DOCTORAL(PhD) THESIS
The effect of Cd stress on plants and its relationship withiron and water deficiency. Protective mechanisms.
LÁSZLÓGÁSPÁR
ELTE Doctoral School of Biology(Dr. AnnaErdei)
Experimental Plant Biology Doctoral Program (Dr. ZoltánSzigeti)
Supervisor: Dr. ÉvaSárvári
Department of Plant Physiology and Plant Molecular Biology
EötvösUniversity
Budapest
2008
INTRODUCTION
Plants are frequently exposed to changes and extremities of environmental factors which potentially restrain their growth and productivity. Among these, so called stressors, are natural (values of light intensity, temperature, etc. outside the optimal range of plants) and artificial factors (heavy metals, xenobiotics, etc.), as well. One of these stressors is soil contamination by the highly toxic heavy metal Cd. Cd contamination of soils is a result of various industrial (mining, metallurgy, burning of fossil fuels) and agricultural activities (use of chemical fertilizers and sewage sludge as fertilizer).
Once absorbed by the plant Cd exerts its effect (among other metabolic processes) on photosynthesis directly (substitution of other essential metal ions, binding to active sites of enzymes resulting in inhibition of activity) and/or indirectly (through disturbances of water and mineral balance), although its exact mode of action is not yet known. In the present work the various aspects of the complexity of the Cd effect on the formation and activity of the photosynthetic apparatus was investigated by comparing the effect of Cd treatment with that of iron and manganese deficiency and water shortage. In order to achieve that, the following points were investigated:
To what extent iron deficiency determines the symptoms of Cd toxicity? – By evaluating Cd symptoms in relation with those of iron deficiency.
What role does have the water balance disturbance generated by Cd treatment in the Cd provoked symptoms related to the photosynthetic apparatus? – By a comparison between the effect of Cd treatment and those ofosmotic/drought stress.
To model the inhibition of photosynthesis (without the water and mineral balance disturbing effect of Cd) the effect of Lincomycin, an inhibitor of prokaryotic type protein synthesis affecting mainly the reaction centres of both PSI and PSII, was investigated.
Considering that Cd affects not only the function of the photosynthetic apparatus but also thestructure of the newly formed photosynthetic apparatus, the investigation covered the functional aspects of the symptoms on photosynthesis, as well as their origin in the structural changes which has received relatively little attention to date.
On the ground of the above mentioned investigationswe expect to evaluate the importance of the different aspects of the stress symptoms generated by Cd, and at the same time to compare the effect of different stressors on photosynthesis and the protective mechanisms triggered by them.
MATERIALS AND METHODS
Plant material
Maize (Zea mays L., Mv NK 333 TC,) seedlings were grown in modified Hoagland solution of ¼ strength (Fodor et al. 1998) in darkness for 5 days and then under continuous white light (100 µmol m-2 s-1) for 72 h. Lincomycin (100 µg ml-1, Sigma) was added into the nutrient solution 16 h before the start of 72-h illumination and was present during the whole greening process.
Heavy metal treatments were performed on micropropagated poplar (Populus glauca L. var Kopetzkii) grown in the above mentioned hydroponic culture. Part of the plants had 10 µM Fe(III)-citrate as iron source instead of 10 µM Fe(III)-EDTA. Plants were treated with 10 µM Cd(NO3)2 or 10% PEG6000 or with the withdrawal of the iron or manganese source.
Drought stress treatments were performed on winter wheat (Triticum aestivum L.) cultivars (Sakha-8, Capelle Desprez) grown in soil watered to 60% of its water capacity. The treatment consisted of the withdrawal of watering.
Pigment determination
Chlorophyll content of leaves and thylakoid suspensions were determined in 80% acetone as in Porra et al. (1989). Carotenoid composition was determined with reverse phase HPLC (CISC, Estación Experimental del Aula Dei, Zaragoza; Debreceni Egyetem, Növénytani Tanszék, Debrecen).
