In Planta Expression of Thermophilic GtGBE Mimics BEIIb and Modifies Rice Starch Structure Without Yield Penalties

Enzymatic modification using glycosyltransferases offers a green route to produce highly branched starch to enhance starch functionality in the food industry, yet this approach faces challenges including the high cost of enzyme production and inefficiency. Here, we developed a viable in planta strategy by heterologously expressing an α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02 (GtGBE) in rice, aiming to tailor starch branching structures directly in a living plant system. Several independent transgenic lines were generated, expressing the GtGBE gene under the control of either the endosperm-specific rice starch branching enzyme IIb (BEIIb) promoter (pBEIIb) or the constitutive maize Ubiquitin promoter (pUbi). GtGBE generated distinct promoter-dependent branching patterns. The pBEIIb-driven construct partially recapitulated BEI-deficient-like profiles through the combined effect of downregulated endogenous BEI and relatively low GtGBE expression levels. In contrast, the pUbi-driven construct enhanced BEIIb-like activity through sustained high GtGBE accumulation and upregulation of endogenous BEIIb. Both strategies increased short amylopectin chains at the expense of intermediate/long chains and amylose, resulting in decreased relative crystallinity, apparent amylose content, gelatinization temperatures, and peak viscosity, alongside improved retrogradation resistance and rheological properties. Notably, GtGBE expression driven by the BEIIb promoter showed greater stability under heat stress. Moreover, pUbi-GtGBE transgenic lines exhibited a significant increase in 1000-grain weight and filled grain number per panicle without compromising other agronomic traits, highlighting the dual potential of this strategy for agricultural and industrial applications. This work establishes GtGBE as an effective molecular tool for the direct and tailored bioengineering of starch in a living plant system.