Lack of evidence for anthocyanins contributing to pigmentation of Chenopodium quinoa

This paper refutes a recent claim that anthocyanins cause red pigmentation in *Chenopodium quinoa* leaves by demonstrating through reanalysis of RNA-seq data that the observed coloration is instead driven by betalain and carotenoid biosynthesis, consistent with the known absence of key anthocyanin genes in this betalain-producing lineage.

The Gist

The Big Picture: A Case of "Wrong Color" in the Plant World

Imagine the plant kingdom as a massive art gallery. For centuries, scientists have known that most plants paint their leaves and flowers using two distinct types of paint:

1. Anthocyanins: The "Red/Blue/Purple" paint (think blueberries or red roses).
2. Betalains: The "Red/Yellow" paint (think beets or prickly pears).

There is a golden rule in this gallery: A plant usually only uses one type of paint. If a plant has the machinery to make Betalains (like the beet family, which includes quinoa), it has permanently deleted the instructions for making Anthocyanins. They are like two rival gangs that never hang out in the same house.

The Controversy: A New Claim

Recently, a study by Zhang et al. (2024) came along and claimed to have found a "hybrid" artist. They said that Quinoa (a member of the beet family) was actually using both paints to turn its leaves red. They claimed that Anthocyanins were the secret ingredient behind the red color.

The Investigation: The "Detective" Paper

The authors of this new paper (Lingemann, Pucker, and their team) decided to play detective. They re-examined the data from the Zhang study and dug into the genetic blueprints of the Quinoa plant. Their conclusion? The claim is wrong. Quinoa is not using Anthocyanins; the red color comes from other sources.

Here is how they proved it, using simple analogies:

1. The Missing Factory (Genetic Evidence)

To make Anthocyanin paint, a plant needs a specific factory machine called arGST.

• The Analogy: Imagine trying to bake a chocolate cake without a chocolate mixer. You can have all the flour and sugar, but without that one specific machine, you can't make the cake.
• The Finding: The authors looked at the Quinoa genome and found that the arGST machine is completely missing. It's not just broken; the factory floor where it should be is empty. Furthermore, the "foreman" (a transcription factor called MYB) who tells the factory to start working is also missing or inactive.
• Result: Without the machine and the foreman, the Anthocyanin factory is shut down. It is physically impossible for the plant to make this paint.

2. The Confused Data (Re-analyzing the RNA)

The Zhang study looked at the plant's "activity logs" (RNA-seq data) to see which genes were turned on.

• The Analogy: Imagine looking at a factory's electricity bill. If the lights are off in the "Anthocyanin Department," but the "Betalain Department" is humming with power, you know which paint is being made.
• The Finding: When the authors re-analyzed the data, they saw that the genes for Anthocyanins were essentially silent (no electricity). However, the genes for Betalains and Carotenoids (another type of pigment, like the orange in carrots) were active.
• The Mix-up: The authors suspect the Zhang study made a mistake in matching their samples. They might have looked at data from young plants (Day 5) while claiming it was from old plants (Day 70), leading to a false conclusion about what was causing the color.

3. The Wrong Test Tube (Metabolomics Critique)

The Zhang study also tried to measure the actual paint in a test tube using a light sensor.

• The Analogy: Imagine trying to identify a specific brand of red paint by shining a light on a bucket of mixed red paint. If the bucket contains both red and orange paint, the light will show "red," but you can't be sure it's only the red paint you are looking for.
• The Finding: The method used by Zhang et al. measured light absorption at a wavelength where both Anthocyanins and Betalains glow. Since we know Quinoa makes Betalains, the "red" signal they saw was almost certainly just the Betalains, not a new discovery of Anthocyanins. Plus, they didn't share their raw data or detailed recipes, making it impossible for others to verify their results.

The Verdict

The authors conclude that Quinoa is still following the old rules.

• It cannot make Anthocyanins because it lacks the essential genetic tools.
• The red color in its leaves is likely a mix of Betalains (its native red paint) and Carotenoids (orange/yellow pigments), which blend together to create the red hue.

Why This Matters

This paper is a reminder that in science, just because a study says something is happening, it doesn't mean it is happening. Sometimes, the "red" we see is just a trick of the light or a misunderstanding of the genetic machinery.

The authors are essentially saying: "Don't be fooled by the color. Check the blueprints. If the factory for Anthocyanins is demolished, the plant isn't painting with Anthocyanins, no matter how red the leaves look."

Technical Summary

1. Problem Statement

The study addresses a conflicting claim in recent literature (specifically Zhang et al., 2024) suggesting that anthocyanins are responsible for the red pigmentation observed in the leaves of Chenopodium quinoa (quinoa).

• Background Context: Plants in the order Caryophyllales (which includes Chenopodiaceae) are historically known for a strict mutual exclusion of pigments: they produce either betalains or anthocyanins, but never both in the same species. This is due to evolutionary losses in the anthocyanin biosynthesis pathway in betalain-producing lineages.
• The Conflict: Zhang et al. (2024) reported anthocyanin presence in red quinoa leaves based on spectrophotometric and LC-MS/MS data. Lingemann et al. argue this contradicts established genetic and evolutionary evidence and likely stems from methodological errors or misinterpretation of data.

