The work, led by Deeksha Singh, Premachandran Yadukrishnan, Puthan Valappil Rahul, and Sourav Datta, focuses on three proteins with long histories in plant biology. ABI5, the ABSCISIC ACID INSENSITIVE 5 transcription factor, is the canonical executor of ABA’s decision to halt seedling growth. HY5, ELONGATED HYPOCOTYL 5, is a light-activated transcription factor that promotes photomorphogenesis, the program by which seedlings open green cotyledons and begin photosynthesis. COP1, CONSTITUTIVELY PHOTOMORPHOGENIC1, is an E3 ubiquitin ligase best known as HY5’s executioner: it tags HY5 for destruction in the dark, and light inactivates it, allowing HY5 to accumulate. What the IISER team shows is that these three proteins form a regulatory module whose behavior during early seedling development is far more intricate than the textbook picture suggests.

The researchers began by tracking ABI5 and HY5 protein levels across the entire arc of early development in Arabidopsis thaliana, from dry siliques and dry seeds through imbibition, stratification, germination, and seedling establishment. The two proteins traced mirror-image trajectories. ABI5 was abundant in mature and dry seeds, stayed high through imbibition and stratification, and then declined as germination proceeded. HY5 did the opposite: it was barely detectable in dry and imbibed seeds and rose steadily during germination and post-germination growth. This inverse pattern makes intuitive sense. High ABA in the seed keeps ABI5 in command and the embryo dormant; as ABA levels fall and light takes over, HY5 rises and the seedling commits to growth.

But the story deepened when the team applied exogenous ABA to germinated seeds. Within three hours, HY5 protein levels dropped measurably at both germinated-seed and post-germination stages, a result confirmed independently with a fluorescent HY5-YFP reporter line. Meanwhile, ABA treatment caused ABI5, which normally fades after germination, to climb back up over a four-day window. The two master regulators were being pushed in opposite directions by the very hormone that governs the checkpoint, raising the question of whether they were also pushing on each other.

To answer that, the researchers exploited loss-of-function mutants. In hy5-215 plants lacking functional HY5, ABI5 protein accumulated more strongly than in wild-type seedlings from day two onward under ABA treatment, even though HY5 is known to bind the ABI5 promoter and activate its transcription. Conversely, in abi5-8 mutants lacking ABI5, HY5 protein built up to higher levels than in wild type across all stages examined. The interpretation is striking: each transcription factor restrains the other’s protein abundance, even as HY5 positively regulates ABI5 at the level of DNA. This arrangement, in which two factors each suppress the other’s accumulation, constitutes a double-negative feedback loop, a circuit motif that can sharpen transitions and create a bistable switch between the quiescent and growth states.

Chromatin immunoprecipitation followed by quantitative PCR added a layer of nuance. When ABA was present, HY5 enrichment at the ABI5 promoter fell, consistent with the protein’s degradation. In four-day-old seedlings, ABI5 bound its target promoters more strongly in the hy5 mutant background, while HY5 bound the RBCS and HY5 promoters more strongly in the abi5 mutant background. In other words, neither factor needs the other to occupy DNA; their mutual antagonism operates at the protein level rather than through competition for binding sites. The authors suggest that the previously documented physical interaction between HY5 and ABI5 proteins may underlie this mutual suppression, though the precise mechanism remains to be worked out.

Genetic epistasis experiments pinned down the hierarchy. The team generated a hy5-215 abi5-8 double mutant and scored seedling establishment, defined by open, expanded green cotyledons, with and without 1 micromolar ABA. Without the hormone, all genotypes established fully by day three. With ABA, hy5-215 was hypersensitive, meaning it arrested more readily, while abi5-8 was hyposensitive, resisting arrest. The double mutant behaved like abi5-8, not like hy5-215. That result places ABI5 genetically downstream of HY5: whatever HY5 does to influence ABA response, it must act through ABI5. It also reconciles an apparent paradox in the literature, since earlier studies had variously described HY5 as a positive regulator of ABA signaling and as a suppressor of ABA-mediated arrest. The resolution is that HY5’s transcriptional activation of ABI5 and its protein-level suppression of ABI5 are two faces of the same module, with the balance between them determining the outcome.

The degradation machinery emerged as the pivot of the whole system. When germinated seeds were treated with MG132, an inhibitor of the 26S proteasome, the ABA-induced decline in HY5 was abolished, while cycloheximide, a translation inhibitor, had no effect. ABA therefore does not stop HY5 from being made; it accelerates its destruction. That destruction depends on COP1, because in cop1-4 mutants ABA failed to reduce HY5 levels. Curiously, total COP1 protein abundance itself did not change with ABA treatment in a YFP-COP1 reporter line. Instead, the hormone changed where COP1 resides. Confocal microscopy of germinated seeds expressing YFP-COP1 showed that a three-hour ABA treatment enhanced the nuclear localization of the ligase. Since COP1 only ubiquitinates HY5 in the nucleus, this relocalization is the mechanistic link between the ABA signal and HY5 destruction.

This finding adds a twist to a complicated picture of COP1 regulation. Darkness is classically known to drive COP1 into the nucleus, degrading light-responsive factors, while light expels it to the cytoplasm. Recent studies had shown that ABA can promote COP1’s cytoplasmic translocation during seed germination in some contexts and enhance its nuclear localization in dark-grown seedlings. The new data show that under normal light-dark cycling, ABA pushes COP1 into the nucleus of germinated seeds, tipping the module toward ABI5 accumulation and growth arrest. The authors also note that endoplasmic reticulum stress and high temperature have been reported to reduce HY5 by enhancing COP1 nuclear localization, suggesting that the COP1-HY5 pair functions as a general stress-responsive rheostat whose output depends on the specific environmental input.

The model that emerges is elegant in its economy. ABA recruits COP1 to the nucleus, COP1 degrades HY5, and with HY5 out of the way, ABI5 protein accumulates unchecked, imposing the developmental arrest that conserves the seedling’s resources until conditions improve. When ABA falls, COP1 retreats from the nucleus, HY5 rebounds, and HY5’s suppression of ABI5 protein helps extinguish the arrest program, allowing growth to resume. The double-negative feedback loop between HY5 and ABI5 likely sharpens this transition, ensuring that the checkpoint flips decisively rather than drifting. For agriculture, the implications are tantalizing: seedling establishment is a vulnerable window for crops facing drought, salinity, and temperature extremes, and understanding the COP1-HY5-ABI5 module at this level of molecular detail may eventually inform strategies for tuning how aggressively seedlings pause or proceed when the environment turns against them. The authors caution that key questions remain, particularly how ABA relocalizes COP1 and whether disrupting the physical HY5-ABI5 interaction alters ABI5 stability, but the framework they have built turns a once-linear signaling pathway into a dynamic switch with a built-in memory of stress.

Subject of Research: Regulation of ABA-mediated post-germination developmental arrest in Arabidopsis by the COP1-HY5-ABI5 module

Article Title: A COP1-HY5-ABI5 module regulates ABA-mediated post-germination developmental arrest

Article References: Singh, D., Yadukrishnan, P., Rahul, P. V., & Datta, S. (2025). A COP1-HY5-ABI5 module regulates ABA-mediated post-germination developmental arrest. Frontiers in Plant Physiology, 3, Article 1675932. https://doi.org/10.3389/fphgy.2025.1675932

Image Credits: AI Generated

DOI: 10.3389/fphgy.2025.1675932

Keywords: abscisic acid, ABI5, HY5, COP1, post-germination arrest, seedling development, Arabidopsis thaliana, ubiquitin ligase, proteasome, light signaling, plant hormone, stress response