Éva Sárvári

Physiological and morphological responses to water stress in Aegilops biuncialis and Triticum aestivum genotypes with differing tolerance to drought

The physiological and morphological responses to water stress induced by polyethylene glycol (PEG) or by withholding water were investigated in Aegilops biuncialis Vis. genotypes differing in the annual rainfall of their habitat (1050, 550 and 225 mm year-1) and in Triticum aestivum L. wheat genotypes differing in drought tolerance. A decrease in the osmotic pressure of the nutrient solution from -0.027 to -1.8 MPa resulted in significant water loss, a low degree of stomatal closure and a decrease in the intercellular CO2 concentration (Ci) in Aegilops genotypes originating from dry habitats, while in wheat genotypes high osmotic stress increased stomatal closure, resulting in a low level of water loss and high Ci. Nevertheless, under saturating light at normal atmospheric CO2 levels, the rate of CO2 assimilation was higher for the Aegilops accessions, under high osmotic stress, than for the wheat genotypes. Moreover, in the wheat genotypes CO2 assimilation exhibited less or no O2 sensitivity. These physiological responses were manifested in changes in the growth rate and biomass production, since Aegilops (Ae550, Ae225) genotypes retained a higher growth rate (especially in the roots), biomass production and yield formation after drought stress than wheat. These results indicate that Aegilops genotypes, originating from a dry habitat have better drought tolerance than wheat, making them good candidates for improving the drought tolerance of wheat through intergeneric crossing.

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and rehydration

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO2 assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO2 assimilation decline was influenced by stomatal closure. Further lowering of net CO2 assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S2QA− takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.

Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings

The effect of some more or less harmful compounds like Cd, Pb, Ni, Ti salts and DCMU at low concentrations on the development of chloroplasts in maize and bean seedlings was investigated. Chlorophyll content, chlorophyll a/b ratio, photosynthetic activity (14CO2 fixation), chlorophyll-protein composition of thylakoid membranes, fluorescence spectra of chloroplasts, fluorescence induction parameters of leaves and electron microscopic structure of maize and bean chloroplasts as well as growth parameters were studied. Stimulation of chlorophyll synthesis and photosynthetic activity was observed at different intervals during all of the treatments, while chlorophyll a/b ratios and fluorescence properties of leaves or chloroplasts did not change considerably except in DCMU treated plants. Heavy metal treatments increased the amount of photosystem I and light-harvesting complex II, while decreased amount of photosystem I and higher amount of light-harvesting complex II was found in DCMU treated thylakoids. Electron microscopy showed only sligth differences in the morphology of chloroplast lamellar system (mostly in DCMU treated plants), while the status of the plasmalemma and tonoplast seemed to be altered as a result of certain metal treatments. Results showed the expression of a cytokinin-like effect on the development of chloroplasts. It is assumed, that these low-dose stressors generate non-specific alarm reactions in plants, which may involve changes of the hormonal balance.

Effects of Pb and Cd on Cucumber Depending on the Fe-Complex in the Culture Solution

Summary Modifying effects of EDTA and citrate as Fe(III)-complexes on Pb-and Cd-toxicity were investigated with cucumber ( Cucumis sativus L.) grown in culture solution. Pb and Cd were applied in lO μM concentration. Pb and Cd inhibited the growth of plants with Fe-citrate but with Fe-EDTA only Cd did. Chlorophyll content of Cd-treated plants-independently on the Fe-complex-was very low similarly to the iron deficient plants. Pb is proved to inhibit the chlorophyll accumulation only in plants supplied with Fe-citrate. Photosynthetic activity of 6-week-old plants was characterized by in vivo CO 2 fixation. The highest activities were observed in the middle (third-fourth) leaf storeys, and decreased in the lower and higher leaf storeys. Cd caused more than 50 % inhibition of the photosynthetic activity when applied with Fe-EDTA. With Fe-citrate, the inhibition exceeded 90 %. The photosynthetic activities in the Pb-treated plants were not significantly different from the control plants. The amount of chlorophyll containing complexes, especially that of PS I was highly affected by Cd particularly in the lower leaves. The results correlate with 59 Fe translocation into the shoot. Fe translocation was stimulated by Pb in the presence of Fe-EDTA but it was inhibited in the presence of Fe-citrate. Cd completely inhibited Fe translocation from the root to the shoot with both chelators. It is concluded that strong iron deficiency has a great but not exclusive role in the observed symptoms, and that the quality of the chelator strongly influences the effects of these polluting metals.

Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis

BACKGROUND AND AIMS Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 micromol m-2 s-1. However, desiccation at irradiance of 350 micromol m-2 s-1 induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery. METHODS Changes in chloroplast structure, CO2 assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 micromol m-2 s-1; ML) irradiance were compared with those under low (30 micromol m-2 s-1; LL) irradiance. KEY RESULTS Well-watered plants (control) at 100 micromol m-2 s-1 were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO2 assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML. CONCLUSIONS Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.

