Table_3_Dry-milled flour rice ‘Seolgaeng’ harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2.pdf

‘Seolgaeng’, an opaque-endosperm rice (Oryza sativa) mutant, is used to prepare high-quality dry-milled rice flour. The mutation causing its opaque-endosperm phenotype was unknown. Map-based cloning identified a missense mutation in the gene FRUCTOSE-6-PHOSPHATE 2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE 2 (OsF2KP2) in Seolgaeng. Transfer DNA insertion and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced f2kp2 mutants exhibited opaque endosperm. Rice harbors another F2KP gene, OsF2KP1. CRISPR/Cas9-induced double mutants of OsF2KP1 and OsF2KP2 (f2kp-d) possessed more opaque endosperm compared to f2kp2 single mutants, whereas the endosperm of the f2kp1 single mutant was normal. Grain hardness and damaged starch content were significantly reduced in f2kp2 mutants compared to the wild type and f2kp1. Amylose content was lower than normal in f2kp2 mutants but not f2kp1. Grain hardness and amylose content were much lower in f2kp-d than in f2kp2. Starch polymerization analysis revealed altered amylopectin structure in f2kp2 and f2kp-d mutants. F2KP activity was lower in f2kp2 and much lower in the double mutants when compared to the wild types, but f2kp1 showed no significant difference. In coleoptiles, hypoxia induced OsF2KP2 expression but downregulated OsF2KP1. These results suggest that OsF2KP2 functions as the main F2KP isoform in endosperm experiencing hypoxia, but OsF2KP1 may partially compensate for the absence of OsF2KP2. We propose that F2KP has a crucial role in inorganic pyrophosphate-utilizing energy metabolism for starch biosynthesis in rice endosperm.

‘Seolgaeng’是一种胚乳不透明的栽培稻（Oryza sativa）突变体，常用于制备高品质干法碾磨米粉。其胚乳不透明表型的致变机制此前尚未明确。通过图位克隆技术，研究人员在Seolgaeng的果糖-6-磷酸2-激酶/果糖-2,6-二磷酸酶2（FRUCTOSE-6-PHOSPHATE 2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE 2，OsF2KP2）基因中鉴定到一处错义突变。经转移DNA（Transfer DNA, T-DNA）插入以及成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关核酸酶9（Cas9）诱导获得的f2kp2突变体，均表现出胚乳不透明表型。水稻中还存在另一类果糖-6-磷酸2-激酶/果糖-2,6-二磷酸酶家族基因OsF2KP1。由CRISPR/Cas9诱导获得的OsF2KP1与OsF2KP2双突变体（f2kp-d），其胚乳不透明程度相较于f2kp2单突变体更为显著；而f2kp1单突变体的胚乳表型则与野生型一致。与野生型及f2kp1突变体相比，f2kp2突变体的籽粒硬度与破损淀粉含量均显著降低。f2kp2突变体的直链淀粉含量低于野生型，但f2kp1突变体无此异常变化。f2kp-d双突变体的籽粒硬度与直链淀粉含量则进一步低于f2kp2单突变体。淀粉聚合度分析结果显示，f2kp2与f2kp-d突变体的支链淀粉结构发生了显著改变。酶活性检测表明，相较于野生型，f2kp2突变体的F2KP酶活性有所下降，而双突变体的酶活性下降更为显著；f2kp1单突变体的酶活性则无显著差异。在胚芽鞘中，低氧胁迫会诱导OsF2KP2的表达，但会下调OsF2KP1的转录水平。上述研究结果表明，OsF2KP2是处于低氧环境的胚乳中主要的F2KP同工型基因，而OsF2KP1可部分补偿OsF2KP2缺失所带来的功能缺陷。本研究提出，F2KP在水稻胚乳淀粉生物合成所依赖的、利用无机焦磷酸的能量代谢过程中发挥关键作用。