The research, led by Tingting Li and Zihan Zhang of Sichuan University under the direction of corresponding author Lin Tang, centers on a gene called LrMYB113, which encodes a member of the R2R3-MYB family of transcription factors. MYB proteins are among the largest families of DNA-binding regulators in plants, and they participate in virtually every aspect of plant development and metabolism, from trichome formation to drought responses. Within this sprawling family, however, certain subgroups have become specialists. The authors used phylogenetic analysis to place LrMYB113 within the S6 subgroup, a branch of the MYB tree whose members across diverse species have repeatedly been implicated in switching on the anthocyanin biosynthesis pathway. That classification alone would have been suggestive, but the team went far beyond sequence comparison, assembling a chain of functional evidence that establishes LrMYB113 as a bona fide master regulator rather than a bystander in the pigment production line.

The first line of evidence came from heterologous expression. When the researchers introduced LrMYB113 into tobacco, a standard testbed for plant gene function, the transgenic plants responded by accumulating visible pigment, a dramatic phenotypic readout indicating that the goji protein could hijack and activate the host’s anthocyanin machinery. Molecular assays confirmed that the anthocyanin pathway genes in these tobacco plants were upregulated, meaning the introduced transcription factor was not simply producing pigment indirectly but was actively recruiting the host’s biosynthetic enzymes into action. This kind of cross-species functionality is significant because it demonstrates that LrMYB113 can recognize the relevant target promoters even in an unrelated genome, pointing to conserved regulatory logic across the Solanaceae, the nightshade family to which both tobacco and Lycium belong.

Encouraged by the tobacco results, the team moved the experiment into the native species itself. Using a hairy root culture system, a technique based on infection with Agrobacterium rhizogenes that allows rapid genetic manipulation of root tissues, they overexpressed LrMYB113 in L. ruthenicum. The outcome was striking at the metabolite level: the engineered hairy roots accumulated elevated levels of four specific acylated anthocyanins, the chemically decorated pigment molecules that give black goji berries their distinctive profile and, importantly, their enhanced stability. Acylation, the attachment of organic acid groups to the anthocyanin backbone, is known to protect these fragile molecules from degradation and to shift their color properties, so the fact that LrMYB113 overexpression boosted precisely these high-value acylated forms underscores the pharmaceutical relevance of the finding. Alongside the metabolite changes, the canonical anthocyanin biosynthetic genes were activated, confirming that the transcriptional cascade had been turned up across the pathway.

With the function of LrMYB113 established, the researchers turned to the question of mechanism: how does this protein exert its control? Decades of work across petunia, maize, Arabidopsis and crop species have established that anthocyanin regulation typically requires a triumvirate of proteins known as the MBW complex, named for its three components: a MYB transcription factor, a bHLH transcription factor and a WD40 repeat protein. The MYB component provides DNA-binding specificity, the bHLH partner stabilizes the interaction and contributes activation potential, and the WD40 protein acts as a scaffold that assembles the whole apparatus. Using yeast two-hybrid assays and bimolecular fluorescence complementation, a technique that reconstitutes a fluorescent protein only when two candidate partners physically interact inside living plant cells, the team demonstrated that LrMYB113 does indeed partner with the bHLH proteins LrJAF13 and LrAN1b and with the WD40 protein LrAN11, assembling a canonical MBW complex in black goji berry.

The next step was to identify the actual targets of this regulatory complex. Promoter binding and transactivation assays revealed that LrMYB113 directly binds to and activates the promoters of two structural genes, LrDFR and LrANS. These are not arbitrary choices. DFR, dihydroflavonol 4-reductase, catalyzes a committed step midway through the anthocyanin pathway, converting dihydroflavonol substrates into leucoanthocyanidins, while ANS, anthocyanidin synthase, performs the subsequent oxidation that yields the colored anthocyanidin skeleton. In other words, LrMYB113 sits at the top of the hierarchy and pulls the levers on the enzymatic bottleneck that determines whether precursor molecules flow into pigment production or are diverted elsewhere in metabolism. Direct promoter binding, rather than indirect influence through intermediate regulators, places LrMYB113 in the category of first-order controllers of the pathway.

