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Fig. 4 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 4. Phylogenetic and exon-intron structure analysis of FBN family genes in rice and Arabidopsis. The phylogenetic analysis was performed using the amino acid sequences of 25 FBN family proteins in rice and Arabidopsis (Left). The unrooted phylogenetic tree was generated by the neighbor-joining method and displayed using MEGA7 software. Numbers above or below branches of the evolution tree indicated the bootstrap values; The exon-intron structures of 25 FBN family genes in rice and Arabidopsis were analyzed (Right), the exons were represented by yellow boxes, the UTR by blue, black lines connecting two exons were introns. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 6. The in vitro lipid binding assays of PAP-domains. A and C, schematic representations of the mutant residues in OsFBN2 and OsFBN4 proteins in the in vitro lipid binding assays. B and D, the in vitro lipid-binding activity assays were performed by a fluorescence lipid probe P-96 (1 mM), fatty acids with C12:0, C14:0, C16:0, C18:0, C20:0, C22:0, with each (0.5 mM) of wild type (Wt) and mutant fusion protein (Mut) of PAP domains in OsFBN2 and OsFBN4 proteins, respectively. Detection experiments were performed with three biological replicates. * and ** symbols indicated the significant difference levels of P <0.05 or P <0.01 between mutant and wild type, respectively.

Fig. 5 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 5. Subcellular localization of CTP domains-fused GFP proteins. These constructs of p35S-FBN1-11-CTP-GFP or p35S-Trxm5-GFP, separately, were transiently expressed in Nicotiana benthamiana mesophyll cells. The co-localizations of fluorescence patterns of CTP domains of ten OsFBN proteins except OsFBN8 fused GFP were observed with the autofluorescence of chlorophyll in chloroplasts in protoplasts. The Trxm5-GFP was used as a chloroplast marker.

Fig. 3 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 3. PKC domain analysis of FBN11 clade in plants. A, Homology analysis of PKC domain sequences in FBN11 clades; B, evolutionary tree analysis of PKC domain in five plant species (Oryza sativa (Os), Selaginella moellendorffii (Smo), Arabidopsis thaliana (At), Glycine max (Gm), and Zea mays (Zm)) and algae (Monoraphidium neglectum (Mon), Chlorella sorokiniana (Chlo), Chlamydomonas sp. UWO 241 (Chla), and Haemato coccus lacustris (Hae); C, the conserved motif analysis of PKC domain in FBN11 protein clades in higher plants.

Fig. 2 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 2. Box-plot graphs and motif analysis of CTP and PAP-domain in plants. The protein sequences of CTP domains and PAP domains were analyzed in six plant species: Selaginella moellendorffii (Smo), Physcomitrella patens (Ppa), Arabidopsis thaliana (At), Glycine max (Gm), Oryza sativa (Os), and Zea mays (Zm), The grand average of hydropathicity (GRAVY) and isoelectric point (pI) of CTP domains of FBN1-11 protein clades were showed in A and B, respectively; The GRAVY and pI of PAP domains in FBN1-11 protein clades were showed in C and D, respectively; The dotted line represented the average values of GRAVY and pI, respectively; The conserved motif sequence of PAP domains in these plants were showed in E.

Fig. 1 in Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice

Fig. 1. Phylogenetic tree analysis of 121 fibrillin family proteins from 10 representative species. The neighbor-joining tree was constructed using the sequences of 121 fibrillin family proteins in ten representative species: Synechococcus sp. PCC 7002 (Ssp), Erysimum siliculosus (Esi), Emiliania huxleyii (Ehu), Thalassiosira pseudonana (Tps), Chlamydomonas reinhardtii (Cre), Volvox carteri (Vca), Physcomitrella patens (Ppa), Selaginella moellendorffii (Smo), Arabidopsis thaliana (At) and Oryza sativa (Os). Bootstrap values were indicated on the branches. Each fibrillin protein was labeled by its name (Table S3). The green and red color meant the proteins present in algae or terrestrial plant, respectively; the blue color represents the proteins present in both algae and terrestrial plants. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 4. Product analysis of LoTPS4 produced from GGPP and FPP. (a) GC-MS analysis (ion chromatogram) of the LoTPS4 enzyme product from GGPP. (b) GC-MS analysis (ion chromatogram) of products obtained from assays with LoTPS4 and FPP. (c, d, e, f) Mass spectra of Peak 1, Peak 2, Peak 3 and Peak 4. Di-epi-α-cedrene (Peak 1), α-cubebene (Peak 2): trans-α-bergamotene (Peak 3) and (E)-β-farnesene (Peak 4).

Fig. 3 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 3. (a) GC-MS analysis of products produced from assays with extracts of the empty vector and GPP. (b) Analysis (total ion chromatogram) of the product of the LoTPS4 enzyme generated from GPP (c) Total ion chromatogram of the D-limonene authentic standard. (d) Mass spectrum of the Peak 3 (e) Mass spectrum of Dlimonene in the floral scent of Lilium 'Siberia' (f) Mass spectrum of the D-limonene authentic standard (g, h, I and j) Mass spectra of Peak 1, Peak 2, Peak 4 and Peak 5. β-Phellandrene (Peak 1), β-myrcene (Peak 2), D-limonene (Peak 3), 3-Carene (Peak 4) and (+)-4-Carene (Peak 5).

