Fig. 7 Peptide mass fingerprinting and microsequencing analysis of recombinant OsAsp1. (A) MALDI-TOF spectrum of 60 kDa thioredoxin–proOsAsp1 fusion protein. A Mascot search indicated that it significantly matched OsAsp1 deposited in GenBank and E. coli thioredoxin. (B) MALDI-TOF spectrum of the 40 kDa fragment of the OsAsp1 mature peptide. A Mascot peptide fingerprint search showed that it significantly matches OsAsp1 deposited in GenBank; the first five cycle N-terminal sequences were DPAKP, corresponding to amino acids 46–50 of preproOsAsp1. (C) MALDI-TOF spectrum of the 19 kDa fragment for the thioredoxin fusion tag plus OsAsp1 propeptide. A Mascot search indicated significant matches for E. coli thioredoxin, and the microsequencing results indicated that the first five cycle amino acid sequences matched SDKII of the tag sequence (see text). From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 1 OsAsp1 gene structure showing the promoter, intron/exon arrangement, 3′- untranslated region. Exons are boxed with shading, and start and stop positions are indicated. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 2 Southern blot analysis and chromosomal location of OsAsp1 Fig. 2 Southern blot analysis and chromosomal location of OsAsp1. (a) Southern blot probed with full-length OsAsp1 gene cDNA. Under low and high wash stringency, only one copy of the OsAsp1 gene was detected in the rice genome when DNA was cut with a variety of enzymes. Here HindIII was used as it does not cut within the OsAsp1 gene and showed restriction fragment length polymorphism between IR64 and Azucena. (b) Chromosomal location of the OsAsp1 gene in rice. Using an IR64×Azucena doubled haploid mapping population, OsAsp1 was mapped on the short arm of chromosome 11, at a distance of 6.5 cM from marker RG118. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 2 Southern blot analysis and chromosomal location of OsAsp1 Fig. 2 Southern blot analysis and chromosomal location of OsAsp1. (a) Southern blot probed with full-length OsAsp1 gene cDNA. Under low and high wash stringency, only one copy of the OsAsp1 gene was detected in the rice genome when DNA was cut with a variety of enzymes. Here HindIII was used as it does not cut within the OsAsp1 gene and showed restriction fragment length polymorphism between IR64 and Azucena. (b) Chromosomal location of the OsAsp1 gene in rice. Using an IR64×Azucena doubled haploid mapping population, OsAsp1 was mapped on the short arm of chromosome 11, at a distance of 6.5 cM from marker RG118. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 3 Relationship of barley nucellin to homologs from rice and other plants. (a) Amino acid sequence alignment of barley nucellin, OsAsp1-3 and homologs from other plants. Gaps were inserted to maximize the similarities. Identical conserved amino acid residues are highlighted in black, asterisks show catalytic motif sites for aspartyl proteases, and the peroxisome targeting motif is boxed. (b) Phylogenetic relationship between OsAsp1, barley nucellin and nucellin-like proteins from other plants. The cladogram illustrates the most parsimonious consensus pattern of relationships obtained using maximum parsimony analysis. Bootstrap values generated with 1,000 replicates are indicated before the nodes. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 3 Relationship of barley nucellin to homologs from rice and other plants. (a) Amino acid sequence alignment of barley nucellin, OsAsp1-3 and homologs from other plants. Gaps were inserted to maximize the similarities. Identical conserved amino acid residues are highlighted in black, asterisks show catalytic motif sites for aspartyl proteases, and the peroxisome targeting motif is boxed. (b) Phylogenetic relationship between OsAsp1, barley nucellin and nucellin-like proteins from other plants. The cladogram illustrates the most parsimonious consensus pattern of relationships obtained using maximum parsimony analysis. Bootstrap values generated with 1,000 replicates are indicated before the nodes. