Lecture Abstracts

Towards understanding desiccation tolerance in higher plants Dorothea Bartels

Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Kirschallee 1, 53115 Bonn, Germany

Limited water availability in some areas of the earth has resulted in evolution of mechanisms to live with restricted water supply. Most higher plants are unable to survive desiccation to an air-dried state and only seeds or pollen can withstand this condition. A small group of vascular angiosperm plants, termed resurrection plants, have evolved unique mechanisms of desiccation tolerance. Resurrection plants can tolerate severe water loss, and mostly adjust their water content with the relative humidity in the environment. The plants grow in ecological niches with seasonal rainfall, they can remain in the desiccated state, comparable to dry seeds. When rainfall occurs, the plants take advantage of the conditions resurrect, grow and produce seeds before other species growing from seeds can do so Our studies are focused on the desiccation tolerant South African resurrection plant Craterostigma plantagineum. We have identified a number of factors which contribute to the phenomenon of desiccation tolerance. Many genes are activated during the dehydration process and several of them encode molecules with a protective function. We try to understand how molecules interact to form a protective barrier against the dehydrating environment and keep cellular components functional. One approach is to understand how regulatory factors and gene expression networks are modulated in C. plantagineum with reference to the genetic model plant A. thaliana to obtain desiccation tolerance. In that context large scale analyses of gene expression patterns have been performed to characterize the transcriptomes at different physiological conditions (Rodriguez et al. 2010). In order to identify essential components of desiccation tolerance we have initiated to study close relatives of C. plantagineum, some of which are desiccation tolerant and others are not (Challabathula and Bartels 2012) This comparative approach should help to define the requirements for a desiccation tolerant phenotype. Desiccation tolerance in resurrection plants shares many components with desiccation tolerance in seeds (Van Zanten et al. 2011).

References: 1. Rodriguez, M. et al. (2010) Transcriptomes of the desiccation tolerant resurrection plant Craterostigma plantagineum The Plant Journal 63, 212-228 2. van Zanten et al. (2011) Seed maturation in Arabidopsis thaliana is characterized by nuclear size reduction and increased chromatin condensation Proc. Natl. Acad. Sci. 108 (50), 20219-20224 3. Challabathula D., Bartels D. (2012) Light response, oxidative stress management and nucleic acid stability in closely related Linderniaceae species differing in desiccation tolerance Planta (in press)

Modification of source/sink relationships to improve crops resistance to

abiotic stresses Zvi Peleg1 and Eduardo Blumwald2

1R. H. Smith Institute of Plant Sciences & Genetics in Agriculture, The Hebrew University of Jerusalem, Isarel. 2Department of Plant Sciences, University of California, Davis, USA

Crop-plants are often grown under unfavorable environmental conditions that prevent the full expression of their genetic yield potential. A significant increase in grain yield of major cereal-crops such as rice (Oryza sativa L.) and wheat (Triticum aestivum L.), is required to fulfill the food supply requirements for the ever increasing population. We generated transgenic rice and wheat plants expressing an IPT (isopentenyltransferase) gene driven by pSARK, a stress- and maturation-induced promoter. Plants were tested for drought tolerance at different developmental stages: (i) pre-anthesis (booting) stage and (ii) post-anthesis stage (two weeks after panicle initiation). In general, both stress treatments had a significant effect on productivity parameters. When compared with the wt plants, the PSARK-IPT rice plants exhibited delayed leaf senescence under both stress treatments. Under the pre-anthesis stress treatment, the transgenic plants produced up to 80% of grain yield (GY) and 98% of 1000-grain weight (TGW) as compared to control plants, whereas the wild-type showed a dramatic reduction in GY (26%). In parallel experiments, plants were tested for salinity tolerance at pre-anthesis stage, by applying NaCl gradually to up to 120mM. Under the salinity treatment, although the transgenic plants displayed a significant yield penalty, they produced 260% more grain yield (GY) than the wt plants. Using a global approach through the employment of transcriptomic, metabolomic and phenomic tools, we gave identified and characterize key components mediating in the enhancement of stress tolerance. The transgenic plants expressing PSARK::IPT exhibited shift in hormonal balance which resulted in alteration of source/sink relationships and improved yield under drought. The expression analysis and enzyme activities of key genes associated with C and N metabolism together with the analysis of protein stability demonstrated that the enhanced stress tolerance displayed by the transgenic PSARK::IPT plants, is associated with alterations of carbon (C) and nitrogen (N) metabolism during the transition to reproductive stage. Our results indicated that alterations in hormone homeostasis resulted in improvement of primary N assimilation, enhancement of biochemical properties of photosynthesis and maintenance of protein function that allowed keeping the energy balance required for the survival of the plant during stress phenomena. Our results clearly demonstrate that the delay of stress-induced senescence can be engineered into cereal-crops with significant yield and quality improvements.

