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

The research, led by Chen Chen and Geng Zhou of the Hunan Vegetable Research Institute at the Hunan Academy of Agricultural Sciences, together with colleagues at Yuelushan Laboratory and Hunan Agricultural University, focused on the gene CsMYB60. This gene encodes an R2R3-MYB transcription factor, a type of DNA-binding protein that acts as a master switch for plant pigment production. Earlier work had already established that CsMYB60 drives the biosynthesis of flavonoids and proanthocyanidins, two classes of plant secondary metabolites, in cucumber fruit spines. More recent studies extended that role to the mature fruit skin, showing that the gene helps control flavonoid accumulation and, with it, the color the peel takes on as the fruit ripens.

To dissect the genetic basis of peel color with precision, the team turned to a powerful experimental resource: a pair of near-isogenic lines. These are two cucumber inbred lines, L14 and L19, that are genetically almost identical except at the region governing peel color. L14 develops a yellow mature peel, while L19, a mutant derived from it, remains white. Because the two lines share nearly all of their genetic background, any difference in peel color can be attributed with high confidence to the small stretch of DNA where they differ, eliminating the noise that usually complicates genetic analysis in diverse varieties.

The researchers combined bulked segregant analysis with high-resolution fine mapping. In bulked segregant analysis, scientists cross the two contrasting lines, then pool the DNA of many offspring that share the same trait, in this case yellow peel, and sequence those pools alongside pools from offspring with the opposite trait. Regions of the genome where the pools diverge sharply are candidate homes for the causal gene. This approach, originally developed for rapid mapping in rice, allowed the team to narrow the peel color locus to a 121-kilobase interval on chromosome 4. Within that interval sat exactly one compelling candidate: CsMYB60.

What the team found when they compared the DNA sequences of the two alleles was the study’s central surprise. The yellow-peel line L14 carries not one but two Mutator-like element insertions, known as MULE1 and MULE2, within the second intron of CsMYB60. The white-peel mutant L19, by contrast, retains only MULE1. Mutator-like elements are a family of transposable elements, mobile DNA sequences that can copy or cut themselves out of the genome and reinsert elsewhere. Far from being genomic junk, transposable elements are now recognized as major drivers of plant evolution, frequently rewiring gene regulation by inserting into promoters, introns, or nearby regulatory regions and altering when, where, and how strongly a gene is expressed.

Here, the double insertion appeared to matter. During the peel color-transition stage, when the fruit’s skin is actively changing, the MULE1 plus MULE2 haplotype showed higher CsMYB60 transcript abundance than the MULE1-only haplotype in the near-isogenic materials examined. In other words, the line carrying both insertions produced more messenger RNA from the pigment-regulating gene, and that line was the one that turned yellow. The authors are careful to note that the causal regulatory contribution of MULE2 itself remains to be determined; the association between the structural haplotype and both expression level and peel color is clear, but precisely how the second insertion boosts transcription is a question for future work.

The team did not stop at correlation. Using CRISPR/Cas9 genome editing, they knocked out CsMYB60 directly in the L14 background. The result was unambiguous: the edited plants lost their yellow pigmentation entirely, confirming that CsMYB60 is required for yellow mature peel formation in this genetic background. This kind of functional validation is the gold standard in plant genetics, because it demonstrates necessity rather than mere association. Delete the gene, and the trait disappears; restore or retain it with the right structural haplotype, and the trait appears.

The findings fit into a broader and rapidly evolving picture of how fruit color is controlled in cucurbits. Cucumber peel and flesh color are governed by a constellation of loci affecting chloroplast development, carotenoid accumulation, and flavonoid biosynthesis. Previous studies have implicated genes such as CsMYB36 in yellow-green immature peel, APRR2 in green immature fruit color, and various chloroplast-related genes in light green phenotypes. The R2R3-MYB family, meanwhile, is famous across the plant kingdom for its role in the MYB-bHLH-WDR transcriptional complexes that regulate flavonoid pathways. What makes the new study distinctive is that it identifies a structural haplotype, a specific physical arrangement of DNA, rather than a simple single-nucleotide change, as the molecular signature associated with the trait.

That distinction has practical consequences for breeding. Structural variation like transposon insertions is often invisible to standard SNP-based genotyping, which means breeders can inadvertently lose or overlook valuable haplotypes when crossing varieties. The authors point out that the molecular marker associated with the MULE1 plus MULE2 haplotype may facilitate tracking of this structural variant in relevant cucumber breeding materials. In practice, that means seed companies and public breeding programs could screen seedlings for the double-insertion haplotype and confidently predict which plants will produce the yellow mature peel that certain markets demand, without waiting for the fruit to ripen.

The study also adds to a growing appreciation of transposable elements as engines of agricultural diversity. In crops ranging from maize to grapevine to tomato, transposon insertions have been shown to create or modulate traits as varied as seed coat color, fruit shape, and ripening behavior. The cucumber peel story is a textbook example of how a mobile element can act as a regulatory rheostat: by landing inside an intron, the MULE insertions appear to tune the output of a transcription factor that sits atop an entire pigment biosynthesis pathway. Whether MULE2 acts by altering chromatin structure, changing intron splicing, or providing novel regulatory sequences remains open, and answering it will likely require additional experiments such as reporter assays and splicing analyses.

For now, the work delivers a complete arc from field observation to molecular mechanism. Two nearly identical cucumber lines, one yellow and one white, led researchers to a 121-kilobase window on chromosome 4, then to a single MYB gene, then to two nested transposon insertions, and finally to a CRISPR experiment that sealed the case. The identification of a previously unrecognized MULE1 plus MULE2 structural haplotype associated with distinct CsMYB60 transcript abundance and mature peel coloration gives cucumber geneticists a new landmark on their maps and gives breeders a practical tool. It also serves as a reminder that some of the most important instructions in a crop’s genome are not written in its genes at all, but in the restless, mobile DNA that has been reshaping those genomes for millions of years.

Subject of Research: Transposon-mediated structural haplotypes at the CsMYB60 gene and their association with mature peel coloration in cucumber

Article Title: Transposon-mediated structural haplotypes at CsMYB60 are associated with peel coloration in cucumber

Article References: Chen, C., Chen, J., Chen, H., Liu, X., Lu, X., Tian, Y., & Zhou, G. (2026). Transposon-mediated structural haplotypes at CsMYB60 are associated with peel coloration in cucumber. Theoretical and Applied Genetics, 139(10), Article 285. https://doi.org/10.1007/s00122-026-05388-2

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05388-2

Keywords: cucumber, peel color, CsMYB60, transposable elements, MULE, structural haplotype, flavonoid biosynthesis, R2R3-MYB transcription factor, CRISPR/Cas9, fine mapping, bulked segregant analysis, plant genetics