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Fig. 5 in MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza

Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3.

Fig. 4 in MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza

Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student's t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines.

Fig. 3 in MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza

Fig. 3. Tissue-specific expression analysis and elicitors-induced analysis of SmMAPK3 in S. miltiorrhiza. (A) Tissue-specific expression of SmMAPK3; the expression levels were normalized to values from roots. (B) SA-induced analysis of SmMAPK3. (C) MeJA-induced analysis of SmMAPK3.

Fig. 1 in MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza

Fig. 1. Identification and autophosphorylation of SmMAPK3 in S. miltiorrhiza. (A) Amplication of SmMAPK3 from S. miltiorrhiza. (B) Phylogenic tree analysis of SmMAPK3 with AtMAPKs. (C) The conserved domains of SmMAPK3. (D) Immunoblotting analysis of SmMAPK3 autophosphorylation in vitro with Phos-tag™ SDS–PAGE. Phosphorylated SmMAPK3 (pSmMAPK3) migrates more slowly in the gel.

Fig. 2. SmMAPK3 in MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza

Fig. 2. SmMAPK3 is associated with the biosynthesis of phenolic acids. (A) Expression patterns of phenolic acid biosynthetic genes in 18 samples. (B) Network built on correlations among kinases, structural genes and TFs. Pearson correlation coefficient (PCC) values were calculated for each pair of genes.

Fig. 8 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 8. The expression level of drought-related genes in NtCOMT1 overexpressing lines, WT, and OE-Empty tobacco after drought treatment. Data are means ±SE of three biological replicates and means followed by different letters are significantly different (p <0.05).

Fig. 7 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 7. Histochemical staining with DAB for detection of H2O2 (A), the content of H2O2 (B) and MDA (C), and the antioxidant enzyme activities of CAT (D) and SOD (E) in NtCOMT1 overexpressing lines, WT, and OE-Empty tobacco after drought treatment. Data are means ± SE of three biological replicates and means followed by different letters are significantly different (p <0.05).

Fig. 5. NtCOMT1 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 5. NtCOMT1 gene expression profiles in different tissues (A) and under abiotic stresses (B). Growth of NtCOMT1 overexpressing lines, WT, and OE-Empty tobacco after drought treatment for 9 days (C). Expression levels in different tissues are shown: root, stem, leaf, flower, and fruit. Expression levels under cold stress (3h, 6h, and 24h), heat stress (3h, 6h, and 9h), drought stress (1day, 5day, and 9day), and cadmium stress treatment (1day, 4day, and 7day). Data represent mean ± SE of three independent biological replicates. Asterisks indicate statistically significant differences determined using Student' s t-test (**, p <0.01; ***, p <0.001).

Fig. 4 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 4. Subcellular localization of the NtCOMT1 proteins in N. benthamiana leaves. (A) 35S-GFP alone was used as the control. (B) NtCOMT1 proteins fused with GFP (35S: NtCOMT1-GFP). (C) Fluorescence of DAPI. (D) merge of 35S: NtCOMT1-GFP and DAPI images.

Fig. 3 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 3. Characterization of NtCOMT1 enzymatic activity. (A) Schematic diagram of melatonin biosynthesis mediated by COMT, converting N-acetylserotonin into melatonin. (B) Purification of His × 6-tagged NtCOMT1, M, molecular mass standards; lane 1, total proteins in 20-μL aliquots of bacterial culture with IPTG; lane 2, 20-μL aliquots of supernatant derived from bacterial cell lysate; lane 3, 20-μL aliquots of precipitated cell lysate; lane 4, purified NtCOMT1 protein. (C) The enzymatic activity of NtCOMT1 under different pH and temperature. (D) Kinetic analysis of purified recombinant NtCOMT1. (E) HPLC analysis of in vitro enzyme activity using N-acetylserotonin as the substrate. The retention time of the new peak is the same as the retention time of the melatonin standard (top: melatonin standard, middle: N-acetylserotonin standard, NtCOMT1: the corresponding protein).

Fig. 2 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 2. Multiple sequence alignment of tobacco COMT as well as well-defined plants COMT proteins. Residues involved in SAM binding (pink), substrate binding (blue), substrate binding in transform the dyad-related polypeptide (green), and catalysis (red) are highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 in NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum

Fig. 1. Phylogenetic relationships (A) and motif compositions (B) of tobacco COMT as well as well-defined plant COMT proteins. The phylogenetic tree was constructed with a bootstrap of 1000 by the maximum-likelihood method. Motif compositions were illustrated by TBtools. The proteins in phylogenetic tree include wellcharacterized OsCOMT (Oryza satica: LOC_Os09g17560), AtCOMT (Arabidopsis thaliana: AT5G54160), SlCOMT (Solanum lycopersicum: XP_004235028), CrCOMT (Carex rigescens: QDF21518), TaCOMT (Triticum aestivum: Traes_1AL_D9035D5E0), and GhCOMT (Gossypium hirsutum: Gh_D12G2714).

Fig. 2 in Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1)

Fig. 2. The structural formulae of products of recombinant enzyme incubations with different substrates. 1, 12-oxo-13-hydroxy-9,15-octadecadienoic acid; 2, 12-oxo-13-hydroxy-9-octadecenoic acid; 3, 9- hydroxy-10-oxo-12,15-octadecadienoic acid; 4, 9-hydroxy-10-oxo-12-octadecenoic acid; 5, 11-hydroxy- 12,13-epoxy-9-octadecenoic acid; 5a, 11-hydroxy- 12,13-epoxy-9,15-octadecadienoic acid; 6, 9,10- epoxy-11-hydroxy-12-octadecenoic acid; 6a, 9,10- epoxy-11-hydroxy-12,15-octadecadienoic acid; 7, 9- hydroxynonanoic acid; 8, (9Z)-12-hydroxy-9-dodecenoic acid; 9, (10E)-12-hydroxy-10-dodecenoic acid; R, -(CH2)7COOH.

