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 first half of the work reads like a meticulous recipe for coaxing a stubborn woody species into life in a petri dish. Working with stem segment explants, the researchers systematically optimized each stage of in vitro regeneration. For shoot induction, they achieved a striking 93.3 percent success rate on Murashige and Skoog medium supplemented with 1.5 milligrams per liter of the cytokinin 6-benzylaminopurine, 0.6 milligrams per liter of the auxin 1-naphthaleneacetic acid, and 30.0 milligrams per liter of adenine sulfate. This combination of a cell-division-promoting cytokinin, an auxin, and a purine precursor proved critical for convincing mature stem tissue to form new shoots rather than callus or die off, a balance that has historically been hard to strike in pomegranate.

Once shoots formed, the team faced the next bottleneck: multiplying them and turning them into whole plants. On medium containing 0.8 milligrams per liter BAP and 0.3 milligrams per liter indole-3-butyric acid, the cultures reached a proliferation coefficient of 5.4, meaning each explant produced more than five new shoots per cycle. Shoots were then strengthened on medium with 1.2 milligrams per liter BAP, 0.3 milligrams per liter NAA, and 0.2 milligrams per liter gibberellic acid, the latter hormone promoting elongation and healthier foliage. Finally, transfer to half-strength MS medium with 1.5 milligrams per liter IBA and 0.5 milligrams per liter NAA produced a rooting rate of 95.3 percent, allowing plantlets to be acclimatized and grown to maturity. Each hormone concentration represents the outcome of dose-response testing, and together they form a complete micropropagation pipeline from stem segment to rooted plant.

With regeneration solved, the researchers turned to Agrobacterium tumefaciens-mediated transformation, the workhorse method of plant genetic engineering. The soil bacterium naturally transfers a segment of its own DNA, the T-DNA, into plant cells, where it integrates into the genome. The team fine-tuned the infection conditions: precultured explants were immersed for thirty minutes in an Agrobacterium suspension adjusted to an optical density of 0.8 at 600 nanometers, containing 20.0 milligrams per liter of acetosyringone, a phenolic compound that activates the bacterial virulence genes responsible for DNA transfer. The explants then spent four days in dark co-culture with the bacteria, a window long enough for gene transfer but short enough to prevent overgrowth that would kill the plant tissue.

Selection was the next challenge, and the authors applied a sequential antibiotic strategy. Kanamycin at 30 milligrams per liter eliminated the cells that had not incorporated the T-DNA, which carried a resistance gene as a selectable marker, while 400 milligrams per liter of timentin suppressed and killed the remaining Agrobacterium without damaging the plant cells. Timentin has been favored in transformation work precisely because it is gentler on regenerating tissue than alternatives such as cefotaxime alone. Under this regime, the team achieved a stable average transformation efficiency of 17.5 percent in ‘Taishanhong’ pomegranate, a figure that stands out for a species previously described in the literature as difficult to transform, where earlier efforts on other cultivars and on the dwarf pomegranate yielded far lower or less reproducible results.

The payoff came when the researchers used the platform to ask a functional question: what does PgMYB10 do? MYB transcription factors are DNA-binding proteins that switch entire metabolic pathways on or off, and the R2R3-MYB family in particular is famous in fruit biology. The apple gene MdMYB10, the pear gene PyMYB10, and the strawberry MYB10 homologs have all been shown to drive red coloration in their respective crops, and a previous pomegranate MYB had been analyzed in transient systems. But correlation from expression studies is not proof of function. By overexpressing PgMYB10 in stable transgenic pomegranate plants, the team could observe the consequences directly in the species where the gene normally operates.

The results were dramatic and visually unmistakable. Transgenic leaves and stems accumulated pigment and turned red, and in three independent transgenic lines the maximum anthocyanin content reached 5.4 times that of wild-type plants, while PgMYB10 transcript levels rose up to 27.2-fold. Quantitative expression analysis showed that six structural genes of the anthocyanin pathway were markedly upregulated: PgCHS (chalcone synthase), PgCHI (chalcone isomerase), PgF3H (flavanone 3-hydroxylase), PgDFR (dihydroflavonol 4-reductase), PgANS (anthocyanidin synthase), and PgUFGT (UDP-glucose flavonoid 3-O-glucosyltransferase). These enzymes catalyze the stepwise conversion of phenylpropanoid precursors into the final colored anthocyanins, with UFGT performing the last glycosylation step that stabilizes the pigment. The coordinated activation of the entire pathway by a single transcription factor is exactly what defines a master regulator, and the data place PgMYB10 firmly in that category for pomegranate.

The significance of the work extends well beyond pigment biology. Anthocyanins are not merely decorative; they are antioxidant compounds with documented nutritional value, and they shape consumer perception of fruit quality, market price, and even the ornamental value of the tree itself. A validated transformation platform means that candidate genes identified by genome sequencing or transcriptomics can now be tested one by one in pomegranate, closing the loop of functional genomics for traits ranging from disease resistance to fruit cracking, a problem the same research group has previously explored at the molecular level. It also opens the door to molecular breeding strategies in which PgMYB10 or its regulatory network could be manipulated to deepen or stabilize fruit coloration in elite cultivars without waiting for decades of conventional crossing.

There are technical caveats worth noting. A transformation efficiency of 17.5 percent, while impressive for pomegranate, still means that most treated explants do not yield transgenic plants, and the protocol was optimized for a single cultivar, ‘Taishanhong’; other genotypes may respond differently to the hormone and antibiotic regimes. The study also relied on overexpression, which demonstrates what PgMYB10 can do when abundant but does not by itself prove it is the sole regulator under normal developmental conditions; anthocyanin control in most plants involves MYB factors acting together with bHLH partners and WD40 proteins, and earlier pomegranate work has implicated a WD40-repeat gene homologous to the Arabidopsis TTG1 in fruit pigmentation. Confirming the native regulatory complex, and testing gene editing rather than overexpression, are logical next steps now that the platform exists.

Even so, the study represents a genuine inflection point for a crop that has lagged behind apple, grape, and strawberry in biotechnology. By combining a fully optimized regeneration pipeline with a reproducible Agrobacterium protocol and an immediate functional demonstration, the Nanjing team has converted pomegranate from a species that could be studied only from the outside into one whose genes can be tested in their native context. For breeders chasing redder arils and richer juice color, and for biologists probing how woody plants control their secondary metabolism, PgMYB10 now stands as the clearest entry point into the pomegranate’s color machinery, and the transformation system behind it as the tool that makes the rest of the genome accessible.

Subject of Research: Establishment of an Agrobacterium-mediated genetic transformation platform in pomegranate and functional validation of PgMYB10 as a regulator of anthocyanin biosynthesis

Article Title: A stable transformation platform in pomegranate uncovers PgMYB10 as a key regulator of anthocyanin biosynthesis

Article References: Zhao, X., Zhang, Y., Teng, Y., & Hao, M. (2026). A stable transformation platform in pomegranate uncovers PgMYB10 as a key regulator of anthocyanin biosynthesis. Plant Cell Reports, 45(9), Article 275. https://doi.org/10.1007/s00299-026-03962-y

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03962-y

Keywords: pomegranate, PgMYB10, anthocyanin biosynthesis, Agrobacterium-mediated transformation, genetic transformation, in vitro regeneration, transcription factors, plant biotechnology, molecular breeding, Punica granatum, tissue culture, functional genomics