# Physiological and morphological responses to osmotic stress in barley (Hordeum vulgare L.)

Hasanuzzaman, Md 2018 , 'Physiological and morphological responses to osmotic stress in barley (Hordeum vulgare L.)', PhD thesis, University of Tasmania.

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## Abstract

Global food production must increase by at least 70% to feed an additional 2.4 billion people by 2050 while world agriculture combats major biotic and abiotic stresses brought by the climate change. Salinity and drought are arguably the two most severe abiotic environmental stresses among these that affect agricultural crop production globally. Therefore, future food security cannot be achieved without a major breakthrough in crop breeding for salinity and drought stress tolerance. The early responses of plants to drought and salinity are similar, as both stresses result in a cellular water deficit. This causes a decrease of the cytosolic and cell vacuolar volumes that inhibit plant growth and productivity. Plants deal with osmotic stress by employing a range of biochemical, morphological and physiological mechanisms. However, it has become clear in recent years that osmotic stress tolerance is highly multifaceted traits, determined by a number of sub-traits, of which the efficient control of stomatal and non-stomatal (residual) transpiration are the most crucial components to increase the efficiency of CO$$_2$$ assimilation. To attain the overall goal of producing robust salinity and drought tolerant cultivars, it is important to quantify the relative contribution of stomatal and residual transpiration in the overall osmotic stress tolerance and to identify their components as a trait determining osmotic stress tolerance. Hence, the major aim of this PhD project was to investigate the stomatal and non-stomatal transpiration and their relative contributions toward salinity and drought stress tolerance, plus the overall plant performance under salinity and drought conditions in contrasting large number of barley genotypes. In this thesis the following specific objectives were addressed: (i) to establish the importance of the residual transpiration as a component of salinity tolerance mechanism; (ii) to reveal the role of cuticular waxes as a determinant of the residual transpiration; (iii) to assess the suitability of different physiological and morphological traits as a proxy for drought tolerance; (iv) to understand the selective physiological and morphological traits contributing to drought tolerance in a large number of barley genotypes.
Four barley (Hordeum vulgare L.) genotypes contrasting in their salinity tolerance were used to evaluate the relationship between residual transpiration to the overall salt tolerance, and also investigated what role of cuticular waxes play in this process. Results revealed that leaf osmolality, osmotic potential, leaf water potential and the amount of total cuticular wax are involved in controlling residual transpiration from barley leaves surface under well irrigated conditions. A significant and negative relationship between the amount of primary alcohols and the residual transpiration implies that some cuticular wax constituents also act as a water barrier on plant leaf surface and thus contribute to salinity stress tolerance. It is suggested that residual transpiration could be a fundamental mechanism by which plant can reduce water use during stress.
We compared different physiological measures of drought stress in six barley genotypes subjected to different drought treatments under glasshouse conditions to find a convenient, reproducible, reliable and rapid screening method to be used a proxy for drought tolerance for a large number of barley genotypes. Genotypes were evaluated by measuring transpiration rate, quantum yield of PSII (chlorophyll fluorescence F$$_v$$/F$$_m$$ ratio), chlorophyll content, dry biomass and shoot water content under drought stress. The transpiration rate and leaf growth/death were quantified after rewatering. In another experiment, the same genotypes were evaluated by applying 18% (w/v) polyethylene glycol (PEG) to germinating seeds grown in paper rolls to induce osmotic stress, using relative root and shoot lengths as a measure of osmotic stress tolerance. The results suggested that transpiration measurements at the recovery stage could be the most sensitive method for evaluating the stress sensitivity of different genotypes. Chlorophyll fluorescence (F$$_v$$/F$$_m$$ ratio) of dark-adapted leaves could be recommended as a suitable proxy for screening tolerance of water stress. Measuring relative root growth rate (length) using PEG-treated paper roll-grown seedlings also seems to be a highly suitable and promising method for screening a large number of genotypes in breeding programs.
Based on our previous work, eighty barley genotypes of different geographical origin and contrasting in salinity stress tolerance were grown under glasshouse conditions and exposed to high salinity stress (300 mM NaCl) for four weeks to investigate the relationship between leaf gas exchange, tissue ionic relations, and overall plant salinity tolerance. Four weeks after the treatment commenced, stomatal conductance, stomatal density, residual transpiration, chlorophyll content, leaf sap Na$$^+$$, K$$^+$$, Cl$$^-$$ concentration and leaf sap osmolality were measured. Responses to salinity stress differed greatly among the genotypes. The overall salinity tolerance significantly correlated with leaf Na$$^+$$ content, osmolality, stomatal density and the residual transpiration. The results suggested that increasing stomatal density as well as minimization of the residual transpiration may be a promising way of improving water use efficiency and increase salinity tolerance in barley. Our data also showed that residual transpiration is strongly affected by the number of stomatal pores on the leaf surface.
To identify the desirable morphological and physiological traits that confer drought stress tolerance, we screened eighty barley genotypes collected from different geographical locations and contrasting in drought stress tolerance. Plants were exposed to continuous drought stress by withholding irrigation for four weeks under glasshouse conditions. Also, root length of the same genotypes was measured from stress-affected plants growing hydroponically. The drought tolerance was scored 30 days after the drought stress commenced based on the degree of leaf damage, fresh and dry biomass and relative water content. These characteristics were related to stomatal conductance, stomatal density, residual transpiration and leaf sap Na$$^+$$, K$$^+$$, Cl$$^-$$ contents measured in control (irrigated) plants. Responses to drought stress differed significantly among the genotypes. The overall drought tolerance was significantly correlated with relative water content, stomatal conductance and leaf Na$$^+$$ and K$$^+$$ concentration. No significant correlations between drought tolerance and root length of 6-day-old seedling, stomatal density, residual transpiration and leaf sap Cl$$^-$$ content were found. Taking together, these results suggest that drought tolerant genotypes have lower stomatal conductance, and lower water content, and lower Na$$^+$$, K$$^+$$ and Cl$$^-$$ contents in their tissue under control conditions than the drought sensitive ones.
In conclusion, the overall studies suggested that residual transpiration is associated with salinity stress tolerance. The total amounts of cuticular wax or cuticular wax components, specifically primary alcohol, act as a water barrier to reduce water loss through the plant leaf surface. Increasing stomatal density and reducing residual transpiration are the promising way of improving water use efficiency under salinity stress contributing to increased salinity tolerance in barley. Interestingly, residual transpiration strongly correlated with the number of stomatal pores on the leaf surface. Measuring chlorophyll fluorescence of dark adapted leaves (F$$_v$$/F$$_m$$ ratio) is recommended as an efficient and promising method for screening a large number of genotypes in breeding programs. Plants with lower stomatal density and stomatal conductance under irrigated conditions showed higher drought tolerance under water deficit conditions. Barley plants with lower Na$$^+$$, K$$^+$$ and Cl$$^-$$ concentration in their tissue showed greater tolerance under drought stress which revealed that tolerant genotypes are more dependent on organic osmolytes than the inorganic ions for osmotic adjustment under drought stress conditions.