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Fig. 3 in Nine new species of Australian Nicotiana (Solanaceae)

Fig. 3. Nicotiana clarksonii based on living plants cultivated at the Royal Botanic Gardens, Kew, from seeds associated with Chase & Christenhusz 18151, Archway Cave, Mungana Caves, Queensland. Drawn by Deborah Lambkin. (a) Carpel, style and stigma. (b) Corolla, split open to show positions of stamens. (c) Flower, side view. (d) Floral limb, face-on. (e) Pubescence on leaf margin. (f) Pubescence on lower portion of stem. (g) Pubescence on calyx. (h) Pubescence on mid-vein. (i) Stem leaf. (j) Mature capsule. (k) Capsule without calyx. (l) Habit. Scale bars: 1.5 cm (a–d, j, k); 3.3 mm (e–h); 3.0 cm (i); plant in (l) is 64 cm tall.

opennotspecifiedJul 2023View details →

Fig. 1 in Nine new species of Australian Nicotiana (Solanaceae)

Fig. 1. Phylogenetic tree for the species of Nicotiana section Suaveolentes, based on the RADseq (nuclear) data matrix of Chase et al. (2021, 2022b), to which we have added the accessions for the new species reported in this paper. Vouchers for these added accessions are indicated under the additional specimens examined for each of the new species. All nodes for which there are no bootstrap percentages indicated are 100. (a) The basal nodes of the RADseq tree. (b) The more derived clades in the same RADseq tree as in (a).

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 Zinc uptake and HMA4 activity are required for micro- and macroelement balance in tobacco (Nicotiana tabacum)

Fig. 2. Classification of gene expression changes observed in both the HMA4 double-knockout mutants (cv. #3) and HMA4 RNAi plants (cv.#1) relative to their respective controls. The genes were classified manually according to the putative function of the closest Arabidopsis Blast hit (see Supplementary Table S2).

Fig. 1 in Zinc uptake and HMA4 activity are required for micro- and macroelement balance in tobacco (Nicotiana tabacum)

Fig. 1. (a) Relative element contents of HMA4-impaired plants compared with their controls. HMA4 RNAi plants in cv.#1 background were compared with their WT controls (n = 3 replicates each), and HMA4 double-mutant plants in cv. #3 background were compared with their null-segregant controls (n = 4). The elements were measured in dry weight of lower leaves of plants grown under greenhouse conditions. The error bars depict the 0.95 confidence interval (CI) corrected according to Satterthwaite approximation. (b) Pictures of representative plants of each group (sticker size = 11.9 cm).

Fig. 6 in Zinc uptake and HMA4 activity are required for micro- and macroelement balance in tobacco (Nicotiana tabacum)

Fig. 6. Relative element contents of different HMA4-double mutants versus the null-segregant WT controls in field experiments. The data were consolidated over 4 years of field experiments in Switzerland and 3 years of field experiments in Poland, as described in the Experimental section. The elements were measured in dry weight of leaves (mid-lower leaf position). Bars indicate estimates and confidence intervals (α = 0.1).

Fig. 4 in Zinc uptake and HMA4 activity are required for micro- and macroelement balance in tobacco (Nicotiana tabacum)

Fig. 4. Relative elemental content of N. tabacum cv. #1 and cv. #2 HMA4 RNAi plants compared with their WT and of HMA4 double-knockout mutants compared with their null-segregants, for four different fertilization conditions: (a) normal fertilization, (b) fertilization containing lower concentrations of phosphate, (c) fertilization containing higher concentrations of Zn, and (d) fertilization with low concentrations of phosphate and high concentrations of Zn. The high Zn condition was achieved by addition of 0.1 g additional Zn per plant. The elements were measured in dry weight of leaves (mid-lower leaf position). Absolute data are listed in Supplementary Table S3. Data represent mean (0.95 CI) of n = 5 replicate plants, corrected according to Satterthwaite approximation. (Three outliers were removed for condition (d): Al and Cu for cv.#2; Ti for cv.#3.).

Fig. 3 in Zinc uptake and HMA4 activity are required for micro- and macroelement balance in tobacco (Nicotiana tabacum)

