MicroRNAs, or miRNAs, are single-stranded RNA molecules typically 21 to 24 nucleotides in length. Although they do not encode proteins, they perform a crucial regulatory function by binding to messenger RNA targets and either cleaving them or blocking their translation. In doing so, they fine-tune the expression of transcription factors, hormone signaling components and transporter genes that govern virtually every aspect of plant life, from root architecture and flowering time to drought tolerance and grain filling. A single miRNA can regulate multiple genes within the same pathway, which makes these molecules extraordinarily powerful levers for crop improvement. Environmental conditions can reshape miRNA expression profiles, and in turn the plant’s stress responses, offering a dynamic regulatory layer that breeders have only begun to exploit.

The review, led by Kasanaboina Krishna of the International Crops Research Institute for the Semi-Arid Tropics and colleagues, synthesizes evidence from across the major cereals and emerging millet studies to build a millet-focused regulatory framework. The authors distinguish between conserved grass-wide miRNA modules and millet-specific candidates, and they are candid about the state of the field: most millet miRNA research to date has been limited to computational prediction and expression profiling, while rigorous functional validation remains scarce. Degradome sequencing evidence, tissue-specific regulatory maps and field-relevant genotype-by-environment analyses are still largely missing, meaning that many of the most exciting candidates remain hypotheses rather than established tools.

Nevertheless, the evidence that does exist is compelling. In foxtail millet, miR394 has been shown to positively regulate drought resistance, with upregulation after treatment with methyl jasmonate, ethephon, salicylic acid and abscisic acid, and improved germination rates and root lengths in response. A member of the miR396 family, SimiR396d, targets the growth-regulating factor gene SiGRF1, and its overexpression enhances both root growth and drought tolerance, directly linking miRNA-mediated developmental regulation to stress adaptation. In pearl millet, one of the most drought-tolerant cereals known, researchers identified 61 novel miRNAs under high vapor pressure deficit, with families including miR167, miR172, miR396 and miR399 implicated in root physiology and abiotic stress responses. Sorghum studies have revealed 80 individual miRNAs responding to drought, heat and combined stress, including eight novel stress-responsive families.

Salinity tolerance offers another striking example. In finger millet, the Eco-miR169–EcNF-YA13 regulatory module has been identified as a key determinant of dehydration and salinity tolerance; the transcription factor EcNF-YA13 supports stress tolerance but is suppressed by Eco-miR169, suggesting that modulating this miRNA could relieve the repression and enhance tolerance. Earlier work in the same species identified 48 conserved and 35 novel salinity-responsive miRNAs, with several families upregulated more than tenfold in tolerant genotypes. In pearl millet, small RNA sequencing revealed 95 salinity-responsive miRNAs targeting 448 genes, many involved in auxin responses, hinting that miRNA-mediated hormone regulation underpins the crop’s remarkable salt resilience.

Beyond stress tolerance, miRNAs appear central to the nutritional traits that make millets so valuable as nutri-cereals. Finger millet is prized for its calcium content, pearl millet for iron and zinc, kodo millet for dietary fiber and phenolics, and fonio for sulfur-containing amino acids. Yet the direct links between specific miRNAs and grain micronutrient accumulation remain under-investigated. One notable exception comes from pearl millet, where pgl-miR159 was identified as a candidate associated with iron metabolism during a broader search for grain iron and zinc genes. In rice, high-iron transgenic lines showed downregulation of root-specific miRNAs that in turn upregulated key transporters such as OsYSL15, OsFRO2 and OsIRT1, boosting iron and zinc uptake. The review argues that identifying orthologous transporter–miRNA modules in millets could provide a mechanistic framework for biofortification, provided the interactions are experimentally validated.

The translational toolkit for moving from candidate miRNAs to improved cultivars is now substantial. The authors propose a roadmap combining tissue- and stage-resolved miRNA atlases with target validation through degradome sequencing, RNA Ligase-Mediated Rapid Amplification of cDNA Ends and reporter assays. Functional intervention platforms include short tandem target mimics, which sequester endogenous miRNAs to relieve repression of beneficial target genes; artificial miRNAs, which deliver highly specific gene knockdowns with minimal off-target effects; and CRISPR/Cas-mediated editing of miRNA loci, promoters or target recognition sites. Proof-of-concept studies in other cereals show the power of these approaches: editing the miR396 binding sites in rice OsGRF4 and OsGRF8 derepressed growth and boosted grain size, while CRISPR-induced mutations in the miR156 recognition element of wheat TaSPL13 improved grain number, size and architecture.

Integration with breeding pipelines is the critical next step. miRNA-derived molecular markers, first developed in Brassica and rice, capture regulatory variation rather than merely neutral structural differences, linking markers directly to traits such as stress tolerance and yield stability. In foxtail millet, researchers designed 66 primer pairs from conserved pre-miRNA sequences with high cross-genera transferability, underscoring their promise as functional genotyping tools. The review also highlights the potential of machine learning and digital miRNA twins, computational models trained on sequence features and expression data to predict stress-responsive miRNAs and simulate how edited or introgressed miRNA modules would perform across drought cycles, heatwaves and nutrient-poor soils before any field trial. Embedding miRNA target interactions into crop simulation platforms could dramatically shorten breeding cycles for climate-resilient varieties.

The regulatory and ecological dimensions are not ignored. India’s 2022 guidelines exempt site-directed nuclease 1 and 2 genome-edited plants from GMO-style environmental risk assessment once vector sequences are segregated, opening a practical pathway for non-transgenic miRNA edits. At the same time, the authors note that plant miRNAs can move within and between organisms, a consideration for environmental safety even in cisgenic edits. Benchmarks for nutritional outcomes already exist: Indian biofortified pearl millet targets of at least 42 milligrams of iron and 32 milligrams of zinc per kilogram of grain, achieved without sacrificing yield. Rapid screening systems built around the model grass Setaria viridis, including spike-dip transformation, protoplast assays and foxtail mosaic virus vectors for virus-induced gene silencing, could accelerate functional validation before the best constructs move into stable millet genotypes for multi-environment testing.

The broader message is one of urgency and opportunity. Millets already possess C4 photosynthesis, deep root systems, compact stature and strong antioxidant defenses that allow them to thrive where major cereals fail. Their inherent micronutrient richness makes them ideal testbeds for miRNA-guided climate-smart breeding. What is missing is the systematic functional validation that would transform descriptive miRNA catalogues into experimentally confirmed regulatory networks. If the roadmap laid out in this review is followed, building comprehensive miRNA atlases, mining landrace and wild-relative diversity for novel miRNA alleles, deploying CRISPR and target mimicry tools, and integrating validated miRNA-trait associations into genomic selection models, the humble millet could become a global model for genetic resilience, nutritional density and smart agriculture, delivering climate-proof, micronutrient-dense grain to the farmers and consumers who need it most.

Subject of Research: MicroRNA regulatory networks controlling climate resilience and nutritional traits in millet crops

Article Title: Harnessing microRNA regulatory networks for engineering climate resilience and nutritional enhancement in millets

Article References: Krishna, K., Habyarimana, E., Jamedar, H. R., VG, I. L., Chavan, S., Prasad, B. V. V., Mohan, Y. C., Edukondalu, B., & Ceasar, S. A. (2026). Harnessing microRNA regulatory networks for engineering climate resilience and nutritional enhancement in millets. Stress Biology, 6(1), Article 57. https://doi.org/10.1007/s44154-026-00332-2

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00332-2

Keywords: microRNAs, millets, climate resilience, drought tolerance, salinity stress, biofortification, CRISPR genome editing, foxtail millet, pearl millet, finger millet, nutritional quality, molecular breeding