Fig 1.
Heterophyllic leaf developments depending on environments are shown in R. trichophyllus but not in sister species, R. sceleratus.
Seedling morphologies and microscopic images of R. trichophyllus (A) and R. sceleratus (B) grown under aerial vs aquatic environments. Seeds of R. trichophyllus or R. sceleratus were germinated on solid MS media for 1 week, then transferred to aerial or aquatic environments. The true leaves produced at 7 days after transference were used for analysis. (a-d) terrestrial and (e-h) aquatic/submerged plants, (b, c) cell shapes of terrestrial leaves and (f, g) those of aquatic/submerged leaves, (d) vasculature of terrestrial and (h) that of aquatic leaves. (i-k) Statistical analyses of leaf indices (i), stomatal densities (j), and number of vessel elements (k) in terrestrial and aquatic/submerged leaves. Data are collected from 24 individual samples and presented as means ± SD from three biological replicates. Black arrowheads denote stomata and white arrowheads denote individual vessel element.
Fig 2.
Transcriptome analysis of aquatic vs terrestrial plants of R. trichophyllus.
(A) Venn diagram of differentially expressed transcripts with two fold changes for three independent experiments. Numbers are up- or down-regulated genes in aquatic plants compared to terrestrial plants. (B) Diagram for large ontology categories showing up-regulation in aquatic plants by BinGo software. Number of genes is represented by relative size of circles that belong to each gene ontology term. (C) Relative expression of genes affiliated to four developmental GO terms for terrestrial vs aquatic plants of R. trichophyllus. Up-regulated genes are painted with red and down-regulated genes are painted with blue. (D) Diagram of up-regulated genes in aquatic plants for ontology categories of plant hormone response genes by the BinGo software. The seedlings, 1 week-old after germination, were transferred to terrestrial or aquatic condition for 10 days. Upper parts of seedlings including leaves and shoot apexes were harvested for RNA sequencing.
Fig 3.
Ethylene and ABA control heterophylly of R. trichophyllus.
(A) Images of seedlings, stomata, and vessel elements for terrestrial leaf and the leaf after ethylene treatment. (B) Images of seedling, stomata, and vessel elements for aquatic leaf and the leaf after treatment of ABA and AgNO3, an ethylene inhibitor. (C-E) Statistical analyses of leaf indices (C), stomatal densities (D), and number of vessel elements (E) after treatment with hormones (ethylene, ABA, and GA) and hormone inhibitors (AgNO3 and PBZ). Data are collected from 16–24 individual samples and presented as means ± SD from three biological replicates. Black arrowheads denote stomata and white arrowheads denote individual vessel element. *P < 0.05; **P < 0.01 (unpaired Student’s t-test).
Fig 4.
Differential expression of ethylene and ABA-related genes under terrestrial and aquatic environments is specific to amphibious R. trichophyllus.
(A and B). Contents of ethylene (A) and ABA (B) in terrestrial vs aquatic leaves. (C and D). Comparison of transcript levels of ethylene- (A) and ABA- (B) biosynthesis and responsive genes following submergence into water. Plants harvested at 7 hours (7h), 1 day (1d), and 2 days (2d) after submergence were compared with terrestrial and aquatic plants for expressions. (E) Effects of submergence on the expression levels of AAO3 genes in R. trichophyllus, Arabidopsis, and R. sceleratus. For submergence, two weeks old plants grown on solid MS media were submerged into water for 5 days for RNA extraction. The data represent means ± standard error from three biological and two technical replicates.
Fig 5.
Expressions of leaf polarity genes, KANs and HD-ZIPIIIs are dependent on the environment.
(A and B) Gene expression analyses of KAN genes (A), and HD-ZIPIII genes in terrestrial and aquatic leaves. (C and D) Transcript levels of leaf polarity genes after chemical treatments. The data are presented as means ± SD from three biological and two technical replicates. ACC, an ethylene precursor, was treated as ethylene. *P < 0.05; **P < 0.01 (unpaired Student’s t-test) (E) Model of heterophyllic developments regulated by ABA and ET activating leaf polarity genes, KANs and HD-ZIPIIIs, respectively. K; KANADIs, H; HD-ZIPIIIs. (F-M) Whole mount in situ hybridization for RtKANa (F-I) and RtHD-ZIPIIIa (J-M). Ab, abaxial side; Ad, adaxial side.
Fig 6.
Effects of ethylene and ABA signalings on the promoter activation of leaf polarity genes and genes critical for stomata and vascular developments.
(A and B) Luminescence analysis of proRtKANa::LUC when added with ACC, an ethylene precursor, or ABA in protoplast solution (A), control; without chemical treatment, and when cotransfected with CFP-RtEIN3 in protoplasts (B), control; cotransfected with CFP construct. (C) Effects of CFP-RtEIN3 transfection on the relative expressions of endogenous KAN genes in protoplasts, control; transfected with CFP construct. (D and E) Luminescence analysis of proHD-ZIPIIIa::LUC when added with ACC or ABA in protoplast solution (D), control; without chemical treatment, and when cotransfected with CFP-RtABI3 in protoplasts (E), control; cotransfected with CFP construct. (F) Effects of CFP-RtABI3 transfection on the relative expressions of endogenous HD-ZIPIII genes in protoplasts, control; transfected with CFP construct. (G) Relative transcript levels of RtSTO and RtVDN7, encoding critical regulators of stomata and vascular developments, when transfected with Rt-EIN3, Rt-ABI3, and RtHD-ZIPIIIa fused with CFP coding sequence. Control; transfected with CFP construct. (H) Comparison of transcript levels of RtSTO and RtVND7 between terrestrial and aquatic leaves of R. trichophyllus. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired Student’s t-test).
Fig 7.
Cold and hypoxia induce aquatic leaf development in R. trichophyllus.
(A) Heterophylly induced by cold and hypoxia. 1 week-old seedlings after germination on the MS media were transferred to cold chamber (4°C) for 1 month or hypoxia chamber (1% O2) for 2 weeks. The column Room Temp is a control at 22°C with 20% O2. (B and C) Gene expression analyses of KAN genes (B), and HD-ZIPIII genes (C) in the leaves after cold for 1 month and hypoxia for 2 weeks treated. *P < 0.05; **P < 0.01 (unpaired Student’s t-test).