Oxidative stress tolerance as a component of the tissue tolerance mechanism in wheat and barley

Soil salinity is a global issue and a major factor limiting crop production worldwide. One side effect of salinity stress is an overproduction and accumulation of reactive oxygen species (ROS), causing oxidative stress and leading to severe cellular damage to plants. While the major focus of the salinity-oriented breeding programs in the last decades was on traits conferring Na\(^+\) exclusion or osmotic adjustment, breeding for oxidative stress tolerance has been largely overlooked. ROS are known to activate several different types of ion channels affecting intracellular ionic homeostasis and thus plant’s ability to adapt to adverse environmental conditions. However, the molecular identity of many ROS-activated ion channels remains unexplored and, to the best of our knowledge, no associated QTLs have been reported in the literature. This work aimed to fill the above knowledge gaps by evaluating a causal link between oxidative and salinity stress tolerance. The following specific objectives were addressed: ➢ To develop MIFE protocols as a tool for salinity tolerance screening in cereals. ➢ To validate the role of specific ROS in salinity stress tolerance, by applying developed MIFE protocols to a broad range of cereal varieties and establish a causal relationship between oxidative and salinity stress tolerance in cereals. ➢ To map QTLs controlling oxidative stress tolerance in barley. ➢ To develop a simple and reliable high-throughput phenotyping method based on above traits. Working along these lines, a range of electrophysiological, pharmacological, and imaging experiments were conducted using a broad range of barley and wheat varieties and barley double haploid (DH) lines. In order to develop the applicable MIFE protocols, the causal relationship between salinity and oxidative stress tolerance in two cereal crops - barley and wheat - was investigated by measuring the magnitude of ROS-induced net K\(^+\) and Ca2\(^+\) fluxes from various root tissues and correlating them with overall whole-plant responses to salinity. No correlation was found between root responses to hydroxyl radicals and the salinity tolerance. However, a significant positive correlation was found for the magnitude of H\(_2\)O\(_2\)-induced K\(^+\) efflux and Ca2\(^+\) uptake in barley and the overall salinity stress tolerance, but only for mature zone and not the root apex. The same trends were found for wheat. These results indicate high tissue specificity of root ion fluxes response to ROS and suggest that measuring the magnitude of H\(_2\)O\(_2\)-induced net K\(^+\) and Ca2\(^+\) fluxes from mature root zone may be used as a tool for cell-based phenotyping in breeding programs aimed to improve salinity stress tolerance in cereals. In the next chapter, 44 barley and 40 wheat (20 bread wheat and 20 durum wheat) cultivars contrasting in their salinity tolerance were screened to validate the above correlation between H\(_2\)O\(_2\)-induced ions fluxes and the overall salinity stress tolerance. A strong and negative correlation was reported for all the three cereal groups, indicating the applicability of using the MIFE technique as a reliable screening tool in cereal breeding programs. Pharmacological experiments were then conducted to explore the molecular identity of H\(_2\)O\(_2\) sensitive Ca2\(^+\) and K\(^+\) channels in both barley and wheat. We showed that both non-selective cation and K\(^+\)-selective channels are involved in ROS-induced Ca2\(^+\) and K\(^+\) flux in barley and wheat. At the same time, the ROS generation enzyme NADPH oxidative was also playing vital role in controlling this process. The findings may assist breeders in identifying possible targets for plant genetic engineering for salinity stress tolerance. Once the causal association between oxidative and salinity stress has been established, we have mapped QTLs associated with H\(_2\)O\(_2\)-induced Ca2\(^+\) and K\(^+\) fluxes, as a proxy for salinity stress tolerance, using over 100 DH lines from a cross between CM72 (salt tolerant) and Gairdner (salt sensitive). Three major QTLs on 2H (QKF.CG.2H), 5H (QKF.CG.5H) and 7H (QKF.CG.7H) were identified to be responsible for H\(_2\)O\(_2\)-induced K\(^+\) fluxes, while two major QTLs on 2H (QCaF.CG.2H) and 7H (QCaF.CG.7H) were for H\(_2\)O\(_2\)-induced Ca2\(^+\) fluxes. QTL analysis for H\(_2\)O\(_2\)-induced K\(^+\) flux by using H\(_2\)O\(_2\)-induced Ca2\(^+\) flux as covariate showed that the two QTLs for K\(^+\) flux located at 2H and 7H were also controlling Ca2\(^+\) flux, while another QTL mapped at 5H was only involved in K\(^+\) flux. According to this finding, the nearest sequence markers (bpb-8484 on 2H, bpb- 5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for salinity tolerance, and annotated genes between 64.45 and 80.95 cM on 2H, 42.99 and 48.38 cM on 5H, 119.83 and 140.86 cM on 7H were deemed to be potential genes. The above findings open previously unexplored prospects of improving salinity tolerance by pyramiding the new trait - H\(_2\)O\(_2\)-induced Ca2\(^+\) and K\(^+\) fluxes - alongside with other (traditional) mechanisms. However, as the MIFE method has relatively low throughput capacity, finding a suitable proxy will benefit plant breeders. Two high-throughput phenotyping methods - viability assay and root growth assay - were then tested and assessed. In viability staining experiments, a dose-dependent H\(_2\)O\(_2\)-triggered loss of root cell viability was observed, with salt sensitive varieties showing significantly more root cell damage. In the root growth assays, relative root length (RRL) was measured in plants under different H\(_2\)O\(_2\) concentrations. The biggest difference in RRL between contrasting varieties was observed for 1 mM H\(_2\)O\(_2\) treatment. Under these conditions, a significant negative correlation in the reduction in RRL and the overall salinity tolerance was reported among 11 barley varieties. Although both assays showed similar results with that of MIFE method, the root growth assay was way simpler that do not need any specific skills and training, and less time-consuming than MIFE (1 d vs 6 months), thus can be used as an effective high-throughput phenotyping method. In conclusion, this project established a causal link between oxidative and salinity stress tolerance in both barley and wheat and provided new insights into fundamental mechanisms conferring salinity stress tolerance in cereals. The high throughput screening protocols were developed and validated, and it was H\(_2\)O\(_2\)- induced Ca\(^{2+}\) uptake and K\(^+\) efflux from the mature root zone correlated with the overall salinity stress tolerance, with salt-tolerant barley and wheat varieties possessed greater K\(^+\) retention and lesser Ca\(^{2+}\) uptake ability when challenged with H\(_2\)O\(_2\). The QTL mapping targeting this trait in barley showed three major QTLs for oxidative stress tolerance conferring salinity stress tolerance. The future work should be focused on pyramiding these QTLs and creating robust salt tolerant genotypes