4A_01_S

Evolution of desiccation tolerance: genomic aspects

Dorothea Bartels

The Scrophulariaceae are one of the most diverse plant families and have been shown to be polyphyletic. Several Scrophulariaceae which belong to the genera Craterostigma and Lindernia desiccation tolerant and are therefore of particular interest. Craterostigma plantagineum has been used extensively to identify molecular mechanisms, which are involved in the acquisition of desiccation tolerance. These studies revealed a large number of genes which are up-regulated in response to dehydration. The genes can approximately be divided into two groups. One group encodes proteins which are directly involved in protection of cellular structures. The other group of genes encodes genes which are involved in regulating the response to dehydration. The dehydration/rehydration cycle is also characterized by a massive sugar conversion from octulose to sucrose.

C. plantagineum belongs to the Linderniae which comprises desiccation tolerant and non-desiccation tolerant plants. Using molecular markers a phylogenetic tree was constructed to determine the molecular relationships of the different plant species. It appears that desiccation tolerant plants cluster together. Comparative studies of these plant species identified conserved genomic structures and very recently evolved mobile signals in stress-relevant genes.

4A_02_S

Functions of tocopherols and other lipid-soluble antioxidants in plants under light- or metal-induced oxidative stress

Michel Havaux¹, Valérie Collin¹, Pascal Rey¹, Christian Triantaphylides¹, Michel Matringe²

2CEA/Grenoble, iRTSV, Laboratoire de Physiologie Cellulaire Végétale, F-38054 Grenoble, France

The term ‘vitamin E’ describes the beneficial biological activity of a group of structurally related compounds, the tocochromanols, in animals and humans. Those compounds are composed of a chromanol head group and a prenyl side chain. Natural vitamin E includes four tocopherols and four tocotrienols, which are synthesized exclusively by oxygenic photosynthetic organisms. In leaves of vascular plants, a-tocopherol is the predominant form of vitamin E. A detailed analysis of tocochromanol distribution in chloroplasts isolated from young tobacco leaves showed that a-tocopherol is predominantly located in the thylakoid membranes. The protective role of vitamin E and the functional interactions between vitamin E and other plastid antioxidants (e.g. xanthophyll carotenoids) were studied using Arabidopsis and tobacco mutant/transgenic plants that lack or over-accumulate vitamin E constituents and/or carotenoids. This genetic approach was combined with the use of new biochemical and biophysical methods that allow characterization, quantification and imaging of lipid peroxidation in vivo. Both tocopherols and tocotrienols were found to protect thylakoid membranes against photooxidative stress. We also found that vitamin E and the xanthophyll zeaxanthin have overlapping functions, with lack of vitamin E being compensated by an increased level of zeaxanthin and vice versa. Lack of both compounds resulted in a very photosensitive phenotype. Vitamin E was also found to be essential for tolerance of Arabidopsis towards oxidative stress induced by stress conditions different from high light, such as heavy metals.

4A_03_S

THE GENETIC BASIS OF SINGLET OXYGEN-MEDIATED SIGNALING OF STRESS RESPONSES IN PLANTS

Rasa Meskauskiene, Klaus Apel

Swiss Federal Institute of Technology (ETH Zurich), Institute of Plant Sciences, Universitaetstr.2, CH - 8092 Zurich, Switzerland. E-mail: kapel@ethz.ch

The evolution of aerobic metabolic processes such as respiration and photosynthesis unavoidably leads to the production of reactive oxygen species (ROS) in mitochondria, chloroplasts and peroxisomes. A common feature among the different ROS types is their capacity to cause oxidative damage by inactivating e.g. proteins, nucleic acids and lipids. These cytotoxic properties explain the evolution of complex arrays of ROS scavengers. In plants, chloroplasts and peroxisomes are the major sites of ROS production. Various abiotic stress conditions may limit the ability of a plant to use light energy for photosynthesis. Under such stress conditions hyper-reduction of the photosynthetic electron transport chain and photo-inhibition of photosynthesis may occur even at moderate light intensities, often causing damages that have been interpreted as unavoidable consequences of injuries inflicted upon plants by toxic levels of ROS. However, this paradigm needs to be modified. Stress responses triggered by ROS are not only due to physicochemical damages but may also be caused by the activation of genetically determined stress response programs. We'll present results of our work showing that ROS may act as signals during stress whose specificities seem to depend on the chemical identity of a given ROS and its intracellular site of generation.

4A_04_S

Molecular mechanisms of photoinhibition of Photosystem II

Imre Vass, Krisztián Cser and Otilia Cheregi

Corresponding author: Imre Vass. Phone: +36-62-599-700, Fax: +36-62-433-434, Email: imre@brc.hu

Light-induced decline of photosynthetic activity, generally called as photoinhibition, is a general phenomenon in all oxygenic photosynthetic organisms under conditions when the metabolic processes can not keep up with the electron flow produced by the primary photoreactions. Although light-induced damage occurs in all pigmented photosynthetic complexes the main site of photodamage is Photosystem II. The main factors, which are responsible for the light sensitivity of Photosystem II are excited pigment molecules, oxygen, manganese, as well as electron donors with high oxidizing potential. Photosystem II can be efficiently protected from photodamage by the combination of harmless dissipation of absorbed light energy, non-radiative charge recombination and repair of damaged reaction center complexes making possible the safe utilization of light, the highly energetic substrate of photosynthesis. The lecture will cover the principles and basic mechanisms of photodamage of photosynthesis, and its repair.

4A_05_S

Mammalian Bax initiates plant cell death through ROS production and organelle destruction

Maki Kawai-Yamada, Keiko Yoshinaga and Hirofumi Uchimiya

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan, mkawai@iam.u-tokyo.ac.jp

Relatively few endogenous plant genes that share sequence homology with the mammalian apoptotic genes have been identified to date. Nonetheless, similarities in PCD exist in plants and animals. Mammalian proapoptotic gene Bax is known to cause cell death when expressed in yeast and plants. We examined transgenic plants expressing both Bax and organelle-targeted green fluorescent protein (GFP) to analyze the cellular event that occur during Bax-induced plant cell death. The results indicated that Bax induced temporal and special cell death events at the organelle level. Such events included ion leakage, DNA fragmentation and cell shrinkage. The mitochondria changed morphologically from being bacilli-shaped to being round. The chloroplasts lost membrane function and their contents leaked out, followed by the disruption of the vacuole. Light was not essential for Bax-induced ion leakage or organelle disruption, but for chlorosis. Furthermore, ROS production was involved in triggering cell death. To compare Bax-induced cell death and other ROS-mediated plant cell death, Arabidopsis leaves expressing mitochondrial-targeted GFP were treated with ROS-inducing chemicals, such as hydrogen peroxide, paraquat and menadione. After 24h treatment, mitochondria showed morphological changes from a bacillus-like to a round shape. The size of mitochondria decreased by half compared with controls. Such cellular events may cause energy depletion, and resulted in plant cell death.

4A_06_S

Reactive carbonyl detoxification and stress resistance in plants

Gábor V. Horváth, Zoltán Turóczy, Petra Kis, Katalin Török, Éva Hideg, László Sass and Dénes Dudits

Institute of Plant Biology, Biological Research Center HAS, Szeged, Hungary. E-mail: hvg@brc.hu

Productivity of plants is greatly affected by environmental stresses, therefore there is a continuous need for the genetic improvement of stress tolerance in the agriculture. During oxidative stress rapid accumulation of reactive oxygen species (ROS) and reactive carbonyl species (RCS, carbonyl stress) significantly contributes to the damage of crop plants. Improvement of intracellular scavenging capacity of such compounds provenly lead to increased stress tolerance. Diverse enzymes can detoxify the reactive carbonyl species (eg. 4-hydoxy-nonenal or methylglyoxal). Their importance is clearly demonstrated by the fact that overproduction of alkenal reductases, aldehyde dehydrogenases or the members of the glyoxalase enzyme family in transgenic plants led to improved tolarance to wide range of stress conditions. We have isolated the MsALR aldose reductase homolog gene from Medicago sativa and showed the accumulation of the transcript at higher levels in response to different stress treatments. Transgenic tobacco plants ectopically expressing the MsALR cDNA were more tolerant to dehydration stress and recovered better from damages caused by water deficit than the untransformed wild type plants and were more tolerant to heavy metal, salt, dehydration and UV-B stress. Results of enzyme activity measurements and in vivo assays for protection against methylglyoxal toxicity strengthened our hypothesis that one important function of aldo/keto reductase proteins is also the elimination of reactive aldehydes and the reduction of the consequences of carbonyl stress in plants.

This work was supported by NKFP Grant No. 4-064-2004, Gábor V. Horváth is grateful for the support of the “János Bólyai” Research Fellowship.

4B_01_S

Use of Gene Silencing and Metabolomics to Characterize Interactive Stress and Defense Pathways in Soybean

T.L. Graham, M.Y. Graham, S Subramanian, O Yu

Ohio State University, Columbus, OH, USA 43210; corresponding author: graham.1@osu.edu

Induced resistance to pathogens can be specific to a given race of a pathogen or effective against a range of pathogens. The former type of resistance involves a form of programmed cell death called the hypersensitive response (HR). The latter type of resistance, called general or basal resistance, is often induced in plants by elicitors from the pathogen called pathogen associated molecular patterns (PAMPs). This form of resistance has some mechanistic similarities to innate immunity in animals. In the work described here we have used RNAi gene silencing and metabolic profiling to study the molecular bases of soybean resistance to the pathogen, Phytophthora sojae. Resistance responses were examined in the context of one of the most highly characterized PAMPs, the cell wall glucan elicitor (WGE) from P. sojae. Silencing either of two biosynthetic enzymes for soybean isoflavonoids (isoflavone synthase or chalcone reductase) led to a complete loss of race-specific resistance and HR cell death in response to P. sojae. Consistent with this, WGE, the major pathogen elicitor of the isoflavonoids in soybean, induced an HR-like cell death in root tissues. Silencing of the endoglucanase thought to release active elicitor fragments from WGE abolished both HR cell death and WGE-induced cell death. Silencing of a unique metallothionein gene (MMT) led to greatly enhanced WGE-induced cell death, suggesting a role of MMT in preventing runaway cell death. Finally, silencing of a pathogenesis-related protein, PR-1a, led to loss of expression of MMT and general resistance. Thus, our studies led to the revelation of very interesting cross-talk between the cell death and general resistance pathways in soybean.