Isolation of thylakoids, subchloroplast fragments and chlorophyll-proteins
Thylakoids were isolated from plastids as in Jansson et al. (1997) and subchloroplast fragments were prepared by the digitonin method (Boardman 1971).Unsolubilized membranes were pelleted with 10 min 2000 g centrifugation, solubilised membranes of predominantly granal origin with 30 min 20000 g, a mixed fraction with 30 min 50000 g and the mainly stromal faction with 60 min 144000 g. Chlorophyll proteins were separated by Deriphat-PAGE after solubilising the thylakoids with glucosidic detergents (dodecil-sucrose:nonil-glukozide:litium-dodecil-sulphate=4,5:4,5:1,0, at 10-15 (w/w) detergent/chlorophyll ratio (Sárvári and Nyitrai 1994). Polypeptide composition of membrane fractions and chlorophyll-proteins was obtained by denaturing PAGE according to Laemmli (1970) except using 10–18% gel gradients. Geles were stained withCoomassie Brilliant Blue R-250. The relative amount of Chl in each band was determined by densitometry of the scanned gels using the Phoretix software(Newcastle, UK). The percentage values of bands identified by their polypeptide pattern as belonging to a given complex were summed up. The absolute amounts of complexes were calculated in μg Chl by dividing the Chl content of 1 g fresh leaf material among the digitonin solubilised fractions and the complexes according to their percentage proportions.
Measurements of fluorescence spectra
Fluorescence emission and excitation spectra of cut leaves and thylakoid suspensions and isolated chlorophyll-proteins were performed at 77K and at room temperature with a FluoroMax-2 spektrofluorimeter (Jobin Yvon, Longjumeau, France).
Fluorescence induction measurements
Fluorescence induction measurements of leaf samples were performed using a PAM Chlorophyll Fluorometer (Walz, Effeltrich, Germany). Rapid fluorescence induction kinetics was performed as described in Morales et al. (1991).
Measurements of photosynthetic activity
Photosynthetic activity of detached leaf discs was determined by 14CO2-fixation in Hg sealed chamber Láng et al.(1985). Radioactivity of the leaf discs was measured with a Beckmann (Fullerton, US)LS 5000 TD scintillation counter. CO2-fixation of intact leaves was measured with an LCA-2 (ADC, UK) infrared gas analyzer.
Determination of metal contents was performed with a TXRF spectrometer (EXTRA IIA) after tissue digestion with concentrated HNO3(Varga és mtsai, 1999).
RESULTS AND CONCLUSIONS
1. The role of disturbances in mineral balance in the effects of Cd on photosynthesis.
– In poplar, as opposed to other plant species, Cd did not inhibit Fe uptake in the roots, but the inhibition of Fe translocation to the shoot (the extent of which depended greatly on the Fe-chelator) was similar to other results. Mn content of plants was decreased by Cd treatment and increased by iron deficiency.
– A linear relationship between the chlorophyll content/photosynthetic activity of plants and the iron content of their leaves was clearly demonstrated irrespective of the cause (Cd stress/Fe deprivation) of the decrease in leaf Fe.
– In leaves where Fe content was greatly decreased both Cd stress and Fe deficiency modified the balance between the different chlorophyll protein complexes, namely, the amount of PSI was most decreased (importance of the [4Fe-4S]-centres in stabilizing the structure of PSI), while the PSIIRC decreased the least. The similarity of the effect of the two stressors underline the primary role of Fe deficiency in the Cd effect. However, there were some difference between the effect of the two stressorsas in the case of Cd stress inhibition of PSII activity and alterations of the chlorophyll protein composition became apparent at a higher leaf Fe content than in the case of iron deprivation which may be in connection with the decrease in leaf Mn content in Cd treated plants.
–Both Cd stress and iron deprivation increased non-photochemical quenching. At higher leaf Fe contents the increase was mainly due to qE type quenching, while in more chlorotic leaves qI type quenching (probably of inactive PSII reaction centres) became more pronounced.
2. The role of water balance disturbance inthe symptoms of Cd stress on the photosynthetic apparatus
– Results of experiments made on poplar clearly demonstrated that water balance disturbance generated by Cd treatment could not have played a major role in provoking Cd related symptoms on the photosynthetic apparatus as 10% PEG treatment did not decrease significantly stomatal opening of leaves developed during the PEGtreatment and as a result there were little change in the structure and function of the photosynthetic apparatus. Although 10-5 M Cd treatment decreased stomatal opening significantly the decrease in CO2-fixation was greater than what could be justified by stomatal closure in itself.
