Molecular Plant Breeding 2024, Vol.15, No.2, 63-69 http://genbreedpublisher.com/index.php/mpb 64 synthesis of ADP glucose, comprising two large (ApL) and two small (ApS) catalytic subunits; starch branching enzyme (SBE) to produce branches connected by a-1,6-glycoside bonds, including SBEI (SBE1), SBEII, and SBEIII; starch debranching enzyme (DBE) hydrolyzing -(1,6)-linkages, with three isoforms of isoamylase-type DBE and one pullulanase-type DBE; soluble starch synthase (SS) catalyzing the transfer of glucose from ADP-glucose to an acceptor glucan chain and involving solely in amylopectin synthesis, with 4 classes of SSI to SSIV; granule bound starch synthase (GBSS) involving amylose biosynthesis, with GBSSI and GBSSII; phosphoglucoisomerase converting fructose 6-phosphate to glucose 6-phosphate; phosphoglucomutase converting glucose 6-phosphate to glucose 1-phosphate; and starch phosphorylase responsible for glucan-elongation reactions (Orzechowski, 2008; Keeling and Myers, 2010; Tetlow and Emes, 2014; Li and Gilbert, 2016; Huang et al., 2021). In these enzymes, AGPase is considered as a rate-limiting enzyme responsible for the synthesis of ADP-glucose in the first and key step of starch biosynthesis (Ihemere et al., 2006). 3 Complexity of Cassava Starch Biosynthesis According to current research in other plants, each enzyme has multiple isoforms (Tappiban et al., 2019), and the enzyme activity and function of the isormors are not entirely the same (Ohdan et al., 2005; Keeling and Myers, 2010; Kötting et al., 2010; Li and Gilbert, 2016; Huang et al., 2021). The expression of some starch biosynthesis genes such as AGPases have been found in both source (leaves) and sink (seeds) organs of rice, and the gene expression modes are tissue and developmental stage-specific (Ohdan et al., 2005). The enzymes’ function depends on the formation of protein complexes (Keeling and Myers, 2010; Cho and Kang, 2020), showing protein–protein interactions. Cassava is considered one of orphan crops that are also known as underutilized crops, lost crops, neglected crops, or crops for the future (Tadele, 2019; Zambrano et al., 2022). The research on genetic background and molecular mechanisms controlling many traits in cassava is still insufficient. The investigation of the mechanism of cassava starch synthesis is currently only in the age of enlightenment. The 98 known wild species of the New World genus Manihot have been found, which are extremely heterogeneous for any particular genotype. Cassava is an outbreeding species (2n=36 chromosomes) and considered to be an amphidiploid or sequential allopolyploids, and asexually propagated by mature woody stem cuttings (El-Sharkawy, 2004). Recently, a total of 45 genes participating in starch biosynthesis in cassava (Tappiban et al., 2019), including AGPase, GBSS, SS, SBE, DBE, and glucan, water dikinase (GWD). The starch synthesis of cassava may be much more complex than expected and may also have its unique characteristics. With 6 field-grown cultivars and 1 wild species, we have found that starch synthesis-related enzymes have multiple active isoforms in cassava. The types of the active isoforms varied depending on the cultivars. The same active isoforms varied greatly with the roots, stems, and leaves of the same and different cultivars with the growth stage. What is even more confusing was that it was hard to associate these corresponding changes with the starch accumulation (unpublished). These factors together will undoubtedly make cassava starch biosynthesis processes more complex than existing paradigms/frameworks proposed in other plant species. 4 Cases of Engineering Cassava Starch Efforts have been made to improve cassava starch yield and alter starch properties by regulating the expression of starch biosynthesis-related genes through gene engineering, with several cases. Expressing AGPase genes usually enhances starch production of plants including cassava in most cases but were found to have no impacts on starch production in rare cases, and even generated unexpected results with respect to yield components including starch content (Tuncel and Okita, 2013). For example, transgenic cassava expressing AGPase-encoding glgC gene of bacterial Escherichia coli showed slight decrease in root starch contents (mg per gram fresh weight) (Ihemere et al., 2006), 151 for wild type cassava and 143 (149 and 138) for transgenic cassava. Overexpressing AGPase gene in cereals increased starch yield, and meantime, resulted in increases in seed number and plant biomass (Tuncel and Okita, 2013). Suppression of GBSSI gene expression caused the reduced amylose content but increased values for clarity, peak viscosity, gel breakdown, and swelling index (Zhao et al., 2011). CRISPR-Cas9-mediated targeted mutagenesis of PROTEIN TARGETING TO STARCHor GBSS gene reduced or eliminated amylose content in root starch of cassava (Bull et al., 2018). Silencing expression of SBE1 and SBE2 by short interfering RNAs-mediated
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