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 4 OsAsp1 transcript expression profile Fig. 4 OsAsp1 transcript expression profile. (a) OsAsp1 gene expression pattern detected by northern blot. OsAsp1 transcripts appeared most abundant in 2 DAF spikelets, detectable at 3–5 DAF in IR64 spikelets and 0–2 DAF in V20B (normal fertile rice), and only at 0 DAF in V20A (cytoplasmic male sterile rice) but not in the 2 DAF spikelets. Samples (20 µg /lane) were separated by agarose gel electrophoresis, transferred to a Hybond N⁺ nylon membrane, and probed with ³²P-labeled OsAsp1 cDNA. The bottom panel indicates an ethidium bromide-stained gel after electrophoresis, which demonstrated that the rRNA bands were of similar intensity among the lanes. (b) OsAsp1 gene expression pattern detected by RT–PCR in different tissues and different rice lines. DNase I-treated total RNA was used as template for RT–PCR. OsAsp1 is expressed in the spikelets from –3 DAF to 6 DAF, with the greatest abundance at 1–4 DAF. The 2 DAF spikelets of V20A (cytoplasmic male-sterile rice) obviously have a lower level expression than those of V20B (normal fertile rice); IR64 spikelets emasculated 1 d before flowering (labeled as 2DAF*) did not yield any detectable transcripts at 2 DAF. (A) OsAsp1-specific primers; the forward primer is from the 5′-UTR, while the reverse primer joins two exons and is therefore able to amplify mRNA but not genomic DNA. (B) Rice GAPDH-specific primers, which amplified IR64 genomic DNA. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 4 OsAsp1 transcript expression profile Fig. 4 OsAsp1 transcript expression profile. (a) OsAsp1 gene expression pattern detected by northern blot. OsAsp1 transcripts appeared most abundant in 2 DAF spikelets, detectable at 3–5 DAF in IR64 spikelets and 0–2 DAF in V20B (normal fertile rice), and only at 0 DAF in V20A (cytoplasmic male sterile rice) but not in the 2 DAF spikelets. Samples (20 µg /lane) were separated by agarose gel electrophoresis, transferred to a Hybond N⁺ nylon membrane, and probed with ³²P-labeled OsAsp1 cDNA. The bottom panel indicates an ethidium bromide-stained gel after electrophoresis, which demonstrated that the rRNA bands were of similar intensity among the lanes. (b) OsAsp1 gene expression pattern detected by RT–PCR in different tissues and different rice lines. DNase I-treated total RNA was used as template for RT–PCR. OsAsp1 is expressed in the spikelets from –3 DAF to 6 DAF, with the greatest abundance at 1–4 DAF. The 2 DAF spikelets of V20A (cytoplasmic male-sterile rice) obviously have a lower level expression than those of V20B (normal fertile rice); IR64 spikelets emasculated 1 d before flowering (labeled as 2DAF*) did not yield any detectable transcripts at 2 DAF. (A) OsAsp1-specific primers; the forward primer is from the 5′-UTR, while the reverse primer joins two exons and is therefore able to amplify mRNA but not genomic DNA. (B) Rice GAPDH-specific primers, which amplified IR64 genomic DNA. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 5 RNA in situ detection of OsAsp1 in rice and nucellin in barley Fig. 5 RNA in situ detection of OsAsp1 in rice and nucellin in barley. (a) OsAsp1 mRNA spatial expression detected by RNA in situ hybridization. Paraffin-embedded callus and ovary sections were probed with DIG-labeled antisense RNA transcribed from OsAsp1 cDNA. Transcripts, shown as purple signals, were detected in the anther, nucellar tissues and immature somatic and zygotic embryos. (A) Anthers at flowering stage. OsAsp1 is expressed in the anther wall and pollen. (B) Embryogenic calli. OsAsp1is expressed in the early somatic embryo. (C) Ovary 1 d before flowering. OsAsp1 is expressed in the nucellar cells, integuments and ovary walls. (D) Ovary at 1 DAF. OsAsp1 is expressed mainly in the embryo and nucellar tissues. (E and F) Ovary at 2–4 DAF. OsAsp1 is most strongly expressed in the immature embryo, and weakly in the nucellar tissues, and aleurone layer. (G) Ovary at 5 DAF. OsAsp1 is expressed in the embryo, being more intensive in the radical region (root primordium). (H) Ovary at 10 DAF. OsAsp1 