Title : The role of antioxidant mechanisms in the tolerance of the Negev desert extremophyte, Anastatica hierochuntica, to abiotic stress
Abstract:
Investigation of stress-tolerant plants is crucial for understanding how plants can tolerate multiple abiotic stresses in the field. The extremophyte desert relative of Arabidopsis thaliana, Anastatica hierochuntica, is tolerant to several abiotic stresses. Because the secondary effect common to most abiotic stresses is massive production of damaging reactive oxygen species, (ROS), we investigated the response of the Anastatica ROS-scavenging machinery in comparison to Arabidopsis during methyl viologen (MV)-induced oxidative stress and heat stress. In vitro-grown Anastatica seedlings exhibited remarkable tolerance to increasing concentrations of MV, and to heat stress, compared to Arabidopsis. Notably, Anastatica was able to maintain total chlorophyll levels in response to high concentrations of MV and to heat stress whereas chlorophyll levels were considerably reduced in Arabidopsis. The severe stress symptoms observed in Arabidopsis were also reflected in the high accumulation of anthocyanins in response to MV and heat stress whereas only low levels of anthocyanins were observed in Anastatica. Nitroblue tetrazolium (NBT) staining of Arabidopsis seedlings indicated massive generation of superoxide radicals over time with increasing MV levels. No staining was apparent in control seedlings. However, Anastatica exhibited NBT staining in both control and MV-treated seedlings, which could reflect either a stress response caused by the experimental procedure that involved transfer of seedlings to treatment plates, or could be due to basal levels of ROS production. 3,3’-diaminobenzidine (DAB) staining of Arabidopsis seedlings for detection of H2O2, increased with elevated MV concentrations suggesting a rise in H2O2 production in response to oxidative stress. In contrast, Anastatica did not display any significant DAB staining in response to MV-induced oxidative stress, which could indicate a more highly active antioxidant system in Anastatica compared to Arabidopsis. ROS production in response to heat stress was different from the direct oxidative stress response. No DAB staining was observed in Anastatica at any time after onset of heat stress while in Arabidopsis, weak staining could only be detected after 3 and 4 h of heat stress. Surprisingly, Anastatica displayed stronger NBT staining that exhibited a clear dose response, compared to Arabidopsis. This result is unexpected and could indicate that Anastatica can tolerate high cellular superoxide levels or/and that high superoxide levels are necessary for heat stress signaling. To obtain further evidence for a more highly active antioxidant system in Anastatica, we have begun to examine expression of genes encoding various ROS-scavenging enzymes. Preliminary QPCR analysis of gene expression during heat stress showed that Anastatica exhibited a larger and more rapid induction of ASCORBATE PEROXIDASE 1 (APX1) expression than Arabidopsis. Overall, our data suggest that Anastatica is highly tolerant to direct oxidative stress and to heat stress. Work is in process to study the expression of a range of antioxidant genes, and antioxidant scavenging enzymes activities in response to oxidative stress and heat.
The study of naturally stress tolerant extremophytes is crucial in understanding how plants can tolerate multiple abiotic stresses in the field. This research provides knowledge concerning the antioxidant machinery in stress tolerant plants, which is a major mechanism allowing these species to cope with the harsh desert conditions.
