Ewa Pociecha

Physiological and biochemical characterisation of watered and drought-stressed barley mutants in the HvDWARF gene encoding C6-oxidase involved in brassinosteroid biosynthesis

Brassinosteroids (BR) are plant steroid hormones that were discovered more than thirty years ago, but their physiological function has yet to be fully explained. The aim of the study was to answer the question of whether/how disturbances in the production of BR in barley affects the plants metabolism and development under conditions of optimal watering and drought. Mutants with an impaired production of BR are one of the best tools in research aimed at understanding the mechanisms of action of these hormones. The study used barley cultivars with a normal BR synthesis (wild type) and semi-dwarf allelic mutants with an impaired activity of C6-oxidase (mutation in HvDWARF), which resulted in a decreased BR synthesis. Half of the plants were subjected to drought stress in the seedling stage and the other half were watered optimally. Plants with impaired BR production were characterised by a lower height and developmental retardation. Under both optimal watering and drought, BR synthesis disorders caused the reduced production of ABA and cytokinins, but not auxins. The BR mutants also produced less osmoprotectant (proline). The optimally watered and drought-stressed mutants accumulated less sucrose, which was accompanied by changes in the production of other soluble sugars. The increased content of fructooligosaccharide (kestose) in optimally watered mutants would suggest that BR is a negative regulator of kestose production. The decreased level of nystose in the drought-stressed mutants also suggests BR involvement in the regulation of the production of this fructooligosaccharide. The accumulation of the transcripts of genes associated with stress response (hsp90) was lower in the watered and drought-stressed BR-deficient mutants. In turn, the lower efficiency of photosystem II and the net photosynthetic rate in mutants was revealed only under drought conditions. The presented research allows for the physiological and biochemical traits of two BR-barley mutants to be characterised, which helps BR function to be understood. The knowledge can also be a good starting point for some breeding companies that are interested in introducing new semi-dwarf barley cultivars.

Barley Brassinosteroid Mutants Provide an Insight into Phytohormonal Homeostasis in Plant Reaction to Drought Stress

Brassinosteroids (BRs) are a class of steroid phytohormones, which regulate various processes of morphogenesis and physiology—from seed development to regulation of flowering and senescence. An accumulating body of evidence indicates that BRs take part in regulation of physiological reactions to various stress conditions, including drought. Many of the physiological functions of BRs are regulated by a complicated, and not fully elucidated network of interactions with metabolic pathways of other phytohormones. Therefore, the aim of this study was to characterize phytohormonal homeostasis in barley (Hordeum vulgare) in reaction to drought and validate role of BRs in regulation of this process. Material of this study included the barley cultivar “Bowman” and five Near-Isogenic Lines (NILs) representing characterized semi-dwarf mutants of several genes encoding enzymes participating in BR biosynthesis and signaling. Analysis of endogenous BRs concentrations in these NILs confirmed that their phenotypes result from abnormalities in BR metabolism. In general, concentrations of 18 compounds, representing various classes of phytohormones, including brassinosteroids, auxins, cytokinins, gibberellins, abscisic acid, salicylic acid and jasmonic acid were analyzed under control and drought conditions in the “Bowman” cultivar and the BR-deficient NILs. Drought induced a significant increase in accumulation of the biologically active form of BRs—castasterone in all analyzed genotypes. Another biologically active form of BRs—24-epi-brassinolide—was identified in one, BR-insensitive NIL under normal condition, but its accumulation was drought-induced in all analyzed genotypes. Analysis of concentration profiles of several compounds representing gibberellins allowed an insight into the BR-dependent regulation of gibberellin biosynthesis. The concentration of the gibberellic acid GA7 was significantly lower in all NILs when compared with the “Bowman” cultivar, indicating that GA7 biosynthesis represents an enzymatic step at which the stimulating effect of BRs on gibberellin biosynthesis occurs. Moreover, the accumulation of GA7 is significantly induced by drought in all the genotypes. Biosynthesis of jasmonic acid is also a BR-dependent process, as all the NILs accumulated much lower concentrations of this hormone when compared with the “Bowman” cultivar under normal condition, however the accumulation of jasmonic acid, abscisic acid and salicylic acid were significantly stimulated by drought.

