The discovery began with a lucky accident of mutagenesis. The researchers, led by Yushan Hou, Xuejiao Kong and Junjie Le of Northeast Agricultural University in Harbin, treated seeds of MK1, a naturally cold-tolerant japonica rice line, with a chemical mutagen that scatters random mutations across the genome. Among the resulting plants, they spotted one seedling that had lost MK1’s prized cold tolerance, wilting under chilling conditions that its parent shrugged off. They named this mutant cs1, for cold-sensitive. Genetic crosses revealed that the vulnerable phenotype was controlled by a single recessive nuclear gene, meaning one broken gene was enough to strip away the tolerance.

To find that gene, the team turned to MutMap, an elegant sequencing strategy that compares the genomes of mutant and parent to locate the causal mutation quickly. The mapping converged on a region of chromosome 9 containing OsERF5, which belongs to the large AP2/ERF family of transcription factors. These proteins bind specific DNA sequences and switch suites of target genes on or off, and in rice they have repeatedly surfaced as players in stress responses to drought, salt, heat and cold. Within OsERF5, the mutant carried a single nonsynonymous SNP in the AP2 domain, the part of the protein that grips DNA. That one-letter change in the genetic code swapped an alanine for a serine, altering the protein’s architecture precisely where it needed to function.

Expression patterns added weight to the suspicion that OsERF5 matters under stress. The gene is broadly active across the plant, but its activity surges in response to cold, salinity, osmotic stress and gibberellin, a plant hormone better known for driving stem elongation and germination. That gibberellin responsiveness turned out to be a crucial clue, hinting that OsERF5 sits at a junction between hormone signalling and the plant’s chemical armour against cold. The researchers then ran the decisive functional tests: knocking OsERF5 out made seedlings more sensitive to chilling, while overexpressing the gene or restoring a functional copy in the mutant enhanced tolerance. The results confirmed that OsERF5 is a genuine positive regulator of cold tolerance at the seedling stage, not a bystander.

How does a single transcription factor protect a seedling from cold? The answer lies in the chemistry of stress. When plants are chilled, their photosynthetic machinery and metabolism leak electrons, generating reactive oxygen species such as hydrogen peroxide. In modest amounts these molecules act as signals, but when they accumulate they oxidise membranes and proteins, causing cellular damage. Malondialdehyde, or MDA, is a classic fingerprint of that membrane damage. The team’s physiological analyses showed that plants with functional OsERF5 accumulated less hydrogen peroxide and less MDA under cold stress, indicating a strengthened antioxidant system that mops up reactive oxygen before it wreaks havoc. In the knockout lines, those protective responses were weakened, and the damage markers climbed.

The gibberellin connection gives this defence system an intriguing twist. Gibberellin is best known as a growth hormone, and breeders have long manipulated it, most famously in the semi-dwarf wheat and rice varieties of the Green Revolution. The new study links gibberellin signalling to reactive oxygen species homeostasis during cold stress, with OsERF5 associated with a GA-mediated antioxidant defence. In practical terms, the hormone does not merely tell the plant to grow; under chilling conditions it appears to help orchestrate the deployment of antioxidant capacity, and OsERF5 is a key intermediary in that command chain. This reframes gibberellin as part of the stress-response toolkit, not just a growth accelerator, and suggests that fine-tuning this pathway could yield seedlings that stay both vigorous and protected when temperatures plunge.

Beyond the lab bench, the study has an evolutionary story to tell. By scanning thousands of rice varieties, the researchers identified twelve haplotypes, distinct versions of the OsERF5 gene sequence carried by different accessions. One of them, Hap_I, stood out as the cold-tolerant version, and it is overwhelmingly predominant in temperate japonica rice, the subspecies grown in cooler regions of Japan, Korea, northern China and beyond. Population genetics analysis revealed that OsERF5 has undergone strong selection during rice domestication, meaning that ancient farmers and natural selection together favoured the cold-tolerant version as rice spread from its tropical origins into harsher climates. The gene thus carries the imprint of a millennia-long breeding programme conducted without anyone knowing the word genetics.

That history makes Hap_I immediately useful for modern breeders. Because the cold-tolerant haplotype already exists in cultivated germplasm, breeders can track it with DNA markers and introgress it into elite varieties that lack it, a strategy known as marker-assisted selection. This is far faster than waiting for plants to grow out under cold conditions, and it avoids dragging along unwanted traits from donor parents. For rice production in high-latitude regions such as northeastern China, where the research team is based, and in high-altitude zones where chilling nights are routine, a validated cold-tolerance haplotype is exactly the kind of resource that turns molecular knowledge into sturdier fields. The authors describe Hap_I as a valuable genetic resource for breeding cold-adapted rice, and the evidence supports that framing.

The study also slots into a rapidly growing catalogue of rice cold-tolerance genes. Earlier work identified COLD1, whose natural variation confers chilling tolerance, and more recent discoveries include COLD11, CTF1, CTB5 and CTB4a, each illuminating a different node of the cold-response network, from ion channels to flowering-stage resilience. OsERF5 adds a transcriptional regulator tied to hormone signalling and antioxidant defence, a layer that had been less well characterised at the seedling stage. Together these genes sketch a picture of cold tolerance as a distributed property, built from sensory machinery, hormonal cross-talk and enzymatic detoxification, any single component of which can be improved by breeding.

For a world where extreme weather increasingly disrupts crop production, the stakes are concrete. Cold damage at the seedling stage forces farmers to delay planting or resow, shrinking the growing season and yields in marginal regions. A gene like OsERF5, validated by knockout, overexpression and complementation, and backed by a clear haplotype signature and a plausible molecular mechanism, gives breeders a precise target rather than a statistical shadow. The work, funded by the National Natural Science Foundation of China and partner programmes, demonstrates how a chemical mutagenesis experiment in a single rice line can end up tracing the evolutionary history of a species and pointing the way to the next generation of cold-proof crops. As chilling temperatures continue to test the limits of the world’s rice paddies, the humble transcription factor on chromosome 9 may prove to be one of the quiet heroes of food security.

Subject of Research: A rice AP2/ERF transcription factor gene, OsERF5, that regulates seedling cold tolerance through gibberellin-mediated antioxidant defence

Article Title: OsERF5 enhances cold tolerance and is associated with GA‑mediated antioxidant defence in rice

Article References: Hou, Y., Kong, X., Le, J., Yang, R., Qi, N., Wang, J., Liu, H., Zheng, H., Xin, W., Zou, D., Lei, L., & Yang, L. (2026). OsERF5 enhances cold tolerance and is associated with GA‑mediated antioxidant defence in rice. Theoretical and Applied Genetics, 139(10), Article 273. https://doi.org/10.1007/s00122-026-05385-5

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05385-5

Keywords: rice, cold tolerance, OsERF5, transcription factor, gibberellin, reactive oxygen species, antioxidant defence, haplotype, japonica rice, plant breeding, abiotic stress, MutMap