4B_02_S

Engagement and modification of the plant host stress machinery: a virulence strategy of the plant pathogen Pseudomonas syringae.

Jean T. Greenberg

The University of Chicago, Chicago, Il 60637, USA jgreenbe@midway.uchicago.edu

Pseudomonas syringae cause disease on a broad range of plant hosts. To have a successful infection, these bacteria utilize a specialized secretion apparatus called the type III secretion system (T3SS). P. syringae secrete a large group (20-40+) of effectors through the T3SS directly into plant cells. There is tremendous diversity in the repertoire of secreted effectors between different P. syringae strains. However, a few effectors are common to all P. syringae strains. We have selected one such common effector and one rare effector to functionally analyze in detail, including their localization in plants and their interaction with potential host targets. We have found that these effectors can modify host functions and alter their stress responses. This points to an interesting intersection between bacterial virulence mechanisms and host stress-coping mechanisms.

4B_03_S

The NDR1-Actin Connection: Linking Gene-for-Gene Resistance and the Actin Cytoskeleton.

Brad Day, Miaoying Tian, Marina Varbanova and Caleb Knepper.

Michigan State Universit, Department of Plant Pathology, East Lansing, MI, USA, bday@msu.edu

Disease resistance in plants involves a molecular surveillance mechanism capable of responding to a myriad of plant derived elicitors. Recent research in this area has revealed a complex genetic and biochemical network required for the regulation of innate immune responses in plants. In total, the coordinated interactions of bacterial effector proteins, plant chaperones and resistance (R)-proteins contribute to the molecular and biochemical events which dictate host susceptibility and resistance. Research in our laboratory focuses on the identification and characterization of protein-protein interactions which not only function in the activation of resistance, but also in the negative regulation of effector-triggered immunity in Arabidopsis thaliana. Using NDR1, a protein required for the activation of R-protein mediated resistance in Arabidopsis, as a molecular and biochemical model for the activation of resistance, we are working towards the elucidation of the dynamic linkages between effector-triggered immunity and the host actin cytoskeleton. To this end, we have begun characterizing several proteins required for reorganization of the actin cytoskeleton in plants, and have identified a genetic interaction between bacterial effector action and the dynamics of the host cytoskeleton. Similarities between the activity of Yersinia pestis effector proteins and P. syringae effectors will be discussed. Our working model suggests that the actin cytoskeleton of plants may be a virulence target for pathogens.

4B_04_S

Elucidation of fumonisin B1-induced cell death signalling in Arabidopsis thaliana

Daniel F. Tomé, John M. Hamilton, Stephen Chivasa, Keith Lindsey, and Antoni R. Slabas

Creative Gene Technology Ltd., Integrative Cell Biology Laboratory, University of Durham, Durham DH1 3LE, United Kingdom. e-mail: j.m.hamilton@durham.ac.uk

Programmed Cell Death (pcd) is a ubiquitous process in plants that is very similar to apoptosis in animals. It occurs in various tissues during plant development, such as death of the tapetum cell layer in mature anthers, the sheading of leaves in autumn in perennial plants, and in the differentiation of xylem. Pcd also occurs as a response to stress such as pathogen attack. However, the mechanism by which plants initiate pcd and the key proteins involved are poorly understood. Recently, we reported that removal of extracellular ATP (eATP) from plant tissues initiates pcd. We also reported that fumonisin B1 (FB1)-induced pcd is mediated via eATP depletion and can be blocked by addition of ATP. Rescue of cells from FB1-induced death by ATP is possible if ATP is supplied before the cells are irreversibly committed to death. Changes in gene expression at or around the time of commitment are of interest as these could be crucial players in cell death. We are now using 2-dimensional difference gel electrophoresis and DNA microarray technologies to identify the key components in this pathway, focusing on time points around commitment. We identified a number of differentially expressed genes and proteins associated with this death pathway. Reverse genetic screening using homozygous knockout mutants has allowed us to identify candidates that could prove critical to the execution of pcd in plants. Moreover, the emerging data is beginning to give us insight into the mechanism by which eATP sustains cell viability in plants.

4B_05_S

Dehydroascorbate uptake is impaired in the early respons of Arabidopsis plant cell cultures to cadmium.

Horemans Nele^*a, Raeymaekers Tine^*, Van Beek Kim^*, D’Haese David^*, Cuypers Ann^** Guisez Yves^*

** Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium

The balance between antioxidants such as ascorbate (ASC) and glutathione and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated on their response to elevated levels of aluminum, zinc or cadmium. For Zn and Al no significant H₂O₂-accumulation was detected. Cd, however, induced a rapid and concentration dependent and initially extracellular H₂O₂-accumulation that could be inhibited by DPI (20 µM). RT-PCR analysis of 3 Rboh genes showed an increased transcription of Rboh F after 15 min. No change in ASC concentration was observed during the first three hours after Cd-addition. In contrast glutathione levels completely diminished within one hour. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate uptake activity (up to 90% inhibition after four hours). This decrease is not present when cells are treated with LaCl_3, before exposure to CdCl₂. LaCL₃ is a typical inhibitor of Ca channels and is known to prevent Cd uptake and Cd induced ROS production in plants. Therefore these results seem to indicate Cd uptake is a prerequisite for the change in DHA transport activity. However DPI did not prevent the drop in DHA uptake activity indicating that this response seems to be independent of the H₂O₂-production. The possible function of the drop in DHA uptake in response to Cd stress will be discussed.

4C_01_S

REGURATORY GENE NETWORK IN DROUGHT STRESS RESPONSE

Kazuo Shinozaki¹ and Kazuko Yamaguchi-Shinozaki ^{2, 3}

sinozaki@rtc.riken.jp

Drought stress induces a variety of genes of which products function in drought stress tolerance and response in plants. Many stress-inducible genes have been used to improve stress tolerance by gene transfer. In this congress, we present our recent studies on molecular mechanism in drought stress response and tolerance. We have identified complex regulatory systems in stress-responsive gene expression: ABA-dependent and ABA-independent systems. In one of the ABA-independent pathways, a cis-acting element (DRE/CRT) and its binding proteins, DREB2s, are important cis- and trans-acting elements in drought-responsive gene expression, respectively. DREB2 is also involved in heat stress response. In the ABA-dependent pathways, bZIP transcription factors (AREB/ABF) are involved in the major process. Protein phosphorylation is important for the activation of AREB proteins. The MYB/MYC and NAC transcription factors are involved in ABA-responsive gene expression and jasmonic acid response In the ABA-dependent pathway, stress-inducible NCED3 is mainly involved in the ABA biosynthesis during drought stress. We analyzed metabolic profiles regulated by ABA using T-DNA tagged mutant and with GC-MS and LC-MS. We analyzed the function of CYP707A3 in the regulation of ABA metabolism during stress responses. We also report the functions of SnRK2 protein kinases in drought and ABA responses using mutants and transgenic overexpressors.

Umezawa et al. Curr Opin Biotech 17:113-122 (2006)

Yamaguchi-Shinozaki and Shinozaki. Ann Rev Plant Bio 57:781-803 (2006)

4C_02_S

Stress-regulated siRNAs and miRNAs

Jian-Kang Zhu

Institute for Integrative Genome Biology, Department of Botany and Plant Sciences, University of California, Riverside

It is often presumed that RNA interference (RNAi) evolved as a cellular surveillance mechanism to silence “foreign” double stranded RNAs (dsRNAs) so that cells can defend against viral infection or transposons. However, recent work suggests a widespread occurrence of dsRNAs unrelated to viral replication or transposons. A significant portion of cellular dsRNAs can be formed by natural antisense transcripts (NATs). Approximately 10-20% of genes in plant and animal genomes encode cis-NATs, i.e. these genes overlap but are on the opposite strands of DNA. In Arabidopsis, dsRNAs generated from cis-NATs can be processed by Dicer-like enzymes into 21-24 nt nat-siRNAs which then direct the cleavage of complementary mRNAs. Evidence indicates that many cis-NAT genes are regulated by biotic and abiotic stresses, and they generate nat-siRNAs only under specific stress conditions. The stress-induced nat-siRNAs are important components in the regulatory circuits leading to stress acclimation. These results suggest that RNAi is not only critical for cellular surveillance but it is also an important cellular gene regulatory mechanism. In this presentation, I will talk about abiotic stress-regulated nat-siRNAs and other endogenous siRNAs in Arabidopsis. In addition, I will discuss the role of miRNAs in plant responses to abiotic stresses.

4C_03_S

From mutants to genes, pathways and networks in plant stress signaling

Hans J. Bohnert

Arabidopsis thaliana transcript profiles can report the effects of abiotic and biotic stresses on tissue- and cell-specific gene expression. Organizing these datasets could reveal the structure and mechanisms of responses and cross-talk between pathways, and in which cells the plants perceive, signal, respond to and integrate environmental inputs.

We have clustered Arabidopsis transcript profiles for >30 treatments, comprising abiotic, biotic and chemical stresses. Ubiquitous stress responses in Arabidopsis, similar to those of fungal and animal cells, employ genes in pathways related to MAP kinases, Snf1-related kinase, vesicle transport, mitochondrial functions, and the fundamental transcription machinery. The induced response to various stresses can be attributed to genes whose promoters are characterized by a small number of common regulatory motifs, while secondary motifs have also been identified. Most genes that are down-regulated by stress show distinct tissue-specific expression patterns and appear to be under strict developmental regulation. The ABA-dependent transcriptome is delineated in the cluster structure, while functions dependent on reactive oxygen species are widely distributed, possibly indicating evolutionary pressures conferring distinction to different stresses in time and space. Cell lineages in the root express stress-responsive genes at different levels. The intersection of stress-responsive and cell-specific profiles identified cell lineages affected by abiotic stress.

In an extension of these studies, the Graphical Gaussian Model (GGM) was used to assemble a gene network for the Arabidopsis transcriptome. Based on partial correlation (pcor), GGM infers co-regulation patterns between gene pairs conditional on the behavior of other genes. We used ‘regularized’ GGM, coupled with iterative random samplings, to expand the network to cover the whole Arabidopsis genome (22,266 genes). This resulted in a network of ~18,000 interactions (edges) among ~7,000 genes (nodes) with high confidence (p < 2.2E-19), where the connections represented ~0.01% of all possible edges.

