Fig 1.
Molecular analysis of transgenic switchgrass lines overexpressing LpP5CS.
(A) Relative expression levels of LpP5CS (TG2 was used as a control), (B) PvP5CS1 and (C) PvP5CS2 in transgenic and WT plants. (D) Proline content in transgenic plants. Switchgrass Ubq1 was used as the reference for normalization, the relative expression levels of genes were calculated using 2−ΔΔCT method, and the expression level of one plant (control) was defined “1”. Value are mean ± SE (n = 6). The significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett’s test).
Table 1.
The characterization of growth and development in transgenic and WT plants.
Fig 2.
LpP5CS regulates growth and development in switchgrass.
(A) Morphological characterization of transgenic switchgrass lines (5 months) overexpressing LpP5CS. (B-E) Relative expression levels of PvFLP3, PvFT, PvFLC and PvMADS18 in transgenic plants and WT plants. Value are mean ± SE (n = 6). Switchgrass Ubq1 was used as the reference for normalization. The significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett’s test). Leaf size is controlled by the complex coordination of cell division and expansion. To interpret the leaf size of group I, group II and WT plants, the leaf epidermal cells were viewed with scanning electron microscopy (SEM). The group II line (TG1) had a 36% reduction and the group I line (TG4) had a 43% increase in cell numbers compared with those of WT plants (Fig 3A–3C). We speculated that the increase in cell number was caused by increased expression of cell cycle-related genes. Thus, the expression level of cell cycle-related genes in TG1, TG4, and WT plants was analyzed. Cyclin-dependent kinases are the master regulators of the eukaryotic cell cycle to stimulate cell division and tissue growth. Therefore, we measured the expression level of the D-type cyclin gene (PvCYCD) (Pavir.J14518.1) and B-type cyclin gene (PvCYCB) (Pavir.J38704.1). The results showed that the expression levels of PvCYCD and PvCYCB were significantly up-regulated in the TG4 plants and significantly down-regulated in the TG1 plants compared with those in WT plants (Fig 3D). Collectively, these findings indicate that proline is crucial to plant growth and development.
Fig 3.
(A) Scanning electron micrograph (SEM) imaging cells on the adaxial surface in the leaf of TG1, TG4 and WT. Bar = 200 um. (B) Leaf width and (C) cell number in TG1, TG4 and WT. Value are mean ± SE (n = 3). (D) Relative expression levels of PvCYCD and PvCYCB in transgenic plants and WT plants. Value are mean ± SE (n = 6). Switchgrass Ubq1 was used as the reference for normalization. The significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett’s test).
Fig 4.
RNA sequencing of the group I, group II and WT plants.
The volcano plot: (A) Group II Vs WT lines, (B) Group I Vs Group II lines, (C) Group I Vs WT lines.
Fig 5.
The salt tolerant evaluation of TG1, TG4 and WT plants.
(A) Phenotypic characterization of TG1, TG4 and WT plants under 400 mM NaCl treatment on the 30th day. (B) Roots of TG1, TG4 and WT under 0 and 400 mM NaCl on the 30th day. (C) The increases of plant height, (D) the increases of leaf length in TG1, TG4 and WT under 0, 200 and 400 mM NaCl treatment for 30 days. Value are mean ± SE (n = 9). The significance of treatment (0, 200 and 400 mM NaCl concentration) an sample type (WT and transgenic plants) was tested at the P < 0.05 level (two way ANOVA), capital letter represents the difference between WT and transgenic plants under the same salt concentration, lowercase represents the difference of WT or transgenic plants under 0, 200 and 400 mM NaCl concentration.
Fig 6.
Physiological analysis of TG1, TG4 and WT plants under salt stress (0, 200 and 400 mM NaCl).
(A) RWC, (B) EL, (C) MDA, (D) proline, (E) total chlorophyll (chlorophyll A + B), (F) carotenoid in TG1, TG4 and WT under salt stress. Value are mean ± SE (n = 3). The significance of treatment (0, 200 and 400 mM NaCl concentration) an sample type (WT and transgenic plants) was tested at the P < 0.05 level (two way ANOVA), capital letter represents the difference between WT and transgenic plants under the same salt concentration, lowercase represents the difference of WT or transgenic plants under 0, 200 and 400 mM NaCl concentration.
Fig 7.
Na+, K+ contents of TG1, TG4 and WT plants under salt stress (0, 200, and 400 mM NaCl).
Value are mean ± SE (n = 3). The significance of treatment (0, 200 and 400 mM NaCl concentration) an sample type (WT and transgenic plants) was tested at the P < 0.05 level (two way ANOVA), capital letter represents the difference between WT and transgenic plants under the same salt concentration, lowercase represents the difference of WT or transgenic plants under 0, 200 and 400 mM NaCl concentration.
Fig 8.
Histological analysis of the leaf in TG1, TG4 and WT plants under 0 and 400 mM NaCl.
(A) The areas of bundle sheath cells, (B) the diameter of bulliform cells, (C) the areas of bulliform cells, (D) the stomatal aperture of the transgenic and and WT under 0 and 400 mM NaCl. Histological analysis in the leaf of internode 3 of TG1, TG4 and and WT under 0 (E) and 400 mM NaCl (F), Bar, 100 um. Scanning electron micrograph (SEM) imaging cells on the adaxial surface in the leaf of internode 3 of TG1, TG4 and WT under 0 (G) and 400 mM NaCl (H), Bar, 50.0 um. Value are mean ± SE (n = 3). The significance of treatment (0, 200 and 400 mM NaCl concentration) an sample type (WT and transgenic plants) was tested at the P < 0.05 level (two way ANOVA), capital letter represents the difference between WT and transgenic plants under the same salt concentration, lowercase represents the difference of WT or transgenic plants under 0 and 400 mM NaCl concentration.
Fig 9.
Proline coordinated events and pathways to regulate plant growth and development and salt stress tolerance.