– The effect of drought stress on photosynthesis was dependent on the leaf RWD in both the drought tolerant and the sensitive wheat cultivar. Although as leaf RWD increased net photosynthesis dropped considerably, the rate of the linear electron transport did not decrease significantly up to 50% RWD. Additional loss of leaf water content led to considerable loss of linear electron transport rate with the antiparallel increase in NPQ. The tolerance of the more tolerant cultivar consisted of the accumulation of sugars (osmotically active substances) and induction of various protective mechanisms (water-water electron transport, photorespiration).
– In wheat under drought stress, as opposed to poplar under Cd stress, in leaves with moderate RWD an increase of PSII (and LHCII) could be detected. In leaves with higher RWD the amount of PSII greatly decreased, but in the tolerant cultivar the stability of PSII increased once more in extremely water deficient leaves, which is probably in connection with the accumulation of inactive PSII reaction centres playing part in non-photochemical quenching.
3. The effect of the decrease in active reaction centres/LHC (Lincomycin treatment)
–Isolation of thylakoid fractions of granal and stromal origin was used to demonstrate reorganization of chlorophyll proteins when the synthesis of reaction centres was inhibited by Lincomycin treatment while the amount of LHCs remained the same. As the composition of photosystems was unchanged the high amount of LHC could not be connected to reaction centres and was present as free LHC in the thylakoids of Lincomycin treated plants. As a result there was a considerable increase in Fo, the long wavelength fluorescence emission at 77K shifted from 735 nm to 730 nm (free LHCI), the intensity of the 679 (free monomeric LHCII) and 700 nm (Lhca2/aggregated LHCII) fluorescence increased. There was a dominance of the long wavelength fluorescence (characteristic of LHCI) in all digitonin fractions, which refer to the energy transfer connection between the free LHCII and LHCI even in the granal fraction.
– Considerable increase in the non-photochemical quenching of variable fluorescence was detected in Lincomycin treated plants. The formation of non-photochemical quenching was significantly faster while its relaxation was considerably slower during dark adaptation than in control plants. The fast formation of non-photochemical quenching is in connection with the constantly high DEPS, while the high steady state quenching and the slow relaxation probably has its origin in the antenna reorganization detected in Lincomycin treated plants.
4. Stress induced structural changes and protective mechanisms in the thylakoids
– On the basis of the results of various studies on different stressors there was a characteristic pattern of changes in the chlorophyll-protein composition (and supposed underlying mechanisms) as a function of the strength of effect on photosynthesis:
(1) increase in PSII (early acceleration of PSII synthesis as a result of the damage caused by ROS),
(2) decrease in PSII, increase (or decrease to a lesser extent) in PSI and LHCII (shift in the balance of PSII damage/regeneration, triggering of protective mechanisms: cyclic electron transport, LHCII related non-photochemical quenching),
(3) decrease of PSI and LHCII relative to PSII (strong decrease in chlorophyll content, non-photochemical quenching by inactivated PSII reaction centres),
(4) stabilization of LHCII (processes leading to senescence).
Iron deficiency was the main cause of the inhibition of photosynthesis provoked by Cd treatment. Iron deficiency related to Cd treatment affected photosynthetic activity and triggered protecting mechanisms against photoinhibition mainly by a control over biogenesis and turnover of various elements of the photosynthetic apparatus. The difference between symptoms of moderate iron deficiency induced by Cd and those brought about by iron deprivation itself implies that the effect of Cd included factors other than iron deficiency (e.g. Mn deficiency) as well. Disturbances of water balance did not play an important role in triggering Cd effect as neither the water deficiency of leaves, nor the stomatal closure were affected to such an extent that they could justify the inhibition of CO2 fixation and photosynthetic electron transport detected in Cd treated plants. Results of lincomycin treatment imply that in the case of strong stresses causing significant damage to the reaction centres and inhibition of the photosynthetic activity (e.g. strong Cd treatment) the resulting disconnection of LHCs and reorganisation of the antenna structure may contribute to the increased non-photochemical quenching capacity protecting against photoinhibition.