When querying for selected genes, locally coherent sub-networks emerged that were predominantly related to metabolic functions and stress responses. Sub-networks for sulfate, phosphate, nitrogen, carbohydrate, tryptophan, cell wall metabolism, and the cold stress response were analyzed in detail. GGM recovered interactions with biological significance that escaped capture by Pearson correlation networks, while eliminating ambiguous interactions inherent in the latter. GGM displayed many known co-regulation pathways as sub-networks and added novel components to known edges. Finally, the network reconciled individual sub-networks in a topology joined at the whole genome level, and provided a general framework that can instruct future studies on plant metabolism and stress responses.

4C_04_S

Regulatory roles of AMP-activated protein kinases and PRL1-CDC5 spliceosome assembly complex in plant stress signalling

Csaba Koncz, Dóra Szakonyi, Gergely Molnár, Mihály Horváth, and Zsuzsa Koncz

(Corresponding author: koncz@mpiz-koeln.mpg.de )

AMP-activated kinases (AMPKs) control essential metabolic and signaling pathways in response to stress stimuli. Trimeric AMPKs, called Snf1-related kinases (SnRK1s) in Arabidopsis, are formed by combinatorial assembly of 3 catalytic alpha (AKIN10, 11 and 12), 3 substrate targeting beta (AKINß1, 2 and3) and one AMP-binding gamma (AKINß?/SNF4) subunits. SnRK1s undergo self-activation by autophosphorylation, their genes show different regulation, and the stability of subunits is controlled by proteasomal degradation. SnRK1? kinases are found in complex with the ?7 subunit of proteasome and SKP1 subunit of SCF ubiquitin ligases suggesting a role in ubiquitination-dependent protein degradation. Genetic dissection of SnRK1 signaling is hampered by the lack of akin10 and akin11 insertion mutations and deficient male transmission of snrk1? mutants. Inducible overexpression of SnRK1? AKIN10 results in hypersensitivity to the stress hormone abscisic acid (ABA). AKIN10 phosphorylates and thereby stabilizes the bZIP transcription factor ABI5, a key regulator of germination response to ABA. SnRK1? subunits interact with the nuclear WD-40 repeat protein PRL1 (Pleiotropic Regulator Locus 1) that functions of SnRK1-inhibitor in vitro. In addition to other defects, prl1 mutants display ABA hypersensitivity, and hyperphosphorylation and stabilization of ABI5. Stability of PRL1 is controlled by proteasomal degradation. PRL1 is found in complex with the Myb3R factor CDC5 that immunoprecipitates the proteasome, CULLIN1, and several unknown ubiquitinated proteins. PRL1 and CDC5 are conserved subunits of Ntc (nineteen-complex) spliceosome-activating complex. Inactivation of CDC5 results in prl1-like phenotype suggesting functional interdependence. Both prl1 and cdc5 mutations cause early flowering and changes in petal and stamen development. Steady-state mRNA level of FLC, a key repressor of flowering, is reduced and FLC pre-mRNA shows defective splicing in prl1. Floral defects of prl1 reflect aberrant splicing of pre-mRNAs of floral homeotic genes AP1, AP3, AG and PI. Alterations in the levels of microRNAs controlling ABA response, leaf development and flowering time in prl1 suggest implication of spliceosome activating complex and SnRK1s in the control of pre-mRNA splicing and biogenesis of small inhibitory RNAs.

4C_05_S

Characterization of biochemical properties of a rice dehydration inducible sucrose nonfermenting1 (SNF1)-related protein kinase 2 (SnRK2) family

Sun Joo Kim ¹, Myung Hee Nam², Dool Yi Kim1, Yeon Hee Lee1, Seok-Cheol Suh¹ and In-Sun Yoon¹*

* Corresponding author; isyoon@rda.go.kr

OSRK1 is a rice SnRK2 protein kinase activated by hyperosmotic stress and ABA. In the present study, we investigated regulatory mechanism and down stream targets of OSRK1. GST-fused recombinant OSRK1 showed strong substrate preference for rice bZIP transcription factors and uncommon cofactor requirement for Mn²⁺ over Mg²⁺. We observed positive relationship between autophosphorylation level and kinase activity. Moreover, OSRK1 could transphosphorylate itself. By deletion of C-terminus 73 amino acids or mutations of Ser-158 and Thr-159 to aspartic acids (Asp) in the activation loop, the activity of OSRK1 was dramatically decreased. Our data suggests that inter-molecular (auto)phosphorylation of catalytic domain of OSRK1 is important for enzyme activation. In an attempt to investigate OSRK1 signaling components, we identified two putative calcium binding proteins by yeast two hybrid screening. In vitro OSRK1 activity was stimulated by those CBPs. By in gel kinase assays, we also showed that ca. 52 kDa and 61 kDa protein kinase activities were highly stimulated in response to salt or ABA in roots of transgenic rice over-expressing OSRK1. They are likely to be downstream target kinases for OSRK1 signaling pathway. This work was supported by On-Site Cooperative Agriculture Research Project, RDA, Republic of Korea.

4C_06_S

DEAD-box helicases in plant stress tolerance

Narendra Tuteja

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi – 110 067, India. Email: narendra@icgeb.res.in, Tel.: +91-11-26189358; Fax: +91-11-26162316

Abiotic stress is an increasing threat in reducing agricultural productivity worldwide. Among abiotic stresses, the high salinity stress is most severe environmental stress, which impairs crop production on at least 20% of irrigated land worldwide. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. Helicases are one of the smallest molecular motors of biological system, which harness the chemical free energy of ATP hydrolysis to catalyze the opening of energetically stable duplex DNA (called DNA helicases) or unfold the secondary structures in RNA (called RNA helicases) and thereby are involved in almost all aspect of nucleic acid metabolism including replication, repair, recombination, transcription, translation, and ribosome biogenesis. Mostly all the helicases contain some conserved signature motifs including DEAD-box, which act as an engine to power DNA unwinding. In plants the role helicases in abiotic stress is just begening to emerge. Here we present the isolation of two pea DNA helicases (PDH45 and PDH47) and their role in salinity stress tolerance.

PDH45: It contains both the DNA and RNA unwinding activities and is homologous to translation initiation factor 4A (eIF4A). Antibodies against the PDH45 inhibit in vitro translation, confirming its role in translation initiation. The enzyme is localized in the nucleus and cytosol and unwinds DNA in the 3’ to 5’ direction. We found that PDH45 mRNA is induced in pea seedlings in response to high salt and its over-expression in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield. Measurement of Na⁺ in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants.

PDH47: It is also a DNA and RNA helicase both and is homologous to eIF4A. It is unique bipolar helicase that contains both the 3’ to 5’ and 5’ to 3’ directional helicase activities. The transcript of PDH47 was induced under salinity stress. ABA treatment did not alter its expression in shoot but induced its mRNA in root indicating the role of PDH47 in both the ABA-independent and-dependent pathways in abiotic stress. This is also localized in the nucleus and cytosol.

The discovery of salinity stress-induced helicases should make an important contribution to our better understanding of DNA and RNA metabolisms and stress signaling in plants. This study suggests a new pathway to engineer to restore crop yield in sub-optimal conditions. The role of some other new helicases in stress tolerance and the possible mechanism of salinity stress tolerance will be discussed.

4D_01_S

Will forest ecosystems continue to remove CO₂ from the atmosphere as climate changes?

P.G. Jarvis* and S. Linder

* margaretsjarvis@aol.com

In almost every forest we look today we find that there is a net removal of CO₂ from the atmosphere and carbon is accumulating in the soil and in the trees. A net gain of carbon by forest ecosystems implies a lack of balance between the processes removing CO₂ from the atmosphere and the processes returning CO₂ to the atmosphere. Ecosystem net primary production (NPP) is the difference between the gross photosynthetic production (GPP) and the losses of carbon resulting from the autotrophic respiration (R_A), i.e., NPP = GPP – R_A. The net ecosystem production (NEP) is the difference between the net primary production (NPP) and the heterotrophic respiration (R_H) associated with mineralisation of organic matter in the soil, i.e., NEP = NPP - R_H. For a forest at equilibrium, we would expect NEP to be zero (i.e., NPP = R_H ). Conversely, when we measure NEP > 0, NPP must exceed R_H. This world-wide disequilibrium in forests today is the reason why forests are a large global carbon sink, removing from the atmosphere close to 40% of CO₂ emissions. It is commonly assumed, and has been shown by some models, that as atmospheric [C0₂] and surface temperature increase in the future, this current disequilibrium will reverse (i.e., RH > NPP). A key question is whether this is likely?

Whilst including a carbon cycle in GCMs and other models has been a major step forward, a carbon cycle without an associated nitrogen cycle is unrealistic. Firstly, correlation across sites shows that NPP is stimulated by the concurrent deposition of atmospheric N. Secondly soil warming experiments show that N released by decomposition of litter and soil organic matter leads to increases in tree leaf area, uptake of CO₂ and tree growth. Thirdly, after an initial enhancement on warming, R_H settles down to the rate prior to the increase in temperature. For these reasons, recent projections by models linking the carbon and nitrogen cycles show NPP of forests increasing over the next 100 years in parallel with R_H, as atmospheric [CO₂] and surface temperature increase. Thus we may expect the ongoing removal of [CO₂] from the atmosphere by forests (NPP > RH) to be maintained.

4D_02_S

Stress to plants and the ecosystem under elevated CO₂

Hartmut K. Lichtenthaler

(Email: hartmut.lichtenthaler@bio.uka.de )

The original stress concept of Hans Selye has also been extended to plants and is today well defined to describe the action of unfavorable environmental constraints and stressors on plants as well as the plants’ response via stress avoiding and stress coping mechanisms including special short-term acclimations and long-term adaptations. Several examples of stress coping mechanisms are presented. Elevated CO₂ is a new challenge for plants and the ecosystem. It may not be a real stress, but it is a continuous strain. This requires particular acclimation and adaptation responses some of which are known. In fact, the plants make use of their general stress coping mechanisms either directly applied or in a modified form to avoid or compensate possible negative effects of elevated CO₂. One has to consider that on top of this all the classical abiotic, biotic and anthropogenic stressors are threatening plant growth and development also under elevated CO₂. Thus, the knowledge of the general stress coping and stress avoiding responses and tolerance mechanisms is needed to understand the regulation of the plants’ metabolism. Emission of volatile isoprenoids, such as isoprene or methylbutenol or the accumulation of mono- or diterpenes, are now understood as a possibility to regulate the internal cell metabolism and to protect the plants against photoinibition and photooxidation.

