Toshihiro Kumamaru

Iron (Fe) deficiency anemia is a significant public health problem worldwide. The development of Fe biofortification in staple food crops, such as rice, through non-transgenic methods is highly anticipated to enhance broad applicability. In this study, we isolated a high Fe-accumulating mutant ( tetsu ) from an N -methyl- N -nitrosourea (MNU) mutagenized ‘Taichung-65 (T65)’ rice population. The tetsu mutant accumulated more than 3-fold higher levels of Fe and significantly higher levels of manganese (Mn) and nickel (Ni) in the shoot than the wild-type (WT) T65, whereas the levels of toxic heavy metals such as cadmium (Cd), lead (Pb), and cobalt (Co) were comparable to those of the WT. In both polished and brown rice of the tetsu mutant, Fe increased by approximately 2-fold, and Zn and Cu also significantly increased compared with those in T65. Perls’ staining revealed that Fe localization in rice grains was not limited to the outer layers and scutellum, but also extended into the endosperm of the tetsu mutant. Concomitant with high Fe accumulation, the tetsu mutant showed remarkable tolerance to alkaline Fe-deficient soil conditions. Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) analysis revealed a novel nonsense mutation in the Hemerythrin motif-containing Ring Zinc-finger protein 1 ( HRZ1 ) gene in the tetsu genome, which is known to govern the negative regulation of the Fe deficiency response and is crucial for normal development. The homozygous tetsu mutation leads to a substantial increase in shoot Fe content, alongside the upregulation of several genes related to Fe uptake and translocation, without causing serious adverse effects on growth. To utilize this novel mutation in Fe-biofortified rice breeding, we created recombinant inbred lines (RILs) derived from crosses between the tetsu mutant and ‘Asamurasaki,’ a nutrient-rich black rice cultivar. During the breeding process, we successfully selected RILs that exhibited normal growth and fertility, resulting in the development of non-transgenic Fe-biofortified rice lines with various waxy/glutinous properties and polyphenol content in brown rice for versatile applications. These results indicate that the identified novel HRZ1 mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars with various beneficial traits.

Key message Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. Abstract In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F₂ lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

Rice (Oryza sativa L.) quality and yield are degraded by high temperature, especially at the ripening stage after the heading of panicles. The effect is lethal when the panicle temperature (Tp) is excessively high; therefore, maintaining a low Tp is important to avoid deleterious impacts on the grains. Microclimatic factors and plant physiological elements determine the Tp. One determining factor is the color (or reflectance) of spikelets that constitute the panicle because it determines the absorption of shortwave radiation energy. An additional factor is the panicle position because it influences heat exchange by the wind and input energy from downward shortwave radiation. In this study, inter-strain differences in spikelet color and panicle height at heading were assessed. The Tp of strains differing in panicle color and panicle height were measured with thermocouples. In addition, to estimate the effect of each trait, we adopted a micrometeorological model. Panicle color was quantified using a hyperspectral sensor. Combining the spectral reflectance and spectral radiation, we assessed the effect of panicle color on Tp. The differences in panicle color and panicle position significantly affected Tp. The strain with a dark panicle had a maximum measured Tp about 1.8 °C higher than that of the strain with a light-colored panicle. The Tp of a strain with panicles at higher positions was up to 2.0 °C higher than that of a strain with panicles at lower positions. These relationships were consistent with the model estimates. When shortwave radiation was strong, the difference in Tp between strains showed a positive correlation, suggesting that the temperature difference was associated with shortwave radiation. Therefore, we concluded that rice strains with a brighter panicle color and low panicle position are less prone to deleterious impacts of high temperature because net radiation is reduced.

Glutelin mRNAs are transported on endosomes through the direct interactions of two RNA-binding proteins with two membrane-trafficking factors.

Localization of mRNAs at the subcellular level is an essential mechanism for specific protein targeting and local control of protein synthesis in both eukaryotes and bacteria. While mRNA localization is well documented in metazoans, somatic cells, and microorganisms, only a handful of well-defined mRNA localization examples have been reported in vascular plants and algae. This review summarizes the function and mechanism of mRNA localization and highlights recent studies of mRNA localization in vascular plants. While the emphasis focuses on storage protein mRNA localization in rice endosperm cells, information on targeting of RNAs to organelles (chloroplasts and mitochondria) and plasmodesmata is also discussed.

In developing rice (Oryza sativa) endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called ‘stay-green’ mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the ‘senescent green leaves’, suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll–protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

Leaf forms are diverse in angiosperms, and different types of cells are differentiated depending on the species. Rice leaves are composed of a leaf blade, a leaf sheath and the junction region between them. Cells with characteristic features, such as bulliform cells and sclerenchyma cells, are differentiated in the leaf blade, together with standard epidermal and mesophyll cells. To understand the genetic mechanism underlying leaf morphogenesis in rice, we focused on a mutant, half-pipe-like leaf1 (hal1), whose leaves are adaxially curled. Histological observation revealed that the bulliform cells, which are responsible for leaf rolling under dry conditions, were small in size and abnormal in shape in a semidominant hal1-d mutant. Bulliform cell files were often ambiguous in semi-transparent hal1-d leaves cleared by the TOMEI method, suggesting that the bulliform cells were undeveloped. Therefore, a reduction in the growth of the bulliform cells seemed to be a major cause of leaf curling in the hal1-d mutant. The hal1-d mutation also affected the size of the leaf blade and the spikelet.