Developing molecular and physiological markers for barley breeding for waterlogging tolerance by targeting root ionic homeostasis

Waterlogging is a serious environmental threat worldwide that severely limits agricultural production. Waterlogging stress adversely affects 10% of the global land area and annual financial losses to agricultural crop production are estimated to exceed €60 billion. Many regions of the world (e.g. Australia, China) are regularly affected by waterlogging stress. Barley ranks fifth amongst all crops in dry matter production in the world. In Australia, most of the barley cultivars are waterlogging sensitive. In most cases, plants are grown on duplex soil, which has a layer of sandy soil over a comparatively water-resistant base of clay soil. Therefore, the continued rainfall events can lead to increasing water tables in the root zone. Waterlogging is a complex trait that conferred by several physiological and biochemical mechanisms. While the major attention of plant breeders was on traits related to oxygen supply and retention (radial oxygen loss; aerenchyma formation; etc), traits related to plant’s ability to maintain ionic homeostasis under waterlogged conditions received much less attention. Therefore, the main objectives of this study were (1) to investigate the effects of oxygen deprivation on intracellular K$^+$ signalling and homeostasis, and its potential roles in acclimation to oxygen-deprived conditions in barley; (2) to develop reliable screening protocols for evaluation of some key physiological traits conferring waterlogging stress tolerance which can be used in breeding programs; (3) to link waterlogging tolerance with K$^+$ retention, membrane potential maintenance and ROS stress tolerance by using a QTL approach; (4) to link these traits with other abiotic stresses such as salinity in order to make the deep understanding of tolerance mechanisms in barley. The major constraint that plants undergo in waterlogged conditions is the inadequate supply of oxygen to submerged parts. Oxygen depletion under waterlogged conditions results in a compromised operation of H$^+$ -ATPase, with strong implications for membrane potential maintenance, excessive ROS accumulation, cytosolic pH homeostasis, and transport of major nutrients across membranes. The above effects, however, are highly tissue-specific and time-dependent, and the causal link between hypoxia-induced changes to cell’s ionome and plant adaptive responses to hypoxia is not well established. This work aimed to fill the above gap and investigate the effects of oxygen deprivation on K$^+$ signalling and homeostasis in plants and potential roles of GORK (depolarization-activated outward-rectifying potassium) channels in plant adaptation to oxygen-deprived conditions in barley. The significant K$^+$ loss was observed in roots exposed to hypoxic conditions; this loss correlated with the cell’s viability. The stress-induced K$^+$ loss was stronger in the root apex immediately after stress onset but became more pronounced in the root base as the stress progressed. The amount ofK$^+$ in shoots of plants grown in waterlogged soil correlated strongly with K$^+$ flux under hypoxia measured in laboratory experiments. Hypoxia-induced membrane depolarization was less pronounced in the tolerant group of cultivars. The expression of GORK was down-regulated by 1.5-fold in mature root while upregulated by 10-fold in the apex after 48 h hypoxia stress. Taken together, our results suggest that GORK channel plays a central role in K$^+$ retention and signalling under hypoxia stress and measuring hypoxia-induced K$^+$ fluxes from the mature root zone may be used as a physiological marker to select waterlogging tolerant varieties in breeding programs. Waterlogging and salinity are two major abiotic stresses that could occur simultaneously and hamper crop production world-wide resulting in multibillion losses. Plant abiotic stress tolerance is conferred by many interrelated mechanisms. Amongst these, the cell’s ability to maintain membrane potential is considered to be amongst the most crucial traits, a positive relationship between the ability of plants to maintain highly negative membrane potential and its tolerance to both salinity and waterlogging stress. However, no attempts have been made to identify quantitative trait loci (QTL) conferring this trait. In this study, the microelectrode MIFE technique was used to measure the plasma membrane potential of epidermal root cells of 150 double haploid (DH) lines of barley (Hordeum vulgare L.) from a cross between a Chinese landrace TX9425 and Japanese malting cultivar Naso Nijo under hypoxic conditions. A major QTL for the membrane potential in the epidermal root cells in hypoxia-exposed plants was identified. This QTL was located on 2H, at a similar position to the QTL for waterlogging and salinity tolerance reported in previous studies. Further analysis confirmed that membrane potential showed a significant contribution to both waterlogging and salinity tolerance. The fact that the QTL for membrane potential was controlled by a single major QTL illustrates the power of the single-cell phenotyping approach and opens prospects for fine mapping this QTL. A reduced concentration of oxygen in waterlogged soils leads to oxygen deficiency in plant tissues, resulting in an excessive accumulation of ROS in plants. This ROS accumulation under waterlogged conditions also contributes to limit agricultural production in low-lying rainfed areas worldwide. To identify QTL for ROS tolerance in barley, 187 double haploid (DH) lines from a cross between TX9425 and Naso Nijo were screened for superoxide anion (O$_2$$^{·−}$) and hydrogen peroxide (H$_2$O$_2$) accumulated under hypoxia stress. In our experiment, we showed that quantifying ROS contents after 48 h hypoxia could be a fast and reliable approach for the selection of waterlogging tolerant barley genotypes. A major QTL on chromosome 2H was identified for both O$_2$$^{·−}$ (QSO.TxNn.2H) and H$_2$O$_2$ (QHP.TxNn.2H) contents. This QTL was located at the same position as the QTL for the overall waterlogging and salt tolerance reported in previous studies, explaining 23% and 24% of the phenotypic variation, for O$_2$$^{·−}$ and H$_2$O$_2$ contents, respectively. The analysis also showed a causal association between ROS production and both waterlogging and salt stress tolerance. The markers associated with this QTL could potentially be used in future breeding programs to improve waterlogging and salinity tolerance. Taken together, the results of this work showed that hypoxic conditions caused a significant loss of K$^+$, in a time- and genotype-specific manner. This has affected cell viability and overall plant tolerance. The genotypic difference in waterlogging stress tolerance in barley was conferred by the differential ability to regulate voltage-gated K$^+$-permeable channels (GORK) in the mature root epidermis. A strong positive correlation between the ability of mature zone cells to retain K$^+$ and the overall waterlogging stress tolerance was found, making it possible to recommend using this method as a physiological marker for breeding plants for waterlogging stress tolerance. A major QTL for membrane potential maintenance under hypoxia stress was identified on chromosome 2H using cell-based phenotyping involving microelectrode MIFE technique. Another important finding of this work is the identification of two major QTL for both O$_2$$^{·−}$ and H$_2$O$_2$ accumulation for hypoxia on Chromosome 2H. Interestingly, the QTL for membrane potential and ROS is located at a similar position to that for waterlogging and salinity tolerance on chromosome 2H. The fact that these QTL are detected at a similar position of chromosome 2H indicates a specific mechanism for different stress tolerances including waterlogging and salinity tolerance. Future work should be focusing to fine map these QTL and use this gene in pyramiding different tolerance mechanisms in breeding programs