Measurements of chlorophyll fluorescence and particularly the imaging of chlorophyll fluorescence and the plants’ blue-green fluorescence provide excellent means to early detect and describe stress symptoms of plants and the ecosystem. Several examples are given.

4D_03_S

Two-face character of the elevated carbon dioxide impacts on the plant metabolism

Michal V. Marek, Otmar Urban

emarek@usbe.cas.cz

The rising atmospheric CO₂ concentration (EC) is an important ecophysiological topic. Because of the important CO₂ functions (effects on carboxylation, RUBISCO activation, stomatal response) on assimilation, it is possible to expect wide range of plants responses to EC. Thus, positive stimulation of photosynthesis or the assimilation depression - photosynthetic adjustment were observed and may be explained by (1) a decrease of RuBPCO amount and/or activity, (2) dilution/redistribution of nitrogen and phosphorus mineral status of assimilatory apparatus, (3) starch, (4) decrease of photosynthetically active pigments content and diminution of light-harvesting complexes, and (5) differences in the new sinks-source status of the plant. All mentioned responses were observed on the example of a dense spruce stand exposed for the long-term affected EC. The pronounced vertical profile of the photosynthetically active radiation led to the differentiation of the photosynthetic apparatus between the shaded (S) and sun needles (E). The prolonged exposure to EC was responsible for the apparent assimilatory activity stimulation observed mainly in deeply S needles. In E needles some signals on a manifestation of the acclimation depression of the assimilation were found. The long-term effect of EC was responsible for the decrease of nitrogen content. Moreover, the prolonged exposure to EC did not cause any stimulation of electron transport rate (ETR) for the E-needles but a strongly positive effect of EC on ETR was observed for the S-needles. The analysis shows that the depression of photosynthetic activity by long-term impact of EC is mainly caused by decreased RUBISCO carboxylation rate. All mentioned modifications to photosynthetic assimilation depend on time during the growing season. The strong depression of assimilation observed in the autumn months was the result of insufficient carbon sink capacity.

4D_04_S

Grassland ecology under elevated air CO₂ concentrations

Zoltán Tuba^1,2, Zoltán Nagy²

2Department of Botany and Plant Physiology, Faculty of Agricultural and Environmental Sciences, Szent István University, Gödöllő, H-2103 Gödöllő, Páter K. u. 1, Hungary, tuba.zoltan@mkk.szie.hu

Global climate change appears to be the greatest ecological problem of the future. For example the CO₂ emitted into the atmosphere will have a long-term effect and this is one of the most influential ecological factor at global scale. Its increasing concentration affects the plants directly, causing changes in their ecophysiological processes. Consequently, the tolerance, reproduction, distribution and abundance of plant species will be altered. Species composition and diversity of plant communities, the vegetation dynamics are all expected to be changed, too. Considering terrestrial ecosystems, grasslands are the second largest vegetation formation after the forests and their area is further increasing with the land use changes (clearance of the forests, urbanisation, extensive agricultural practices and abandonment of agricultural land). On a global scale grassllands cover 24% of the Earth's vegetated area. They occur over a broad range of climatic and soil conditions and vary from intensively managed sown pastures to natural grassland communities. Despite their importance, the potential effects of climate change on grasslands have received much less attention than effects on other ecosystems such as forests. The main objective of the proposed talk is to discuss the effects of elevated air CO₂ concenrations on the structure (botanical, species composition, floral diversity, canopy and below ground/root architecture) and function (CO₂,and H₂O exchange carbon cycling, dry matter production responses, etc.) of grasslands and to quantify the carbon storage in grassland ecosystems.

4D_05_S

How do plants survive extreme climatic events?

Ivan Nijs

University of Antwerp, Department of Biology, Universiteitsplein 1, B-2610 Wilrijk, Belgium, ivan.nijs@ua.ac.be

Climatic extremes, such as heat waves and extreme droughts, are predicted to become more frequent and severe in a future atmosphere with more greenhouse gases. However, untill now nearly all experimental studies on the impact of climate warming on plants have concentrated on changes in mean temperature, and studies on extremes are only beginning to emerge. Here I report on six recent field studies concerning the ecophysiological effects of heat waves, including three studies on temperate grassland and three on arctic tundra. In all experiments, the heat waves were generated with computer-controlled infrared irradiation in the field. In some cases we allowed natural precipitation, in others we excluded rainfall completely. We investigated the following questions: (1) is resistance to extremes in individuals plants coupled to specific plant traits? (2) does resistance vary with the complexity of the community? (3) do climatic extremes exert physiological after-effects long after the event has passed, e.g. on stomatal functioning, photosynthetic efficiency, chlorophyll synthesis/breakdown, etc.? (4) does tundra react differently than temperate grasslands? The main findings can be summarized as follows. In grassland species, morphological and ecophysiological indicators that best explained plant resistance to extreme drought, were different from known indicators of resistance to moderate stress. Plant survival in complex grassland communities was different from survival in monocultures, which can be explained by differences in resource use (water). In arctic communities, heat waves alleviated stress during exposure, but stress symptoms aggravated after the heat waves had ended. Because plant responses were species-specific in most communities, future shifts in species composition when current regimes of extreme events change, can be anticipated.

4D_05_6

Automatic plant leaf temperature monitoring for stress detection

Module 4 – Poster lectures:

4A_01_P

Cytochromes b561, new players in plant iron metabolism and oxidative stress metabolism

ASARD Han, BÉRCZI Alajos, VAN BEEK Kim, GRIESEN Daniel

han.asard@ua.ac.be

Cytochromes b561 (Cyt b561) are a newly identified class of trans-membrane proteins, using ascorbate as an electron donor. These proteins have been demonstrated to transfer electrons across the membrane in which they are embedded, but their physiological role remains unclear. We have identified four Cyt b561 isoforms (AtCytb1-4) in Arabidopsis and are characterizing their mechanism of action and using biochemical, molecular biological and physiological approaches. Several lines of evidence suggest that the plant Cyts b561 are involved in iron metabolism and in oxidative stress responses: 1) A knock-out in one of the four Cyts b561 identified from Arabidopsis demonstrates a particular phenotype under iron deficiency. 2) The recombinant AtCytb1 protein can be oxidized by iron chelates. 3) The AtCytb1 gene appears upregulated under iron-deficiency conditions. And 4) In vivo experiments with AtCytb1 expressed in yeast demonstrate its ferric-reductase capability. Recent experiments however also demonstrate that the AtCytb1 knock-out plants show a particular phenotype under oxidative stress conditions. Strongly reduced root development is observed in the mutant plants when treated with paraquat. These results suggest that Cyts b561 may provide a link between plant iron metabolism and oxidative stress phenomena, using ascorbate as the electron donor.

References: Bérczi et al. (2007) An Arabidopsis cytochrome b561 with trans-membrane ferrireductase capability. FEBS Lett. 581: 1505-08. Griesen et al. (2004) Localization of an ascorbate-reducible cytochrome b561 in the plant tonoplast. Plant Physiol. 134:726-34.

Acknowledgements: this work was supported by grants from the Belgian Fund for Scientific Research (FWO) and the Hungarian Scientific Research Fund (OTKA T-034488)

4A_02_P

Selenium-induced oxidative stress in coffee cell suspension cultures

Ricardo A. Azevedo¹, Priscila L. Gratao¹, Paulo Mazzafera², Peter J. Lea³, Rui A. Gomes-Junior⁴

1ESALQ, Universidade de Sao Paulo, Brazil; ²Universidade Estadual de Campinas, Brazil, ³University of Lancaster, U.K., ⁴Instituto Agronômico do Paraná, Brazil. E-mail: raazeved@esalq.usp.br.

Selenium (Se) is an essential element for humans and animals that is required for key antioxidant reactions, but can be toxic at high concentrations. We have investigated the effect of Se in the form of selenite on coffee cell suspension cultures over a 12 day period. The antioxidant defence systems were induced in coffee cells grown in the presence of 0.05 and 0.5 mM sodium selenite (Na₂SeO₃). Lipid peroxidation and alterations in antioxidant enzymes were the main responses observed, including a severe reduction in ascorbate peroxidase activity, even at 0.05 mM sodium selenite. Ten superoxide dismutase (SOD) isoenzymes were detected and the two major Mn-SOD isoenzymes (bands V and VI) responded more to 0.05 mM selenite. SOD band V exhibited a general decrease in activity after 12 h of treatment with 0.05 mM selenite, whereas band VI exhibited the opposite behavior and increased in activity. An extra isoenzyme of glutathione reductase (GR) was induced in the presence of selenite, which confirmed our previous reports obtained with Cd (Gomes-Junior et al.: Chemosphere 65, 1330-1337, 2006) and Ni (Gomes-Junior et al.: Plant Physiology and Biochemistry 44, 420-429, 2006) indicating that this GR isoenzyme may have the potential to be a marker for oxidative stress in coffee. Funding by FAPESP and CNPq, Brazil.

4A_03_P

Response of Brazilian cultivars of soybean (Glycine max) expose to ozone under controlled conditions

Patrícia Bulbovas^a*, Silvia Ribeiro de Souza^b, Marisa Domingos^b and Ricardo Antunes Azevedo^a

* e-mail: patibul@usp.br

Ozone (O₃) can enter the leaves and react with water forming reactive oxygen species (ROS). ROS is able to cause physiological and biochemical damage to plants. This study was carried out to investigate the O₃ effects on two distinct Brazilian cultivars of soybean (Glycine max): ‘Tracajá’ and ‘Sambaíba’. Plants were grown in two chambers, one with filtered air (FA) and the other with filtered air plus 40 ppb of ozone (FA+O₃) for five days. Components of the antioxidative defense system such as ascorbic acid (AA), glutathione reductase (GR), ascorbate peroxidase (APX), guayacol peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) were analyzed and the relative growth rate (RGR) and biomass production were also determined. APX and GPX in ‘Sambaíba’ and AA and GPX in ‘Tracajá’ decreased under FA+O₃. AA and CAT in ‘Sambaíba’ and CAT in ‘Tracajá’ exhibited similar levels under both treatments. GR in ‘Sambaíba’ and GR and APX increased in ‘Tracajá’ exposed to FA+O₃. CAT and SOD activity staining by non-denaturing PAGE revealed the same isoforms numbers for both cultivars, but different isoforms of GR. ‘Tracajá’exhibited variations of some CAT and SOD isoforms in FA+O₃. The root/shoot ratio in ‘Tracajá’ and root/shoot ratio, leaf RGR and root biomass production in ‘Sambaíba’ were lower under FA+O₃. O₃ treatment induced distinct antioxidative responses by the distinct antioxidant systems in response to O₃ (AF+O₃). The antioxidant defense system variations compensated the lower O₃ interference on growth parameters and biomass production.

