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

The drama of germination unfolds around a tug-of-war between two plant hormones. Abscisic acid, or ABA, is the guardian of dormancy. It accumulates during seed maturation, keeps the quiescent embryo safe from germinating at the wrong moment, and maintains that arrested state even after the dry seed takes up water. Gibberellins, or GAs, are the antagonists. They promote the weakening of the tissues surrounding the embryo, boost the growth potential of the embryonic axis, and ultimately drive the radicle through the seed coat. The balance between these two hormones determines whether a seed sleeps or wakes, and environmental temperature is one of the most powerful levers on that balance. What the new study reveals is how a specific temperature signal rewires the internal wiring of the hormone network rather than merely tipping the amounts of the hormones themselves.

To dissect the mechanism, the researchers assembled a panel of Arabidopsis thaliana mutants, each defective at a different node of the ABA and GA pathways. Among them were cyp707a2, which cannot degrade ABA and therefore accumulates abnormally high levels of the hormone; sleepy1, which lacks the F-box protein that tags the repressor RGL2 for destruction and so blocks GA signaling downstream; ga3ox1, which cannot synthesize gibberellins in the cold; and rgl2, which lacks the master dormancy repressor altogether. They also built a cyp707a2 sleepy1 double mutant to combine the two defects. Freshly harvested wild-type seeds of Arabidopsis are strongly dormant, and all of these mutants either deepened or altered that dormancy in informative ways, giving the team a genetic toolkit for asking precisely which steps of the pathway cold treatment can and cannot override.

The answer to the first question came quickly: twenty-four hours of stratification at four degrees Celsius completely broke the dormancy of wild-type seeds, and, strikingly, it also rescued the dormant phenotypes of cyp707a2, sleepy1, and the cyp707a2 sleepy1 double mutant. In other words, even seeds flooded with ABA, or unable to destroy the RGL2 repressor, could be roused from dormancy by cold. But stratification failed to rescue ga3ox1, the mutant that cannot make gibberellins. That single asymmetry was the study’s linchpin. It meant that the dormancy-breaking power of cold depends on the seed’s ability to biosynthesize GA. Consistent with this, when the researchers blocked GA biosynthesis pharmacologically with paclobutrazol, stratification lost its effect entirely, and exogenous GA reversed the inhibitor completely. Cold, in essence, works by turning the GA tap back on.

But the deeper story lies in what GA then unlocks. Germination is an energy-intensive undertaking, and a dry seed’s most important fuel reserve is the starch packed into its endosperm. Mobilizing that starch requires alpha-amylase, a starch-digesting enzyme whose activity has long served as a proxy for germination vigor. ABA suppresses alpha-amylase; GA induces it. The team found that stratification dramatically boosted both the transcript abundance and the enzymatic activity of alpha-amylase in freshly harvested wild-type seeds and in mutants carrying elevated endogenous ABA or treated with exogenous ABA. Even in cyp707a2 seeds drowning in ABA, and even in sleepy1 seeds in which RGL2 degradation is blocked, the cold treatment raised AMY1, AMY2, and AMY3 expression and pushed alpha-amylase activity sharply upward, peaking at the radicle emergence stage.

How could cold override a repressor that cannot be degraded? Here the study delivers its most novel insight. Under warm imbibition, ABA stabilizes and amplifies the RGL2-ABI5 module: the DELLA protein RGL2, the dominant germination repressor among Arabidopsis’s five DELLA proteins, cooperates with the ABA-responsive transcription factor ABI5 to form a joint inhibitory complex that clamps down on alpha-amylase genes. The researchers measured a steep, dose-dependent interplay between the hormones: ABA enhanced RGL2 expression while GA suppressed it, and the antagonism extended directly to alpha-amylase activity and AMY gene transcription. Yet stratification substantially suppressed RGL2 expression in wild-type seeds, cutting it by about 66 percent, and it also suppressed ABI5 in wild-type and sleepy1 seeds. Even where ABA levels stayed high and RGL2 and ABI5 transcript levels remained elevated, as in the cyp707a2 double-mutant background, the cold treatment still drove alpha-amylase up.

