Opportunities and prospects for increasing zinc in barley

Zinc (Zn) is a vital micronutrient for all living organisms, with essential roles in numerous biological and physiological processes. Zinc deficiency in humans is of worldwide concern and it is primarily associated with a plant-based diet of crops grown on Zn-deficient soils. It is estimated that approximately half of the soils where cereals are grown are lacking adequate amount of plant-available Zn. The lack of sufficient Zn in food crops contributes greatly to the ‚ÄövÑv¿hidden hunger‚ÄövÑvp with approximately 17% of the world‚ÄövÑv¥s population severely affected by Zn deficiency. Enhancing the Zn levels in food crops could be a solution to alleviate Zn malnutrition in humans. Although investigations into the potential for Zn biofortification of cereals has previously been performed, further in-depth understanding of mechanisms related to uptake, transport and sequestration of Zn is needed in order to better develop Zn- biofortified crops. Moreover, there is the need for a rapid and cost-effective analytical method to quantify Zn in plant samples as well as further elucidation of the beneficial roles of Zn in increasing plant growth and tolerance to abiotic stresses. The aim of this thesis is to provide a deeper understanding of Zn additive effect on the growth and abiotic stress tolerance in barley. Barley is an economically important plant species ranking fourth among cereals in farming acreage. It is a crop that is consumed widely by both humans (as cereal foods and beverages) and animals (as a livestock feed). Consequently, the research findings presented here provide information that is relevant to future breeding programmes and biotechnological strategies for crop improvement. Additionally, being a diploid grass, it is a natural model for the genetics and genomics of the Triticeae tribe, which includes highly important crops such as wheat. The following specific objectives were addressed in this work: ‚Äövª¬¢ Reveal what is already known regarding the effect of available substrate Zn content on the plant Zn concentration in barley ‚Äövª¬¢ Identify the quantitative trait loci (QTL) associated with increased Zn accumulation efficiency in barley ‚Äövª¬¢ Describe a cheap, quick and reliable method using pXRF to quantify Zn in barley shoots ‚Äövª¬¢ Investigate the role of Zn in amelioration of salinity stress in barley The meta-analysis clearly demonstrated that the accumulation of Zn in shoots of barley varies significantly between genotypes while also depending on Zn availability in soil. In addition, this study found that the genotypes with higher shoot Zn concentrations also had the higher grain Zn. Therefore, shoot Zn concentration was used as a selection criterion for increased grain Zn levels for the subsequent trials. Determination of elemental concentrations in plant tissue samples is critical for analysing its nutritional content. Analytical analysis of micronutrients in plants mostly relies on either inductively coupled plasma mass spectrophotometry (ICP-MS) or ICP-optical emission spectrometry (ICP-OES). Both methods are expensive and time-consuming, with complicated sample preparation, including the use of concentrated inorganic acids. Furthermore, such techniques are not widely available and are costly to establish, requiring skilled technicians. Therefore, a rapid, cheap, and easier analytical method for elemental measurements in plant samples would be beneficial to breeding projects, which normally involve large numbers of samples. Portable X-ray fluorescence (pXRF) devices are widely used in soil science, archaeology, metallurgy and geology to examine elemental concentrations. In this thesis, pXRF method was used to measure elemental concentrations of four elements Zn, iron (Fe), copper (Cu) and cadmium (Cd) in barley shoot samples. The dried plant material was ground to fine powder, with exactly 1 g being used for scanning with a pXRF instrument. Moreover, the pXRF method proved to be reliable only for Zn, but not for Fe, Cu or Cd investigated in the research presented. Thus, the work demonstrates the potential of pXRF as a reliable and cost-effective alternative to ICP methods for determining Zn in plant samples. In the subsequent experiment, initially ten barley genotypes were screened for shoot Zn concentration. A semi-hydroponics experiment was conducted in a glasshouse using 2.5-inch pots filled with sand and perlite by placing them into large trays, containing Hoagland‚ÄövÑv¥s nutrient solutions. Plants were subjected/placed in either full-strength Hoagland‚ÄövÑv¥s containing Zn (considered as adequate Zn level; Zn concentration > 0.5 