Fig. 10 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 10. Subcellular localization of LoTPS2 and LoTPS4. Confocal laser scanning microscopy of LoTPS2 and LoTPS4 was performed by using EGFP fusion proteins in Arabidopsis protoplasts. The full-length coding region and the N-terminal 80 amino acids of the coding region of LoTPSs were fused to the GFP reporter gene in the p35 S vector to generate the p35 S-LoTPSs/GFP construct. The red column shows chlorophyll autofluorescence; the green column shows GFP fluorescence; the merged columns show combined GFP fluorescence and chlorophyll autofluorescence; and the BF columns represent bright field images. The names of the constructs are given on the left. HcTPS7 from H. coronarium was used as a marker. Scale bars: 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 2. GC-MS analysis of products generated by LoTPS2. (a) GC-MS analysis (total ion chromatogram) of the products obtained by incubating extracts of the empty vector (control) with geranyl pyrophosphate. (b) GC-MS analysis (total ion chromatogram) of the LoTPS2 enzyme with GPP. (c) GC–MS analysis (total ion chromatogram) of the LoTPS2 enzyme with farnesyl pyrophosphate showing (E, E)-α- farnesene as the sole product. (d) Mass spectrum of the peak. (e) Mass spectrum of (E, E)-α-farnesene in the NIST08 library.

Fig. 7 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 7. Relative expression analysis of LoTPS2 and LoTPS4 during different flower development performed by quantitative real-time PCR. (a, b) Transcript levels of the LoTPS2 and LoTPS4 genes during flower development. (c, d) Relative expression analysis of LoTPS2 and LoTPS4 at different flower developmental stages. Flower development was divided into 5 different stages: D1 (bud stage), D2 (little open), D3 (half open), D4 (full-bloom), D5 (senescence). GAPDH was used as an internal control. The root was set as 1. Data are presented as the mean ± SEM (n = 3).

Fig. 8 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 8. Relative expression analysis of LoTPS genes and the emission of (E, E)-α-farnesene and D-limonene from Lilium 'Siberia' at different time intervals. (a, b) Expression pattern of LoTPS2 and LoTPS4 for 3 days postanthesis. (c) Emission pattern of (E, E)-α-farnesene from Lilium 'Siberia' (d) Emission pattern of D-limonene from Lilium 'Siberia' flowers over 3 days after full-bloom. The plants were kept under a 12 h light, 12 h dark photoperiod. GAPDH was used as an internal control. Each point is the average of 3 replicates.

Fig. 6 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 6. (a) A labeled diagram of a fullbloom Lilium 'Siberia' flower. (b) Pictorial view of Lilium 'Siberia' flowers at different flower developmental stages.

Fig. 5 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 5. Relative gene expression analysis of LoTPS2 and LoTPS4 in floraland vegetative tissues of Lilium 'Siberia' (a) relative expression levels of LoTPS2 in different tissues of Lilium 'Siberia' (b) Relative expression levels of LoTPS4 in different tissues of Lilium 'Siberia' analyzed by qRT-PCR. (c, d) Relative expression levels of LoTPS2 and LoTPS4 in full-bloom flowers of different Lilium species. GAPDH was used as an internal control. The highest expression level was set as 1 (100%). Lon: Longiflorum; Bru: Brunello; Sib: Siberia; Aca: Acapulco; Sor: Sorbonne; Man: Manissa. Data are presented as the mean ± SEM (n = 4).

Fig. 1 in Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'

Fig. 1. (a) Alignment of the amino acid sequences of LoTPS2 and LoTPS4, with AdAFS1 from Actinidia deliciosa (FJ265785) and (+)-limonene synthase (Q8L5K3) from Citrus limon. The protein sequences were aligned using ClustalX 2.1 and edited with GeneDoc. RRX8W motifs were present in LoTPS4 and (+)-limonene synthase but were missing in both LoTPS2 and AdAFS1. The conserved RRX8W, DDXXD, and RxR domains are underlined. (b) N-terminal sequence alignment of TPS-f clade terpene synthases. AdAFS1; CbLIS2 (C. breweri, AAD19840); CbLIS (C. breweri, AAC49395); CcLIS (Clarkia concinna, AAD19839). The CDIS (conifer diterpene internal sequence) is indicated by a dotted line. Dashes indicate gaps inserted for optimal alignment. (c) Phylogenetic analysis of LoTPS2 and LoTPS4 from Lilium 'Siberia' with amino acid sequences of other selected terpene synthases. The alignment was performed using ClustalX 2.1, and the tree was built via the neighborjoining method using the MEGA 6 program and iTOL (http://itol.embl.de/). The dot size at the branches of the tree shows bootstrap values. Accession numbers are given in a Supplementary Table 1.

Fig. 8. Proposed structures for 1 in Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Fig. 8. Proposed structures for 1:1:1 complexes between genistin, iron, and galacturonic acid (left) or digalacturonic acid (right).

Fig. 5 in Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Fig. 5. UHPLC separation of color-enriched seed coat extracts from event DAS-411Ø4-7 and non-transgenic control with UV absorbance detection at 272 nm. Red trace, DAS-411Ø4-7; blue trace, non-transgenic control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6 in Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Fig. 6. LC-MS quantitation of genistin (brown) and genistein (blue) content of color-enriched seed coat fractions from event DAS-411Ø4-7 and non-transgenic control. Samples were either untreated in water, heated at 95 °C in water or heated at 95 °C in 2 N HCl. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3 in Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Fig. 3. UV–visible absorption spectra of color-enriched fractions. (A) Absorbance spectra of 3 mg/mL color-enriched seed coat extracts (brown, event DAS-411Ø4-7; blue, non-transgenic control) dissolved in water (solid lines), or with the addition of 100 mM acetate (long dashes) or EDTA (short dashes). (B) Difference spectra of event DAS-411Ø4-7 (brown) and non-transgenic control (blue) samples dissolved in water or with 100 mM EDTA added. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Fig. 2. Enrichment of seed coat coloration (SCC) from non-transgenic Maverick and event DAS-411Ø4-7 soybean seeds.