GIGANTEA-ENHANCED EM LEVEL complex initiates drought escape response via dual function of ABA synthesis and flowering promotion

ABSTRACT

Plants use the regulation of their circadian clock to adapt to daily environmental challenges, particularly water scarcity. During drought, plants accelerate flowering through a process called drought escape (DE) response, which is promoted by the circadian clock component GIGANTEA (GI). GI up-regulates the flowering inducer gene FLOWERING LOCUS T (FT). Phytohormone Abscisic acid (ABA) is also required for drought escape, and both GIGANTEA and Abscisic acid are interdependent in the transition. Recent research has revealed a new mechanism by which GIGANTEA and the protein ENHANCED EM LEVEL form a heterodimer complex that turns on ABA biosynthesis during drought stress by regulating the transcription of 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3). This highlights the close connection between the circadian clock and ABA regulation and reveals a new adaptive strategy for plants to cope with drought and initiates the DE response.

Introduction

Physiological and developmental plasticity in plants occurs at every level of complexity to cope with environmental stressors.¹ Understanding the molecular, cellular and behavioral plant response mechanisms for adaptation to environmental challenges is crucial for solving world food insecurity problems and increasing global crop yield, ^[1,2] Circadian biology plays a crucial role in stress signaling in plants by coordinating the expression of genes involved in the response to stress conditions.³ The central component of the plant circadian clock is a set of genes known as clock genes, which form three interconnected loops known as the morning, central, and evening loops. The morning loop consists of the genes LHY (LATE ELONGATED HYPOCOTYL) and CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1), expressed in the morning, promoting the expression of TOC1 (TIMING OF CAB EXPRESSION 1), a central component of the circadian rhythm. The central loop, composed of TOC1, PRR (PSEUDORESPONSE REGULATOR) genes, and LUX (LIGHT-REGULATED) genes, controls the expression of the evening loop genes and its own expression, forming a feedback loop to maintain the circadian rhythm. The evening loop, consisting of GI (GIGANTEA), ELF3 (EARLY FLOWERING 3), and ELF4 (EARLY FLOWERING 4), expressed in the evening, regulates LHY and CCA1 expression, which in turn regulate TOC1 expression. These interlinked circadian loops work together to generate a rhythmic pattern of gene expression, driving the circadian rhythm.^4–7 Approximately 30% of the plant transcriptome, including transcripts involved in hormone biosynthesis pathways,^8–10 shows diurnal expression and is regulated by circadian oscillation.³ In particular, abscisic acid (ABA), a phytohormone with a diurnal biosynthesis pattern and accumulation, regulates various physiological processes including seed dormancy, seed germination, post-germination seedling growth, abscission acceleration, and stomatal movement.¹¹ Although various studies explain the link between ABA (biosynthesis, accumulation, and signaling) and circadian rhythm, the underlying mechanisms are still unclear.^8,12 Adams et al. (2018) suggested that LHY, a core circadian clock transcription factor, plays a complex role in regulating the expression of a rate-limiting enzyme in the ABA biosynthesis pathway.⁸ In addition to LHY, other clock genes like TOC1, PRRs and GI also take part in the rhythmic accumulation of ABA through the indirect regulation of the key enzymes in ABA biosynthesis.^8,13,14 The initial steps of ABA synthesis take place in plastids, where the carotenoids are converted to xanthoxin in a series of reactions by 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting enzyme family in ABA biosynthesis.¹¹ Xanthoxin is relocated to the cytoplasm, where it is converted to active ABA by two catalytic reactions.¹¹ The NCED3 transcript is the most expressed among the NCED enzymes in plant stems and roots.¹⁵ NCED3 exhibits a diurnal transcription oscillation matching the diurnal pattern of ABA that peaks during daytime and declines at night.¹³ We have recently shown that ENHANCED EM LEVEL (EEL), a bZIP transcription factor, and GI binds to the ABRE motif of the NCED3 promoter and promotes its transcription.¹³ These findings could explain the regulation of ABA biosynthesis by the circadian clock components that transcriptionally regulate the key enzymes of this process¹³ (Figure 1).

Figure 1.

Schematic diagram showing GI-EEL regulation of ABA biosynthesis pathway.

In the nucleus, GI forms a complex with EEL on NCED3 promoter bearing ABRE motif; this positively regulates NCED3 expression. In plastids, NCED3 enzyme is responsible for converting neoxanthin to xanthoxin, which relocates to the cytoplasm and undergoes catalytic reactions to form ABA.