appears in the coleoptile, shoot and root primordia region (plumule and radicle). (b) Barley nucellin gene spatial expression detected by in situ RNA hybridization. HvNucellin transcripts appeared in the early embryo as well as the nucellus before and after pollination. A barley antisense nucellin cDNA fragment generated by RT–PCR from the ovary before pollination was in vitro transcribed, labeled with DIG and detected with NBT and BCIP. (A) Ovary before pollination hybridized with the antisense RNA probe. Hybridization signals were detected in nucellar cells. (B) Ovary 2 days after pollination hybridized with the antisense RNA probe. Strong hybridization signals were detected in the nucellar cells and early embryo. AW, anther wall; AL, aleurone layer; Co, coleoptile; Em, embryo; En, endosperm; ES, embryo sac; In, integument; Nu, nucellus; Ov, ovule; OW, ovary wall; Pl, plumule; Ra, radicle; Sc, scutellum; Se, somatic embryo; STM, shoot meristem; Bar, 50 µm. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 5 RNA in situ detection of OsAsp1 in rice and nucellin in barley Fig. 5 RNA in situ detection of OsAsp1 in rice and nucellin in barley. (a) OsAsp1 mRNA spatial expression detected by RNA in situ hybridization. Paraffin-embedded callus and ovary sections were probed with DIG-labeled antisense RNA transcribed from OsAsp1 cDNA. Transcripts, shown as purple signals, were detected in the anther, nucellar tissues and immature somatic and zygotic embryos. (A) Anthers at flowering stage. OsAsp1 is expressed in the anther wall and pollen. (B) Embryogenic calli. OsAsp1is expressed in the early somatic embryo. (C) Ovary 1 d before flowering. OsAsp1 is expressed in the nucellar cells, integuments and ovary walls. (D) Ovary at 1 DAF. OsAsp1 is expressed mainly in the embryo and nucellar tissues. (E and F) Ovary at 2–4 DAF. OsAsp1 is most strongly expressed in the immature embryo, and weakly in the nucellar tissues, and aleurone layer. (G) Ovary at 5 DAF. OsAsp1 is expressed in the embryo, being more intensive in the radical region (root primordium). (H) Ovary at 10 DAF. OsAsp1 appears in the coleoptile, shoot and root primordia region (plumule and radicle). (b) Barley nucellin gene spatial expression detected by in situ RNA hybridization. HvNucellin transcripts appeared in the early embryo as well as the nucellus before and after pollination. A barley antisense nucellin cDNA fragment generated by RT–PCR from the ovary before pollination was in vitro transcribed, labeled with DIG and detected with NBT and BCIP. (A) Ovary before pollination hybridized with the antisense RNA probe. Hybridization signals were detected in nucellar cells. (B) Ovary 2 days after pollination hybridized with the antisense RNA probe. Strong hybridization signals were detected in the nucellar cells and early embryo. AW, anther wall; AL, aleurone layer; Co, coleoptile; Em, embryo; En, endosperm; ES, embryo sac; In, integument; Nu, nucellus; Ov, ovule; OW, ovary wall; Pl, plumule; Ra, radicle; Sc, scutellum; Se, somatic embryo; STM, shoot meristem; Bar, 50 µm. From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |

Fig. 6 SDS–PAGE analysis of recombinant OsAsp1 expression and its autolysis in vitro. Recombinant OsAsp1 expressed as a thioredoxin–proOsAsp1 fusion protein (60 kDa) was absent in media without IPTG (lane 1), but present in the induction media with 1 mM IPTG (lane 2). Affinity-purified recombinant thioredoxin–proOsAsp1 fusion protein contaminated with bacterial proteins (lane 3); after dialysis in 50 mM acetate buffe (pH 3.5) overnight at 4°C, the 60 kDa fusion protein thioredoxin–proOsAsp1 was autocleaved into 40 and 19 kDa fragments (lane 4). More mature peptide (40 kDa) and fusion tag plus propeptide (19 kDa) were generated when autolysed at room temperature for 24 h in 100 mM acetate buffer at pH 3.5 (lane 5). From: The Rice Nucellin Gene Ortholog OsAsp1 Encodes an Active Aspartic Protease Without a Plant-specific Insert and is Strongly Expressed in Early Embryo Plant Cell Physiol. 2005;46(1):87-98. doi:10.1093/pcp/pci002 Plant Cell Physiol |