Polyacylated anthocyanins: nature's strategy for blue flower coloration and their application in genetic engineering

True blue flower coloration remains a rare and highly coveted trait in ornamental horticulture. Its natural occurrence is less than 10% of angiosperm species. The delphinidin derivatives provide the chromophoric basis for blue coloration; however, their structural stability is governed by intricate intramolecular and intermolecular interactions. To preserve blue hues, plants have evolved sophisticated stabilization mechanisms, including the formation of polyacylation, copigmentation, and metalloanthocyanin, as observed in Gentiana, Lamiaceae, and Delphinium. In this review, we integrate the current knowledge of biochemical mechanisms underlying natural blue pigments and evaluate the genetic engineering strategies aimed at creating blue flowers. These efforts to create blue flowers can be classified into three paradigms: (1) First-generation substrate engineering focused on introducing flavonoid 3',5'-hydroxylase (F3'5'H), which often yielded violet rather than true blue phenotypes due to vacuolar instability; (2) second-generation stabilization engineering incorporated endogenous copigments and successfully achieved stable blue coloration, as demonstrated in chrysanthemum; and (3) the emerging third-generation strategy seeks the autonomous reconstruction of complete stabilization modules. We highlight recent advances in synthetic biology that underpin this transition, including mitochondrial pH regulation via PhDC, manipulation of metal ion homeostasis, and the de novo assembly of polyacylation complexes, such as the Cineraria module. Together, these developments mark a shift from single-gene introduction toward rational metabolic design, providing a promising roadmap for overcoming evolutionary constraints in traditionally nonblue crops, such as roses and orchids.