Fig. 3. Working model of metabolic changes in HMA4-impaired plants based on the gene expression changes observed in the roots and leaves of HMA4 RNAi and HMA4-mutant N. tabacum plants relative to their controls. The lack of HMA4 function causes Zn deficiency in leaves, leading to a local Zn-deficiency response. In roots, a Fe-deficiency response is observed, caused either by a high Zn:Fe ratio in the roots or by a systemic Zn-deficiency signal. The Fe-deficiency signal leads—via the transcription factor FIT1—to upregulation of the Fe- and Mn- transporter genes IRT1 and NRAMP1 and also induces the synthesis and export of iron-binding compounds (IBC), thus additionally assisting Fe uptake. At the same time, a P-deficiency signal involving the transcription factor HHO2 is generated in the leaves. In roots, the P-deficiency signal leads to increased expression of genes encoding acid phosphatases, enabling increased phosphate uptake from the rhizosphere. Consequently, Fe, Mn, Cu, and P accumulate in the leaves. P is stored in vacuoles (via PHT5; 1, PHT5; 3) and also exported to mitochondria and chloroplasts. Cu is detoxified by transport to the chloroplasts, which are a major site of Cu use because of Cu-containing plastocyanin production. However, high levels of Cu are toxic for the photosynthetic electron transport system, especially for photosystem II, which is reflected in the upregulation of PII light harvesting complex genes (LHCB). At the same time, Cu uptake to the cells is limited by downregulation of COPT1. Ion uptake might cause a lower osmotic potential, leading to water influx via various aquaporin genes. High turgor pressure or Fe excess ultimately affects the cell wall, as observed by the increased expression of genes involved in cell wall biosynthesis, expansion, and crosslinking. bHLH115: basic helix-loop-helix transcription factor; CA: carbonic anhydrase; CESA: cellulose synthase; COPT: copper transporter; DMRL: dimethyl-8-ribityllumazine synthase; EXPA: expansin; FIT: FER-like regulator of iron uptake (transcription factor); HHO2: hypersensitivity to low phosphate-elicited primary root shortening 1 homolog 2 (myb-like transcription factor); HMA: heavy metal ATPase; IBC: iron binding compound; IRT: iron-regulated transporter; LAC: laccase; LHCB: photosystem II light harvesting complex; NA: nicotianamine; NAS: nicotianamine synthase; NRAMP: natural resistance-associated macrophage protein (metal ion transporter); PDR: pleiotropic drug resistance (ATP-binding cassette transporter); PHT: phosphate transporter; PIP: plasma membrane intrinsic protein; PME: pectin methyl esterase; PMEI: pectin methyl esterase inhibitor; PRX: peroxidase; RBCS: ribulose bisphosphate carboxylase small chain; TIP: tonoplast intrinsic protein; TBL: TRICHOME BIREFRINGENCE-LIKE (xylan acetylation); ZIP: zinc transporter.

Fig. 6 in Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 6. Differentially expressed genes related to the plant hormone signal transduction pathway in the comparison (NaCl vs NaCl + ACh). (A) diagram of auxin, gibberellin, brassinosteroid and salicylic acid signalling transduction pathways; (B) information and expression patterns of differentially expressed genes involved in auxin, gibberellin, brassinosteroid and salicylic acid signalling transduction pathways. Red means upregulated expression of genes, and green means downregulated expression of genes. The number in each sample name represents the sample order. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3 in Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 3. Functional annotation of differentially expressed genes (DEGs) based on gene ontology categorization. The left Y-axis represents the significantly enriched GO terms (p <0.05) pathways. The Xaxis represents the percentage of DEGs belonging to the corresponding pathway. The sizes of bubbles represent the number of DEGs in the corresponding pathway, and the colours of the bubbles represent the enrichment p-value of the corresponding pathway. The left y-axis shows the Gene Ontology terms. Biological process, cellular component and molecular function are indicated by different colours. Only significantly enriched GO terms (p <0.05) are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8 in Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 8. Quantitative real-time PCR (RT-qPCR) validation of selected differentially expressed genes detected in Nicotiana benthamiana leaves. The expression levels obtained by RT-qPCR are represented in black lines, RT-qPCR data showed the mean values from three replicates, and the error bars represent the SE of the means, while the corresponding expression data for RNA-seq are represented in the white histogram. CN, control; CN + ACh, 10 μM acetylcholine; NaCl, 150 mM NaCl stress; NaCl + ACh, 150 mM NaCl stress plus 10 μM acetylcholine.

Fig. 5 in Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 5. Heatmap representing the differentially expressed genes (DEGs) involved in cell wall extensibility of Nicotiana benthamiana leaves as influenced by NaCl alone or in combination with acetylcholine treatment (NaCl + ACh). Red means upregulated expression of genes, and green means downregulated expression of genes. The number in each sample name represents the sample order. (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 Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 1. Changes in reactive oxygen species accumulation and production and antioxidant enzyme activity in Nicotiana benthamiana leaves 5 days after treatment with acetylcholine (ACh) under salt stress. (a) leaves were stained with NBT and DAB, (b) Fv/Fm, (c) Superoxide content, (d) Hydrogen peroxide content, (e) Ascorbate peroxidase activity and (d) Catalase activity as influenced by salt stress alone or in combination with ACh treatment. Data are means of three replications ±SE. Means with the same lowercase letters are not significantly different at p <0.05, according to Duncan's multiple range test. CN: control; CN + ACh, 10 μM acetylcholine; NaCl, 150 mM NaCl stress; NaCl + ACh, 150 mM NaCl stress plus 10 μM acetylcholine.

Fig. 7 in Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Fig. 7. Heatmap representing the differentially expressed genes involved in transcription factors extensibility of Nicotiana benthamiana leaves as influenced by NaCl alone or in combination with acetylcholine treatment (NaCl + ACh). Red means upregulated expression of genes, and green means downregulated expression of genes. The number in each sample name represents the sample order. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)