Modulation of root nodule antioxidant systems by nitric oxide : prospects for enhancing salinity tolerance in legumes
Thesis (PhD(Agric) (Plant Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Salinity is one of the major limiting abiotic stresses on legume plant yield, leading to early senescence of root nodules. This occurs because of accumulation of reactive oxygen species (ROS) in plant cells under salinity stress. Concurrent with the increase in cellular reactive oxygen species levels is the increase in cellular antioxidants and corresponding antioxidant enzymes. This feature is observed mostly in the shoots and roots of more tolerant genotypes compared to the susceptible genotypes. It is accepted that the mechanism of plant tolerance to stress is dependent upon the response of the antioxidant systems. Most studies carried out on shoot tissues suggest that scavenging of ROS by the plant antioxidant system is modulated by nitric oxide (NO). However, the pathways by which NO mediates such antioxidant responses are not fully understood. For legumes, salinity stress has adverse effects on yield and this is in part due to inhibition of nitrogen fixation in the root nodules of the legumes, which causes severe nitrogen starvation in nitrogen-deficient soils. Nodules are specialized organs comprising of both the rhizobia and the plant tissue, hence the physiological aspects may vary from the findings from the leaves. It was therefore deemed necessary to establish the role of NO on the nodule antioxidant system in the absence and presence of salinity stress. For the purposes of this study, the effect of both exogenously applied NO and endogenous NO on superoxide dismutase, glutathione peroxidase and glutathione content was determined. The studies involved the use of nitric oxide donors like sodium nitroprusside (SNP) and diethylenetriamine/nitric oxide adduct (DETA/NO), their respective fixed controls potassium ferricyanide and diethylenetriamine (DETA), plus a nitric oxide synthase inhibitor (to inhibit nitric oxide production by the enzyme nitric oxide synthase) on nodulated roots. The data obtained in this work points out specifically at roles played by nitric oxide in regulating superoxide dismutases, glutathione peroxidase and glutathione during salinity stress and proposes a link between nitric oxide-mediated changes in these antioxidant systems and salinity stress tolerance. Both the exogenously applied and endogenous nitric oxide increases the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR). However, there is both time dependency and nitric oxide concentration dependency on the enzyme activities. The total SOD enzyme activity increases upon nitric oxide exposure and with time of exposure. The individual SOD isoforms identified and studied in the root nodules all contribute to this increase in SOD activity upon nitric oxide treatment except for MnSOD I. This increase in activity is regulated at transcriptional level as the RT-PCR results targeting the individual isoforms reveals an increase in transcript levels after 6 hours of nitric oxide treatment. However, the CuZn SOD I isoform transcripts are reduced upon nitric oxide treatment. A similar response was also observed in GPX enzyme activity in which nitric oxide increased the GPX activity above all the controls. The GR enzyme activity exhibits an opposite response because the activity decreases with time of exposure to NO and concentration of NO. In order to determine the effect of NO under saline conditions, an experiment was set up that involved incubation of nodulated roots in solutions containing 150 mM NaCl. The stressed nodules exhibited generally higher levels of enzyme activities than the non-stressed nodules. Furthermore, exposure to nitric oxide donor in combination with NaCl induced even higher activities of SOD and GPX than NaCl or nitric oxide donor alone. There were also higher levels of reduced glutathione and total glutathione recorded under stress compared to optimal conditions. Nitric oxide increased the concentration of these forms of glutathione, suggesting an improved redox status based on the GSH/GSSG ratios under salinity stress in the presence of nitric oxide. Attenuation of nitric oxide synthesis with L-Nω-Nitroarginine methyl ester (L-NAME) reverses all the recorded effects of nitric oxide on antioxidant enzymes and glutathione pool. This was observed in salinity stressed nodules and non-stressed nodules. This work further establishes that NO plays a pivotal role in modulating the enzymatic activities through a pathway that is mediated by guanosine 3,5-cyclic monophosphate (cGMP). The experiment involving the inhibition of soluble guanylyl cyclase (sCG) (an enzyme that catalyzes the biosynthesis of cGMP), cell-permeable cGMP anaologue and L-NAME revealed that GPx activity is modulated through a cGMP-dependent pathway and NO is positioned up-stream of cGMP in the pathway leading to improved GPX activity. Cyclic GMP also modulates the GPX activity in a concentration dependent manner. NO improves the redox status of the cell under both saline conditions and non-saline conditions and this effect is modulated through a cGMP-dependent pathway. It is thus rational to conclude that; in the root nodules of legumes, like in other plant tissues, the increased accumulation of antioxidants and the increased activity of their corresponding enzymes, as modulated through the cGMP-dependent pathway by nitric oxide, confer root nodule tolerance to salinity. This concept directly points out at an attractive strategy for developing legumes that are genetically improved for enhanced root nodule tolerance to salinity; via differential regulation of antioxidants and antioxidant enzyme genes in the root nodules under abiotic stress. Towards attaining the goal for such genetic improvement, experiments involving construction of an abiotic stress-responsive and nodule-specific chimeric promoter were carried out. By fusing the 5-untranslated (5-UTR) region of the LEA gene that contains an abiotic stress-responsive cis-acting element (from theGmPM9 promoter) to the nodulin N23 promoter bearing the highly functional cluster of motifs for nodule specificity, the candidate nodule specific promoter that is abiotic stress responsive (ASREF/NSP) was constructed. The construct harbouring this ASREF/NSP chimeric promoter was fused to the -glucuronidase (GUS) reporter gene so as to study the functionality of the promoter in Medigaco truncatula plants. The construct was delivered into the Medicago plants through Agrobacterium rhyzogenes mediated transformation to produce composite Medicago plants. The transgenic roots have been cultured for futher manipulation and to confirm the functionality of the promoter. Furthermore several strategies can be deployed via the use of this chimeric promoter so as to enhance the nodular antioxidant system. This would involve either gene regulator-chimeric promoter fusion or the use of a single gene approach. As part of this work, the MtNOA gene homologous to AtNOAs, has been cloned from Medicago trancatula and put as ASREF/NSP fusion in a binary vector pBINPLUS and delivered into Medicago trancatula for nodule-specific and abiotic stress-induced nitric oxide synthesis. Since there is no plant NOS identified to date, the possibility of the use of a regulatory gene in this aspect is still limited. There are other options involving the use of the chimeric promoter with the individual genes encoding the antioxidant enzyme genes such as genes encoding SOD, GPX and the glutathione synthatase to enhance the plant antioxidant system during abiotic stress.
AFRIKAANSE OPSOMMMING: Geen opsomming was ingedien met die tesis