AUTHOR=Kang Ju-Won , Lee Sang-Kyu , Shim Su-Hyeon , Shin Dongjin , Cho Jun-Hyeon , Lee Ji-Yoon , Ko Jong-min , Ji Hyeonso , Park Hyang-Mi , Ahn Eok-Keun , Lee Jong-Hee , Jeon Jong-Seong 

TITLE=Dry-milled flour rice ‘Seolgaeng’ harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1231914

DOI=10.3389/fpls.2023.1231914

ISSN=1664-462X

ABSTRACT=<p>‘Seolgaeng’, an opaque-endosperm rice (<italic>Oryza sativa</italic>) 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 <italic>FRUCTOSE-6-PHOSPHATE 2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE 2</italic> (<italic>OsF2KP2</italic>) in Seolgaeng. Transfer DNA insertion and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced <italic>f2kp2</italic> mutants exhibited opaque endosperm. Rice harbors another <italic>F2KP</italic> gene, <italic>OsF2KP1</italic>. CRISPR/Cas9-induced double mutants of <italic>OsF2KP1</italic> and <italic>OsF2KP2</italic> (<italic>f2kp-d</italic>) possessed more opaque endosperm compared to <italic>f2kp2</italic> single mutants, whereas the endosperm of the <italic>f2kp1</italic> single mutant was normal. Grain hardness and damaged starch content were significantly reduced in <italic>f2kp2</italic> mutants compared to the wild type and <italic>f2kp1</italic>. Amylose content was lower than normal in <italic>f2kp2</italic> mutants but not <italic>f2kp1</italic>. Grain hardness and amylose content were much lower in <italic>f2kp-d</italic> than in <italic>f2kp2</italic>. Starch polymerization analysis revealed altered amylopectin structure in <italic>f2kp2</italic> and <italic>f2kp-d</italic> mutants. F2KP activity was lower in <italic>f2kp2</italic> and much lower in the double mutants when compared to the wild types, but <italic>f2kp1</italic> showed no significant difference. In coleoptiles, hypoxia induced <italic>OsF2KP2</italic> expression but downregulated <italic>OsF2KP1</italic>. 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.</p>