Mn2\\(^+\\) tolerance in barley (Hordeum vulgare L.) and its contribution to waterlogging tolerance

Vast agricultural areas are affected by flooding causing up to 80% yield reduction and resulting in multibillion dollar losses. In recent years, the occurrence of flooding has increased though human activities, especially in agricultural systems with poor drainage. Therefore, waterlogging stress is becoming one of the main challenge of the modern agriculture. Of all the cereals, barley (Hordeum vulgare L.) is a widely adaptable crop, which is ranked fourth among grains in quantity produced behind maize (Zea mays L.), rice (Oryza sativa L,) and wheat (Triticum aestivum L,). The less complex barley genome also makes it a useful species to understand physiological and molecular aspects of plant adaptation to the flooding stress and to improve their adaptive abilities to confront environmental constrains. Waterlogging tolerance is a complex trait affected by various factors including soil characteristics, temperature, plant developmental stage, microbe activities and oxygen availability. Understanding the mechanisms of waterlogging tolerance make it possible for plant breeders to target individual physiological traits and create barley breeding materials with enhanced waterlogging tolerance. Up to now, the focus of plant breeders was predominantly on alleviating detrimental effects of anoxia, while other (potentially equally important) traits were essentially neglected. One of these is the soil elemental toxicity. Excess water triggers a progressive decrease in the soil redox potential, thus increasing the concentrations of Mn2\\(^+\\) and Fe2\\(^+\\) that can be toxic to plants, when exceeding a threshold concentration. Cellular detoxification and exclusion are the main strategies for plants to resist excess ion concentration in soil. However, specific details of their coordination and the relative contribution of these components towards manganese toxicity tolerance in barley have not been fully revealed. Besides, the linkage between ion toxicity tolerance and waterlogging stress tolerance is still poorly understood, although tolerance to one or more elemental toxicities can be an essential trait to improve plant performance in waterlogged soils. Accordingly, the major aim of this PhD project was to investigate the physiological and molecular aspects of manganese toxicity tolerance associated with waterlogging stress tolerance. The following specific objectives were addressed: To quantify the relative contribution of Mn2\\(^+\\) toxicity to waterlogging stress tolerance; To develop a rapid screening method and screen a large number of barley varieties; To identify QTLs controlling tolerance to manganese toxicity in barley associated with tolerance of waterlogging stress; To investigate physiological and molecular mechanisms conferring manganese tolerance. Working along these lines, a broad range of barley (Hordeum vulgare and Hordeum spontaneum L.) genotypes contrasting in waterlogging stress tolerance were used to investigate its linkage with manganese toxicity tolerance. In total, twenty barley genotypes (including three wild barleys) contrasting in waterlogging stress tolerance were studied for their ability to cope with the toxic (1 mM) amounts of Mn2\\(^+\\) in the root rhizosphere. Under Mn2\\(^+\\) toxicity, chlorophyll content of most waterlogging-tolerant genotypes (TX9425, Yerong, CPI-71284-48 and CM72) remained above 60% of the control value, whereas sensitive genotypes (Franklin and Naso Nijo) had a chlorophyll content less than 35% of the control. Manganese concentration in leaves was not related to visual Mn2\\(^+\\) toxicity symptoms, suggesting that various Mn2\\(^+\\) tolerance mechanisms might have operated in different tolerant genotypes, i.e. avoidance versus tissue tolerance. The overall significant (r = 0.60) correlation between tolerances to Mn2\\(^+\\) toxicity and waterlogging in barley suggests that plant breeding for tolerance to waterlogging traits may be advanced by targeting mechanisms conferring tolerance to Mn2\\(^+\\) toxicity, at least in this species. Direct selection (using only agronomic traits) for stress tolerance is easily affected by environments thus less effective. Marker assisted selection (MAS) could provide an indirect selection process which can effectively reveal distinct genetic differences but not merely on trait