2. Methodology

The authors performed a comprehensive re-analysis of the datasets and claims made by Zhang et al. (2024) using a multi-omics approach:

• Genomic Analysis (Gene Presence/Absence):

  • Microsynteny Analysis: Compared genomic regions surrounding the arGST (anthocyanin-related glutathione S-transferase) locus in C. quinoa against anthocyanin-producing species (Vitis vinifera, Solanum lycopersicum) and other Amaranthaceae.
  • Phylogenetic Screening: Conducted BLASTp searches and phylogenetic tree construction (using IQ-TREE) to identify orthologs of key anthocyanin genes (arGST, MYB transcription factors) in C. quinoa and related betalain-producing species.
  • Orthology Detection: Used tools like KIPEs and MYB_annotator to identify flavonoid and betalain pathway genes.

• Transcriptomic Re-analysis (Gene Expression):

  • Data Source: Re-processed raw RNA-seq data (FASTQ files) from the Sequence Read Archive (SRA) linked to Zhang et al. (2024).
  • Discrepancy Check: Noted a mismatch between the sample metadata in the manuscript (days 70, 90, 110) and the actual SRA data (days 5, 20, 35).
  • Quantification: Used kallisto for transcript quantification against the C. quinoa reference genome (Cquinoa_392_v1.0).
  • Pathway Analysis: Performed differential expression analysis (using PyDESeq2) and KEGG pathway enrichment analysis (using clusterProfiler and pathview) to map expression levels of anthocyanin, betalain, and carotenoid biosynthesis genes.

• Metabolomic Critique:

  • Critically evaluated the spectrophotometric methods (absorbance at 530/657 nm) and LC-MS/MS parameters reported by Zhang et al., highlighting missing methodological details (e.g., column types, collision energies) and the non-specificity of absorbance peaks for betalains vs. anthocyanins.

3. Key Contributions

• Genetic Evidence of Pathway Blockage: Demonstrated that the anthocyanin biosynthesis pathway in C. quinoa is genetically blocked due to the absence of the arGST gene (confirmed via microsynteny and phylogeny) and the lack of a functional orthologous MYB transcription factor required to activate the MBW complex.
• Re-interpretation of RNA-seq Data: Showed that the RNA-seq data from Zhang et al. actually supports the absence of anthocyanin biosynthesis (low/absent expression of DFR and ANS) while showing activity in betalain and carotenoid pathways.
• Identification of Data Mismatch: Revealed a critical inconsistency between the sample descriptions in the original manuscript and the actual data deposited in public repositories, suggesting the observed "differential expression" might be an artifact of comparing mismatched developmental stages.
• Methodological Critique: Provided a detailed critique of the metabolomic methods used by Zhang et al., arguing that standard spectrophotometry cannot distinguish betalains from anthocyanins without specific chromatographic separation and MS/MS validation, which was lacking in the original study.

4. Results

• Absence of Key Genes:
  • arGST: No orthologs were found in C. quinoa or other Amaranthaceae. Microsynteny analysis showed the locus is disrupted or missing.
  • MYB: No clear ortholog of the anthocyanin-activating MYB was found in C. quinoa. The ancestral MYB in this lineage appears to have been co-opted for betalain regulation.
• Expression Profiles:
  • Anthocyanin Pathway: DFR (dihydroflavonol 4-reductase) was absent; ANS (anthocyanidin synthase) showed very low transcription. High expression of FLS (flavonol synthase) suggests flux is directed toward flavonols/proanthocyanidins, not anthocyanins.
  • Betalain & Carotenoid Pathways: Genes for betalain synthesis (CYP76AD1, DODA) and carotenoid synthesis were expressed. However, the expression levels did not perfectly correlate with the reported red/green phenotypic differences in the original study, further suggesting the original phenotypic interpretation was flawed.
• KEGG Enrichment: While the original study claimed anthocyanin pathway enrichment, the re-analysis showed that the enrichment was limited to upstream flavonoid genes (e.g., CHS, CHI, F3H) but lacked the terminal enzymes (DFR, ANS) required for anthocyanin production.
• Metabolite Ambiguity: The study concludes that the "anthocyanin" signal detected by Zhang et al. is most likely betalains, which share similar absorbance maxima (532–550 nm) and were previously confirmed to exist in quinoa.

5. Significance

• Reinforcement of Evolutionary Theory: The study strongly supports the long-standing hypothesis of mutual exclusion between anthocyanins and betalains in Caryophyllales. It provides robust genetic evidence that the transition to betalain pigmentation involved the irreversible loss of specific anthocyanin pathway components (arGST and specific MYBs).
• Correction of Scientific Literature: It serves as a necessary correction to a recent publication (Zhang et al., 2024), preventing the propagation of incorrect biological conclusions regarding quinoa pigmentation.
• Methodological Standards: The paper highlights the dangers of relying on "black box" bioinformatics services or superficial metabolomic analyses without deep genetic validation. It emphasizes the need for:
  • Verification of sample metadata in public repositories.
  • Rigorous reporting of LC-MS/MS parameters.
  • Integration of genomic, transcriptomic, and metabolomic data to validate pigment identity.
• Future Research Direction: It calls for in-depth investigation of specific genes rather than relying on broad pathway enrichment analyses, which can be misleading due to database biases toward anthocyanin genes.

In summary, Lingemann et al. demonstrate that the red pigmentation in Chenopodium quinoa leaves is not caused by anthocyanins, but is likely due to betalains and/or carotenoids, and that the genetic machinery required for anthocyanin production is fundamentally absent in this species.