The variegated pattern in P0429 is unlike the familiar mottling seen in ornamental plants, where chlorophyll defects create white or yellow sectors. Instead, the red zones owe their color to betacyanins, a class of nitrogen-containing pigments characteristic of the plant order Caryophyllales, to which quinoa belongs. Betalains, the broader pigment family that includes red-violet betacyanins and yellow betaxanthins, functionally replace anthocyanins in most members of this lineage and are synthesized from the amino acid L-tyrosine. Beyond their role as natural colorants, these pigments are potent antioxidants that help plants cope with salinity, drought, and intense light by scavenging reactive oxygen species and providing photoprotection, making them doubly interesting for a crop prized for its stress tolerance.

When the researchers examined cross-sections of variegated leaves, they found something unexpected: the pigmentation was confined almost entirely to the epidermal bladder cells, or EBCs, that blanket most of the plant’s aerial surfaces. These large, two-celled structures, consisting of a stalk and a swollen bladder, are often considered the simplest form of salt glands and are known to help quinoa manage salinity, deter herbivores, and regulate osmotic pressure. When the team mechanically brushed the EBC layer off the leaf surface, the red sectors became visually indistinguishable from the green ones, proving that the entire mosaic resides in this specialized epidermal layer. The boundary between red and green EBC territories was sharp, developmentally stable, and occasionally aligned with the leaf’s vascular tissue.

Quantitative measurements drove the point home. Spectrophotometric analysis at 538 nanometers, the absorption maximum of betacyanins, revealed that red EBCs contained 6.23 micrograms of betacyanins per gram of tissue, roughly fifty times the 0.12 micrograms per gram found in colorless EBCs from green sectors. Extracts from red EBCs displayed a deep purple-red hue that rapidly turned yellow upon the addition of sodium hydroxide, a classic chemical signature of betacyanins. By contrast, the underlying leaf lamina retained only low pigment levels, and the difference between red and green EBCs was an order of magnitude larger than any difference measured in the lamina beneath. No significant variation in yellow betaxanthins was detected between the two cell types, confirming that betacyanin accumulation in the bladder cells is the dominant driver of the variegation.

To find the molecular switch behind this dramatic difference, the team performed RNA sequencing on red and colorless EBCs isolated from the very same variegated leaves. Because the cells were harvested from adjacent sectors of a single organ, the genetic background was held constant, and the differential expression analysis identified only seven genes, all upregulated in the red EBCs. Quantitative PCR validation narrowed the field further. Among the candidates, one gene stood out: Cqu0091301, a member of the CYP76ADα lineage of cytochrome P450 enzymes, showed both the highest absolute mRNA level and the largest fold change between red and colorless EBCs. The CYP76ADα family catalyzes the first committed step of betalain biosynthesis, the hydroxylation of tyrosine to L-DOPA, and is widely regarded as a rate-limiting component of the pathway.

But the story took a twist when the researchers inspected the gene model of Cqu0091301 in the available quinoa reference genomes. In those assemblies, the gene’s second exon appeared truncated, deleting a conserved P450 domain and rendering the predicted enzyme non-functional. Clues from the sequencing data suggested otherwise: no reads aligned to the short second exon, while a homologous gene, Cqu0091280, showed a peculiar enrichment of mismatched reads over part of its second exon. De novo transcriptome assembly recovered a transcript matching Cqu0091301 but with an extended three-prime region, hinting that the reference genomes were missing something. Whole-genome resequencing of P0429, followed by targeted PCR, revealed the answer: a 3,957-base-pair genomic insertion, absent from the reference assemblies, that restores the full-length second exon and the complete, conserved P450 domain of Cqu0091301.

This structural variation proved to be the linchpin of the entire phenotype. When the team re-analyzed their RNA sequencing data using the corrected gene model, the expression difference between red and colorless EBCs became even more pronounced, while the apparent differential expression of the homolog Cqu0091280 diminished, indicating that reads previously misassigned to the homolog actually belonged to the restored gene. Other betalain pathway genes, including tyrosine decarboxylase, DODA, and several glucosyltransferases, showed extremely low expression in EBCs and no differential upregulation in red sectors. The only genes consistently and markedly elevated in red EBCs were the three B-subgenome CYP76ADα copies, Cqu0091280, Cqu0091301, and Cqu0091310, cementing the conclusion that upregulation of this P450 lineage, and Cqu0091301 in particular, is the key regulatory step driving betacyanin synthesis in the pigmented bladder cells.

Genomic context added yet another layer of intrigue. The quinoa genome harbors seventeen CYP76AD family members distributed across three subfamilies, and all five CYP76ADα genes are physically linked to DODA genes, forming adjacent CYP76ADα–DODA pairs on two homoeologous chromosomes. Such metabolic gene clusters are a recurring theme in plant secondary metabolism and are thought to facilitate coordinated regulation of enzyme production, conferring evolutionary advantages and enhanced environmental adaptability. Expression profiling across tissues showed that CYP76ADα genes and their linked DODA partners share highly similar relative expression patterns, consistent with transcriptional co-regulation, even though the P450 genes are generally expressed at an order of magnitude higher basal levels than their DODA neighbors.

Perhaps the most striking finding concerns the division of labor between quinoa’s two subgenomes. As an allotetraploid, quinoa carries A and B subgenomes derived from distinct ancestral species, and the study revealed that their CYP76ADα copies have evolved non-overlapping tissue preferences. The three B-subgenome genes dominated expression in EBCs, while the A-subgenome copies Cqu0282960 and Cqu0282940 were undetectable in bladder cells; instead, Cqu0282960 was preferentially expressed in seedlings and Cqu0282940 in stems and flowers, both upregulated in red tissues of those organs. Across most pigmented organs, the B-subgenome pairs made the greater overall contribution to betalain biosynthesis, a clear demonstration of subgenome functional divergence. Because the reference variety Real carries a structurally incomplete Cqu0091301, the functional burden in its tissues likely falls on the second-highest expressed B-subgenome pair, underscoring how a single insertion can reshape the architecture of pigment regulation in a polyploid crop.

The implications extend well beyond explaining a curious color pattern. The study provides the first report of genomic structural variation driving betalain biosynthesis and links it directly to cell-type-specific pigmentation in a crop of growing global importance. It also highlights a practical lesson for the genomics era: reference genomes, however high quality, can silently truncate genes and mislead expression analyses, and resequencing diverse accessions can recover functional alleles hidden in the pan-genome. For breeders, the restored Cqu0091301 allele offers a potential target for tuning betalain accumulation, with consequences for stress tolerance and natural colorant production. For evolutionary biologists, the work illuminates how duplicated genes in polyploid genomes partition their duties across tissues, and how a single insertion of roughly four kilobases can tip the balance between green and red, one bladder cell at a time.

Subject of Research: Genomic structural variation controlling cell-type-specific betalain pigment biosynthesis in allotetraploid quinoa

Article Title: Genomic structural variation underlies cell type-specific betacyanin variegation in Chenopodium quinoa

Article References: Zhang, Z., Wang, Y., Hu, X., Yu, T., Feng, Y., Zhang, J., Zhang, T., Feng, G., & Zhang, H. (2026). Genomic structural variation underlies cell type-specific betacyanin variegation in Chenopodium quinoa. Stress Biology, 6(1), Article 15. https://doi.org/10.1007/s44154-025-00284-z

Image Credits: AI Generated

DOI: 10.1007/s44154-025-00284-z

Keywords: quinoa, betacyanin, betalain, epidermal bladder cells, CYP76ADα, structural variation, variegation, subgenome, gene cluster, polyploidy, plant pigments, Stress Biology