Glutathione reductase in leaves of cowpea: cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment

Abstract

Background and aims: Reactive oxygen species are frequently produced when plants are exposed to abiotic stresses. Among the detoxication systems, two enzymes, ascorbate peroxidase and glutathione reductase (GR) play key roles. GR has also a central role in keeping the reduced glutathione pool during stress thus allowing the adjustments on the cellular redox reactions. The aim of this work was to study the variations in cytosolic and dual-targeted GR gene expression in the leaves of cowpea plants submitted to progressive drought, rapid desiccation and application of exogenous abscisic acid (ABA).

Methods: Two cowpea (Vigna unguiculata) cultivars, one drought-resistant ('EPACE-1'), the other drought-sensitive ('1183') were submitted to progressive drought stress by withholding irrigation. Cut-off leaves were air-dried or treated with exogenous ABA. Two GR cDNAs, one cytosolic, the other dual-targeted to chloroplasts and mitochondria were isolated by PCR and cloned in plasmid vectors. Reverse-transcription PCR was used to study the variations in GR gene expression.

Key results: Two new cDNAs encoding a putative dual-targeted and a cytosolic GR were cloned and sequenced from leaves of V. unguiculata. Drought stress induced an up-regulation of the expression of the cytosolic GR gene directly related to the intensity of the stress in both cultivars. The expression of dual-targeted GR was up-regulated by the drought treatment in the susceptible cultivar only. Under a fast desiccation, the '1183' cultivar responded later than the 'EPACE-1', although in 'EPACE-1' it was the cytosolic isoform which responded and in '1183' the dual-targeted one. Exogenous ABA enhanced significantly the activity and expression levels of GR in both cultivars after treatment for 24 h.

Conclusions: These results demonstrate a noticeable activation in both cultivars of the antioxidant metabolism under a progressive water stress, which involves both GR genes in the case of the susceptible cultivar. Under a fast desiccation, the susceptible cultivar responded later than the resistant one, suggesting a weaker capacity of response versus the resistant one. Exogenous ABA probably acts on GR gene expression via a mediated signal transduction pathway.

Fig. 1

GR-specific activity in cowpea (Vigna unguiculata) ‘EPACE-1’ and ‘1183’ leaves. GR-specific activity was measured in control and drought-stressed plants (top), under a desiccation experiment (middle) and after ABA-treatment (bottom). Values are means ± s.d. of three to five independent experiments. GR activity was assayed by following the oxidation of NADPH (decrease in absorbance at 340 nm) and expressed in nmol mg⁻¹ protein min⁻¹. Control plants (C), Ψw = −0·5 ± 0·1 MPa; S, droughted plants: S1, Ψw = −1·0 ± 0·1 MPa; S2, Ψw = −1·5 ± 0·2 MPa; S3, Ψw = −2·0 ± 0·2 Mpa; D, desiccation treatment for 0·5 h (D1), 2 h (D2), 5 h (D3); A, ABA treatment for 0·5 h (A1), 2 h (A2), 24 h (A3).

Fig. 2

Alignment of cytosolic GR (cGR) amino acid sequences from Vigna unguiculata (Vu) and Pisum sativum (Ps, Genebank accession no. X98274) and of dual-targeted GR (dtGR) from Vigna unguiculata and Glycine max (Gm, no. L11632) amino acid sequences. The alignment was obtained using the program Clustalw (Thompson et al., 1994). ^*, identity; ‡, strongly similar; †, weakly similar; ↓, cleavage site for dual-targeted isoform (the targeting signal peptide is in italics); a, conserved arginine residues for NADPH binding; b, glutathione binding residue; the redox-active disulfite bridge is underlined (from Stevens et al., 1997).

Fig. 3

Effect of progressive drought on mRNA level of dtGR, cGR and S19 in ‘EPACE-1’ and ‘1183’ Vigna unguiculata leaves. Control plants (C), Ψw = −0·5 ± 0·1 MPa; S, droughted plants: S1, Ψw = −1·0 ± 0·1 MPa; S2, Ψw = −1·5 ± 0·2 MPa; S3, Ψw = −2·0 ± 0·2 MPa. (A) Gel analysis of GR isoenzyme transcripts; RT-PCR was carried out on 100 ng total RNA with 35 cycles, results were visualized using a UV light transilluminator. (B) Relative mRNA level. The mRNA levels were quantified with the Imager 1D/2D software and normalized to the respective S19 gene. Relative transcript levels were calculated with reference to the controls taken as 1 (100 %). Results are means ± s.d. of three independent experiments.

Fig. 4

Effect of desiccation (D) on mRNA level of dtGR and cGR and 18S rRNA in ‘EPACE-1’ and ‘1183’ Vigna unguiculata leaves. Desiccated leaves were air-dried at 24 °C under dim light for 0·5 h (D1), 2 h (D2) or 5 h (D3). Leaves from control plants (C) were taken prior to treatments. (A) Gel analysis of GR isoenzyme transcripts; RT-PCR was carried out on 100 ng total RNA with 35 cycles; results were visualized using a UV light transilluminator. (B) Relative mRNA level. The mRNA levels were quantified with the Imager 1D/2D software and normalized to the respective S19 gene. Relative transcript levels were calculated with reference to the controls taken as 1 (100 %). Results are means ± s.d. of three independent experiments.

Fig. 5

Effect of exogenous abscisic acid (ABA) on mRNA level of dtGR and cGR in ‘EPACE-1’ and ‘1183’ Vigna unguiculata leaves. Exogenous ABA (100 μm) was applied by immersing petioles of excised leaves at 24 °C under dim light for 30 min (A1), 2 h (A2) and 24 h (A3). (A) Gel analysis of GR isoenzyme transcripts. RT-PCR was carried out on 100 ng total RNA with 35 cycles; results were visualized using a UV light transilluminator. (B) Relative mRNA level. The mRNA levels were quantified with the Imager 1D/2D software and normalized to the respective S19 gene. Relative transcript levels were calculated with reference to the controls taken as 1 (100 %). Results are means ± s.d. of three independent experiments.