Genetic diversity and regulatory mechanism of anthocyanin-related glutathione S-transferase-encoding gene GaGST in Gossypium arboreum

Glutathione S-transferases (GSTs) play an important role in the anthocyanin transport. Several studies have reported that cotton GST genes modulate the red petal and petal-base spots phenotype, however, the genetic diversity and regulatory mechanisms involved in GSTs remain largely unclear. Using bulk segregant analysis sequencing (BSA-seq), map-based cloning, and virus-induced gene silencing (VIGS), we demonstrate that the red petal-base spots phenotype is controlled by a single dominant gene, GaGST , in Gossypium arboreum . GaGST encodes a glutathione S-transferase. Two natural GaGST structural variations, a 15-bp insertion at the first exon (named GaGST +15 ) and a 14-bp deletion at the third exon (named GaGST -14 ), lead to the loss of gene function and the absence of red petal-base spots. Silencing orthologs of GaGST in the flowers of G. barbadense acc. Hai7124 plants resulted in the lighter red spots, further confirming that GaGST is causal gene of the red petal-base spots. Dual-luciferase reporter assays showed that two previously reported MYB transcription factors, GaPC and GaBM, which individually controlled colorations of cotton petals and petal-base spots, could bind to the GaGST promoter and activate its transcription, thereby promoting the red phenotype at the specialized positions in the cotton flower. This study identifies GaGST as a key transporter of red pigment glycosides, characterizes two natural loss-of-function variations of GaGST in G. arboreum accessions, and provides new insight into the genetic mechanism underlying GaGST -associated color patterns regulated by MYB anthocyanin activators in cotton flowers.