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. 2019 Oct 28;10(11):853.
doi: 10.3390/genes10110853.

Comparative Physiological and Transcriptomic Analyses Reveal Mechanisms of Improved Osmotic Stress Tolerance in Annual Ryegrass by Exogenous Chitosan

Junming Zhao  1 , Ling Pan  2 , Man Zhou  3 , Zhongfu Yang  4 , Yu Meng  5 , Xinquan Zhang  6
Affiliations

Comparative Physiological and Transcriptomic Analyses Reveal Mechanisms of Improved Osmotic Stress Tolerance in Annual Ryegrass by Exogenous Chitosan

Junming Zhao et al. Genes (Basel). .

Abstract

Water deficit adversely affects the growth and productivity of annual ryegrass (Lolium multiflorum Lam.). The exogenous application of chitosan (CTS) has gained extensive interests due to its effect on improving drought resistance. This research aimed to determine the role of exogenous CTS on annual ryegrass in response to water stress. Here, we investigated the impact of exogenous CTS on the physiological responses and transcriptome changes of annual ryegrass variety "Tetragold" under osmotic stress induced by exposing them to 20% polyethylene glycol (PEG)-6000. Our experimental results demonstrated that 50 mg/L exogenous CTS had the optimal effect on promoting seed germination under osmotic stress. Pre-treatment of annual ryegrass seedlings with 500 mg/L CTS solution reduced the level of electrolyte leakage (EL) as well as the contents of malondialdehyde (MDA) and proline and enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbic acid peroxidase (APX) under osmotic stress. In addition, CTS increased soluble sugars and chlorophyll (Chl) content, net photosynthetic rate (A), stomatal conductance (gs), water use efficiency (WUE), and transpiration rate (E) in annual ryegrass seedlings in response to three and six days of osmotic stress. Transcriptome analysis further provided a comprehensive understanding of underlying molecular mechanisms of CTS impact. To be more specific, in contrast of non-treated seedlings, the distinct changes of gene expressions of CTS-treated seedlings were shown to be tightly related to carbon metabolism, photosynthesis, and plant hormone. Altogether, exogenous CTS could elicit drought-related genes in annual ryegrass, leading to resistance to osmotic stress via producing antioxidant enzymes and maintaining intact cell membranes and photosynthetic rates. This robust evidence supports the potential of the application of exogenous CTS, which will be helpful for determining the suitability and productivity of agricultural crops.

Keywords: Lolium multiflorum Lam.; antioxidant enzymes; exogenous chitosan; osmotic stress; physiological and photosynthetic characterizes; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chitoscan (CTS) pretreatment improved annual ryegrass resistance to osmotic stress tolerance. (A) Phenotype of annual ryegrass after polyethylene glycol (PEG) treatment with or without CTS. Effect of CTS on changes of annual ryegrass relative water content (B), chlorophyll content (C,D), and protein content (E) before and after osmotic stress treatment. Vertical bars represent mean values ± SD for each mean. Different letter indicates significant differences at given day (P ≤ 0.05).
Figure 2
Figure 2
Effect of chitoscan (CTS) pre-treatment on changes of annual ryegrass EL value (A), MDA content (B), H2O2 content (C), and O2−· content (D) before and after osmotic stress treatment. Vertical bars represent mean values ± SD for each mean. Different letter indicates significant differences at given day (P ≤ 0.05).
Figure 3
Figure 3
Effect of chitoscan (CTS) pre-treatment on changes of antioxidant enzymes superoxide dismutases (SOD) (A), POD (B), CAT (C), APX (D), soluble sugars (E), and proline (F) content in annual ryegrass under osmotic stress condition. Vertical bars represent mean values ± SD for each mean. Different letter indicates significant differences at given day (P ≤ 0.05).
Figure 4
Figure 4
Effect of chitoscan (CTS) pre-treatment on photosynthetic characteristics net photosynthetic rate (A), water use efficiency (B), intercellular CO2 concentration (C), stomatal conductance (D), and transpiration rate (E) of annual ryegrass suffered from osmotic stress. Vertical bars represent mean values ± SD for each mean. Different letter indicates significant differences at given day (P ≤ 0.05).
Figure 5
Figure 5
Effect of chitoscan (CTS) pre-treatment on expression level of genes LmFeSOD (A), LmCyt-Cu/ZnSOD (B), LmPOD (C), LmCAT (D), and P5CS1 (E) under osmotic stress condition. The relative expression levels were normalized to that of LmActin and presented using the 2−ΔΔCt method. Vertical bars represent mean values ± SD for each mean. Different letter indicates significant differences at given day (P ≤ 0.05).
Figure 6
Figure 6
Gene ontology database (GO) and kyoto encyclopaedia of genes and genomes database (KEGG) classifications of differentially expressed genes (DEGs). (A) GO analysis of the upregulated genes in CTS vs. mock. (B) GO analysis of the downregulated genes in CTS vs. mock. (C) KEGG classifications of all genes between the without and with 3 d of CTS pre-treatment in annual ryegrass plants.
Figure 7
Figure 7
Effect of CTS on DEGs of carbon fixation in photosynthetic organisms in leaves of annual ryegrass. Definition of genes encoding enzymes: (1) 2.2.1.1, transketolase (TKT); (2) 1.2.1.13, glyceraldehyde-3-phosphate dehydrogenase (GAPDH/GAPA); (3) 5.3.1.6, ribose-5-phosphate isomerase 2 (RPI2); (4) 4.1.1.39, ribulose bisphosphate carboxylase small chain (rbcS); (5) 2.7.2.3, phosphoglycerate kinase (PGK); (6) 4.1.1.31, phosphoenolpyruvate carboxylase 1(PPC1). Red means an up-regulation.
Figure 8
Figure 8
The metabolic pathway involved in photosynthesis. Definition of genes encoding proteins: (1) PsbP, photosystem II oxygen-evolving enhancer protein 2; (2) Psb27, photosystem II Psb27 protein; (3) PsaN, photosystem I subunit PsaN; (4) PetF, ferredoxin; (5) PetH, ferredoxin-NADP+ reductase. Red boxes indicated an up-regulation.
Figure 9
Figure 9
A proposed model for CTS-mediated osmotic stress responses in annual ryegrass.
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