GI, a multifunctional protein, regulates plant development and stress response

Over the last two decades, GI has received significant attention from researchers due to its multifunctional characteristics. GI regulates several key physiological processes including plant growth and development and plant responses to environmental stresses, such as salt, drought, cold and oxidative stress.^13,15–17 Unlike other circadian clock proteins, GI has no DNA binding domain, but it modulates the plant transcriptome through interaction with other transcription factors.^18–21 GI interactions with other proteins are versatile. On the one hand, GI binds to ZEITLUPE (ZTL) and acts as a co-chaperone protein that facilitates ZTL maturation and stability;¹⁸ on the other hand, it interacts with FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) to promote its ubiquitination function.²¹ GI plays a crucial role in regulating the temporal expression of CONSTANT (CO) protein in the nucleus under long-day conditions. The expression of CO is characterized by bimodal peaks in the early morning and late afternoon. ZTL plays a role in mediating the degradation of CO in the morning through direct binding to it. However, the expression of FKF1 and GI in the afternoon disrupts this process. The proteins form an active complex, with GI preferentially interacting with ZTL and inactivating its function. This leads to sequestration of CO from ZTL. Meanwhile, FKF1 stabilizes CO through forming a protein complex with it. ZTL also interacts with FKF1, inhibiting the FKF1-mediated CO stabilization, leading to destabilization of CO. GI’s preferential binding to ZTL also interferes with the complex formation between FKF1 and ZTL. These complex and sophisticated regulatory mechanisms allow for the highly accumulated expression of CO in the late afternoon of long days to control FLOWERING LOCUS T (FT) transcription.^21–23 Although GI has contradictory molecular functions, it has a consistent physiological function in regulating the photoperiodic flowering pathway through modulating the florigen genes expression.^24,25 Baek and colleagues (2020) explained the mechanistic regulation of ABA biosynthesis and accumulation by GI that seems inconsistent with the occurrence of flowering inhibition by exogenous ABA treatment,^13,26 as the application of exogenous ABA to Arabidopsis thaliana plants significantly delays their floral transition.²⁶ However, an increased level of endogenous ABA was observed during flowering in short-term drought stress in Arabidopsis.²⁷ This phenomenon is known as the drought escape (DE) response.²⁸ Recent reports have indicated the importance of GI in DE response via regulating ABA biosynthesis, suggesting that GI is involved in flowering promotion by both ABA synthesis and florigen genes regulation in response to drought stress.^13,27 In contradiction, recent research article suggests that the highest accumulation of GI at noon plays a crucial role in establishing a phase of decreased ABA concentration and is associated with a negative regulation of ABA transcriptional responses and sensitivity.²⁹ Thus, here we show unlike the wild-type plants, the loss-of-function mutant of GI (gi-1) exhibits an ABA insensitive phenotype (Figure 2); and this is additional evidence for the less endogenous ABA concentration in gi-1 measured previously in Beak et al. and explains the close link between GI and ABA. In summary, upon binding to FKF1 and ZTL, GI regulates flowering, while its association with EEL promotes ABA biosynthesis and DE responses^13,21 (Figure 1).

Figure 2.

Eel, gi-1, and eel/gi-1 double mutants show insensitivity toward ABA.

(A) Seeds of WT (Col-0), eel (SALK_021965), gi-1,²³ and eel/gi-1 double mutants were germinated on 1/2 MS medium (1.5% [w/v] Suc, 0.6% [w/v] agar, pH 5.7) at 23°C with or without 0.5 μM ABA in a long-day chamber at 22°C. Photographs were taken 5 days after germination. (B) Green cotyledon rate was scored for different concentrations of ABA at 5 days. Error bars represent means ± SD of 36 seeds from three independent experiments.

EEL, a bZIP type transcription factor, regulates ABA biosynthesis

EEL is an ABI5/AtDPBF family member of the bZIP transcription factors in Arabidopsis and is involved in ABA signaling.^30,31 The ABI5/AtDPBF proteins including EEL display significant localization in the embryo during the maturation phase.^30,32 Accordingly, this could be additional evidence for the importance of EEL as a transcriptional regulator for the ABA-dependent stress signals during embryogenesis and seed maturation. Moreover, the transcriptional function of EEL pivots on forming either a homodimer or a heterodimer complex with other proteins.^13,30 GI-EEL, a recently discovered heterodimer, positively regulates ABA synthesis in drought stress.¹³ Previous research attempted to address the fundamental question of how ABA signals are integrated into the photoperiodic flowering network. It provided evidence for ABA’s control of FT gene expression under normal and drought stress conditions by impacting photoperiodic signaling through GI. It also highlighted ABA’s negative effect on the floral transition of Arabidopsis that is independent of the photoperiodic pathway.³³ However, our study reveals a different type of ABA regulation by the photoperiodic clock genes GI and EEL. As it is commonly observed that mutants with lower endogenous ABA content display an ABA-insensitive phenotype,^34–38 it is interesting to note that the loss-of-function EEL mutant, along with the gi-1 mutant and the eel/gi-1 double mutant, also exhibit ABA-insensitive phenotypes (Figure 2). These phenotypes could be explained by the low level of endogenous ABA in these mutants due to the lack of ABA biosynthesis, along with the dual desensitization and degradation response toward exogenous ABA.³⁹ In addition, it was observed that the expression of both GI and EEL is significantly reduced by exogenous ABA (Figure 3), which can be related to a negative feedback regulation of the increased exogenous ABA treatment. The reduced significant difference in the transcript levels after 16 h treatment (Figure 3) could be related to the ABA desensitization and degradation processes. This accumulated evidence strongly supports the critical role of the GI-EEL complex in manipulating ABA response.

Figure 3.

Expression response of GI and EEL to ABA stress response.

Transcript levels of GI (A) and EEL (B) expressed in 10-day-old WT seedlings germinated in 1/2 MS medium sprayed with 50 μM ABA or Mock for indicated time points. TUBULIN2 was used as an internal control for normalization. Bars represent means ± SD of three biological replicates with three technical replicates each. RNA extraction, cDNA synthesis, qRT condition and primer used in this report as indicated in Baek et al. ¹³

Conclusion

In conclusion, despite conflicting research on ABA regulation of the floral transition under stress conditions,⁴⁰ the role of the GI-EEL complex in regulating the diurnal oscillation of ABA biosynthesis is clear. This complex enhances the transcription of NCED3 by binding to its promoter, thus regulating the DE response. GI, a well-known regulator of the circadian clock and photoperiodic flowering, and EEL play a crucial role in helping plants adapt to short-term water shortages by regulating endogenous ABA levels. This highlights the interplay between phytohormones, particularly ABA, and the circadian clock as a key adaptive strategy for plants facing environmental challenges.

Funding Statement

This paper was supported by the National Research Foundation of Korea (NRF) funded by the Korean Government (2022R1A2C3004098).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

D-J.Y designed the study, D.B performed the experiments, Z.E.B, M.B, J.P, A.A and D-J.Y performed the literature search and wrote the manuscript.