Betalains, a distinctive group of nitrogen-containing pigments exclusive to the Caryophyllales order, possess diverse biological activities such as antioxidant, anti-inflammatory, and antimicrobial properties, making them highly valuable for applications in food, nutraceutical, and pharmaceutical industries. This Review provides a comprehensive analysis of betalain biosynthesis, structural diversity, and ecological significance, highlighting their roles in enhancing stress resilience, adaptation mechanisms, and plant evolutionary strategies. The evolutionary development of betalains is explored, revealing their emergence through gene duplication events and providing insights into their mutual exclusivity with anthocyanins. This study utilizes comparative genetics and advanced molecular tools to uncover the intricate regulatory networks involving transcription factors such as MYB, bHLH, WRKY, and SPL, which govern betalain biosynthesis. Furthermore, the Review discusses innovative transgenic studies that introduce betalains into non-native species, demonstrating their potential to enhance stress tolerance and boost agricultural productivity. While significant progress has been made in understanding betalain biosynthesis pathways, the evolutionary relationships with anthocyanins and the specific ecological functions of betalains in plants remain areas of ongoing exploration. Future research directions include integrating chemotaxonomic studies, molecular phylogenetics, and multiomics approaches to unravel the full spectrum of betalain functions and regulatory mechanisms. Such studies are essential to deepening our understanding of these vibrant pigments and their evolutionary implications, offering new opportunities for biotechnological innovations and sustainable agricultural practices. This Review stands out by combining genetic, ecological, and evolutionary perspectives, providing novel insights into the multifunctionality of betalains and their potential to drive future advancements in plant science and biotechnology.
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