Overexpression of the Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) gene results in morphological alterations and salinity tolerance in switchgrass (Panicum virgatum L.)

Abstract

In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5C S ( Lolium perenne L.) gene into switchgrass by Agrobacterium -mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.

Expression of Arabidopsis Ornithine Aminotransferase (AtOAT) encoded gene enhances multiple abiotic stress tolerances in wheat

“…Glutamate pathway of proline biosynthesis is predominantly active during abiotic stress conditions. An increased expression of P5CS under osmotic stress was reported in wheat, barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), switchgrass (Panicum virgatum), and Lepidium draba (Rana et al 2016;Bandurska et al 2017;Guan et al 2019;Lopez-Galiano et al 2019;Goharrizi et al 2020). Our present study also reveals the activation of glutamate pathway in wheat under drought, salt and heat stresses as an increased expression of TaP5CS1 and TaP5CR was observed in AtOAT expressed transgenic plants.…”

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

“…These results indicate that proline generates the suppression of NaCl-induced physiological damage as shown by the lower electrolyte leakage and ROS levels which further explain that proline significantly improves the physiological status of stressed plants (Figures 4 and 6). Similarly, our previous results also showed that overexpressing heterologous P5CS genes from Lolium perenne and Puccinellia chinampoensis in switchgrass improve the salt tolerance by reducing the electrolyte leakage and ROS levels (Guan et al, 2018;Guan et al, 2019). Therefore, no matter overexpressing heterologous or endogenous P5CS genes in switchgrass, which can increase the proline content and improve the salt stress.…”

Proline improves switchgrass growth and development by reduced lignin biosynthesis

“…1B). Additionally, group II overexpression lines showed relatively lower proline content, and the proline content of group I transgenic plants was higher compared with WT plants 16 . Combined with the proline levels, these results suggested that proline synthesis is reduced and proline degradation is enhanced in the group II plants compared with the WT plants, and while in group I, there is an increased proline synthesis and decreased proline degradation.

Figure 1Proline metabolism related enzyme activities.

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“…Switchgrass callus generated from mature seeds (Alamo) was transformed with a pCAMBIA 1301- LpP5CS overexpression cassette using the Agrobacterium-mediated transformation method 16 . Group I consists of two independent overexpression lines (TG4 and TG6) while group II contains TG1 and TG2.…”

“…Total RNA was extracted from transgenic and WT leaves at the E5 stage, and RNA samples included three groups: TG1 and TG2 lines (group II), TG4 and TG6 lines (group I), and WT1 and WT2 plants. The RNA-seq data were downloaded from NCBI, the SRA number is SRP130275 16 . The paired-end reads were aligned to the reference genome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvirgatum_er) using TopHat v2.0.12 26 .…”