Proline Biosynthesis Enzyme Genes Confer Salt Tolerance to Switchgrass (Panicum virgatum L.) in Cooperation With Polyamines Metabolism

Abstract

Understanding the regulation of proline metabolism necessitates the suppression of two Δ₁-pyrroline-5-carboxylate synthetase enzyme (P5CS) genes performed in switchgrass (Panicum virgatum L.). The results reveal that overexpressing PvP5CS1 and PvP5CS2 increased salt tolerance. Additionally, transcript levels of spermidine (Spd) and spermine (Spm) synthesis and metabolism related genes were upregulated in PvP5CS OE-transgenic plants and downregulated in the PvP5CS RNAi transformants. According to salt stress assay and the measurement of transcript levels of Polyamines (PAs) metabolism-related genes, P5CS enzyme may not only be the key regulator of proline biosynthesis in switchgrass, but it may also indirectly affect the entire subset of pathway for ornithine to proline or to putrescine (Put). Furthermore, application of proline prompted expression levels of Spd and Spm synthesis and metabolism-related genes in both PvP5CS-RNAi and WT plants, but transcript levels were even lower in PvP5CS-RNAi compared to WT plants under salt stress condition. These results suggested that exogenous proline could accelerate polyamines metabolisms under salt stress. Nevertheless, the enzymes involved in this process and the potential functions remain poorly understood. Thus, the aim of this study is to reveal how proline functions with PAs metabolism under salt stress in switchgrass.

Keywords: PvP5CS1 and PvP5CS2; polyamines; proline; salt stress; switchgrass.

Figure 1

Expression pattern of two PvP5CS genes (PvP5CS1 and PvP5CS2) in switchgrass. Relative expression level of two PvP5CS genes in different tissues (A). Relative expression level of two PvP5CS genes in different development stages (B). Analysis of PvP5CS genes expression under different NaCl concentrations (0 mM, 150mM, 250mM, and 350mM) after 24h salt treatment (C). Transcripts of PvP5CS genes under 350 mM NaCl at the indicated time points (D). Switchgrass UBQ1 was used as the reference for normalization. At the first stage of elongation, there in none internode, so it is defined as E0. When switchgrass grows the internode, E1 to E5 are defined by the number of internodes present. At the beginning of the reproductive stage, flag leaf comes out, and it is defined as R1. When switchgrass has the fully emerged spikelets without peduncle, it arrived at R2 stage. The R3 stage is defined by fully emerged spikelets and visible peduncle. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test). WT: wild type control.

Figure 2

P5CS, ProDH activity and proline content in PvP5CS OE and RNAi transgenic plants. P5CS (A) and ProDH (B) activity and Proline content (C) in PvP5CS OE and RNAi transgenic plants. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 3

Phenotypic comparation and biomass analysis among PvP5CS OE transgenic plants. Phenotypes of three independent transgenic lines overexpressing PvP5CS1 (A) and PvP5CS2 (B). Total above ground dry weight of transgenic lines overexpressing PvP5CS genes (C). Flowering time in OE-PvP5CS1 and OE-PvP5CS2 transgenic switchgrass lines (D). Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 4

The salt tolerant evaluation of PvP5CS OE and RNAi transgenic plants under salt treatment. Phenotypes of PvP5CS OE and RNAi transgenic plants under 0 mM NaCl (A) and 350 mM NaCl (B) treatment for 15 days. Relative leaf water content (C), Electrolyte leakage (D), proline (E) and Chlorophyll (F) of transgenic lines OE-PvP5CS1, OE-PvP5CS2 and RNAi-PvP5CS in (B) after 350 mM NaCl treatment for 15 days. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 5

Phenotype and ion (Na⁺ and K⁺) contents of roots in PvP5CS OE and RNAi transgenic plants under salt stress. Phenotype of roots in PvP5CS OE and RNAi transgenic plants under 350 mM NaCl salt stress (A). Root vigor in PvP5CS OE and RNAi transgenic plants under 350 mM NaCl salt stress (B). Na⁺ (C) and K⁺ (D) contents of roots in PvP5CS OE and RNAi transgenic plants under 350 mM NaCl salt stress. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 6

Overexpression of PvP5CS genes in switchgrass positively regulates the induction of ROS scavengers and membrane transporters, and reduces H₂O₂ accumulation under 350 mM NaCl treatment. Color of detached leaves after staining with diaminobenzidine (DAB) in wild type, PvP5CS OE and RNAi transgenic plants under 0 mM (A) and 350 mM (B) NaCl treatment. H₂O₂ content in the leaves of wild type, PvP5CS OE and RNAi transgenic plants under 0 mM NaCl (C) and 350 mM NaCl (D) treatment. Relative expression levels of PvCAT (E) and PvSOD (F) in wild type, PvP5CS OE and RNAi transgenic plants under 0 and 350 mM NaCl treatment. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 7

Transcript levels of the genes involved in polyamine synthesis (A–C) and catabolism (D) in PvP5CS OE and RNAi transgenic plants under control and salt stress conditions. Switchgrass UBQ1 was used as the reference for normalization. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 8

Exogenous proline can largely improve transcript levels of the genes involved in polyamine synthesis (A–C) and catabolism (D) in PvP5CS RNAi transgenic plants under 350 mM NaCl salt stress. Switchgrass UBQ1 was used as the reference for normalization. Data are mean values of three biological repeat, and significance of treatments was tested at the P < 0.05 level (one way ANOVA, Dunnett's test).

Figure 9

Schematic illustration of a working model depicting the relationship between proline and polyamine in wild type, PvP5CS OE and RNAi transgenic plants under control and salt stress conditions. In summary, proline metabolism is in cooperation with polyamines metabolism in switchgrass. If switchgrass has enough proline to confer salt stress, ornithine could be as substrate involved in PAs biosynthesis. However, under less proline accumulation to improve the salt tolerance of switchgrass, ornithine will be used as a precursor entering the pathway of proline synthesis. Altogether, proline and polyamine metabolism cooperated to improve salt tolerance in switchgrass.