That observation forced the authors to a conclusion with real conceptual weight: stratification does not simply neutralize the repressors, it uncouples them from their target. The RGL2/ABI5 module can remain transcriptionally active, and ABA can remain abundant, yet the alpha-amylase genes escape their grip. The team’s proposed model describes the cold signal as acting in two coordinated arms. First, low temperature induces GA biosynthetic genes, raising GA content, which relieves the RGL2-mediated repression of alpha-amylase expression. Second, and more importantly, stratification decouples the RGL2/ABI5 checkpoint from its control over alpha-amylase activity, allowing starch hydrolysis and the resulting energy supply to proceed even in the presence of high ABA. Environmental signals, in other words, do not just change hormone levels; they rewire the connectivity of the signaling network itself.

Just as telling is what stratification could not do. Even after cold treatment, high concentrations of exogenous ABA still blocked green cotyledon emergence, the visible milestone of seedling establishment. Stratification only weakly reversed ABA’s inhibition of cotyledon greening at low hormone concentrations and failed entirely at high ones. The authors interpret this as evidence of multiple checkpoints along the journey from dormancy to seedling. Germination proper depends on starch mobilization and radicle elongation, processes that stratification can restore by freeing alpha-amylase. Cotyledon greening, by contrast, involves plastid differentiation, chlorophyll synthesis, and the assembly of the photosynthetic apparatus, processes that likely rely on additional ABA-controlled programs that a short cold shock cannot reach. Dormancy release and seedling establishment, the study shows, are governed by separable molecular switches.

The findings also settle a lingering genetic question about RGL2 itself. Because sleepy1 mutants accumulate RGL2 protein yet can still germinate after stratification, the data indicate that RGL2 degradation is not strictly required for the cold effect; rather, cold acts upstream of GA biosynthesis and then bypasses the repressive influence of whatever RGL2 remains. The rgl2 mutant, lacking the repressor entirely, showed reduced ABA sensitivity and higher GA content, while sleepy1 showed the opposite, corroborating RGL2’s role as the central crosstalk node between the two hormones. The authors propose several candidate mechanisms for how the cell senses cold and conveys the message to the RGL2/ABI5 loci: cold-induced GA gene expression, temperature-driven changes in membrane fluidity and kinase cascades, and epigenetic modifications such as DNA methylation and histone changes that could remodel chromatin at the RGL2 and ABI5 promoters.

Beyond the elegance of the mechanism, the work carries practical promise. Stratification is already a mainstay of horticulture and nursery practice for species from Echinacea to apple and hazel, and seed priming strategies against salt and chilling stress in crops hinge on precisely the GA-ABA-amylase axis this study illuminates. Understanding that cold works by disarming a specific transcriptional checkpoint, rather than by globally draining ABA, suggests new ways to engineer germination vigor: modulating RGL2 or ABI5 function, or their connection to AMY genes, could allow breeders to produce seeds that germinate readily even under the elevated ABA conditions that accompany stress. For now, the study reframes an ancient agricultural practice in modern molecular terms. A night in the cold, it turns out, is not merely a nudge to the seed’s hormone balance; it is a targeted intervention that severs the repressor’s grip on the seed’s fuel supply and lets the embryo eat its way into life.

Subject of Research: How cold stratification overcomes ABA-mediated seed dormancy through RGL2/ABI5 and alpha-amylase regulation in Arabidopsis

Article Title: Stratification overcomes ABA-mediated seed dormancy by uncoupling RGL2/ABI5 inhibition from α-amylase expression

Article References: Tian, Y., Liu, Q.-L., Chen, M.-X., & Liu, Y.-G. (2026). Stratification overcomes ABA-mediated seed dormancy by uncoupling RGL2/ABI5 inhibition from α-amylase expression. Stress Biology, 6(1), Article 43. https://doi.org/10.1007/s44154-026-00312-6

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00312-6

Keywords: stratification, seed dormancy, abscisic acid, gibberellins, RGL2, ABI5, alpha-amylase, Arabidopsis thaliana, SLEEPY1, CYP707A2, starch hydrolysis, plant hormones