HSP Transcript and Protein Accumulation in Brassinosteroid Barley Mutants Acclimated to Low and High Temperatures

In temperature stress, the main role of heat-shock proteins (HSP) is to act as molecular chaperones for other cellular proteins. However, knowledge about the hormonal regulation of the production of the HSP is quite limited. Specifically, little is known about the role of the plant steroid hormones—brassinosteroids (BR)—in regulating the HSP expression. The aim of our study was to answer the question of how a BR deficit or disturbances in its signaling affect the accumulation of the HSP90, HSP70, HSP18, and HSP17 transcripts and protein in barley growing at 20C (control) and during the acclimation of plants at 5 C and 27 C. In barley, the temperature of plant growth modified the expression of HSPs. Furthermore, the BR-deficient mutants (mutations in the HvDWARF or HvCPD genes) and BR-signaling mutants (mutation in the HvBRI1 gene) were characterized by altered levels of the transcripts and proteins of the HSP group compared to the wild type. The BR-signaling mutant was characterized by a decreased level of the HSP transcripts and heat-shock proteins. In the BR-deficient mutants, there were temperature-dependent cases when the decreased accumulation of the HSP70 and HSP90 transcripts was connected to an increased accumulation of these HSP. The significance of changes in the accumulation of HSPs during acclimation at 27 C and 5 C is discussed in the context of the altered tolerance to more extreme temperatures of the studied mutants (i.e., heat stress and frost, respectively)

The Impact of Mutations in the HvCPD and HvBRI1 Genes on the Physicochemical Properties of the Membranes from Barley Acclimated to Low/High Temperatures

1) Background: The study characterized barley mutants with brassinosteroid (BR) biosynthesis and signaling disturbances in terms of the physicochemical/structural properties of membranes to enrich the knowledge about the role of brassinosteroids for lipid metabolism and membrane functioning. (2) Methods: The Langmuir method was used to investigate the properties of the physicochemical membranes. Langmuir monolayers were formed from the lipid fractions isolated from the plants growing at 20 C and then acclimated at 5 C or 27 C. The fatty acid composition of the lipids was estimated using gas chromatography. (3) Results: The BR-biosynthesis and BR-signaling mutants of barley were characterized by a temperature-dependent altered molar percentage of fatty acids (from 14:0 to 20:1) in their galactolipid and phospholipid fractions in comparison to wild-type (WT). For example, the mutants had a lower molar percentage of 18:3 in the phospholipid (PL) fraction. The same regularity was observed at 5 C. It resulted in altered physicochemical parameters of the membranes (Alim, coll, Cs1). (4) Conclusions: BR may be involved in regulating fatty acid biosynthesis or their transport/incorporation into the cell membranes. Mutants had altered physicochemical parameters of their membranes, compared to the WT, which suggests that BR may have a multidirectional impact on the membrane-dependent physiological processes

Mutations in the HvDWARF, HvCPD and HvBRI1 Genes-Involved in Brassinosteroid Biosynthesis/Signalling : Altered Photosynthetic Efficiency, Hormonal Homeostasis and Tolerance to High/Low Temperatures in Barley

Brassinosteroids (BR) are steroid phytohormones that are involved in the growth and stress response in plants, but the precise mechanisms of their action are still being discovered. In our study we have used BR-deficient barley mutants 522DK and BW084 (which carry missense mutations in the HvDWARF and HvCPD genes, respectively). We have also used a BR-signalling mutant that harbors missense substitutions in the HvBRI1 gene. Our aim was (1) to find out if the content of phytohormones in the mutants grown at 20 °C is different than in the wild types and whether/how the content of phytohormones changes after plant acclimation at temperatures of 5 °C and 27 °C?, (2) to characterise the effectiveness of the light reactions of photosynthesis of the barley mutants in comparison to wild types at various temperatures, and (3) to verify the impact of mutations on the tolerance of barley to high and low temperatures. Hormonal characteristics of the BR mutants of barley show the complexity of the interactions between BR and other plant hormones that are additionally modified by temperature and possibly by other factors. The results suggest the participation of BR in auxin catabolism. Further, BR appears to play a role in maintaining the ABA–ABAGlc balance. As for the gibberellin content in plants at a temperature of 20 °C, more in-depth studies will be required to explain the contradictory effects regarding the accumulation of GA3, GA4 and GA5, which appears to be dependent on the type of mutation and connected to the BR level. A fast-kinetic chlorophyll a fluorescence analysis has revealed that the mutants had lower values of energy absorption than the wild types, but the values of the energy transferred via the electron-transport chain was maintained at the wild-type level. We presumed that BR are involved in regulating plant acclimation to extreme (low/high) temperatures, thus the BR-deficient and BR-signalling mutants should be less tolerant to low/high temperatures when compared to the wild types. Unexpectedly, all of the mutants showed a higher tolerance to high temperatures than the wild types. The BW084 and BW312 mutants were less tolerant to frost than the wild type, but 522DK had a similar frost tolerance as the reference wild-type cultivar