Effect of nanosilver in wheat seedlings and Fusarium culmorum culture systems

The purpose of this study was to examine the effect of silver nanoparticles (AgNPs) produced using the high-voltage arc discharge method on the growth and metabolism of common wheat seedlings. Additionally, a simultaneous assessment of the AgNP-induced reduction in seedling infection by Fusarium culmorum (Fc) was performed. AgNP- and Fc-treated seedlings indicated that both factors considerably inhibited their growth. A significant Fc-induced reduction in seedling blight was observed following treatment with AgNPs; however, treatment with nanoparticles was also accompanied by a serious disintegration of the cell membranes of roots. Moreover, treatment with AgNPs increased the quantum efficiency of energy trapping in the PSII reaction centre (Fv/Fm) with a simultaneous decrease in energy dissipation in the form of heat. Induction of photosynthesis in the presence of AgNPs did not affect height but was reflected in higher total dry weight. Moreover, analysis of antioxidant enzyme activity typical for the stress response indicated the toxicity of AgNPs treatment compared to Fc treatment. Seedlings exposed to AgNP activity demonstrated accumulation of Ag in roots and its translocation to aerial parts, while the pathogen reduced both accumulation and translocation of this element.

Endogenous progesterone and its cellular binding sites in wheat exposed to drought stress.

Progesterone is a basic hormone that regulates the metabolism in mammals. The presence of this compound has also been found in certain plants. It is believed that progesterone can regulate growth processes and resistance to stress, however, its precise role in plants remains unknown. The research conducted in this study was aimed at analyzing the content of endogenous progesterone and its cellular binding sites in the leaves of spring wheat exposed to drought. Changes were studied in two cultivars of wheat - a cultivar sensitive to drought (Katoda) and tolerant cultivar (Monsun). Plants had undergone periodic droughts during the seedling stage or in the phase of heading. The occurrence of free progesterone as well as its conjugated forms was observed in wheat studied. The amount of progesterone ranged from 0.2 to 5.8pmolgFW(-1) and was dependent on the cultivar, age of the plants, stage of development and fluctuated as a result of the exposure to drought. Cv. Katoda responded to a water deficit by lowering the amount of progesterone and cv. Monsun by increasing its level. Progesterone in plants grown in limited water conditions occurred primarily in a free form. While in the optimal watering conditions, some of its pool was found in the form of conjugates. In the spring wheat the occurrence of binding sites for progesterone was detected in cell membranes, cytoplasm and nuclei in the range of 10-36fmol/mg of protein. The wheat cultivars tested, Monsun and Katoda, differ in their concentration of cellular binding sites for progesterone. This number varied in the individual fractions during different stages of plant development and due to the effect of drought stress. The number of binding sites for progesterone located in the membrane fraction of seedlings and flag leaves increased significantly under drought in the cv. Katoda (35-46%), but did not change in the cv. Monsun. Whereas the number of cytoplasmic progesterone binding sites increased during the drought in the cv. Monsun (about 50%), they did not change in the cv. Katoda. Changes in the amount of progesterone and its binding sites in the cell under the influence of drought were then different depending on whether the cultivar was tolerant or sensitive to drought. The possibility of utilizing these changes as markers of drought resistance is discussed. The results obtained suggest that progesterone is a part of wheat response to stress factors (drought).

Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne

Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold.

Progress of snow mould infection in crowns of winter rye (Secale cereale L.) is related to photosynthetic activity during cold acclimation.

Resistance to snow mould is a feature determined by multiple genes. Therefore, determining the phenotype of resistant plants is difficult as it requires an investigation over a long period of time from cold acclimation through pathogenesis. The aim of the present study was (i) to determine the characteristics of the resistant genotype and (ii) to clarify the connections between photosynthesis during cold acclimation and then pathogenesis caused by Microdochium nivale. Two inbred lines of winter rye (Secale cereale L.) differing in their susceptibility to snow mould were used in the study. After cold acclimation snow mould resistant (SMR) line was characterised by higher values of CO2 assimilation and electron transport efficiency but did not differ from snow mould susceptible (SMS) line in carboxylation rate of RuBisCO (Vcmax). Higher soluble carbohydrate accumulation, due to higher photosynthesis intensity, as well as an ABA increase at 5 days post infection (DPI) in leaves and crowns were found in SMR line during the pathogenesis period. Callose deposition was found around non-infected bundle sheets and in cortex cells at 5 DPI (at the same time point as ABA peak) only in SMR line, which probably prevented the infection of leaf initials. Early leaf initials infection in SMS line may be responsible for inhibiting leaf growth and plant regeneration after stress cessation. The results show different physiological and biochemical characteristics of the investigated lines, which can be applied in the selection of resistant genotypes and identifying genomic regions responsible for metabolic pathways increasing pathogen resistance.