Fig. 1 in Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1)

Fig. 1. The multiple alignment of the CYP74L1, CYP74L2, and CYP74L3 sequences of S. moellendorffii. Conservative structures are marked as follows: the I-helix groove region (SRS-4) is numbered 1–6, two positions after the I-helix groove region are marked by ▾ symbol, the F/L toggle, the ERR-triad, the PPV domain, and the cysteinyl ligand are marked by ◆, ●, □, and ◊ symbols, respectively.

Fig. 4 in Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1)

Fig. 4. The TIC GC–MS chromatograms of products (Me/TMS) of incubations of the recombinant CYP74L2 with 13-HPOT (A), 13-HPOD (B), 9-HPOT (C), and 9-HPOD (D). Conditions of incubation, extraction, derivatisation and analysis are described in Materials and Methods. 1a, 1b (threo and erythro isomers of 12,13-dihydroxy-9,15-octadecadienoic acid (the products of the α-ketol 1 reduction with NaBH4)); 2a, 2b (threo and erythro isomers of 12,13- dihydroxy-9-octadecenoic acid (the products of the α-ketol 2 reduction with NaBH4)); 3a, 3b (threo and erythro isomers of 9,10-dihydroxy-12,15-octadecadienoic acid (the products of the α-ketol 3 reduction with NaBH4)); 4a, 4b (threo and erythro isomers of 9,10- dihydroxy-12-octadecenoic acid (the products of the α-ketol 4 reduction with NaBH4)); 7, 9-hydroxynonanoic acid; 5, 11-hydroxy-12,13-epoxy-9-octadecenoic acid; 5a, 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid; 6, 9,10-epoxy-11-hydroxy-12-octadecenoic acid; 6a, 9,10-epoxy-11-hydroxy-12,15-octadecadienoic acid; 7, 9-hydroxynonanoic acid; 8, (9Z)-12-hydroxy-9-dodecenoic acid; 9, (10E)-12-hydroxy-10- dodecenoic acid. The structural formulae of products are present at Fig. 2. 9-HOT, (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid.

Fig. 6 in Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1)

Fig. 6. The multiple alignment of the CYP74L1, CYP74L2, and CYP74L3 sequences of S. moellendorffii with other CYP74s described earlier. Conservative structures are marked as follows: the I-helix groove region (SRS-4) is numbered 1–6, two positions after the I-helix groove region are marked by ◆ symbol, the F/L toggle is marked by ▾ symbol.

Fig. 6 in Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny

Fig. 6. The content of Chl a, Pchl-GG and Chl-GG in the primary leaves of 11- day-old etiolated Cucurbita maxima 'Bambino' seedlings during illumination. The data are means ± SE, n = 3–4. The pigments were extracted and analyzed using HPLC as described in the Experimental section.

Fig. 7 in Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny

Fig. 7. (a) The unrooted phylogenetic tree of the nucleotide sequences of the chlorophyll synthase genes. The tree shows topology only. Seven different clades are distinguished. The subtree representing Clade 7 is shown with the branches occupied by two isoforms of Cucurbita pepo, C. maxima and C. moschata: C1 and C2. (b) Alignment of the chlorophyll synthase sequences of Cucurbita isoform C1, C2 and the remaining sequences of the Clade 7. The size of the letters is scaled proportionally to the numbers of the sequences that have a given amino acid in the given position. The letters are arbitrarily colour-coded to highlight similarities and differences: grey - the indicated residue is found in at least 80% of the sequences in this position in every group; green – conserved residues unique to only one group of sequences; black – variable residues, less than 80% of sequences are present in this position. The residues previously shown to play a catalytic role are highlighted in red. The position of the transmembrane helices suggested by the Uniprot database is highlighted in blue, while that predicted by the homology modelling is marked with a darkblue rectangle. (c) A 3D model of chlorophyll synthase based on the primary sequence of C. pepo subsp. pepo (NCBI: XP 023543340.1). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4 in Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny

Fig. 4. The content of Chl-GG (A), Chl a (B) and Pchl-GG (C) in the primary leaves of 11-day-old etiolated Cucurbita pepo 'Miranda' seedlings during illumination. In the case of ALA-treated plants, the 11-day-old seedlings were incubated for additional 3 days in 10 mM ALA solution in the dark before illumination. The data are means ± SE, n = 3–4. FW = fresh weight. The pigments were extracted and analyzed using HPLC as described in the Experimental section.

Fig. 2 in Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny

Fig. 2. (a) A simplified scheme of the biosynthetic pathway of protochlorophylls and non-enzymatic routes leading to protopheophytins. (b) The composition of protochlorophylls and protopheophytins in the seed coats of the species and varieties under investigation. The phylogenetic relationships between these species is represented. The size of each dot corresponds to the relative content of a given compound and is normalized so that the sum of all protochlorophylls in a given species is 100%. The dots are color-coded and arranged as protochlorophylls and protopheophytins in the framed part of panel a. The pigment composition corresponds to that shown in Fig. S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)