4A_04_P

Glutathione half-cell reduction potential: a universal stress marker from plants to humans?

Ilse Kranner, Simona Birtić and Hugh W. Pritchard

E-mail address of corresponding author: i.kranner@kew.org

Ageing phenomena and programmed cell death (PCD) are typically studied in human or animal cells rather than in plants. However, the seeds of higher plants represent excellent models for the study of ageing because viability loss can be easily induced experimentally. ‘Orthodox’ seeds are desiccation tolerant and ‘recalcitrant’ seeds are desiccation sensitive. Lethal damage, as judged by germination tests, can be induced by artificial ageing in orthodox, and by drying in recalcitrant seeds. We have investigated mechanisms of viability loss to find a reliable marker that quantifies ‘stress’. Oxidative damage has previously been correlated with degenerative processes and death, but how exactly this contributes to viability loss is unknown. We show in four species subjected to ageing or desiccation, that seed viability decreased by 50% when the half-cell reduction potential of glutathione (E_GSSG/2GSH), a major cellular antioxidant and redox buffer, increased to a zone of -180 to -160 mV, in agreement with a model that has been suggested for human cells (1). In a meta-analysis of data representative of 13 plant and fungal orders we show that stress generally becomes lethal when E_GSSG/2GSH exceeds -160 mV. We put forward that this change in E_GSSG/2GSH is one of the ‘death triggers’ that initiate PCD, finally causing inter-nucleosomal DNA fragmentation in the final, or execution phase, of PCD. E_GSSG/2GSH is therefore a universal marker of plant cell viability and allows us to predict whether a seed will live, germinate and produce a new plant, or if it will die.

1. Schafer, F.Q. & Buettner, G.R. (2001) Free Radical Biol. Med. 30, 1191-1212.

4A_05_P

Expression analysis of phenylalanine ammonia lyase in relation to ozone stress in yellow poplar plants (Liriodendron tulipifera)

A. Francini, E. Pellegrini, G. Lorenzini, C. Nali*

Department of Tree Science, Entomology and Plant Pathology “G. Scaramuzzi”, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy. *cristina.nali@agr.unipi.it 

Tropospheric ozone is a widespread phytotoxic pollutant that causes significant damage to plants, but relatively little is known about ozone stress in urban trees. The phenylpropanoid pathway is one of the most important pathways for the synthesis of natural products in plants such as phenols and lignin. In this study, the hypothesis that phenylalanine ammonia lyase (PAL) may be affected by ozone treatment (120 ppb, 5 h d^-1 for 45 consecutive days) in Liriodendron tulipifera (yellow poplar) plants was examined. At the end of exposure, the material showed characteristic symptoms of injury in the form of severe minute roundish dark-blackish necrosis localised in the interveinal areas of the adaxial surface of leaves. Treated plants increase PAL activity after ozone fumigation (+43%) and total phenols (+41%) compared to controls. Total leaf RNA protocol was optimised for yellow poplar. Degenerate primers were designed on highly conserved sequences available on GenBank, that encode for this enzyme. The primers yielded 679 bp cDNA fragments. Sequences obtained were translated in aminoacids and compared in the GenBank. Blast results showed high homology at aminoacids level with PAL, confirming that the cDNA fragments isolated effectively encode for this gene. In particular, the sequences showed high homologies with Ulmus pumila (93%) and with Pyrus comunis (96%). The northern analysis of PAL gene expression lead to support the view of ozone as an abiotic elicitor of defence responses in yellow poplar.

4A_06_P

Influence of antioxidants on some stages of oxidative stress in plant cells of wheat and barley seedlings

N. Shkute, M. Savicka, N. Zatinajchenko, N. Rjabovola

Institute of research and management of biological resources, Daugavpils University, Vienibas iela 13, Daugavpils, LV-5401, Latvia

The production of superoxide is crucial for normal morphogenesis at early stages and is causal of apoptosis and senescence at late stages of development of etiolated cereals seedlings (wheat, barley and others). It was shown, that cyclic change of rate of superoxide production is correlate with changes of nuclear DNA synthesis and apoptotic fragmentation in cells of some development and senescence separated organs (leaf, coleoptile) of this plants. It was found, that antioxidant defence systems: superoxide dismutase and peroxidase are activated in apoptotic cells of senescence organs. The influence of endogenic antioxidant (ascorbic acid) and synthetic antioxidant (? - Ionol) on superoxide production, on activation of antioxidant defence systems, on nuclear DNA synthesis and nuclear DNA apoptotic fragmentation and on structure of cellular organelles was investigated. It was found, that cyclosporin A, inhibitor of the mammalian permeability transition pore, inhibits of peroxide formation and nuclear DNA apoptotic fragmentation. Taking this date mitochondria are likely to be involved in plant programmed cell death, induced by oxidative stress, but the molecular mechanisms may be different from those found in animals.

4A_07_P

Seasonal variation in the activity of antioxidant enzymes peroxidase, superoxide dismutase and catalase in an open and a shaded population of Iris pumilas

Branka Tucić*, Ana Vuleta and Darka Šešlija

Department of Evolurionary Biology, Institute for Biological Research “Siniša Stanković“, Belgrade, Serbia, *btucic@ibiss.bg.ac.yu

Seasonal variation in the activity of peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), and catalase (CAT, EC 1.11.1.6) was determined in the leaves of two Iris pumila populations, one naturally growing at an open dune site and the other in the understory of a Pinus stand. A repeated-measures profile analysis revealed that the average level, as well as the mean change in the activity of these enzymes varied significantly between contrasting light habitats. POD activity was significantly greater at exposed dune site than under vegetation canopy, and reached its maximum in the summer but only in plants experiencing full sunlight. Conversely, in woodland understory, POD activity gradually increased from spring to autumn. The mean activity of SOD and CAT was consistently greater in plants inhabiting vegetation shade compared to those exposed to full irradiance. Throughout the growing season, the variation pattern of CAT activity was the same in both light habitats, while the response curve of SOD activity changed the shape with environmental conditions, particularly in the period from summer to autumn. At open dune site in the spring, the activity of POD appeared to be inversely related to SOD, suggesting that plants with lower SOD production up-regulate their POD level to compensate for SOD reduction under given environmental setup. The observed results imply that abiotic stress can disrupt the redox homeostasis in I. pumila plants, changing the balance between POD, SOD and CAT activities according to the extent of oxidative stress in cells.

4A_08_P

Battle against reactive compounds-AKRs and stress

Zoltán Turóczy, Mátyás Cserháti, Petra Kis, Dénes Dudits, Gábor V. Horváth

Institute of Plant Biology, Biological Research Center HAS, Temesvári krt. 62, H-6726 Szeged, Hungary, E-mail: turoczy@brc.hu

Plants exposed to abiotic stress are subjected to oxidative damage. Reactive compounds produced under such conditions significantly increase the cytotoxic effect of environmental stress factors. Aldo/keto reductases (AKRs) have been considered as effective enzymes for the detoxification of lipid peroxidation- and glycolysis-derived reactive aldehydes. The promoters of targeted rice AKR genes were screened for stress-related transcription factor binding sites (TFBS’s) by a new, enumeration based method algorithm. QRT-PCR was used to determine the transcript profile for the selected genes in abscisic acid (ABA), H₂O₂, NaCl and mannitol treated rice cell suspensions. Among the genes, OsALR1 showed the highest inducibility and transcript level during the treatments, suggesting its important role in stress tolerance. Furthermore the Q-PCR experiments substantiated, that the number of motifs found in the promoters and the stress response of the genes were in close correlation. In addition, the photosynthetic parameters of the tobacco lines, overexpressing rice OsALR1 and OsALR4 were better then those of the wild type plants after paraquat and methylglyoxal (MG) treatments. The in vitro enzyme kinetic constants of the GST-OsALR1 fusion protein revealed a high reducing activity for toxic aldehydes like MG in the presence of NADPH. Results of in vivo assays in E. coli for protection against MG toxicity have also made it clear, that one important function of these AKR proteins is the elimination of reactive aldehydes from plant cells expletively besides the glyoxalase system. This work was supported by NKFP Grant No. 4-064-2004, Gábor V. Horváth is grateful for the support of the “János Bólyai” Research Fellowship.

4A_09_P

Influence of Cadmium on the Antioxidant System of Tobacco Plants

Cardoso, A.I.⁽¹⁾, Mourato, M.P. ^*(1), Martins, L.L. ⁽¹⁾, Pinto, A.P. ⁽²⁾, Mota, A.M. ⁽³⁾, Gonçalves, M.L. ⁽³⁾, Varennes, A. ⁽¹⁾

* Corresponding author: mmourato@isa.utl.pt

In this work we studied the effect of cadmium (0, 0.010, 0.025, 0.050, 0.100 mM) on enzymatic and non-enzymatic parameters of tobacco plants grown in hydroponic culture, in order to get a further insight in the detoxification processes and cadmium absorption mechanisms of that plant. Tobacco has been chosen since it has been referred as a cadmium accumulator plant. Leaf dry weight percentage increased with cadmium concentration indicating lower water absorption possibly due to affected root development. Chlorophyll a and b levels decreased markedly (down to 16 % of the control value for the highest cadmium concentration of 0.100 mM) showing that the photosynthetic system was affected by cadmium. Hydrogen peroxide measurements showed no significant variations, which could indicate that the antioxidant system of this plant is capable of quenching the possible excess of hydrogen peroxide induced by cadmium. In fact, both guaiacol peroxidase and ascorbate peroxidase activities were enhanced with increasing cadmium concentration in nutrient solution. Another enzyme usually involved in the antioxidant system of plants, superoxide dismutase, showed no difference in its activity levels compared to the control. A slight increase on malondialdehyde levels, both in leaves and roots, indicated that lipid peroxidation should occur as a result of increased ROS formation induced by cadmium toxicity.

