Allene oxide cyclase (AOC) is a key enzyme in the jasmonic acid (JA) biosynthetic pathway in plants, during which it catalyzes stereospecific conversion of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT) to cis(+)-12-oxophytodienoic acid. Here, rice allene oxide cyclase (OsAOC) was localized to the chloroplast and its native oligomeric structure was analyzed by gel electrophoresis in the absence and presence of a protein-crosslinking reagent. The results suggest that OsAOC exists in solution as a mixture of monomers, dimers, and higher order multimers. OsAOC preferentially exists as dimer at room temperature, but it undergoes temperature-dependent partial denaturation in the presence of SDS. A heteromeric 2:1 complex of OsAOC and rice allene oxide synthase-1 (OsAOS1) was detected after cross-linking. The yield of cis(+)-12-oxophytodienoic acid reached maximal saturation at a 5:1 molar ratio of OsAOC to OsAOS1, when OsAOC and OsAOS1 reactions were coupled. These results suggest that the OsAOC dimer may facilitate its interaction with OsAOS1, and that the heteromeric 2:1 complex may promote efficient channeling of the unstable allene oxide intermediate during catalysis. In addition, conceptual similarities between the reaction catalyzed by AOC and Nazarov cyclization are discussed.

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (k_cat/K_m) and the Michaelis–Menten constant (K_m) of immobilized lipoxygenase were 21% and 49% of k_cat/K_m and K_m of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.