Rice Genomics and Genetics 2025, Vol.16, No.4, 219-236 http://cropscipublisher.com/index.php/rgg 221 2.2 Key enzymes and their functions The synthesis of rice starch involves multiple enzymes, each of which plays a different role in the pathway. A large number of studies on the genes encoding these enzymes have clarified their influence on the formation of rice quality. Adp-glucose pyrophosphorylase (AGPase) is the rate-limiting enzyme at the pathway initiation, consisting of a small subunit (such as the AgpS gene encoding) and a large subunit (AgpL encoding). Enhancing AGPase activity usually increases starch production, but excessively high activity may disrupt carbon allocation. Particle-binding starch synthase (GBSS), encoded by the Waxy (Wx) gene, is the sole enzyme for amylose synthesis. GBSS is located inside starch granules and is dedicated to catalyzing the extension of amylose. Therefore, the loss of Wx gene function will result in rice grains containing almost no amylose (i.e., sticky, commonly known as "glutinous rice"). The natural variation of the Wx gene directly determines the level of amylose content in rice and is an important genetic factor affecting the taste. The soluble starch synthase (SS) family includes various isoenzymes such as SSI, SSII, SSIII, and SSIV, and their functions are divided: SSI mainly extends short chains, SSIIa prefers to synthesize medium-length chains, and SSIIIa is responsible for the synthesis of longer chains, etc. Among them, SSIIa, also known as the ALKgene in rice, controls the distribution of amylose and amylopectin side chain lengths and has a significant impact on properties such as gel consistency and gelatinization temperature. Allelic variations in SSIIa among different rice varieties (such as the ALK difference between indicarice andjaponicarice) lead to differences in cooking properties, which are utilized in breeding. Amyloidase (SBE) in rice includes SBEI and SBEIIb, etc. SBEIIb is the main branching enzyme in endosperm. The deficiency of SBEIIb will significantly increase the content of amylose and change the structure of amylopectin, resulting in a high amylose/resistant starch phenotype. However, it may also lead to seed shrinkage and poor quality. Starch debranching enzymes (DBE) include two types of enzymes: ISA and PUL. ISA (isoamylase) mainly prunes the branch points of amylopectin to form the double helix chains required for crystallization. Mutations with ISA defects are characterized by loose starch granule structure and powdery endosperm. PUL (pullulanase, also known as rate-limiting starch debranching enzyme) can also remove side chains, but its effect is more limited than that of ISA. Starch phospholysis enzymes may play a role in the initiation or degradation of starch synthesis, and dismutase (DPE) is involved in the transfer equilibrium of short chains such as maltotriose (Sun et al., 2017). The synergistic action of these enzymes ensures normal starch accumulation. Once the function of a key enzyme is lost, it often leads to abnormal composition and structure of starch, which in turn affects the appearance and quality of grains. For instance, mutations in the Wx gene cause the grains to appear opaque and pinkish white, and the absence of ISA also results in a milk-like endosperm. Therefore, these starch synthase coding genes are also major genes affecting rice quality, and their functional analysis and allelic variation utilization are the basis for molecular improvement of rice quality. 2.3 Expression patterns of starch biosynthesis-related genes during grain development Rice starch is mainly synthesized during the grain filling process, and the spatiotemporal expression patterns of related genes determine the changes in enzyme activity at different developmental stages. Overall, most starch synthase genes start to be expressed early after pollination and reach their peak in the middle and late stages to ensure that a large amount of starch accumulates before the grains mature. For instance, the mRNA of the Wx gene begins to accumulate in large quantities 57 days after pollination and maintains high expression until the later stage of grouting. The GBSS protein encoded by it can be detected throughout the grouting period. This is consistent with the process in which amylose is mainly synthesized continuously in the middle and later stages (Ying et al., 2022; Ma, 2024). Among SS genes, SSI and SSIIIa genes are usually strongly expressed in the early and middle stages of grouting, providing enzymatic activity for the rapid synthesis of amylopectin. The expression of the SSIIa gene often persists in the middle and later stages, thereby affecting the formation of longer side chains and the binding of amylose. The expression peak of the branched enzyme SBEIIb gene also occurred in the middle of grouting, and its protein activity was relatively high at 715 days after grouting, and then gradually decreased. This indicates that during the critical period when starch accumulates in large quantities, the activity of branching enzymes is sufficient to ensure the reasonable branching of amylopectin. The expression of the de-branching enzyme ISA gene is relatively early, reaching its peak in the early stage of grouting. The presence of ISA protein
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