Relationship between Changes in Ion Content of Leaves and Chlorophyll-Protein Composition in Cucumber under Cd and Pb Stress

Hydroponically cultured cucumber plants supplied with 4 μᴍ Fe chelated either with EDTA or citrate and treated with Cd (10 μᴍ) and Pb (10, 50 μᴍ) from their one- or fourleaf stage were grown up to five-week-old age. The decrease in the chlorophyll content was the most pronounced in plants treated with Cd from a younger age, and in the case of Fecitrate. The chlorophyll a/b ratio of Cd stressed plants was also significantly lowered. In later treated plants the accumulation of chlorophyll was inhibited and the chlorophyll a/b ratio decreased only in the vigorously growing young leaves. Pb treatment had only a slight effect on both parameters. The changes in the chlorophyll-protein pattern of thylakoids were strongly related to their chlorophyll content but the response of each complex was different. Cd reduced the amount of chlorophyll containing complexes in the order of photosystem I > light-harvesting complex II > photosystem Il-core, while light-harvesting complex II appeared somewhat more sensitive than photosystem I in Pb treated plants. In accordance, a decline or blue shift of the long wavelength fluorescence emission band of chloroplasts was observed referring to disturbances also in photosystem I antenna assembly. The accumulation of chlorophyll and chlorophyll-proteins did not show close relationship to the heavy metal content of leaves which was the highest in the first of the intensively expanding leaves in the time of the treatment. The extraordinary sensitivity of photosystem I, and the relative stability of photosystem II under Cd treatment were similar to the case found in iron deficient plants. However, the pattern of chlorophyll content of leaf storeys of Cd treated plants rather followed the changes in their Mn content

Apparatus and Method for Rapid Determination of Photosynthetic CO2 Fixation of Leaves

Summary A simple apparatus and a rapid and sensitive isotope method for determination of photosynthetic CO2 fixation of seedlings, whole leaves or leaf segments is described. The apparatus consists of two main parts: the 14CO2 evolving system and a closed illuminated glass chamber for 14CO2 fixation. The samples are introduced into the glass chamber through a Hg-blockade without ceasing the closeness of the inner gas space. The apparatus is very useful in cases when rapid determination of CO2 fixation in great number of samples is needed.

Differences in root functions during long-term drought adaptation: comparison of active gene sets of two wheat genotypes

In an attempt to shed light on the role of root systems in differential responses of wheat genotypes to long-term water limitation, transcriptional differences between two wheat genotypes (Triticum aestivum L., cv. Plainsman V and landrace Kobomugi) were identified during adaptation to moderate water stress at the tillering stage. Differences in organ sizes, water-use efficiency and seed production were detected in plants grown in soil, and root functions were characterised by expression profiling. The molecular genetic background of the behaviour of the two genotypes during this stress was revealed using a cDNA macroarray for transcript profiling of the roots. During a 4-week period of moderate water deficit, a set of up-regulated genes displaying transiently increased expression was identified in young plantlets, mostly in the second week in the roots of Kobomugi, while transcript levels remained constantly high in roots of Plainsman V. These genes encode proteins with various functions, such as transport, protein metabolism, osmoprotectant biosynthesis, cell wall biogenesis and detoxification, and also regulatory proteins. Oxidoreductases, peroxidases and cell wall-related genes were induced significantly only in Plainsman V, while induction of stress- and defence-related genes was more pronounced in Kobomugi. Real-time qPCR analysis of selected members of the glutathione S-transferase gene family revealed differences in regulation of family members in the two genotypes and confirmed the macroarray results. The TaGSTZ gene was stress-activated only in the roots of Kobomugi.

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration. In contrast to the great differences in the morphology of plants, the ultrastructure and the organization of thylakoids were surprisingly similar in well-hydrated shade and sun populations. A high ratio of photosystem (PS)I binding light harvesting complex (LHC)II, important in low- and fluctuating light environment, was characteristic to both shade and sun plant, and the ratios of the main chlorophyll-protein complexes were also similar. The intensive protective mechanisms, such as shading by steep leaf angle and accumulation of protective substances, probably reduced the light intensity at the chloroplast level. The significantly increased ratio of monomer to oligomer antennae in well-hydrated sun plants may be connected with the temporary high light exposure of chloroplasts. During desiccation, LHCII was removed from PSI and part of PSII supercomplexes disassembled with some loss of PSII core and LHCII. The different reorganization of antennae, possibly connected with different quenching mechanisms, involved an increased amount of monomers in shade plants but unchanged proportion of oligomers in sun plants. Desiccation-induced responses were more pronounced in sun plants which also had a greater capacity to recover due to their stress-acclimated attitude.

Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

Real-time RT-PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.