Disturbances in production of progesterone and their implications in plant studies

Progesterone is a mammalian hormone that has also been discovered in plants but its physiological function in plants is not explained. Experiments using inhibitors of progesterone synthesis and binding would be useful in studies on the significance of this compound in plants. Until now, trilostane and mifepristone have been used in medical sciences as progesterone biosynthesis and binding inhibitors, respectively. We tested these synthetic steroids for the first time in plants and found that they reduced the content of progesterone in wheat. The aim of further experiments was to answer whether the potential disturbances in the production/binding of progesterone, influence resistance to environmental stress (drought) and the development of wheat. Inhibitors and progesterone were applied to plants via roots in a concentration of 0.25-0.5mg/l water. Both inhibitors lowered the activity of CO2 binding enzyme (Rubisco) in wheat exposed to drought stress and trilostane additionally lowered the chlorophyll content. However, trilostane-treated plants were rescued by treatment with exogenous progesterone. The inhibitors also modulated the development of winter wheat, which indicated the significance of steroid regulators and their receptors in this process. In this study, in addition to progesterone and its inhibitors, brassinosteroid (24-epibrassinolide) and an inhibitor of biosynthesis of brassinosteroids were also applied. Mifepristone inhibited the generative development of wheat (like 24-epibrassinolide), while trilostane (like progesterone and an inhibitor of biosynthesis of brassinosteroids) stimulated the development. We propose a model of steroid-induced regulation of the development of winter wheat, where brassinosteroids act as inhibitors of generative development, while progesterone or other pregnane derivatives act as stimulators.

Mechanisms involved in the regulation of photosynthetic efficiency and carbohydrate partitioning in response to low- and high-temperature flooding triggered in winter rye (Secale cereale) lines with distinct pink snow mold resistances.

In terms of climate changes and global warming, winter hardiness could be determined by unfavorable environmental conditions other than frost. These could include flooding from melting snow and/or rain, coincident with fungal diseases. Therefore, we designed an experiment to identify potential common mechanisms of flooding tolerance and snow mold resistance, involving the regulation of photosynthetic efficiency and carbohydrate metabolism at low temperatures. Snow mold-resistant and susceptible winter rye (Secale cereale) plants were characterized by considerably different patterns of response to flooding. These differences were clearer at low temperature, thus confirming a possible role of the observed changes in snow mold tolerance. The resistant plants were characterized by lower PSII quantum yields at low temperature, combined with much higher energy flux for energy dissipation from the PSII reaction center. During flooding, the level of soluble carbohydrates increased in the resistant plants and decreased in the susceptible ones. Thus increase in resistant line was connected with a decrease in the energy dissipation rate in PSII/increased photosynthetic activity (energy flux for electron transport), a lower rate of starch degradation and higher rates of sucrose metabolism in leaves. The resistant lines accumulated larger amounts of total soluble carbohydrates in the crowns than in the leaves. Irrespective of flooding treatment, the resistant lines allocated more sugars for cell wall composition, both in the leaves and crowns. Our results clearly indicated that studies on carbohydrate changes at low temperatures or during anoxia should investigate not only the alterations in water-soluble and storage carbohydrates, but also cell wall carbohydrates. The patterns of changes observed after low and high-temperature flooding were different, indicating separate control mechanisms of these responses. These included changes in the photosynthetic apparatus, starch accumulation and cell wall carbohydrate accumulation.

Assessment of candidate reference genes for the expression studies with brassinosteroids in Lolium perenne and Triticum aestivum

Quantitative PCR studies need proper reference genes with expression stability exclusively validated under certain experimental conditions. The expression stability of several genes commonly used as references was tested under 24-epibrassinolide (EBR) and temperature treatment. Different statistical approaches (qBase(PLUS), BestKeeper, NormFinder) were used to prepare rankings of expression stability in two species of an economic importance: common wheat (Triticum aestivum) and perennial ryegrass (Lolium perenne). Candidate reference genes were shown to be regulated differentially in these two plant species. The maximum stability values indicated that the expression stability was higher in T. aestivum. Taking into account of all ranks it seems that TBP-1 and UBI in ryegrass and ACT, ADP and EF1A in wheat should be used as reference genes in the brassinosteroids and temperature involving studies.

Variation in waterlogging-triggered stomatal behavior contributes to changes in the cold acclimation process in prehardened Lolium perenne and Festuca pratensis

According to predicted changes in climate, waterlogging events may occur more frequently in the future during autumn and winter at high latitudes of the Northern Hemisphere. If excess soil water coincides with the process of cold acclimation for plants, winter survival may potentially be affected. The effects of waterlogging during cold acclimation on stomatal aperture, relative water content, photochemical activity of photosystem II, freezing tolerance and plant regrowth after freezing were compared for two prehardened overwintering forage grasses, Lolium perenne and Festuca pratensis. The experiment was performed to test the hypothesis that changes in photochemical activity initiated by waterlogging-triggered modifications in the stomatal aperture contribute to changes in freezing tolerance. Principal component analysis showed that waterlogging activated different adaptive strategies in the two species studied. The increased freezing tolerance of F. pratensis was associated with increased photochemical activity connected with stomatal opening, whereas freezing tolerance of L. perenne was associated with a decrease in stomatal aperture. In conclusion, waterlogging-triggered stomatal behavior contributed to the efficiency of the cold acclimation process in L. perenne and F. pratensis.