Literature cited
Boardman NK (1971) Subchloroplast fragments: digitonin method. In: Methods in Enzymol. (San Pietro A, ed), Vol XXIII. Photosynthesis, Part A, pp. 268-276: Acad. Press, New York
Fodor F, Cseh E, Varga A, Zárai Gy (1998) Lead uptake, distribution and remobilization in cucumber. Journal of Plant Nutrition 21:1363–1373.
Jansson S, Stefánsson H, Nyström U, Gustafsson P, Albertsson P-Å (1997) Antenna protein composition of PS I and PS II in thylakoid sub-domains. Biochmica et Biophysica Acta 1320: 297-309.
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685.
Láng F, Sárvári É, Szigeti Z (1985) Apparatus and method for rapid determination of photosynthetic CO2 fixation of leaves. Biochemical Physiology Pflanzen 180: 333-336.
Morales F, Abadía A, Abadía J (1991) Chlorophyll fluorescence and photon yield of oxygen evolution in iron-deficient sugar beet (Beta vulgaris L.) leaves. Plant Physiology 94: 607–613.
Porra RJ, ThompsonWA, KriedemannPE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochmica et Biophysica Acta 975: 384-394.
Sárvári É, Nyitrai P (1994) Separation of chlorophyll-protein complexes by Deriphat polyacrylamide gradient gel electrophoresis. Electrophoresis 15: 1067-1071.
Varga A, Martinez RMG, Záray G, Fodor F (1999) Investigation of effects of cadmium, lead, nickel and vanadium contamination on the uptake and transport processes in cucumber plants by TXRF spectrometry. Spectrochimica Acta B 54:1455-1462.
Publications forming part of the doctoral thesis
1. Full articles in scientific journals
1. Nyitrai P, Bóka K, Gáspár L, Sárvári É, Lenti K, Keresztes Á „Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings” Journal of Plant Physiology 160 (2003): 1175-1183. IF: 1,149
2. Nyitrai P, Bóka K, Gáspár L, Sárvári É, KeresztesÁ „Rejuvenation of ageing bean leaves under the effect of low-dose stressors” Plant Biology 6 (2004): 708-714. IF: 1,420
3. Molnár I, Gáspár L, Sárvári É, Dulai S, Hoffmann B, Molnár-Láng M, Galiba G „Physiological and morphological responses to water stress in Aegilops biuncialis and Triticum aestivum genotypes with differing tolerance to drought” Functional Plant Biology 31 (2004): 1149-1159. IF: 2,398
4. Fodor F, Gáspár L, Morales F, Gogorcena Y, Lucena J J, Cseh E, Kröpfl K, Abadía J, Sárvári É „The effect of two different iron sources on iron and cadmium allocation in cadmium exposed poplar plants (Populus alba L.)” Tree Physiology 25 (2005): 1173-1180. IF: 2,087
5. Gáspár L, Sárvári É, Morales F, Szigeti Z „Presence of ‘PSI free’ LHCI and monomeric LHCII and subsequent effects on fluorescence characteristics in lincomycin treated maize” Planta 223 (2006): 1047-1057. IF: 3,053
6.Czövek P, Király I, Páldi E, Molnár I, GáspárL „Comparative analysis of stress tolerance in Aegilops and Triticum wheat varieties to detect different drought tolerance strategies” Acta Agronomica Hungarica 54 (2006): 49-60.