Reactive oxygen species (ROS) mediates rapid systemic acquired acclimation to abiotic stresses

Nobuhiro Suzuki1, Gad Miller2, Karen Schlauch3, Joel Shoman4, Vladimir Shulaev2, Ron Mittler2,5 1 Department of Biological Sciences, University of North Texas, Denton, TX USA 2 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.; 3Department of Biochemistry and Molecular Biology, University of Nevada, Mail Stop 200, Reno NV 89557, USA; 4Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; 5Department of Plant Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel.

Systemic acquired protection of plants against pests, pathogens and extreme environmental conditions requires cell-to-cell communication and long distant signaling. We recently described a rapid systemic signal in Arabidopsis thaliana that is dependent on the respiratory burst oxidase homolog D (RbohD) gene and mediated by ROS. The rapid systemic signal is triggered by wounding, heat, cold, high-intensity light, and salinity stresses and is accompanied by accumulation of ROS in distal tissues. Furthermore Ca2+- and ROS-inhibitors can delay and even inhibit the propagation of the systemic signal to distal parts of the plant. We further show that this systemic signal is required for the systemic acquired acclimation (SAA) of Arabidopsis plants. SAA to heat stress is rapidly triggered by the pretreatment (i.e acclimation treatment) of distal leaves to heat, high-light or wounding stress stimuli in a Rboh-dependent manner, suggesting large degree of overlap between the systemic signal(s). In contrast, transcriptional and metabolic profiling performed on systemic tissue of wounded, heat- and HL-pretreated plants revealed little overlap in gene expression pattern in the response to these acclimation treatments. Our results reveal a great degree of complexity in the stress-triggered systemic signals mediated by ROS and suggest specialization of SAA in response to divergent stress stimuli.

Genomic studies of biotic and abiotic stress responses in wild emmer wheat: from genetic diversity to gene cloning

Tzion Fahima1, Dina Raats1, Elitsur Yaniv1, Zeev Frenkel1, Assaf Distelfeld12, Roi Ben-David1, Hanan Sela1, Boulos Chalhoub3, Alan H. Schulman4, Jorge Dubcovsky2, Abraham

Korol1

1Department of Evolutionary and Environmental Biology, University of Haifa, Mt. Carmel, Haifa 31905, Israel. 2Department of Plant Sciences, University of California, Davis, CA 95616,

USA. 3INRA-URGV, Evry, France. 4MTT Agrifood Research and University of Helsinki, Helsinki, Finland. Email address: fahima@research.haifa.ac.il.

Wild emmer wheat, Triticum dicoccoides, the tetraploid ancestor of domesticated wheat, was discovered in 1906 by A. Aaronsohn in Israel. Aaronsohn had the pioneering vision that wild wheat germplasm will be used for crop improvement. Nevertheless, traditional approaches for utilization of wild alleles are usually very slow. The advanced genomic technology available today may help to increase the efficiency of utilization of wild germplasm. Our studies are focused on unraveling the genetic basis of biotic and abiotic stress tolerance in wild emmer wheat. Stripe rust and powdery mildew are devastating diseases in many wheat-growing regions of the world. New strategies to reduce yield losses are required to satisfy the increasing world demand for cereals. Wild emmer wheat, is a promising source of resistance to stripe rust and powdery mildew. The availability of wheat genetic maps, ESTs mapped to deletion bins, and BAC libraries enabled us to achieve significant progress towards positional cloning of several disease resistance genes derived from wild emmer wheat (e.g. Yr15, YrH52, PmG3M, and Yr36). Since wheat genome is not sequenced yet, we are using synteny with rice and Brachypodium distachyon genomes as a model for positional cloning in wheat. Genetic maps were developed by crossing resistant T. dicoccoides genotypes with susceptible T. durum cultivars. DNA markers were used to assign the target genes to chromosome deletion bins and ESTs assigned to these bins were used to establish colinearity with contigs located on rice and B. distachyon chromosomes. This comparative genomics approach enabled us to develop sub-centiMorgan maps for Yr36, Yr15 and YrH52. Using this strategy, we have cloned the slow rusting gene Yr36 (Science 323:1357-60). Yr36 has a novel architecture with a kinase and a START lipid-binding domain. This gene, which was lost during the domestication of wheat, provides a new tool for controling this disease. Further work is underway to clone Yr15 and YrH52 located on chromosome 1BS, and PmG3M located on 6BL. Physical map of chromosome 1BS was constructed within the framework of the European consortium TriticeaeGenome. The assembled 1BS physical map is being utilized for positional cloning of Yr15 and YrH52. These studies demonstrate the potential of wild emmer wheat gene pool for improvement of cultivated wheat, and emphasize the contribution of the recently developed genomic tools for the utilization of wild wheat germplasm.

