An aspartic acid protease from common bean is expressed 'on call' during water stress and early recovery.

A cDNA encoding a putative aspartic acid protease precursor (PvAP1) was cloned from the leaves of common bean (Phaseolus vulgaris). Sequence analysis showed that PvAP1 presents all the characteristic features of phytepsins, the typical plant APs. PvAP1 gene expression was tightly regulated by water stress, being significantly up-regulated under mild water stress (Ψ(w)=-1.0 MPa) for the drought-susceptible cultivar (Carioca) and moderate water stress (Ψ(w)=-1.5 MPa) for the more drought-tolerant cultivar (IPA). Protein gel blotting analysis under water stress revealed the presence of two main bands of calculated MW of 46 and 38 kDa, suggesting proteolytic processing of the enzyme precursor form under drought in both cultivars. Taken together, our results suggest that water stress regulates PvAP1 activity both at the transcriptional and post-transcriptional levels, and that the response occurs earlier and is stronger in the drought-susceptible cultivar.

Homoglutathione synthetase and glutathione synthetase in drought-stressed cowpea leaves: expression patterns and accumulation of low-molecular-weight thiols.

Glutathione (GSH) is an abundant metabolite and a major antioxidant in plant cells. However, in the Leguminosae, homoglutathione (hGSH) may replace glutathione (GSH) partially or completely. To date, cowpea (Vigna unguiculata) has been considered a non-hGSH-producing species, and no hGSHS cDNA has been isolated. Here we report on the cloning of a full-length cDNA coding for a hGSHS (EC 6.3.2.23) and the cloning of a partial cDNA coding for a putative glutathione synthetase (GSHS; EC 6.3.2.3) in cowpea leaf extracts. These cDNAs possess, respectively, the leucine/proline hGSHS signature and the alanine/alanine GSHS signature at the 3' end. Expression analysis showed a significant up-regulation of hGSHS during progressive drought stress that could be directly related to the drought tolerance of the cowpea cultivar used, while GSHS was mainly constitutively expressed. Nevertheless, quantification of low-molecular-weight thiols confirmed the previous findings that cowpea is essentially a GSH producing plant, as no hGSH was detected in the leaves. These findings raise new questions regarding the function, activity and substrate specificity of the cloned hGSHS cDNA. These questions are discussed.

(h)GR, beans and drought stress.

Reactive oxygen species (ROS) are commonly found in plants as natural by-products of the metabolism but their production is greatly enhanced under abiotic stresses. Particular metabolites and enzymes belonging to the ascorbate-glutathione cycle are able to scavenge these deleterious molecules and modulate the cellular redox-status. In the March issue of Journal of Plant Physiology, we have shown that drought stress induces a raise in glutathione reductase (GR) activity and gene expression that could be related to the intensity of the drought treatment and the drought susceptibility of the bean cultivar (cowpea and/or common bean). In the present addendum we show new data on GR specific activity during progressive drought stress and recovery of the drought-susceptible bean cultivar which can be related to the previously found dual-targeted GR gene expression. Furthermore, since in leguminous plants homoglutathione (hGSH) is generally the most abundant low molecular weight thiol form, we discuss on the occurrence of a (homo)glutathione reductase activity in beans.

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress.

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.

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

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.

Isolation and characterization of four ascorbate peroxidase cDNAs responsive to water deficit in cowpea leaves.

BACKGROUND AND AIMS: Abiotic stresses stimulate formation of active oxygen species in plant tissues. Among antioxidant mechanisms, H2O2 detoxication by ascorbate peroxidases (APX) plays an important role. Several APX isoforms exist in plant cells, and they have rarely been studied separately. The aim of this work was to study changes in cytosolic, peroxisomal, stromatic and thylakoid APX gene expression in response to progressive drought, rapid desiccation and application of exogenous abscisic acid in the leaves of cowpea (Vigna unguiculata) plants. METHODS: Two cowpea (V. unguiculata) cultivars, 'EPACE-1' which is drought-tolerant and '1183'which is drought-sensitive, were submitted to drought stress by withholding irrigation. Detached leaves were air-dried or treated with exogenous abscisic acid. APX cDNAs were isolated by PCR and cloned in plasmid vectors. Changes in gene expression were studied using reverse-transcription PCR. KEY RESULTS: Four new V. unguiculata cDNAs encoding putative cytosolic, peroxisomal and chloroplastic (stromatic and thylakoidal) APX were isolated and characterized. In response to the different treatments, higher increases in steady-state transcript levels of the cytoplasmic and peroxisomal APX genes were observed in '1183' compared with 'EPACE-1'. On the other hand, the expression of the chloroplastic APX genes was stimulated earlier in the tolerant cultivar when submitted to progressive drought. CONCLUSIONS: Water deficit induced differences in transcript accumulation of APX genes between the two cultivars that were related to their respective tolerance to drought. Chloroplastic APX genes responded early to progressive water deficit in the tolerant plant, suggesting a capacity to efficiently detoxify active oxygen species at their production site. The more sensitive '1183' was also able to respond to drought by activating its whole set of APX genes.