Additional file 4: Figure S4. of Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice

Total Pchlide and photoactive Pchlide levels determined by UV/VIS spectrophotometry. (PDF 175 kb

Additional file 2: Figure S2. of Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice

Expression levels of OsPORA in the 2nd leaves of OPAO T1 lines. (PDF 125 kb

Additional file 3: Figure S3. of The rice zebra3 (z3) mutation disrupts citrate distribution and produces transverse dark-green/green variegation in mature leaves

Amino acid alignment of Z3 homologs in higher plants. The amino acid sequences of Z3 homologs in higher plants were acquired from NCBI ( http://www.ncbi.nlm.nih.gov /), and the amino acid alignment was obtained using the Clustal Omega EMBL-EBI ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) and BoxShade 3.21 Server ( https://www.ch.embnet.org/software/BOX_form.html ). O. brachyantha, Z. mays_1, Z. mays_2, H. vulgare, S. bicolor, S. tuberosum, G. max, and A. thaliana have 99, 96, 95, 87, 72, 65, 64, and 62% sequence similarity to Z3, respectively. The mutated residue (S542P in z3) is indicated by a red arrowhead. O. sativa_ZEBRA3 (Oryza sativa ZEBRA3, LOC_Os03g05390, NP_001048962.1); O. brachyantha (Oryza brachyantha, XP_006649400.1); Z. mays_1 (Zea mays, NP_001151517.1); Z. mays_2 (Zea mays, ACG43196.1); H. vulgare (Hordeum vulgare, BAK05230.1); S. bicolor (Sorghum bicolor, XP_002467148.1); S. tuberosum (Solanum tuberosum, XP_006363328.1); G. max (Glycine max, XP_003533988.1); A. thaliana (Arabidopsis thaliana, NP_171728.2). (PDF 2499 kb

Additional file 1: Figure S1. of The rice zebra3 (z3) mutation disrupts citrate distribution and produces transverse dark-green/green variegation in mature leaves

Late flowering phenotypes of the z3 mutant. a Flowering phenotypes of the 117-day-old WT and z3 mutant grown under natural long day conditions (14 h light/day, 37o N latitude) in the paddy field. b Days to heading of the WT and the z3 mutant in natural long day conditions. Means and SD were obtained from 15 plants of each genotype. Error bars indicate SD. Differences between means were compared using Student’s t-test (*** P < 0.001). c Comparison of leaf emergence rates between the wild type and the z3 mutants grown under long-day conditions (14.5 h-light/9.5 h-dark) in the growth chamber. Mean and standard deviation values are shown (n = 10). Leaf emergence rate was calculated according to the methods described by Itoh et al. (1998). The average heading dates of the wild type and the z3 mutants are shown by closed and open arrows, respectively. (PDF 961 kb

Additional file 6: Figure S6. of The rice zebra3 (z3) mutation disrupts citrate distribution and produces transverse dark-green/green variegation in mature leaves

Analysis of reactive oxygen species (ROS) in the z3 mutant leaves. a-b Hydrogen peroxide (H2O2) and superoxide anion radicals (O2-) in flag leaves of the WT and z3 mutants at 160 DAS grown under natural long day conditions were visualized by staining with DAB (a) and NBT (b), respectively. Leaves before (left) and after (right) staining are shown. (PDF 497 kb

Additional file 7: Figure S7. of The rice zebra3 (z3) mutation disrupts citrate distribution and produces transverse dark-green/green variegation in mature leaves

Transmission electron microscopy analysis of chloroplasts in the z3 mutant leaves. a-c Chloroplasts in the green leaves of the WT (a) and in the dark-green (b) and green (c) sectors of the z3 mutant leaves. Flag leaves of the 150-day-old WT and z3 mutant grown under natural long day conditions were sampled for analysis. G, grana thylakoid. Scale bars = 0.5 μm. (PDF 955 kb

