František Hnilička

The Physiology and Proteomics of Drought Tolerance in Maize: Early Stomatal Closure as a Cause of Lower Tolerance to Short-Term Dehydration?

Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.

Trace elements present in airborne particulate matter—Stressors of plant metabolism

Changes of amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, proline, tryptophan, alanine, glycine, valine and serine), gas-exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO(2) concentration) and nitrate levels in Lactuca serriola L. under airborne particulate matter (PM) contamination reported here reveal their role in plant chronic stress adaptation. Results of the pot experiment confirmed the toxic effect of trace elements present in PM for lettuce. PM applied to soil or on the lettuce leaves were associated with the strong inhibition of above-ground biomass and with the enhancement of plant trace element contents. The significant changes of amino acid levels and leaf gas-exchange parameters of the plants showed strong linear dependences on PM contamination (R(2)=0.60-0.99). PM application on leaves intensified toxic effect of trace elements (As, Pb, Cr and Cd) originating from PM by shading of the leaf surface. The plant accumulation of nitrate nitrogen after PM contamination confirmed to block nitrate assimilation.

Nitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis

Increased endogenous plant cytokinin (CK) content through transformation with an isopentyl transferase (ipt) gene has been associated with improved plant stress tolerance. The impact of zinc (tested levels Zn1=250, Zn2=500, Zn3=750mgkg(-1)soil) on gas exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration) and nitrogen utilization by plants resulted in changes of free amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, glycine, serine, cystein) and differed for transformed and non-transformed tobacco plants. For pot experiments, tobacco plants (Nicotiana tabacum L., cv. Wisconsin 38) transformed with a construct consisting of SAG12 promoter fused with the ipt gene for cytokinin synthesis (SAG plants) and its wild type (WT plants as a control) were used. Physiological analyses confirmed that SAG plants had improved zinc tolerance compared with the WT plants. The enhanced Zn tolerance of SAG plants was associated with the maintenance of accumulation of amino acids and with lower declines of photosynthetic and transpiration rates. In comparison to WT plants, SAG plants exposed to the highest Zn concentration accumulated lower concentrations of asparagine, which is a major metabolic product during senescence.

The evaluation of photosynthetic parameters in maize inbred lines subjected to water deficiency: Can these parameters be used for the prediction of performance of hybrid progeny?

The response of selected photosynthetic and morphological parameters of plants to drought was examined in 5 inbred lines of maize (Zea mays L.) and their 10 F1 hybrids. The aim of the study was to establish whether the photosynthetic performance of parental genotypes under drought conditions correlates with the performance of their progeny and whether the net photosynthetic rate, the chlorophyll fluorescence parameters or the content of photosynthetic pigments could be used as reliable physiological markers for early breeding generations. The relative importance of the additive and the nonadditive (dominance, maternal) genetic effects in the inheritance of these parameters was also assessed by means of the quantitative genetics analysis. The results showed that the nonadditive genetic effects associated with a particular combination of genotypes or a particular direction of crossing are at least equally and often even more important as the additivity and that these genetic effects almost totally change with the exposure of plants to drought conditions. This was reflected in the inability to predict the response of F1 hybrids to drought on the basis of the photosynthetic performance of their parents, which indicates that the practical usability of such parameters in maize breeding programs is rather limited.

24-Epibrassinolide and 20-hydroxyecdysone affect photosynthesis differently in maize and spinach

The aim of the work was to examine the effect of brassinosteroid (24-epibrassinolide; 24E) and ecdysteroid (20-hydroxyecdysone; 20E) on various parts of primary photosynthetic processes in maize and spinach. Additionally, the effect of steroids on gaseous exchange, pigment content and biomass accumulation was studied. The efficiency of the photosynthetic whole electron-transport chain responded negatively to the 24E or 20E treatment in both species, but there were interspecific differences regarding Photosystem (PS) II response. A positive effect on its oxygen-evolving complex and a slightly better energetical connectivity between PSII units were observed in maize whereas the opposite was true for spinach. The size of the pool of the PSI end electron acceptors was usually diminished due to 24E or 20E treatment. The treatment of plants with 24E or 20E applied individually positively influenced the content of photosynthetic pigments in maize (not in spinach). On the other hand, it did not affect gaseous exchange in maize but resulted in its reduction in spinach. Plants treated with combination of both steroids mostly did not significantly differ from the control plants. We have demonstrated for the first time that 20E applied in low (10nM) concentration can affect various parts of photosynthetic processes similarly to 24E and that brassinosteroids regulate not only PSII but also other parts of the photosynthetic electron transport chain - but not necessarily in the same way.

The influence of drought and the application of 24-epibrassinolide on the formation of dry matter and yield in wheat

Winter wheat varieties: Ebi, Estica, Samanta were grown in three experimental variants (control, drought stress and high temperature, stress and the application of 24-epibrassinolide). The plants were monitored for the weight of the dry matter and the straw and grain yields. The stressors were found to have a negative effect on the amount of dry matter in the above-ground wheat biomass and the yield of grain and straw. Drought and high temperature lowered the amount of dry matter in the grain and straw in all varieties. For all monitored varieties the application of 24-epibrassinolide reduced the negative effect of the monitored stressors. The best reaction to the application of 24-epibrassinolide was seen in the variety Estica, the worst in the variety Samanta. Of the varieties monitored the variety Samanta exhibited the tolerance to drought and high temperatures. On the contrary Ebi is a sensitive variety.

