Betalain, an economically valuable water-soluble natural plant pigment, is prized for its strong antioxidant activity, making it popular as a dietary supplement and a visual marker for plant transformation. However, market demand significantly outstrips current production capacity. This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize (Zea mays L.) endosperm. We engineered a novel enhanced RUBY (eRUBY) system derived from the original polycistronic RUBY construct (CYP76AD1_P2ADODA1_P2ADOPA5GT unit, abbreviated CDG) by introducing arogenate dehydrogenase (ADHα) to increase the L-tyrosine substrate supply. All the genes were driven by the endosperm-specific promoter. Fusion of ADHα into a single polycistronic eRUBY construct (CDGA) produced significantly higher betanin (6.88 mg g⁻¹ dry weight) and isobetanin (1.81 mg g⁻¹ dry weight) levels than in CDG + A, which stacked the ADHα cassette independently with CDG. The high betalain accumulation in CDGA lines (which also exhibited higher transgene copy number) resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT (versus 2.48–2.80-fold in CDG + A). Importantly, transgenic plants maintained a normal phenotype. Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine, alanine, aspartate, and glutamate contributes to betalain production. Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety. Collectively, these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.

A growing global population and the increasing prevalence of diet-related health issues such as “hidden hunger”, obesity, hypertension, and diabetes necessitate a fundamental rethinking of crop design and breeding. Synthetic metabolic engineering offers a method to modify and redesign metabolic pathways to increase the nutritional value of crops. We summarize recent advances in the biofortification of key nutrients including provitamin A, vitamin C, vitamin B9, iron, zinc, anthocyanins, flavonoids, and unsaturated fatty acids. We discuss the potential of multi-gene stacking, gene editing, enzyme engineering, and artificial intelligence in synthetic metabolic engineering. We propose future research directions and potential solutions centered on leveraging AI-driven systems biology, precision gene editing, enzyme engineering, agrobacterium-mediated genotype-independent transformation, and modular metabolic engineering strategies to develop next-generation nutritionally enhanced super crops and transform global food systems.

Grain number and seed-setting rate are components of crop yield. Cytokinin influences grain yield. However, emerging studies suggest that high cytokinin signals often lead to reduced branching or seed-setting rate, leading to reduced grain yield, although the mechanisms remain unclear. In this study, we identified and characterized the rice (Oryza sativa L.) gene LONELY GUY-LIKE 8 (LOGL8), based on analysis of the LOGL8-pm (promoter mutant of LOGL8) mutant, which harbors a T-DNA insertion in the promoter of this gene. The mutation in LOGL8-pm causes ectopic hyperexpression of LOGL8 in inflorescence organs, resulting in plants with smaller panicles and defective ovules lacking archesporial cells and integuments. Knockout of LOGL8 caused pollen abortion, leading to a reduced seed-setting rate. LOGL8 encodes a putative cytokinin-activating enzyme. Our results showed that LOGL8 directly catalyzes the biosynthesis of bioactive cytokinins. Therefore, we propose that the ectopic expression of LOGL8 disrupts cytokinin spatiotemporal distribution and causes inhibition of LONELY GUY (LOG), which affects panicle branching and female organ development. These findings reveal the important role of LOGL8 in male development, and highlight the delicate balance of local cytokinin levels during panicle branching and female organ development.