ppm) or Zn-depleted Hoagland‚ÄövÑv¥s (considered as low Zn level; Zn concentration < 0.5 ppm). Above-ground vegetative part of plants (i.e., shoots) were harvested prior to the anthesis stage and assessed for their total Zn accumulation (shoot Zn concentration x shoot biomass). The amount of shoot Zn accumulation recorded in cv Franklin was almost half (50 %) when plants were grown at ‚ÄövÑv¨Zn conditions as compared to +Zn level, whereas this percentage in cv Yerong was only 18.3 %. Therefore, cv Franklin (as low Zn accumulator) and cv Yerong (as high Zn accumulator) were selected as parent material for further study based on largest and lowest differences in the accumulation of Zn in shoots between the adequate and low Zn treatments. A double-haploid population derived from a cross between Yerong x Franklin and comprising of 176 lines was further selected to determine the accumulation of Zn in shoots. The plants were grown in the same type of semi-hydroponics system under glasshouse conditions and supplemented with low and adequate Zn Hoagland‚ÄövÑv¥s nutrient solution. Shoot samples were assessed for their Zn and other mineral concentrations using ICP-MS to determine major QTLs for mineral accumulations in shoots of barley. One major QTL was identified for Zn accumulation in shoots of the mapping population, which was stable under both +Zn and -Zn conditions. This QTL was located on chromosome 2H between flanking markers of bPb-4875 (44.04 cM) and bPb-6088 (83.62 cM). The total phenotypic variances explained (PVE) by the QTL ranged from 9.4 ‚ÄövÑv¨ 12.1 for shoot Zn accumulation. This would facilitate researchers to develop new barley cultivars in the future with greater low Zn tolerance as well as increased Zn-accumulation efficiency. It is also known that Zn plays a pivotal role in a plant through regulating major physiological functions presumably as a signalling factor. For example, it triggers the cellular antioxidant mechanism, which is important for plant defence against various stresses. Reactive oxygen species (ROS) play a significant role in plants regulating their growth, cells cycle and programmed cells death. However, excessive ROS production in plants during extreme abiotic stresses such as salinity and drought, causes DNA damage, cell death and eventually cease plant growth. This leads to significant losses to the production and nutritional quality of agricultural produce. Agronomic Zn fertilisation was found to be effective in decreasing the ROS-induced stress in rice and wheat, which could be further investigated using barley by studying its physiological aspects in addition with ion flux kinetics. Therefore, a glasshouse experiment was performed by growing barley in semi-hydroponics system. It was identified in this study that addition of Zn increased the plant fresh and dry weight under salinity stress (200 mM NaCl). Zinc supplementation allowed plants to reduce excessive Na+ accumulation and therefore, maintain a higher shoot K+/Na+ ratio as compared to the no Zn application (control) plants, which indicated increased tolerance due to Zn application. This study demonstrated that antioxidant activity increased in shoots of plants with supplemented Zn such as 21 % in CAT and 7% in SOD, as compared to plants without supplemented Zn under salinity stress conditions. Furthermore, this study used 3-days old seedlings, which were supplemented with basic solution medium (BSM), in addition with two different doses of Zn (10 and 100 ˜í¬¿M ZnSO4) to carry out micro-electrode flux estimation (MIFE) analysis. The changes in K+ and Ca2+ flux in roots due to oxidative stress (10 mM H2O2) application were found to be decreased in seedlings supplemented with Zn as compared to those with no Zn. These results showed that the Zn supplementation may increase the tolerance of barley by enhancing ROS detoxification mechanism in barley during unfavourable abiotic conditions. In conclusion, this thesis helps to understand the essential role of Zn in plants with also a focus on increasing Zn accumulation in barley. This study demonstrated that shoot Zn concentration can be a useful indicator for grain Zn and could be used as a selection criterion to diagnose Zn status in barley genotypes. QTL analysis revealed the presence of Zn associated candidate genes on chromosome 2H. The use of pXRF technology was validated for its use in agricultural and plant science as a cost-effective and efficient way of measuring Zn concentration in shoots of plants. Finally, the thesis provides strong evidence that Zn supplementation can mitigate the damaging effects of soil salinity - a serious threat to agricultural productivity and quality.