Plasma membrane ATPase and the aquaporin HvPIP1 in barley brassinosteroid mutants acclimated to high and low temperature

The integral parts of the cell membranes are the functional proteins, which are crucial for cell life. Among them, proton-pumping ATPase and aquaporins appear to be of particular importance. There is some knowledge about the effect of the temperature during plant growth, including stress-inducing temperatures, on the accumulation of the membrane proteins: plasma membrane H+-ATPase and aquaporins, but not much is known about the effect of the phytohormones (i.e. brassinosteroids (BR)) on control of accumulation of these proteins. The aim of our study was to answer the question of how a BR deficit and disturbances in the BR perception/signalling affect the accumulation of plasma membrane H+-ATPase (PM H+-ATPase), the aquaporin HvPIP1 transcript and protein in barley growing at 20 °C and during its acclimation at 5 °C and 27 °C. For the studies, the BR-deficient mutant 522DK (derived from the wild-type Delisa), the BR-deficient mutant BW084 and the BR-signalling mutant BW312 and their wild-type Bowman were used. Generally, temperature of growth was significant factor influencing on the level of the accumulation of the H+-ATPase and HvPIP1 transcript and the PM H+-ATPase and HvPIP1 protein in barley leaves. The level of the accumulation of the HvPIP1 transcript decreased at 5 °C (compared to 20 °C), but was higher at 27 °C than at 20 °C in the analyzed cultivars. In both cultivars the protein HvPIP1 was accumulated in the highest amounts at 27 °C. On the other hand, the barley mutants with a BR deficiency or with BR signalling disturbances were characterised by an altered accumulation level of PM H+-ATPase, the aquaporin HvPIP1 transcript and protein (compared to the wild types), which may suggest the involvement of brassinosteroids in regulating PM H+-ATPase and aquaporin HvPIP1 at the transcriptional and translational levels

Isolation and purification of diadinoxanthin cycle pigments and their influence on molecular dynamic of artificial membranes

Okrzemki to glony, u których zachodzi cykl diadinoksantynowy (cykliczne przemiany diadinoksantyny (Ddx) w diatoksantynę (Dtx)). Przedmiotem niniejszej pracy magisterskiej jest izolacja i oczyszczanie barwników cyklu diadinoksantynowego oraz zbadanie ich wpływu na dynamikę molekularną błon modelowych. W ramach pracy przeprowadzono badania mające na celu optymalizację warunków hodowli okrzemek P. tricornutum, polegającą na doborze temperatury, intensywności światła, składu pożywki oraz czasu po jakim je wirowano. Z otrzymanych osadów izolowano barwniki (HPTLC). Niezależnie od warunków prowadzenia hodowli, pozyskiwano czystą Dtx. Z hodowli wzrastających w T = 15 – 23 ±1⁰C, których osady zbierano po upływie 7 – 10 dni otrzymywano zanieczyszczoną Ddx. W przypadku osadów uzyskanych z hodowli przechowywanych w T = 12 – 15⁰C i wirowanych po 5 dniach, pozyskiwano czystą Ddx. Kolejnym krokiem było przeprowadzenie próby oczyszczenia zanieczyszczonej Ddx. Badania wstępne wykazały, że jest to możliwe stosując jako mieszaninę ekstrakcyjną 60% aceton oraz płytki TLC, żel krzemionkowy, 60 F254. W badaniach wpływu otrzymanych barwników na dynamikę molekularną błon modelowych wykorzystano spektrometrię elektronowego rezonansu paramagnetycznego (EPR). Dla błon ze znacznikiem spinowym SASL-5 wstępnie stwierdzono brak wpływu Ddx i Dtx na jej dynamikę molekularną w obszarze hydrofilowym. Gdy błona zawierała znacznik SASL-16, stwierdzono jej usztywnienie pod wpływem Dtx. Ponieważ proces wbudowywania znacznika SASL-16 był utrudniony, nie można było określić wpływu Ddx na dynamikę molekularną błony.Diatoms are group of algae which proceed diadinoxanthin cycle (cyclical conversions of diadinoxanthin (Ddx) and diatoxanthin (Dtx)). The subject of this thesis refers to the isolation and purification of diadinoxanthin cycle pigments and their influence on the molecular dynamics of artificial membranes. During the study, conditions of diatom P.tricornutum culture such as temperature, light intensity, composition of the medium and time of cultivation was optimized. Then cultures were centrifuged and the sediment was collected. Isolation of pigments was performed (HPTLC). From every sediment pure Dtx was obtained. Ddx isolated from sediments derived from cultures which grown in T = 15–23 ±1⁰C and which was collected after 7-10 days was contaminated. In case of sedminents from cultures that grown in T= 12–15⁰C and collected after 5 days, pure Ddx was obtained. The next step was purification of contaminated Ddx. Preliminary studies have shown that this is possible with 60% acetone as extraction mixture and with Silica gel 60 F254 TLC plates. During the examination of influence of diadinoxanthin cycle pigments on molecular dynamics of artificial membranes electron paramagnetic resonance spectrometry (EPR) was used. In membranes with SASL-5 spin label preliminary test have shown that Ddx and Dtx have no effect on molecular dynamics of this membrane. In case of SASL-16 spin label, influence of Dtx was observed but there is no evidence for Ddx effect because of problems connected with incorporations of SASL-16 in membranes and with limited time for research