Additional file 2: Figure S2. of The rice zebra3 (z3) mutation disrupts citrate distribution and produces transverse dark-green/green variegation in mature leaves

Statistical analysis of agronomic traits in the z3 mutant. a–f Agronomic traits were investigated between the WT and the z3 mutant plants grown under natural conditions. Investigated traits are as follows: a main panicle length, b number of panicles per plant, c number of spikelets per main panicle, d seed setting rate, e 500-grain weight, and f yield per plant. Ten plants were used to measure each trait. Values are shown as means. Error bars indicate SD. Student’s t-test was performed for statistical analysis (* P < 0.05, ** P < 0.01, *** P < 0.001). (PDF 2110 kb

Table_1.DOCX

<p>In rice (Oryza sativa), moderate leaf rolling increases photosynthetic competence and raises grain yield; therefore, this important agronomic trait has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism remains unclear. Here, we isolated and characterized Rolled Fine Striped (RFS), a key gene affecting rice leaf rolling, chloroplast development, and reactive oxygen species (ROS) scavenging. The rfs-1 gamma-ray allele and the rfs-2 T-DNA insertion allele of RFS failed to complement each other and their mutants had similar phenotypes, producing extremely incurved leaves due to defective development of vascular cells on the adaxial side. Map-based cloning showed that the rfs-1 mutant harbors a 9-bp deletion in a gene encoding a predicted CHD3/Mi-2 chromatin remodeling factor belonging to the SNF2-ATP-dependent chromatin remodeling family. RFS was expressed in various tissues and accumulated mainly in the vascular cells throughout leaf development. Furthermore, RFS deficiency resulted in a cell death phenotype that was caused by ROS accumulation in developing leaves. We found that expression of five ROS-scavenging genes [encoding catalase C, ascorbate peroxidase 8, a putative copper/zinc superoxide dismutase (SOD), a putative SOD, and peroxiredoxin IIE2] decreased in rfs-2 mutants. Western-blot and chromatin immunoprecipitation (ChIP) assays demonstrated that rfs-2 mutants have reduced H3K4me3 levels in ROS-related genes. Loss-of-function in RFS also led to multiple developmental defects, affecting pollen development, grain filling, and root development. Our results suggest that RFS is required for many aspects of plant development and its function is closely associated with epigenetic regulation of genes that modulate ROS homeostasis.</p

Image_1.PDF

<p>In rice (Oryza sativa), moderate leaf rolling increases photosynthetic competence and raises grain yield; therefore, this important agronomic trait has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism remains unclear. Here, we isolated and characterized Rolled Fine Striped (RFS), a key gene affecting rice leaf rolling, chloroplast development, and reactive oxygen species (ROS) scavenging. The rfs-1 gamma-ray allele and the rfs-2 T-DNA insertion allele of RFS failed to complement each other and their mutants had similar phenotypes, producing extremely incurved leaves due to defective development of vascular cells on the adaxial side. Map-based cloning showed that the rfs-1 mutant harbors a 9-bp deletion in a gene encoding a predicted CHD3/Mi-2 chromatin remodeling factor belonging to the SNF2-ATP-dependent chromatin remodeling family. RFS was expressed in various tissues and accumulated mainly in the vascular cells throughout leaf development. Furthermore, RFS deficiency resulted in a cell death phenotype that was caused by ROS accumulation in developing leaves. We found that expression of five ROS-scavenging genes [encoding catalase C, ascorbate peroxidase 8, a putative copper/zinc superoxide dismutase (SOD), a putative SOD, and peroxiredoxin IIE2] decreased in rfs-2 mutants. Western-blot and chromatin immunoprecipitation (ChIP) assays demonstrated that rfs-2 mutants have reduced H3K4me3 levels in ROS-related genes. Loss-of-function in RFS also led to multiple developmental defects, affecting pollen development, grain filling, and root development. Our results suggest that RFS is required for many aspects of plant development and its function is closely associated with epigenetic regulation of genes that modulate ROS homeostasis.</p