MicroRNAs are short RNA molecules, typically around 21 nucleotides long, that do not encode proteins. Instead, they act as guides, binding to complementary sequences in messenger RNAs and triggering their degradation or blocking their translation. In plants, the miR156 family is one of the most ancient and influential of these regulators, targeting a group of transcription factors known as SPL proteins, which control everything from flowering time to leaf shape. Because SPL transcription factors sit at the top of gene regulatory hierarchies, the miR156-SPL module functions as a powerful hub: by tuning how much SPL protein a cell produces, miR156 indirectly influences hundreds of downstream genes. Previous work in annual crops and model plants such as Arabidopsis, alfalfa, apple, and rice had implicated this module in drought and salt responses, but its role in long-lived woody species remained largely uncharted territory.

The Harbin team, led by corresponding authors Zhang Huihui and Liu Xuemei, set out to map this circuit in birch, an economically and ecologically important tree of northern forests. Using transgenic birch lines engineered to overexpress either bp-miR156c or BpSPL2, the researchers subjected the plants to drought stress and compared their performance against wild-type controls. The results were striking and, at first glance, counterintuitive. Plants with extra bp-miR156c fared worse under drought, showing reduced tolerance, while plants overexpressing BpSPL2, the very gene that bp-miR156c silences, became notably more drought resistant. This inverse relationship confirmed that BpSPL2 is a positive regulator of drought tolerance, and that bp-miR156c, by cleaving the BpSPL2 transcript, dampens the tree’s drought defenses rather than boosting them.

GUS staining experiments provided direct evidence of the regulatory relationship, showing that BpSPL2 is indeed a target gene of bp-miR156c and is subject to its cleavage. With that hierarchy established, the researchers dug into what BpSPL2 actually does when water becomes scarce. Under drought conditions, the BpSPL2-overexpressing lines displayed significantly alleviated photodamage in both photosystem II and photosystem I, the two light-harvesting complexes at the heart of photosynthesis. Drought typically forces plants to close their stomata to conserve water, which starves the photosynthetic apparatus of carbon dioxide and causes absorbed light energy to spill into dangerous side reactions. The BpSPL2 lines also showed reduced oxidative damage, suggesting that the transcription factor helps the tree keep its cellular chemistry in balance even as its water supply dwindles.

To understand the molecular basis of these protective effects, the team performed RNA sequencing on the transgenic and wild-type plants under drought stress. The analysis revealed that differentially expressed genes in the BpSPL2-overexpressing plants were significantly enriched in several key pathways: photosynthesis-related processes, tryptophan metabolism, redox processes, and glutathione metabolism. This pattern pointed toward two parallel mechanisms. The enrichment of redox and glutathione-related genes suggested that BpSPL2 bolsters the tree’s antioxidant arsenal, while the activation of tryptophan metabolism hinted at a connection to auxin, the plant hormone that drives root development and is synthesized from tryptophan.

The researchers then used a combination of yeast one-hybrid assays, chromatin immunoprecipitation followed by PCR, and dual-luciferase reporter assays to identify the direct targets of BpSPL2. These experiments demonstrated that the BpSPL2 transcription factor recognizes a specific DNA sequence motif, known as the GTAC motif, and binds to the promoters of two critical genes. The first is BpGSTF3, which encodes a glutathione-S-transferase, an enzyme family famous for its role in detoxifying reactive oxygen species and xenobiotic compounds. The second is BpASA1, which encodes anthranilate synthase, the rate-limiting enzyme in tryptophan biosynthesis and therefore a gatekeeper of auxin production. By binding to both promoters, BpSPL2 enhances the transcription of these genes, setting in motion the two-pronged drought defense.

The consequences of this dual activation are elegant. On one hand, upregulating BpGSTF3 increases glutathione-S-transferase and antioxidant enzyme activities, allowing the leaves to mop up the reactive oxygen species that accumulate during drought-induced photosynthetic stress. This mitigates the photodamage to photosystems II and I and reduces oxidative injury to cellular membranes and proteins. On the other hand, activating BpASA1 ramps up tryptophan synthesis, which feeds into the production of indole-3-acetic acid, the principal naturally occurring auxin. Elevated auxin stimulates the formation of lateral roots, the branching side roots that dramatically expand a plant’s absorptive surface area. Indeed, overexpression of BpSPL2 significantly promoted root system development in birch, with particularly pronounced effects on lateral root growth, giving the trees a better architectural foundation for scavenging moisture from drying soil.

The study’s findings fit into a broader and rapidly evolving picture of the miR156-SPL module as a versatile stress-regulation toolkit in plants. Research in alfalfa has shown that miR156 improves drought tolerance by silencing SPL13, while work in apple has linked the module to both salt stress tolerance and flavonoid synthesis. In rice, variations in the SPL gene OsSPL10 confer drought tolerance by regulating reactive oxygen species production, and recent studies in poplar have implicated a miR156g-SPL module in drought responses. The birch work adds an important dimension because trees face drought over years and decades rather than a single growing season, and their survival depends on integrating root architecture, photosynthetic resilience, and antioxidant capacity in a way that annual crops do not. Demonstrating that a single transcription factor can simultaneously tune all three of these systems in a woody species is a significant conceptual advance.

There are also practical implications. As climate change intensifies drought frequency and severity across the boreal and temperate forests where birch thrives, understanding the genetic levers of drought tolerance becomes essential for both conservation and forestry. The bp-miR156c-BpSPL2 module identified in this study provides a theoretical foundation for breeding or engineering birch trees with enhanced drought resistance, whether through marker-assisted selection for favorable alleles or through biotechnological approaches that modulate SPL activity. Because the module acts upstream of both antioxidant defense and root development, manipulating it could deliver coordinated improvements that would be difficult to achieve by targeting either trait alone. The work was supported by the National Key Research and Development Program of China, the National Natural Science Foundation of China, and related institutional funds, reflecting the strategic importance of forest resilience research in the region.

For now, the Harbin team’s contribution is a detailed molecular map: a microRNA that silences, a transcription factor that activates, two target genes that execute, and a physiological outcome that keeps a tree alive when the rain stops. It is a reminder that in the quiet chemistry of a leaf and the hidden architecture of a root system, plants have evolved regulatory circuits of remarkable sophistication. Decoding them, one module at a time, may prove essential as forests around the world confront a hotter, drier future.

Subject of Research: The bp-miR156c-BpSPL2 regulatory module controlling drought tolerance in birch through reactive oxygen species scavenging and lateral root development

Article Title: The bp-miR156c–BpSPL2 module positively regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla

Article References: Peng, H., Hongrui, Z., Jiaqian, A., Zhongjia, Y., Huilei, D., Huihui, Z., & Xuemei, L. (2026). The bp-miR156c–BpSPL2 module positively regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla. Plant Cell Reports, 45(10), Article 282. https://doi.org/10.1007/s00299-026-03924-4

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03924-4

Keywords: drought tolerance, birch, Betula platyphylla, miR156, SPL transcription factor, BpSPL2, reactive oxygen species, lateral roots, auxin, glutathione-S-transferase, tryptophan metabolism, plant molecular biology