The disadvantages of being a hybrid during drought: A combined analysis of plant morphology, physiology and leaf proteome in maize

A comparative analysis of various parameters that characterize plant morphology, growth, water status, photosynthesis, cell damage, and antioxidative and osmoprotective systems together with an iTRAQ analysis of the leaf proteome was performed in two inbred lines of maize (Zea mays L.) differing in drought susceptibility and their reciprocal F1 hybrids. The aim of this study was to dissect the parent-hybrid relationships to better understand the mechanisms of the heterotic effect and its potential association with the stress response. The results clearly showed that the four examined genotypes have completely different strategies for coping with limited water availability and that the inherent properties of the F1 hybrids, i.e. positive heterosis in morphological parameters (or, more generally, a larger plant body) becomes a distinct disadvantage when the water supply is limited. However, although a greater loss of photosynthetic efficiency was an inherent disadvantage, the precise causes and consequences of the original predisposition towards faster growth and biomass accumulation differed even between reciprocal hybrids. Both maternal and paternal parents could be imitated by their progeny in some aspects of the drought response (e.g., the absence of general protein down-regulation, changes in the levels of some carbon fixation or other photosynthetic proteins). Nevertheless, other features (e.g., dehydrin or light-harvesting protein contents, reduced chloroplast proteosynthesis) were quite unique to a particular hybrid. Our study also confirmed that the strategy for leaving stomata open even when the water supply is limited (coupled to a smaller body size and some other physiological properties), observed in one of our inbred lines, is associated with drought-resistance not only during mild drought (as we showed previously) but also during more severe drought conditions.

Growth and photosynthesis of Upland and Pima cotton: response to drought and heat stress

The effects of drought and heat stress on physiology of two common cotton species, Gossypium hirsutum L. (Upland) and G. barbadense L. (Pima) were investigated in this study. Four consecutive 28-day pot experiments were carried out in a fully controlled growth chamber under four temperatures (25, 30, 35 and 40°C), while two water treatments (well-watered and drought) were imposed for 10 days to both cotton species. Growth according to dry matter accumulation of G. barbadense was more limited by sole drought conditions (50%), whereas heat stress was more remarkable on growth of G. hirsutum (64%). Chlorophyll (Chl) a and b content were more temperature-sensitive in G. hirsutum than G. barbadense under well-watered conditions. The most noticeable decrease in droughtinduced rates of photosynthesis rate (P n ), transpiration rate (E) and stomatal conductance (g s ) were recorded under 35°C, although genotypic variation was found under 25°C. Higher water use efficiency was associated with higher temperature. Relative decrease in Chl a content, g s and maximal quantum efficiency of PSII exhibited by both spe cies were distinctive physiological traits for heat and drought tolerance.

Gas exchange and Triticum sp. with different ploidy in relation to irradiance

Different species of Triticum were grown during a greenhouse experiment, including T. monococcum L., T. dicoccum Schrank, T. durum Desf., T. spelta L. and T. aestivum cv. Vanek. The goal was to establish the influence of irradiance on the parameters of photosynthetic performance in relation to their ploidy. Photosynthetic rate (Pn), transpiration (E) and stomatal conductance (gs) were measured at irradiance ranging from 217–1305 μmol/m2/s. In all monitored species, saturation irradiance for photosynthesis at the level of 609 μmol/m2/s was reached. The highest average Pn was measured in the diploid T. monococcum (32.5 μmol CO2/ m2/s) while the lowest Pn occurred in the hexaploid T. spelta (22.0 μmol CO2/m 2/s). The Pn in hexaploid T. aestivum (29.6 μmol CO2/m 2/s) was comparable with the tetraploid T. durum. Similarly, E also decreased with the increase of ploidy. The highest gs was measured in T. durum (1.03 mol CO2/m 2/s) and T. aestivum (0.99 mol CO2/ m2/s). In all monitored species a close linear dependency was recorded between Pn and gs. Species with lower ploidy reach maximum values of Pn with lower gs. Hexaploid T. aestivum and tetraploid T. durum, require higher gs in order to achieve higher Pn and yet they do not reach the Pn values of species with lower ploidy.

Use of combustion methods for calorimetry in the applied physiology of plants

As a thermodynamics method, combustion calorimetry has a wide spectrum of uses in the applied physiology of plants. During evaluations, it suitably combines its physical relations with properties of living organisms, thus providing a solid overview concerning the exchange of energy between a plant and its environment. Measuring photosynthetic rate, expressed through changes of energy content, is comparable with gasometric methods, because the course of photosynthesis and the energy content in leaves dependent on their ontogenetic development is similar. Combustion calorimetry enables monitoring the changes of accumulation of assimilated nutrients and their translocation between the individual organs of the plant. The close relation between the creation of dry matter and the accumulation of energy-rich substances into the plant body is apparent from the acquired experimental data. Based on the methods of combustion calorimetry, it is possible to determine the relationship between the source and the sink. It is further possible to use this method as one of the detection methods to determine changes in metabolism and the degree of resistance of plants, as well as ecosystems, under stress conditions.