itself. Therefore, further studies were conducted in specific doubled-haploid populations to determine whether the same genes are responsible for Mn2\\(^+\\) and waterlogging tolerance. A total of 177 lines from Yerong/Franklin population were used to identify QTL conferring Mn2\\(^+\\) tolerance and another 188 DH lines from TX9425/Naso Nijo were used to validate the QTL identified in the Yerong/Franklin population. Seven QTLs were identified from these two populations. Among all, four QTL controlling plant survival under manganese toxicity determined almost 40% of phenotypic variation. Two significant QTL for leaf chlorosis were identified at a similar position as those for plant survival on chromosome 3H and 6H, explaining 22.1% and 7.5% of phenotypic variation respectively. In the TX9425/Naso Nijo DH population, only one significant QTL accounting for plant survival was identified which was located at a same position on chromosome 3H as the major QTL identified in the Yerong/Franklin DH population. Based on the major QTL on chromosome 3H, three candidate genes (POD, KAT3, HMA) for this QTL were identified, suggesting antioxidant system and potassium transport might play a substantial role in coping with manganese toxicity. We then used the MIFE (microelectrode ion flux measurement) technique to study some aspects of manganese stress signalling, focusing on K\\(^+\\) transport (as per about QTL findings). K\\(^+\\) retention plays a pivotal role in conferring many abiotic stress tolerances in plants. In this work, ten selected barley genotypes were used to study Mn-induced changes in K\\(^+\\) transport. These fluxes were then related to appropriate changes in fluxes of Ca2\\(^+\\)+ (a known second messenger) and H\\(^+\\) (a proxy for H\\(^+\\)-ATPase activity). All genotypes responded to Mn treatment by net K\\(^+\\) influx, while net Ca2\\(^+\\) and H\\(^+\\) efflux was observed after adding 1 mM Mn2\\(^+\\). No significant difference among genotypes was found. Several inhibitors were used to understand the specific signal pathway affected by manganese. Manganese-induced K\\(^+\\) uptake and Ca2\\(^+\\) efflux were significantly inhibited by TEA (a blocker of K\\(^+\\) channels) and vanadate (H\\(^+\\)-ATPase inhibitor). However, a significant K\\(^+\\) and Ca2\\(^+\\) leakage was measured in DPI-pretreated root when applied Mn treatment, suggesting that NADPH-oxidase may play an essential role in regulating Mn uptake. High manganese concentration did not significantly affect net Ca2\\(^+\\) flux and net K\\(^+\\) flux in Gd3\\(^+\\), La3\\(^+\\) or TG (thapsigargin, endomembrane Ca2\\(^+\\) channel inhibitor) pretreated roots. The above results suggest that both non-selective cation channels and Ca2\\(^+\\)/H\\(^+\\) exchangers contribute to manganese uptake and transport in barley roots. Hypoxic conditions also trigger a burst in reactive oxygen species (ROS), resulting in a significant K\\(^+\\) efflux. This work has shown that hypoxia enhanced sensitivity to exogenous H\\(_2\\)O\\(_2\\) while additional Mn ion efficiently alleviated the impact of hypoxia on intracellular K\\(^+\\) homeostasis. The reported up-regulated expression of HAK gene also suggests that manganese may play an important role by signalling K\\(^+\\) deficiency and enabling plants with mechanisms for better K\\(^+\\) retention to confront stress. In conclusion, this project has found that different barley genotypes adopt different strategies to resist manganese toxicity. Both exclusion and internal tolerance mechanisms contribute to Mn2\\(^+\\) tolerance. Tolerance to Mn2\\(^+\\) showed a significant positive correlation with waterlogging tolerance. However, Mn2\\(^+\\) does not appear to be toxic in roots. The ability of roots to retain K\\(^+\\) was proven to be one of the key traits conferring tolerance to numerous stress. Mn was able to trigger a hyperpolarisation of the plasma membrane, leading to significant K\\(^+\\) uptake. NADPH oxidase-mediated apoplastic H\\(_2\\)O\\(_2\\) production may be causally related to ROS inducible Ca2\\(^+\\) uptake systems contributing to Mn uptake. Also, HvHAK5 transporter was found to be involved in maintenance of K\\(^+\\) content in root, which was identified at the major QTL on chromosome 3H associated with manganese toxicity. This QTL could therefore be used in breeding programs to enhance bothmanganese toxicity tolerance and waterlogging tolerance.