Perhaps the most mechanistically revealing result came from the dual-luciferase assays, a sensitive reporter system that quantifies how strongly a transcription factor activates a target promoter. These experiments showed something unexpected: LrMYB113 alone was sufficient to strongly activate the LrDFR and LrANS promoters, without requiring its bHLH or WD40 partners. Adding the partner proteins to form the complete MBW complex did enhance activity relative to the bHLH or WD40 components acting individually, but not relative to LrMYB113 on its own. This is a notable departure from the classical MBW model, in which the MYB protein typically depends heavily on its bHLH partner for full transcriptional output. In black goji berry, LrMYB113 appears to function as an unusually autonomous activator, with the MBW complex providing modulatory refinement rather than essential partnership. The finding suggests that the regulatory architecture of anthocyanin synthesis in this species may have evolved distinctive features that could be exploited independently of the standard complex.

The study also fits into a broader and increasingly detailed picture of pigment regulation in Lycium. Previous work from the same research group had shown that anthocyanin biosynthesis in goji berry can be inactivated by a deletion in the promoter of a bHLH transcription factor called LrLAN1b, establishing that cis-regulatory changes in partner genes can silence the pathway. More recently, an R3-type MYB factor, LrMYB30, was reported to act as a negative regulator of fruit coloration in the same species. Taken together with the new results, these findings sketch a regulatory network in which positive activators like LrMYB113, negative regulators like LrMYB30, and partner proteins like LrAN1b and LrAN11 interact to fine-tune pigment output. Understanding both the accelerators and the brakes of this system is essential for any serious breeding or metabolic engineering effort, because pigment levels reflect the balance of opposing forces rather than the strength of any single component.

The practical implications extend well beyond basic biology. Black goji berry anthocyanins have been documented in pharmacological studies to ameliorate memory impairment, oxidative stress and neuroinflammation in animal models, and reviews of the species highlight a growing portfolio of potential health applications. Yet the medicinal development of L. ruthenicum has been hindered precisely by the gap in knowledge that this study addresses: without knowing which transcriptional regulators control flavonoid accumulation, breeders and biotechnologists have lacked rational genetic targets. LrMYB113 now provides one. Overexpressing this gene, editing its regulatory regions, or selecting natural allelic variants with enhanced activity could all plausibly increase the anthocyanin content of cultivated berries, raising the potency of a crop whose value lies almost entirely in its phytochemistry. Because the hairy root system used in the study responds robustly to LrMYB113 manipulation, the gene also offers a tool for producing acylated anthocyanins in controlled culture settings, an approach that could reduce pressure on wild populations harvested from fragile desert ecosystems.

There remain open questions. The authors note that the transcriptional regulation of flavonoid biosynthesis in L. ruthenicum is still incompletely understood, and the interplay between LrMYB113 and the other known regulators of the species has yet to be mapped in full. Whether LrMYB113 also influences the acyltransferase enzymes that decorate the anthocyanins, or the transporters that sequester them into vacuoles, remains to be tested. What is already clear, however, is that the study delivers a rare and satisfying arc of evidence, from phylogenetic prediction through cross-species functional validation to native-system metabolite analysis, protein interaction mapping and direct promoter targeting. For a plant whose pigments have colored traditional medicine and modern nutraceutical markets alike, the identification of LrMYB113 transforms a chemical curiosity into an engineering opportunity, and it offers a textbook demonstration of how a single transcription factor can orchestrate an entire branch of plant specialized metabolism.

Subject of Research: Transcriptional regulation of anthocyanin biosynthesis by the R2R3-MYB factor LrMYB113 in Lycium ruthenicum

Article Title: R2R3-MYB transcription factor MYB113 specifically regulates anthocyanin accumulation in Lycium ruthenicum

Article References: Li, T., Zhang, Z., Wang, J., Qin, H., & Tang, L. (2026). R2R3-MYB transcription factor MYB113 specifically regulates anthocyanin accumulation in Lycium ruthenicum. Plant Cell Reports, 45(9), Article 274. https://doi.org/10.1007/s00299-026-03953-z

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03953-z

Keywords: Lycium ruthenicum, black goji berry, LrMYB113, R2R3-MYB transcription factor, anthocyanin biosynthesis, MBW complex, flavonoids, LrDFR, LrANS, acylated anthocyanins, hairy root culture, metabolic engineering