Molecular adaptation of plants to the environment: changes in root

architecture and in ion and protein homeostasis Dudy Bar-Zvi, Doron Shkolnik-Inbar, Guy Adler and Petra Peharec-Stefanic

Ben Gurion University of the Negev, Israel

Plants respond to changes in the environment by physiological, biochemical, molecular and developmental changes leading to adaptation to the altered conditions. This reponse includes global changes in the profile of expressed genes and proteins. Mutants of the ABSCISIC ACID INENSITIVE (ABI) 4 gene, encoding an AP2-domain transcription factor, were isolated by screening of Arabidopsis seedlings germinating in the presence inhibitory chemicals such as ABA, sucrose or NaCl. ABI4, was assumed to play role mainly in seed maturation and germination. We show that ABI4 is also expressed in roots of mature plants. We have recently shown that ABI4 modulate the ABA and cytokinin inhibition of lateral root formation by inhibiting the polar transport of auxin (Shkolnik-Inbar and Bar-Zvi (2010) Plant Cell 22, 3560–3573). We have also further studied the effects of ABI4 on plant performance under salinity. abi4 mutants display increased salt tolerance also at mature stages. Overexpressing ABI4 resulted in enhanced sensitivity to NaCl. Upon exposure to salt, the abi4 mutants accumulated reduced levels of sodium, compared with WT plants. The ABI4-target gene whose change in expression results in enhancing salt-tolerance of the abi4 mutants was identified. We have shown by chromatin immunoprecipitation (ChIP) that ABI4 binds in planta the promoter of this gene. The ABI4 DNA-binding sites on the promoter were characterized using electrophoretic mobility shift assay (EMSA) of WT and mutated sequences. This study demonstrates that ABI4 has a central role in the modulation of root development and root activities in response to abiotic stresses. Abiotic stresses also lead to changes in the protein profiles within plant cells. This includes hundreds of proteins that are either increased or decreased. Controlled protein degradation is thus required to eliminate proteins whose levels should be reduced under stress conditions, and to remove proteins that are irreversibly damaged due to the stress conditions. The Ubiquitin-Proteasome System (UPS) is the central mechanism for controlled protein degradation in the cytosol and nucleus. In this pathway, proteins that are designated for degradation are first tagged with short oligomer of ubiquitin. The resulting ubiquitinated proteins are than being degraded by the proteasome. We have identified a number of mutants in genes encoding ubiquitin-ligases that display increased drought sensitivity. These mutants as well as plants overexpressing these genes were studied. We have used yeast-2-hybrid screening to identify potential targets of these enzymes.

Molecular processes behind the tolerance of germinating seeds to stress

Bai B, Rosenthal L, Kazachkova Y, Quansah L, Nevo N and Fait A.

The French Associates Institute for Agriculture and Biotechnology of Drylands, Institutes for Desert Research, Ben-Gurion University, Midreshet Ben-Gurion

Seeds in the seed-bank experience diurnal cycles of imbibition followed by complete dehydration. These conditions pose a challenge to the regulation of germination. The effect of recurring hydration-dehydration (Hy-Dh) cycles were tested on seeds from four Arabidopsis thaliana accessions [Col-0, Cvi, C24 and Ler]. Diurnal Hy-Dh cycles had a detrimental effect on germination rate and on the final percentage of germination in Col-0, Cvi and C24 ecotypes, but not in Ler ecotype, showing improved vigor following the treatments. Membrane permeability measured by ion conductivity was generally increased following each Hy-Dh cycle. Among the ecotypes Col-0 seeds displayed highest membrane permeability, whilst Ler was characterized by the greatest increase in electrical conductivity following Hy-Dh cycles. Following Dh, the respiratory activity of Ler seeds significantly increased in contrast to the other ecotypes, indicative of a dramatic shift in metabolism. These differences were associated with accession-specific content and patterns of change of (i) cell wall related laminaribiose and mannose, (ii) fatty acid composition, specifically of the unsaturated oleic acid and α-linoleic acid, (iii) Asn, ornithine and related polyamine putrescine. Furthermore, in Ler ecotype the content of TCA cycle intermediate fumarate, succinate and malate increased in response to dehydration, as opposed to a decrease in the other three ecotypes. These findings suggest contrasting regulatory mechanisms between arabidopsis ecotypes in response to stress. Comparative metabolomics analysis identified potentially relevant processes for the improvement of crop performances under un-predictable environmental conditions.