Investigation of arsenate phytotoxicity in cucumber plants

V. Czech¹, E. Cseh¹, F. Fodor¹, P. Czövek¹, J. Fodor²

2Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest, P.O. Box 102

The effect of arsenate on the stress-sensitive vegetable plant, cucumber (Cucumis sativus cv. Joker F1) grown in hydroponic culture (modified Hoagland solution, 10?M KH₂PO₄, 10?M FeCl₃ and 10?M KH₂AsO₄) was investigated.

The presence of arsenic in soils and drinking water and its consequent appearance in the food chain has a potential health damaging effect. The arsenic load of the Hungarian population in the various parts of the country is substantially different, it exceeds 50 µg/l in the Southern part of the Great Plain. The arsenic allowance of drinking water in the European Union is 10 µg/l.

As(V) treatment influences the water household of cucumber depending on its stage of development. When the first leaf is developing at the time of treatment turgor loss occurs first in the roots then in the shoot. As(V) inhibits transpiration. This effect is significantly reduced by 100?M phosphate because As(V) is taken up through the phosphate transport system. Arsenate increases the water saturation deficit of the leaf and reduces the exudation rate of the root to 25% of the control. As(V) also significantly reduces the dry matter of plant parts and causes an ion leakage in the roots leading up to 50% higher conductivity in the washing solution. These findings imply that the turgor loss is caused by the loss of membrane semipermeability.

As(V) is reduced to As (III) in the roots that may lead to the formation of reactive oxygen species. Lipid peroxidation occurs in the hypocotyls in which the malondialdehyde concentration increases due to the oxidative stress. Cucumbers grown on Fe-ascorbate (10?M FeCl₃ + 30?M ascorbate) had healthy hypocotyls in spite of the As(V) treatment so that Fe-ascorbate prevented lipid peroxidation. Further measurements on the stress indicator H₂O₂ level and the redox processes of ascorbate in the hypocotyls are discussed.

4B_01_P

Stress reactions occurring during bird cherry infestation by bird cherry-oat aphid

Hubert Sytykiewicz, Iwona Łukasik, Bogumił Leszczyński

University of Podlasie, Department of Biochemistry and Molecular Biology, B. Prusa 12 St.,
08-110 Siedlce, Poland, e-mail: leszczb@ap.siedlce.pl

Quite often biochemical interactions between host-plants and herbivorous insect pests are related to stress reactions within infested plant tissues and feeding herbivores. In the present paper we report on such dual stress reactions within tissues of the bird cherry-oat aphid (Rhopalosiphum padi L.) fed on the bird cherry (Prunus padus L.).The carried out experiments showed that the aphid infestation had elucidated an increase in activity of leucylaminopeptidase and other proteinases isolated at pH 5 and pH 7 from the bird cherry leaves. It is important since these enzymes are involved in induction of the plant defensive mechanisms towards various pathogens. It has been recognized that the one of the earliest plant responses to herbivores involve generation of the reactive oxygen species and induction of oxidative stress within insect tissues. It appears to be real for the studied aphid species since depletion of total thiol groups and increase in lipid peroxidation was demonstrated within the bird cherry-oat aphid tissues exposured to plant phenolics. However, R. padi showed an ability to neutralize the free oxygen radicals with help of enzymatic and non-enzymatic antioxidants. For example, an induction of the aphid superoxide dismutase and catalase, neutralizing toxic oxygen forms was found during the spring migration period, when the highest level of the proteinases activity within the bird cherry leaves was noted. The role of the stress reactions in biochemical interactions between the bird cherry and the bird cherry-oat aphid is discussed.

Proteomic analysis of Leaf Rust-infected wheat

V Pós¹, K Manninger², K Halász¹, É Hunyadi-Gulyás³, E Szájli³, M Cserháti⁴, K Medzihradszky³, J Györgyei⁴, N Lukács¹

1Dept. of Plant Physiology and Biochemistry, CUB, Budapest, ²Plant Protection Inst., Budapest, ³BRC Proteomics Res. Group, Szeged, ⁴BRC Inst. of Plant Biol., Szeged, e-mail: noemi.lukacs@uni-corvinus.hu

The extracellular matrix plays an important role in plant defense against biotic stress. Presuming that the genetic background of near-isogenic wheat lines differing in their resistance gene(s) has an influence on the protein composition of extracellular matrix, we analyzed their intercellular washing fluid (ICF) to identify fungal infection-associated changes in the protein pattern and to detect secreted proteins which may contribute to resistance reaction. Seedlings of the susceptible ’Thatcher’ and the corresponding near-isogenic Lr1 and Lr9 wheat lines resistant to the pathotype 43722 of leaf rust (P. recondita fsp. tricii) were inoculated and apoplastic fluid was collected over a week p.i. Proteins were separated on 1- and 2D PAGE and identified by MALDI-TOF and LC-MS-MS. In the Lr1-resistant line three apoplastic proteins, a glucan-endo-1,3-beta-D-glycosidase (35413 Da, pI 8.8), a chitinase 1 enzyme (27077 Da, pI 8.7) and a pathogenesis-related protein, PR1.1 (17651 Da, pI 8.7) were induced earlier and reached higher concentration than in the Thatcher line. In addition, the ICF of both Lr1 and Lr9 showed much higher 1,3-endoglucanase activity and different induction kinetics as that of the ‘Thatcher’ line. Since it is well known that these proteins play an important role in the antifungal defence of resistant as well as of sensitive plants, their differential expression might be caused by differences in gene regulation. Results of the promoter analysis of the corresponding homologous genes in rice will be shown to point out potential common motifs in the promoter region of the most homologous proteins.

4B_03_P

Role of maize phenolics in the genotypic resistance to Gibberella Stalk Rot (Fusarium graminearum Schwabe)

Rogelio Santiago, Lana M. Reid, John T. Arnason, German Sandoya, and Rosa A. Malvar

Email: rsantiago@mbg.cesga.es

Six maize inbred lines known to represent a wide spectrum of susceptibility to Gibberella stalk rot were investigated for a relationship between their phenolic contents in the pith and their resistance to Gibberella stalk rot. The phenolic acid profiles were evaluated from silking to grain maturity. Four different fractions of phenolic compounds were extracted from inoculated and non-inoculated (control) pith tissues: insoluble cell wall-bound phenolics, free phenolics, soluble ester-bound phenolics, and soluble glycoside-bound phenolics. Analysis by HPLC revealed that p-coumaric and ferulic acid were the most abundant compounds in the soluble and cell wall bound fractions. The quantity of free, glycoside-bound and ester-bound phenolics in the pith was lower than the level required for inhibition to of Fusarium growth and/or mycotoxins production; however, significant negative correlations between diferulic acids contents in the cell walls and disease severity rating four days after inoculation were found. According to these results previous studies showed significant negative correlations between disease severity and diferulic acid contents in the maize grain. Special attention should be therefore given to levels of diferulic acids during the early infection process. Diferulates may play a role in genotypic resistance of maize to Gibberella stalk rot as barriers preformed prior to infection.

4C_01_P

Induction of lectin expression in rice as a reponse to stress treatments

Elke Fouquaert, Godelieve Gheysen and Els J.M. Van Damme

Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium, Elke.Fouquaert@Ugent.be

Numerous wild and cultivated plant species contain carbohydrate-binding proteins called lectins. Most plant lectins are involved in the recognition and binding of glycans from foreign organisms, and accordingly are believed to play a role in plant defense. However, in recent years evidence has accumulated that plants synthesize well-defined carbohydrate binding proteins upon exposure to stresses like drought, high salt, wounding, treatment with some plant hormones or pathogen attack.

As early as 1990 Claes et al. (1) demonstrated that a protein called SalT is specifically induced when rice plants are subjected to salt stress. Later SalT was identified as a dimeric mannose-binding jacalin-related lectin composed of 15 kDa subunits (2). More recently we have identified two more lectins in rice, belonging to the class of OSR40 proteins and a Galanthus nivalis-related lectin, respectively. Expression of the OSR40 proteins is induced by salt stress or ABA treatment (3).

Localization studies of the rice lectins demonstrated that they are exclusively located in the cytoplasm and the nucleus. The occurrence of the genes encoding the three different lectins in the rice genome was studied by screening different rice species and cultivars with a different genetic background. Lectin induction in response to different stress conditions was also analyzed.

Since these lectins are synthesized only as a response to specific physical, chemical and biotic stress factors, it can be assumed that they play a specific physiological role in the plant.

(1) Claes et al., 1991. Plant Cell 2, 19-27.

(2) Zhang et al., 2000. Planta 210, 970-978.

(3) Moons et al., 1997. Planta 202, 443-454.

4C_02_P

Effect of low-concentration stressors in barley seedlings

Erika Kovács^1,2, Péter Nyitrai¹, Pálma Czövek¹, Mihály Óvári³, Áron Keresztes²

k-erika@elte.hu

The effects of some chemical stressors in high concentration have been thoroughly investigated in plants, but information about the same stressors in low concentration is very limited. In our experiments the effects of low-concentration Cd²⁺ (5x10^-8 M) and a herbicide, DCMU (10^-7 M) were investigated in barley seedlings. Chlorophyll accumulation indicated a stimulative effect of both stressors in treated plants. Our aim was to reveal the mechanism of this stimulation. The amount of active cytokinins increased in treated roots (prior to their transport to the leaves). This seems to be the key event in stimulation. In order to identify the signaling pathway involved in the increased cytokinin synthesis of roots, the PIP₂-IP₃/DAG and MAPK pathways were tested with different inhibitors added together with the stressors to the nutrient solution. It seems that the signal is transmitted through DAG and protein kinase C to the MAPK pathway. It is known that higher amounts of Cd²⁺ or DCMU cause oxidative stress in plants, upon which the activity of antioxidant enzymes (e.g. SOD) increases, and malonyl dialdehyde (MDA) might be finally produced by peroxidation of membrane lipids. After application of Cd²⁺ and DCMU, the amounts of SOD and MDA were measured to ascertain whether also low-concentration stressors may cause oxidative stress. We found a slight transient increase in the amount of SOD in roots.

This work was supported by grant T-047243 from OTKA.

