Step outside and spring is in full bloom, from red tulips, to pink magnolias to purple lilacs, but how do plants create all that color? The alluring hues that attract pollinators and provide beautiful bouquets begin with the formation of pigments known as anthocyanins.
Anthocyanins are part of a plant’s communication and protection plan. Synthesized by specialized metabolic pathways, they filter harmful radiation and corral cell-damaging free radicals. Scientists know a lot about how anthocyanins are assembled in the central metabolism, but very little about the final leg in their metabolic journey to the vacuole where they finally reveal their true colors.
Erich Grotewold, chair of MSU’s Department of Biochemistry and Molecular Biology, and Nan Jiang, postdoctoral research associate, designed experiments to test the hypothesis that an enzyme from the glutathione S-transferase (GST) family, found in all eukaryotic cells including the human liver, protects anthocyanins during their journey from the endoplasmic reticulum to the vacuole.
“The role of the GST in the formation of anthocyanin pigments has remained very elusive,” Grotewold explained. “There is good evidence that what they are doing is not chemically modifying the pigment, but rather binding to the pigments and helping them move to the vacuole without being degraded.”
During the study, recently published in Nature Communications, they intercepted a connection between the central and specialized metabolism of the model plant, Arabidopsis thaliana, that may lead to a new, mechanistic understanding of how these two aspects of plant metabolism coordinate.
It all began with tt19, a genetic mutant of a GST that accumulates no anthocyanin pigments making the seed and seedlings appear white. By mutagenizing the entire tt19 genome, Grotewold and Jiang hoped to identify a mutated gene involved in degrading the anthocyanins, thereby recovering pigmentation without the protection potentially furnished by the GST.
“If the hypothesis is that this GST binds to anthocyanins and stabilizes the compound because another enzyme wants to come along and catabolize it, then if we knock out the catabolizing enzyme, the GST should no longer be needed, and the pigment should be recovered,” explained Grotewold. “Nan designed a beautiful assay in which he could screen tens of thousands of really tiny seedlings at the same time to identify which ones might be able to recover the pigmentation.”
In genetic terms, Jiang created suppressors, mutants that suppressed the pigment-free tt19 mutant phenotype. He painstakingly identified and coddled the tiniest suppressor seedlings into full grown plants whose progeny was necessary for everything that followed.
“I transferred potential suppressor seedlings into a media for normal plant growth to let them adapt,” said Jiang, who went through seven generations of plants during the study. “The purple color would slowly disappear, and the green leaves would show up with chloroplast recovered after 10-15 days before I transferred the seedlings to soil.”
Of the thousands of seedlings, Jiang identified and propagated a total of six suppressors. Whole genome re-sequencing revealed that all six of the suppressor lines had nothing to do with the GST escort as they had hypothesized.
Instead, all of them were involved in the biogenesis of a class of small interfering RNAs (siRNAs) from the RDR6-SGS3-DCL4 pathway already known to participate in controlling the expression of hundreds of genes.
“We were very puzzled because abolishing this siRNA pathway usually does not lead to any major phenotypes,” Grotewold explained. “Here we found six independent mutations and all of them hit on this siRNA system, the RDR6-SGS3-DCL4 pathway.”
The researchers discovered the suppressors were, in fact, restoring partial levels of anthocyanin, but they were also making massive amounts of another type of flavonoid—flavonols. Carbon from central metabolism was being pushed into the specialized pathway, but this only happened when both the tt19 GST and RDR6 mutations existed together, which is why Jiang’s painstaking cultivation of suppressor line progeny was so valuable.
“What we think is happening is that under normal conditions, siRNAs from specialized metabolism control the central metabolism like stabilizers on a plane, adjusting to small winds or stress conditions and maintaining balance,” explained Grotewold, who noted they have yet to investigate how applicable to other plants and pathways their findings will be. “But when there is a disfunction in both specialized metabolism and siRNA biogenesis pathways, the plant cannot stay balanced and chaos comes.”
“This is a beautiful example of how investments in basic research can lead to unexpected outcomes that open the door for future exploration,” said Karen Cone, a program director in NSF’s Division of Molecular and Cellular Biosciences that funded the study.