and RT-qPCR analysis was performed. UBQ5 was used as the internal control. Error bars indicate SD.

HOS15 functions as an adaptor protein in the CUL4-based E3 ligase machinery

In recent years, WD40-repeat proteins have received considerable attention owing to their key roles in regulating several physiological processes, including plant growth and development, flowering time, salinity, ABA, and drought stress responses.^2,20-23 HOS15 is one of the 85 WD40-repeat containing proteins previously described in Arabidopsis that function as substrate receptors for the DDB1-CUL4 E3 ligase complex.²⁴ HOS15 interacts with and degrades histone deacetylase (HD2C) in a proteasome-dependent manner, subsequently protecting the plant against cold stress.²⁵ More recently, HOS15 was found to be involved in ABA signaling through its association with and negative effect on OST1 protein stability.¹⁹ In addition to its role as a component of the E3 ligase machinery, HOS15 functions as a transcriptional co-repressor.^25-27 Since HOS15 is a multifunctional protein,²⁸ we only focused on its involvement in the regulation of ABA signaling in this review.

HOS15 plays a critical role in the fine tuning of ABA signaling

HOS15 is a WD40-repeat protein; however, unlike other WD40-repeat proteins (DWA1, DWA2, and ABD1), it does not interact with ABA-responsive TFs.^2,19,20 By contrast, HOS15 has been shown to interact with SnRK2s (SnRK2.3/OST1) and ABI1/2.¹⁹ Interestingly, the application of exogenous ABA was found to completely inhibit the interaction between HOS15 and OST1, whereas that between HOS15 and ABI1/2 remained unaltered.¹⁹ These findings demonstrate that HOS15 may have a crucial role in the ABA signaling pathway, depending on the presence or absence of ABA. OST1 protein is degraded very rapidly in a proteasome-dependent manner upon treatment with cycloheximide (CHX).^18,19 However, in contrast to the wild type, OST1 is highly stable and exhibits greater accumulation in hos15-2 plants, indicating that the OST1 protein is tightly regulated by HOS15.¹⁹ These observations demonstrate that HOS15 fine-tunes the ABA signaling pathway by controlling the OST1 protein abundance. Beside ABA signaling, HOS15 together with PWR and HDA9, functions as a chromatin-remodeling complex, regulating the transcriptional abundance of a wide range of genes, including those involved in leaf morphology, pathogenesis, flowering time, and freezing stress.^23,25-27

HOS15 as a missing component in ABA signaling

Among the SnRK2s, SnRK2.6 (OST1) is specifically expressed in guard cells and regulates ABA-mediated stomatal movement.²⁹ Fujita et al. (2009) found that the three SnRK2s, SnRK2.2, SnRK2.3 and SnRK2.6 (OST1) function redundantly and only their double or triple mutants are hypersensitive to drought stress, indicating that all of these three SnRK2s are important for the drought stress responses.¹² Interestingly, HOS15 only interacts with two of them (OST1 and SnRK2.3) and affects their protein stability upon transient co-expression in tobacco and Arabidopsis.¹⁹ Furthermore, OST1 was found to be excessively accumulated in hos15-2 plants, suggesting that the ABA hypersensitivity and drought-stress tolerance of hos15-2 plants are largely due to stabilized OST1 (Figure 1). In accordance with this observation, drought-resistant phenotypes of hos15-2 were dramatically suppressed by ost1-3 mutation, indicating that OST1 is downstream of HOS15 and that drought-tolerant phenotypes of hos15-2 are OST1 dependent.¹⁹ These pieces of evidence further suggest that like the activation of ABA signaling, the deactivation of ABA signaling post stress is also very critical and needs to be properly regulated. The activation of SnRK2s in ABA signaling has been well studied; however, the manner in which SnRK2s are regulated or turned-over in post-ABA conditions has not yet been properly elucidated.^{10,12,16,17,29,30} In this regard, the identification of HOS15 as a potential regulator of SnRK2 protein abundance has resolved this long-standing mystery.¹⁹ Indeed, HOS15 functions as one of the major switches for the activation and de-activation of ABA signaling (Figure 3).

Figure 3.

Hypothetical model of the study. Under normal conditions, ABI1/2 and HOS15 interact with OST1. ABI1/2 inhibits OST1 activity by dephosphorylation, and HOS15 degrades OST1 to maintain it in a resting state. In response to ABA, PYR1 binds to ABA, thus interacting with and inhibiting ABI1 and releasing OST1 sequestered to ABI1/2. The interaction between HOS15 and OST1 is also inhibited by ABA, which leads to the activation of OST1. OST1 is first autophosphorylated and then transphosphorylates the target TFs. After the removal of ABA from the system (4 hours later, as shown by Ali et al., 2019), ABI1/2 again interacts (reverse reaction) with and dephosphorylates OST1, recruiting HOS15 to OST1 for degradation. HOS15 degrades OST1, leading to the fine-tuning of ABA signaling desensitization.

Conclusion

Owing to its major role, HOS15 was found to be one of the most important missing components in the ABA signaling network. As described earlier, HOS15 plays a central role in the ABA signaling network; hence, it was of interest to determine its exact position in the current model of the ABA signaling pathway. Under normal conditions, PP2Cs interact with and inhibit OST1 activity through its dephosphorylation.³¹ In the presence of ABA, ABA receptors inhibits PP2Cs, thus releasing OST1, which first autophosphorylates and then transphosphorylates the target TFs.¹¹ ABA also impairs the interaction between HOS15 and OST1,¹⁹ suggesting that, in the presence of ABA, all inhibitory components stay away from OST1 and the SnRK2s that phosphorylate and activate the ABA-responsive components. In contrast, ABA has no clear effect on the interaction of HOS15 with ABI1 and ABI2.¹⁹ This shows that once the ABA pathway is activated, OST1 is released from the HOS15-ABI1/2 complex and is free to interact with its targets. Toward the end, when the ABA pathway is about to turn-over, ABI1/ABI2 promotes HOS15 and OST1 interaction, resulting in a much stronger complex, and finally HOS15 degrades OST1.¹⁹ In summary, HOS15 plays a crucial role in regulating ABA signaling through the degradation of OST1, thus maintaining a balance between the active and inactive states. Future work should focus on determining the manner in which 1) HOS15 is signaled to interact with OST1 and 2) ABI1/ABI2 stabilizes the HOS15-OST1 complex.

Funding Statement

This work was supported by the Next Generation Bio-Green21 Program, Rural Development Administration (RDA), Republic of Korea (SSAC, PJ01318201), the National Research Foundation of Korea (NRF), funded by the Korean Government (2019R1A2C2084096), and Global Research Lab (2017K1A1A2013146).

Author contributions

D-J.Y. designed the work, A.A. performed the experiments, and D-J.Y. and A.A. analyzed the data, reviewed the literature, and drafted the manuscript.

Disclosure of Potential Conflicts of Interest

The authors report no conflict of interest.