4C_03_P

Calcium sensors are regulated by different stress signals

Ana Lima, Edgar Cruz e Silva, Etelvina Figueira

agusmaolima@gmail.com

In order to adapt to environmental stresses, plants use diverse signalling strategies. Calcium functions as a versatile messenger in mediating responses to biotic/abiotic stress signals and a variety of developmental cues in plants. The signal-specific Ca signature is readily decoded by an array of Ca-binding proteins or Ca sensor. However, little is known about the specificity of calcium signatures and how the signal is decoded. Several families of Ca²⁺ sensors have been identified in higher plants, such as calmodulin (CaM) and CBL (calcineurin B-like) proteins. Recent investigations have demonstrated that this protein variety may be are related to the specificity of stress signal perception and to the activation of the corresponding stress tolerance mechanisms. Therefore, the knowledge of which calcium sensors are triggered by specific stress signals is of major importance. Calcium-binding proteins were isolated from Vitis vinifera cells, after exposure to several stresses, with the aim to understand the specificity and regulation of each individual stress. Vitis vinifera cell cultures were exposed to salt, heavy metal, heat and cold stresses. Calcium-binding proteins were purified by hydrophobic interaction chromatography. Results demonstrated that exposure to different stresses induced alterations in the proteins eluted. Protein profiles were separated by SDS-PAGE and Native PAGE in the presence and absence of calcium. The eluted proteins comprised not only calcium binding proteins, but also their targets, since only few proteins presented calcium-induced alterations in their conformations.Results provide new insights in stress signal transduction and corroborate the importance of calcium signaling in plant stress responses.

4C_04_P

Adaptation mechanisms in Rice (Oryza sativa L.) under salt stress

Md A. Kader and S. Lindberg

Department of Botany, Stockholm university, 106 91 Stockholm, Sweden. E-mail: Sylvia.Lindberg@botan.su.se

The project focuses on two important aspects of Na⁺ toxicity in salt-tolerant rice cv. Pokkali and salt-sensitive cv. BRRI Dhan29, namely i) how Na⁺ stress induces a change in cytosolic Ca²⁺, [Ca²⁺]_cyt, and pH, [pH]_cyt, and ii) how cells could maintain a low cytosolic Na⁺ and/or Na⁺/K⁺ ratio. The salt-induced changes in [Ca²⁺]_cyt and [pH]_cyt and their sources were monitored in single rice protoplasts by fluorescence microscopy. The expression of the transporter genes OsHKT1, OsHKT2 and OsVHA, which are thought to play a significant role in maintaining correct cytosolic Na⁺ and or Na⁺/K⁺ ratio, were examined in both rice cultivars under salt stress condition by real time RT-PCR and in situ PCR. The results show that Na⁺ must be sensed inside the cytosol, before any changes in [Ca²⁺]_cyt and [pH]_cyt occur. Sensing of Na⁺ induced different changes in [Ca²⁺]_cyt and [pH]_cyt in the two rice cultivars with different sources for the changes. The [pH]_cyt changes were coupled to different H⁺-ATPases in the two cultivars. The expression analysis of OsHKT1, OsHKT2 and OsVHA showed variable and cell- specific induction in these cultivars under salt stress condition. The important mechanism for salt tolerance in cv. Pokkali was to keep cytosolic Na⁺ at a low level, by reducing Na⁺-influx (through down-regulation of OsHKT1) and compartmentalizing cytosolic Na⁺ into the vacuole (through the induction of vacuolar H⁺ATPase OsVHA, an energizer for the tonoplast Na⁺/H⁺ antiporter). Pokkali might also induce increased uptake of K⁺ through the induction of OsHKT2, as evident in this study. Vacuolar compartmentalization of Na⁺ is also present in salt-sensitive cv. BRRI Dhan29, but to a lesser extent and much later than in cv. Pokkali. The results suggest that the signaling and subsequent adaptive responses in the salt-tolerant rice cv. Pokkali are different from that in the salt-sensitive cv. BRRI Dhan29.

4C_06_P

Alternative electron donors substitute the inactivated oxygen-evolving complex in different plant species

Szilvia Z. Tóth, Jos Thomas Puthur and Győző Garab

E-mail: Sztoth@brc.hu, jtputhur@rediffmail.com

Electron transport processes were investigated in different plant species in vivo under the conditions in which the oxygen-evolution was fully inhibited by a heat pulse (48-51 °C, 40 s in water bath). This treatment induces no visible symptoms and the plants recover in 1-2 days. Under these conditions, the K peak (~F_{400 µs}) appears in the fast chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Additional fluorescence increase occurs in the 0.2-2 s time range of the fluorescence kinetics indicating additional Q_A^- accumulation. Recently we have shown that this Q_A^- accumulation was due to naturally occurring alternative electron donors at the donor side of photosystem II. The donation probably originates from ascorbate, which provides electrons with a halftime of ~30 ms in barley (Tóth et al. 2007, Biochim Biophys Acta 1767: 295-305). In this study, we investigated several other plant species, including Pisum sativum, Arabidopsis thaliana, Agropyron elongatum, Marchantia polymorpha, as well as the cyanobacterium Synechocystis PCC 6803 and the green alga Chlamydomonas reinhardtii. We established the functioning of the alternative electron donors in all the investigated species. Preliminary data indicate that the inactivation temperatures of the oxygen-evolving complex and the halftimes of electron donation may vary from species to species, and may also depend on the growth conditions. We will discuss the physiological significance of this alternative electron donation to photosystem II in light and heat stress.

4C_07_P

Molecular and physiological impact of cold stress on Coffea sp.

EICHLER, P.^(1,2,*), FORTUNATO, A.^(1,3), GOUVEIA, M.M.⁽⁴⁾, PARTELLI, F.⁽⁵⁾, CHAVES, M.M.^(2,3), RAMALHO, J.C.^(1,6), RIBEIRO, A.^(1,6)

⁽¹⁾ Centro de Ecofisiologia, Bioquímica e Biotecnologia Vegetal / Instituto de Investigaçao Científica Tropical, Quinta do Marques, 2784-505 Oeiras, Portugal
⁽²⁾ Inst. Sup. Agronomia / UTL, Tapada da Ajuda, 1349-017 Lisboa, Portugal
⁽³⁾ Inst. Tecnol. Química Biológica / UNL, Quinta do Marqu?s, 2781-901, Oeiras, Portugal
⁽⁴⁾ Dept. Biologia and Centro de Estudos da Macaronésia,, Univ. Madeira, Campus da Penteada, 9000-390 Funchal, Portugal
⁽⁵⁾ Universidade Estaual do Norte Fluminense Darcy Ribeiro, CCTA/LFIT, Campos dos Goyatacazes, RJ, Brazil
⁽⁶⁾ Unidade de Biotecnologia Ambiental / FCT / UNL, 2829-516 Monte de Caparica, Portugal.

* E-mail: p.eichler@clix.pt

Among different abiotic stresses, low positive temperature (chilling) limits plant metabolism and is often associated to yield reductions, particularly in tropical plants, such as Coffea sp. In order to contribute to the understanding of the molecular and physiological aspects of coffee tolerance to cold we are currently analysing the expression of genes related to the oxidative stress response and to photosynthesis by semi-quantitative and real-time RT-PCR, using photosynthesis as an integrative metabolism and a sensor to acclimation ability. This study involves a multidisciplinary analysis of the impacts of chilling exposure in some coffee genotypes with different cold tolerance potential, submitted to an acclimation period, to three chilling cycles (4^oC) and to a rewarming period. Results of gene expression are in accordance with physiological and biochemical data previously obtained by our group (Campos et al., 2003; Ramalho et al., 2003) and suggest that the transcriptional activity of the genes under study changes according to the degree of cold tolerance/susceptibility of coffee genotypes.

Campos et al., (2003). J. Plant Physiol. 160: 283-292.
Ramalho et al., (2003). Plant Biol. 5: 631-641.

4E_01_P

Structure-Function Studies of tomato ASR1, a Water Stress- and Salt Stress-Regulated Plant-Specific Protein

Dudy Bar-Zvi¹, Zvia Konrad¹, Slava Rom¹, Doron Shkolnik¹, Natalia Shadrin², Rodolfo Ghirlando³, and Yehuda Goldgur²

Departments of Life Sciences¹, and Chemistry², Ben-Gurion University, Beer-Sheva 84105, Israel. ³Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA. E-mail of corresponding author: barzvi@bgu.ac.il

ASR1 (abscisic acid stress ripening) is a low molecular weight plant specific protein encoded by an abiotic-stress regulated gene. The ASR1 protein possesses a zinc-dependent DNA-binding activity. The DNA binding site was suggested to reside in the central part of the polypeptide by using truncated forms of the protein. Two additional zinc-binding sites were shown to be localized at the amino terminus of the polypeptide. ASR1 protein is presumed to be a natively unfolded protein using a number of prediction algorithms. The degree of order of ASR1 was determined experimentally using non-tagged recombinant protein expressed in E. coli and purified to homogeneity. Purified ASR1 was shown to be unfolded using dynamic light scattering, gel filtration, microcalorimetry, circular dichroism and Fourier transform infrared (FTIR) spectrometry. The protein was shown to be monomeric by analytical ultracentrifugation. Addition of zinc ions resulted in a global change in the ASR1 structure, from monomer to homodimer. Upon binding of zinc ions, the protein becomes ordered as shown by FTIR and microcalorimetry, concomitant with dimerization. Cytosolic ASR1 is unfolded whereas nuclear ASR1 is ordered. The effect of zinc binding on ASR1 folding and dimerization, and activity of cytosolic and nuclear ASR1 protein in is discussed.

H-1117, Budapest Pázmány sétány 1/C Hungary, E-mail: ikiraly@ludens.elte.hu

2Research Institute for Fruitgrowing and Ornamentals H-1223 Budapest, Park u. 2.

