Polyamines mitigate the destructive impacts of salinity stress by enhancing photosynthetic capacity, antioxidant defense system and upregulation of calvin cycle-related genes in rapeseed (Brassica napus L.)

Abstract

Salinity is widespread environmental stress that poses great obstacles to rapeseed development and growth. Polyamines are key plant growth regulators that play a pivotal role in regulating salt tolerance. Rapeseed (Brassica napus L.) seedlings were treated by spermine (Spm) and spermidine (Spd) versus untreated control under salt stress conditions. It was detected that the Spd-treated plants had significantly elevated chlorophyll and proline content and maintained higher photosystem II (PSII) activity than those treated with Spm as well as untreated control under salt-stressed conditions. Similarly, Spd alleviated the devastating effects of NaCl stress on CO₂ assimilation and significantly elevated Rubisco activity (ribulose 1,5-bisphosphate carboxylase/oxygenase). The application of Spd also enhanced the activities of different antioxidant enzymes under NaCl stress. It modulated their respective transcription levels, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and dehydroascorbate reductase (DHAR). In addition, exogenously sprayed Spd enhanced the polyamine pathway as observed by upregulated transcription of polyamine oxidase (PAO) and diamine oxidase (DAO). The Spd application enhanced expressions of Calvin cycle enzyme related genes such as Rubisco small subunit, Rubisco large subunit, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglyceric acid kinase (PGK), triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate aldolase (FBA), sedoheptulose-1,7-bisphosphatase (SBPase), and fructose-1,6-bisphosphate phosphatase (FBPase). Consequently, this study demonstrates that exogenous application of Spd has a valuable role in regulating antioxidant enzyme activity, polyamine pathway, and Calvin cycle enzyme-related genes to alleviate salt stress damage in the plants.

Keywords: Gene expression; Photosynthesis; Rapeseed; Salinity stress; Spermidine; Spermine.

Fig. 1

Growth rate and photosynthesis parameters. A) Fresh mass (FM), B) Dry mass (DM), C) Total chlorophyll, D) Photosynthetic quantum yield (φPSII), E) Net photosynthetic rate, and F) intercellular CO₂ concentration (Ci) in rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. The bars on the columns correspond to the SE, and distinct letters differ significantly by LSD (p < 0.01).

Fig. 2

Rubisco activity and transcript level of RbcL and RbcS Genes. A) Rubisco activity, B) Transcript amounts of RbcL, and C) Transcript amounts of RbcS genes in rapeseed seed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. qPCR experiments were repeated thrice with two replications each to quantify transcript levels. Transcript levels were normalized to actin and GAPDH transcript levels. The bars on the columns correspond to the SE, and distinct letters differ significantly by LSD (p < 0.01).

Fig. 3

Malondialdehyde, H₂O₂, and Proline Contents. A) Thiobarbituric acid reactive substances (TBARS) (MDA), B) H₂O₂ and C) Proline contents in leaves of rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. The bars on the columns correspond to the SE, and distinct letters differ substantially by LSD (p < 0.01).

Fig. 4

The activities of antioxidant enzymes in rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. A) superoxide dismutase (SOD); B) catalase (CAT); C) ascorbate peroxidase (APX); D) glutathione reductase (GR); and E) dehydroascorbate reductase (DHAR). The bars on the columns correspond to the SE, and distinct letters differ substantially by LSD (p < 0.01).

Fig. 5

Transcript levels of antioxidant enzymes encoding genes in leaves of rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. A) CuZnSOD2, B) CAT1, C) APX, D) DHAR, and E) GR. Transcript levels were determined by qPCR and standardized against actin and GAPDH transcript levels. The experiments of qPCR were replicated three times with two technical replications. The bars on the columns correspond to the SE, and distinct letters differ substantially by LSD (p < 0.01).

Fig. 6

Expression of polyamine biosynthesis-related genes and polyamine pathway catabolic-related genes. A) spermine synthase (SpmS), B) spermidine synthase (SpdS), C) diamine oxidase (DAO), and D) polyamine oxidase (PAO) in rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. Transcript levels were determined by qPCR and normalized against actin and GAPDH transcript levels. The qPCR experiments were repeated three times with two technical replications. The bars on the columns correspond to the SE, and distinct letters differ substantially by LSD (p < 0.01).

Fig. 7

Transcriptions of key genes involved in carbohydrate metabolism related to the Calvin cycle in rapeseed plants treated with 150 mM NaCl, 0.25 mM Spd + 150 mM NaCl or 0.25 mM Spm + 150 mM NaCl compared to untreated plants. A) ribulose-bisphosphate carboxylase/oxygenase activase (RCA), B) ribulose-5-phosphate kinase (PRK), C) fructose-1,6-bisphosphate aldolase (FBA), D) sedoheptulose-1,7-bisphosphatase (SBPase), E) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), F) fructose-l,6-bisphosphate phosphatase (FBPase), G) 3-phosphoglyceric acid kinase (PGK), and H) triose-3-phosphate isomerase (TPI). Transcript levels were determined by qPCR and standardized against actin and GAPDH transcript levels. The experiments of qPCR were replicated thrice with two replications each. The bars on the columns correspond to the SE, and distinct letters differ substantially by LSD (p < 0.01).

Fig. 8

PC-biplot based on physiological and molecular parameters of salt-stressed rapeseed plants treated with NaCl versus non-stressed conditions, compared with exogenously sprayed Spd and Spm.