2. Articles in congress issues of scientific journals
1. Láng F, Sárvári É, Gáspár L, Fodor F, Cseh E (2001) „Influence of light intensity on thylakoid development under Cd stress in poplar” In: Proc. 12th Congress on Photosynthesis (online), S3-23, Brisbane, Australia
2. Gáspár L, Sárvári É, Molnár I, Stéhli L, Molnár-Láng M, Galiba G (2002) „Structural changes of the photosynthetic apparatus under osmotic stress in different Triticum aestivum and Aegilops biuncialis genotypes” Acta Biologica Szegediensis 46: 91-93., Szeged
3. Molnár I, Gáspár L, Stéhli L, Dulai S, Sárvári É, Király I, Galiba G, Molnár-Láng M (2002) „The effects of drought stress on the photosynthetic processes of wheat and of Aegilops biuncialis genotypes originating from various habitats” Acta Biologica Szegediensis 46: 115-116., Szeged
4. Gáspár L, Molnár I, Stéhli L, Terstyánszky A, Galiba G, Sárvári É (2003) „Structural and functional changes of the photosynthetic apparatus in wheat and Aegilops biuncialis genotypes under osmotic stress” In: Proc. 4th International Congress of PhD students, pp. 223-227., Miskolc
5. Hakmaoui A, Gáspár L, Sajnani C, Sárvári É, Ater M, Baron M (2004) „Approches physiologiques et biochimiques pour la sélection dés plantes utiles en phytoremédiation” In: Proc. Congrés International de Biochimie, pp. 487-490., Marrakech, Marocco
6. Gáspár L, Czövek P, Ferenc F, Hoffmann B, Nyitrai P, Király I, Sárvári É (2005) „Greenhouse testing of new wheat cultivars compared to those with known drought tolerance” Acta Biologica Szegediensis 49: 97-98., Szeged
7. Kovács S, Gáspár L, Cseh E, Kröpfl K, Sárvári É (2005) „Protective effects of phosphonomethyl-sarcosine against the copper and cadmium induced inhibition of leaf development in poplar”Acta Biologica Szegediensis 49: 61-63., Szeged
3. Abstacts in congress issues of scientific journals
1. Gáspár L, Sárvári É, Szigeti Z (2000): „Lincomycin induced changes in the organisation of light-harvesting antennae” Plant Physiology and Biochemistry, 38S: 105 (P). IF: 1.414
2. Sárvári É, Gáspár L, kröpfl K, Fodor F, Cseh E (2004) „Comparison of the effects of Cd toxicity with iron and manganese deficiency on thylakoid development in poplar” Acta Physiologia Plantarum 26S: 210 (Abstracts of 14th FESPB Congress) Cracow, Poland IF: 0.433
3. Gáspár L, Nyitrai P, Terstyánszky A, Sárvári É(2004) „Physiological parameters of a sensitive and a tolerant wheat genotype under drought stress”, Acta Physiologia Plantarum 26S: 193 (Abstracts of 14th FESPB Congress) Cracow, Poland IF: 0.433
4. Láng F, Szegi P, Basa B, Solti Á, Gáspár L,Tamás L, Mészáros I, Sárvári É (2007) „Time scale of the appearance of Cd effects on photosynthetically competent poplar leaves” Photosynthesis Research 91: 32214th International Congress of Photosynthesis, Glasgow, UK IF: 2.193
5. SoltiÁ, SzegiP, GáspárL, Lévai L, SzigetiZ, SárváriÉ (2007) „Cd-induced inhibition of photosynthesis can be recovered by elevated Fe supply”Cell Stress & Chaperones 12: 4G-07-P. 2nd World Congress of Stress, Budapest IF: 3.097
6. Szegi P, Solti Á, Gáspár L, Mészáros I, Sárvári É (2007) „Kinetics of photosynthetic responses and development of protective mechanisms during Cd stress in poplar”Cell Stress & Chaperones 12: 4G-06-P. 2nd World Congress of Stress, Budapest IF: 3.097
4. Abstracts in congress publications
1. Sárvári É, Gáspár L, Szigeti Z, Láng F (2001) „A klorofill-proteinek szerveződése stresszkörülmények közt” Fotoszintéziskutatók találkozója, Szeged
2. Gáspár L, Sárvári É, Szigeti Z (2003) „Cadmium toxicity and iron deficiency in poplar leaves detected by fluorescence imaging” In: Proc. 10th Congress of the European Society for Photobiology, p. 159. Vienna, Austria
3. Sárvári É, Gáspár L, Fodor F, Cseh E (2003) „Cd és Pb hatása a klorofill-proteinek akkumulációjára magasabbrendű növényekben” Fotoszintéziskutatók konferenciája, Noszvaj
4. Gáspár L, Láng F, Fodor F, Cseh E, Sárvári É (2004) „Comparison of the effects of water deficiency and Cd stress on the photosynthetic apparatus of poplar” In: Book of abstracts, p. 174, 14th Congress on Photosynthesis, Montreal, Canada
5. Sárvári É, Gáspár L, Szegi P, Basa B, Solti Á, Nyitrai P, Mészáros I (2006) „Photosynthetic acclimation under Cd stress in poplar” In: Abstracts of 15th. Congress of FESPB, p. 171, Lyon, France
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