Biocontrol is an environmentally sound method used for the prevention and control of plant diseases. Advisedly selected fungal and bacterial strains are marketed worldwide promoting to spread of this inexpensive and effective technology. The effect of biocontrol species is composed of (1) direct interaction with potential plant pathogens, (2) eliciting host-mediated defense reactions, (3) other beneficial effects as enhanced root growth. Trichoderma harzianum is a soil fungi living in the close vicinity of plant roots. As a hyper parasite it kills many pathogenic fungi generally by hydrolyzing mycelia walls. The T22 T. harzianum strain has also a strong chitinase activity and many beneficial effects on the host plant, including enhanced root growth. We used the commercially available T22 strain to defend fruit trees against drought stress profiting its effect on the plant defense reactions. The changes of the superoxide dismutase activity and the raise of malondialdehyde level as a consequence of far gone lipid peroxidation were monitored during the stress treatment. The physiological state of the control and stressed fruit trees was characterized also by the chlorophyll content, the fresh weight-dry weight ratio and the stomatal conductancy. The results obtained clearly demonstrate that T22 treated fruit trees resisted well even the vigorous drought stress applied. The enhanced resistance correlated with the changing of the activity of the reactive oxygen scavenging system. This work was supported by GVOP-3.1.1-2004-05-0061/3.0.

pigsy17@freemail.hu, tari@bio.u-szeged.hu

Imposition of salt stress reduced the net CO₂ assimilation rate, chlorophyll (Chl) and carotenoid contents, stomatal conductance and biomass production of tomato (Lycopersicon esculentum Mill. L. cv. Rio Fuego). Pre-treatments of plants with 10^-4 M, but not with 10^-7 M salicylic acid (SA), could partially restore the CO₂ fixation rate, Chl a/b ratio, carotenoid levels under 100 mM NaCl exposure. Accumulation of soluble sugars, a biochemical marker of salinity tolerance of tomato, could be detected in pre-treated plants. After SA application, concentrations of hexoses (glucose and fructose) remained high in the leaves and that of sucrose in the roots during salt stress. Both SA and salt stress increased the H₂O₂ production of tissues. Activities of superoxide dismutase and catalase (CAT), a H₂O₂ generating and scavenging enzymes, respectively, decreased significantly under salt stress, but in 10^-4 M SA pre-treated plants, CAT activity was significantly induced both in the root and shoot tissues. The improved photosynthetic performance, the accumulation of soluble sugars as compatible osmolytes and the effective elimination of H₂O₂ by CAT contributed to the successful acclimation of 10^-4 M SA pre-treated tomato plants to high salinity. – This work was supported by OTKA T038392.

guotha@bio.u-szeged.hu

We investigated the effect of water deficit on the photosynthetic parameters and the abscisic acid (ABA) levels in wheat. A comparison was made between the changes of the parameters mentioned above, in seedling stage under osmotic stress and in reproductive growth phase under soil drought in two Hungarian wheat cultivars Triticum aestivum L. cv. MV Emese (resistant) and GK Élet (sensitive). The water status parameters, CO₂ assimilation, chlorophyll a (chla) fluorescence, pigment content and ABA levels were determined as a function of the development of water deficit. The tolerant genotype cv. Emese maintained higher water potential in upper leaves under osmotic or drought stress than the sensitive cv. Élet. In spite of this, the tolerant line exhibited an earlier ABA accumulation in the grains (DPA 9) than the sensitive line (DPA 24). The characteristics of the CO₂ assimilation and chla fluorescence parameters measured in flag leaf (Yield, qP, NPQ) did not differ between the two varieties and compared to the control, the Yield changed similarly to the CO₂ fixation. But in seedling stage under osmotic stress the CO₂ assimilation declined significantly and in contrast to the flag leaf the qP decreased and the NPQ increased significantly in both varieties, and the tendencies were the same in both genotypes. The chla fluorescence parameters and the efficiency of photosynthesis measured on the seedlings did not correspond to those measured on the flag leaf in the reproductive growth phase.-This work was supported by Grant No. NKFP 4/064/2004

Institute of Cytology and Genetics, Novosibirsk 630090, Russia; ak@bionet.nsc.ru

Investigation of proline metabolism is important both to understand stress response mechanisms and to improve crops. Earlier evaluations of stress resistance of transgenic arabidopsis plants bearing antisense suppressor of proline dehydrogenase resulted in some controversial observations (Nanjo et al., 1999; Mani et al., 2002; Ribarits, 2007). We obtained tobacco plants bearing a fragment of arabidopsis proline dehydrogenase gene in antisense orientation. The usage of heterologous antisense suppressor resulted in a mild increase in proline content in non-stressed plants (by a factor of 2 to 3 in different lines). Under the stress conditions (200 mM of NaCl) the content of proline was increased to similar levels in both transgenic and control plants. According to the results obtained transgenic plants were characterized by a mild increase in general (non-specific) stress tolerance: (1) they survive better in the presence of NaCl or PEG6000 in growth media and contain a smaller quantity of MDA; (2) they tolerate higher concentrations of various heavy metals (Pb, Cd, Ni, Hg); (3) the detached leaves of transgenic plants lose water at a lower rate; (4) the electrolyte leakage experiments showed lower membrane permeability of cells of transgenic plants; (5) after the exposition at high temperature, seeds of transgenic plants were characterized by a higher germination rate. It may be assumed that an increased level of proline under normal conditions could partially alleviate the damage at stress onset and facilitate rapid induction of stress response mechanisms. Transgenic plants were characterized by normal phenotype and development.

Biological Research “Siniša Stanković“, 11060 Belgrade, Serbia, ¹ manitas@ibiss.bg.ac.yu

Physically connected ramets of clonal plants are able to reciprocally exchange essential resources (i.e., water, assimilates and nutrients) if they are unevenly distributed over space. We tested the hypothesis that translocation of resources between source and sink ramets increases stress tolerance of the whole clone growing in a patchy environment. This study was conducted in an exposed population of Iris pumila growing in the wild. In early spring, circle-shaped clonal genotypes were cut into two equal parts with different integration status: C - with physically connected rhizome segments and NC - with ceased connections. One half of each clone was shaded with a neutral screen in such a way that both C and NC parts consisted of exposed (high-light stressed) and shaded (up to 50% of ambient irradiance) quarters. During the summer, the leaf tissues were sampled from each quarter within every clone. Variation pattern of the following traits was examined: the level of stress proteins Hsp70 and Hsp90 (Hsp90a and Hsp90b isoforms), as well as relative water content (RWC) and specific leaf area (SLA). While the value of SLA decreased with irradiance, Hsp90a expression and RWC increased, regardless of their integration status. Conversely, the amount of Hsp70 and Hsp90b dramatically elevated exclusively in the exposed quarter interconnected with shaded one. These results suggest that resource-sharing among interconnected ramets could provide a powerful strategy in I. pumila to enhance the tolerance to stressful abiotic heterogeneity commonly occurring within its natural habitats.

Corresponding author: mjquiles@um.es

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of the development. One, B. fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, B. oleracea, is an agricultural species that is widely cultivated throughout the wold. The B. fruticulosa plants grown under high illumination and heat showed a greater reduction of the foliar area and biomass than the B. oleracea plants in relation with the respective control plants,. The quantum yield of the PS II, the capacity of photosynthetic electron transport and photochemical quenching, decreased in B. oleracea plants when grown under stress conditions, indicating the inhibition of PS II. However, in B. fruticulosa the values of these parameters were similar to control plants. PS I was more stable than PS II, probably because it plays a protective role though cyclic electron flow. The PS I activity increased in B. oleracea and B. fruticulosa plants exposed to heat and high illumination. The activities of plastidial NADH deshydrogenase complex and terminal oxidase were much higher in B. fruticulosa than in B. oleracea, and both were stimulated in plants grown in stress conditions. Acclimation and tolerance to high illumination and heat of the photosystem activities was higher in the wild species than in the agricultural species, indicating that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that, the chlororespiration process is probably involved in both adaptation and acclimation to heat and high illumination. This work was supported by the Spanish MEC (BFU2005-09243-C02-01).

Soil Science Department, Agriculture College, Vali-Asr University, Rafsanjan, Iran. azamrazavinasab@yahoo.com

Soil salinity is an important growth limiting factor for most plants. Salinity inhibits plant growth by osmotic stress, nutritional imbalance and some specific toxicity and it is being progressively exacerbated by fertilization, especially in arid regions. In other hands, nitrogen (N) is an essential element for plants. It has a fundamental role in amino acids, proteins, nucleoic acids and lots of enzyme structure. The proper use of N fertilizers in all soils is important, but particularly so in saline soils, where N might reduce the adverse effects of salinity. In high salinity levels, the antagonistic relationships are attributed to the negative membrance potential of root cells and the negative charge of both nitrate and chloride. Most plants respond to salinity stress in their environment by osmoregulation of their cellular content. Synthesis and accumulation of organic solute of carbohydrate and N compounds (e.g. proline) are used for this purpose. The present study, therefore, was initiated to evaluate the effects of N and salinity on proline and reducing sugars content in pistachio. Pistachio seedlings were grown in potted soils exposed to four salinity levels (0, 800, 1600, and 2400 mg NaCl kg^-1 soil) and four N levels (0, 60, 120, and 180 mg kg^-1 soil as urea). As the salinity levels increased, the leaf proline content significantly increased while concentration of reducing sugars decreased. A significant increase in proline and reducing sugars was observed with N application. The evidence presented in this study suggests that a function of proline under salt stress is that of osmoregulation. Also, the results show that the provision of the optimal nutritional regimes, therefore, has an important contribution to the determination of pistachio reaction to soil salinity.

Department of Soil Science, College of Agriculture, Rafsanjan University, Rafsanjan, Iran. Roghayeh_shahriari2371@yahoo.com

Salinity is a world-wide problem which adversely affects the growth of many plants through a series of interacting factors including some specific ion toxicities, ion imbalance and suppression of water potential. Most plants respond to changing osmotic potentials in their external environment by osmoregulation of their cellular content. Synthesis and accumulation of organic solutes (e.g proline) are utilized for this purpose. The amount of proline accumulation correlates well with the degree of stress in plants subjected to different salts. Soil fertilization appears to be beneficial in reducing the plant growth depression due to soluble salts. The present study, therefore, was initiated to evaluate the main and interactive effects of zinc, phosphorous and salinity levels on proline and reducing sugars content in pistachio. Pistachio plants were grown in potted soils exposed to four salinity levels (0, 1000, 2000, 3000 mg NaCl Kg^-1 soil), four phosphorous levels (0, 60, 120 and 180 mg P Kg^-1 soil as Ca(H₂PO₄)₂) and three zinc (Zn) levels (0, 5, 10 mg Zn Kg^-1 soil as ZnSo₄.7H₂O). As the salinity levels increased, the leaf proline content significantly increased. However, Zn application declined the proline content but the application of phosphorous had not a significant effect on proline and reducing sugars content. T