Brassinosteroids and the Tolerance of Cereals to Low and High Temperature Stress: Photosynthesis and the Physicochemical Properties of Cell Membranes

Cereals, which belong to the Poaceae family, are the most economically important group of plants. Among abiotic stresses, temperature stresses are a serious and at the same time unpredictable problem for plant production. Both frost (in the case of winter cereals) and high temperatures in summer (especially combined with a water deficit in the soil) can result in significant yield losses. Plants have developed various adaptive mechanisms that have enabled them to survive periods of extreme temperatures. The processes of acclimation to low and high temperatures are controlled, among others, by phytohormones. The current review is devoted to the role of brassinosteroids (BR) in cereal acclimation to temperature stress with special attention being paid to the impact of BR on photosynthesis and the membrane properties. In cereals, the exogenous application of BR increases frost tolerance (winter rye, winter wheat), tolerance to cold (maize) and tolerance to a high temperature (rice). Disturbances in BR biosynthesis and signaling are accompanied by a decrease in frost tolerance but unexpectedly an improvement of tolerance to high temperature (barley). BR exogenous treatment increases the efficiency of the photosynthetic light reactions under various temperature conditions (winter rye, barley, rice), but interestingly, BR mutants with disturbances in BR biosynthesis are also characterized by an increased efficiency of PSII (barley). BR regulate the sugar metabolism including an increase in the sugar content, which is of key importance for acclimation, especially to low temperatures (winter rye, barley, maize). BR either participate in the temperature-dependent regulation of fatty acid biosynthesis or control the processes that are responsible for the transport or incorporation of the fatty acids into the membranes, which influences membrane fluidity (and subsequently the tolerance to high/low temperatures) (barley). BR may be one of the players, along with gibberellins or ABA, in acquiring tolerance to temperature stress in cereals (particularly important for the acclimation of cereals to low temperature)

Molecular dynamics of chloroplast membranes isolated from wild-type barley and a brassinosteroid-deficient mutant acclimated to low and high temperatures

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 C, whereas in the hydrophobic core, they were visible at both 20 and 5 C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts

Insights into Metabolic Reactions of Semi-Dwarf, Barley Brassinosteroid Mutants to Drought

The roles of endogenous brassinosteroids (BRs) in the modulation of reaction to drought and genetic regulation of this process are still obscure. In this study, a multidirectional analysis was performed on semi-dwarf barley (Hordeum vulgare) Near-Isogenic Lines (NILs) and the reference cultivar “Bowman” to get insights into various aspects of metabolic reaction to drought. The NILs are defective in BR biosynthesis or signaling and displayed an enhanced tolerance to drought. The BR metabolism perturbations affected the glucose and fructose accumulation under the control and stress conditions. The BR metabolism abnormalities negatively affected the sucrose accumulation as well. However, during drought, the BR-deficient NILs accumulated higher contents of sucrose than the “Bowman” cultivar. Under the control conditions, accumulation of transcripts encoding antioxidant enzymes ascorbate peroxidase (HvAPX) and superoxide dismutase (HvSOD) was BR-dependent. However, during drought, the accumulation of HvAPX transcript was BR-dependent, whereas accumulations of transcripts encoding catalase (HvCAT) and HvSOD were not affected by the BR metabolism perturbations. The obtained results reveal a significant role of BRs in regulation of the HvAPX and HvCAT enzymatic activities under control conditions and the HvAPX and HvSOD activities during physiological reactions to drought

Brassinosteroid-lipid membrane interaction under low and high temperature stress in model systems

Background: In earlier studies [1], we indicated that applying brassinosteroids (BRs) to lipids that had been isolated from plants altered the physicochemical properties of the monolayers. A continuation of these dependencies using the defined model lipid systems is presented in this paper. The influence of homocastasterone (HCS) and castasterone (CS) (BRs for which the increase in concentration were characteristic of plants grown at low temperatures) on the membrane properties of their polar and the hydrophobic parts were studied. Results: Changes in the electrokinetic potential indicate that both BRs decreased the negative charge of the surface, which is an important factor in modifying the contacts with the polar substances. This property of BRs has not yet been described. The studies of the interactions that occur in the hydrophobic part of the membrane were investigated using the EPR methods and Langmuir techniques. The physicochemical parameters of the lipid structure were determined, and the excess of Gibbs free energy was calculated. Conclusion: We conclude that examined BRs modify both the hydrophilic and hydrophobic properties of the membranes, but to a greater extent HCS. The consequence of these changes may be the attempt to maintain the stability of the membranes in stressful temperature conditions and/or to the possibility of adsorption of other substances on membranes surfaces. The change of plant metabolism towards increasing the amount of BR, mainly HCS (under cooling) may by an important factor for maintaining optimal structural properties of membranes and their functionality despite temperature changes