Role of auxin in flower development and fruit set under temperature stress

Hagai Yasuor

Agricultural Research Organization, Gilat Israel Abiotic stress results in damage flower and poor fruit set in vegetable and field crops. Reduced fruit set in tomato grown under high temperatures results from inhibited pollen development, anther release, viability and stigma elongation which were correlated with altered auxin metabolism in these organs. High temperature reduced the expression auxin response factor DR5 in the developing anthers and stigma (region of papillae formation). Reduced DR5:GFP signals indicate that auxin is depleted in the stomium formation region as well (Fig. 1). In addition alteration in auxin response factor expression was observed in developing embryos, mainly in the placenta and integuments. Similarly yield reduction also found when glyphosate was applied after flower bud initiation to cotton. Glyphosate application to cotton grown at a high temperature regime caused a complete loss of pollen viability and inhibition of anther dehiscence, while at a moderate temperature regime only 50% of the pollen grains were disrupted and anther dehiscence was normal. Elevated auxin content in cotton anthers resulted in male sterile flowers, in which the orientation of secondary cell wall thickening (CWT) in the endothecium was altered and stomium formation was inhibited. The reorientation of CWT in cotton was accompanied by a similar change in microtubule orientation. Fig. 1. Influence of high temperature on auxin response factor (DR5::VENUS) in tomato flower organs before anthesis (A) and in the developing embryo (B). Arrows indicate DR5 expression in anther tips, stomium region, stigma and in the developing embryo, placenta and integuments.

An artificial capillary barrier to improve root-zone conditions for horticultural crops

Naftali Lazarovitch, Eviatar Ityel, Moshe Silberbush and Alon Ben-Gal

The French Associates Institute for Agriculture and Biotechnology of Drylands, Institutes for

Desert Research, Ben-Gurion University, Midreshet Ben-Gurion Capillary barriers (CBs) occur at the interface of two soil layers having distinct differences in textural and hydraulic characteristics. The primary objective of this study was to introduce an artificial CB, created by a layer of gravel below the root zone substrate, in order to optimize conditions for the cultivation of horticultural crops. A secondary objective was to test the hypothesis that the increased soil matric head created by CB would improve plant production, especially when irrigated with brackish water. Potential root zone formats were analyzed with and without the gravel CBs for variables including: depth of CB; barrier separating the root zone from the surrounding soil; and root zone soil texture. Field and lysimeter studies were conducted with bell pepper (Capsicum annuum) plants, comparing root-zones with and without an underlying CB and irrigated at various rates with either desalinated (DW, EC = 0.2 dSm-1)) or brackish (SW, EC = 3.8 dS m-1) water. Field and simulated results revealed that artificial CBs increased root zone water content and changed water flow dynamics. Plants grown above a CB, when irrigated with SW, yielded 24% higher biomass than control plants for all irrigation applications, but when DW was irrigated, only a 6% advantage for the CB root-zone was observed. Biomass yield for the root-zone containing a CB was only slightly affected by water salinity, while yields significantly decreased when irrigated with SW in the control. The extent of water uptake, plant growth and yield responses to the presence of a CB were found to be climate dependent. When VPD was low, smaller responses were measured, while more pronounced response was found when VPD increased. References: Ityel, E. et al. (2010) An introduced capillary barrier to improve root zone conditions for horticultural crops: physical effects on water content. Irrig. Sci., 29:171-180. DOI: 10.1007/s00271-010-0227-3. Ityel, E. et al. (2011) An artificial capillary barrier to improve root zone conditions for horticultural crops: response of pepper, lettuce melon and tomato. Irrig. Sci., DOI: 10.1007/s00271-011-0281-5. Ityel, E. et al. (2012) An artificial capillary barrier to improve root-zone conditions for horticultural crops: response of pepper plants to matric head and irrigation water salinity. Agric. Water Manage. 105:13-20. DOI:10.1016/j.agwat.2011.12.016

Poster Abstracts

The Negative Regulators of Arabidopsis Abiotic Stress Responses, STRS1 and

STRS2 are DEAD-box RNA Helicases that Exhibit Nucleolar/Nucleoplasmic

Re-partitioning in Response to Stress

Asif Khan1, Yuval Kaye1, Giorgia Batelli2, Jian-Kang Zhu2, Gideon Grafi1 and Simon Barak1

1French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus, 84990, Israel. 2Department

of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA Plants exhibit complex transcriptional, post-transcriptional and post-translational regulation of

stress-responsive gene expression. We previously identified two Arabidopsis negative regulators

of abiotic stress responses, which we designated STRESS RESPONSE SUPPRESSOR (STRS)1

and STRS2 that both encode DEAD-box RNA helicases. Here, we show in an in vitro assay that

both STRS1 and STRS2 possess an RNA-dependent ATPase activity intrinsic to RNA helicases.

Examination of the sub-cellular localization of the GFP-STRS fusion proteins demonstrated that

the STRS proteins localize to the nucleolus with a lower level in the rest of the nucleoplasm.

Closer examination of GFP fluorescence revealed that the STRSs may also localize to the

perinuclear chromocenters. FRAP assays measuring the recovery of GFP fluorescence in the

nucleolus after bleaching suggested that the STRSs are highly mobile. Consistent with this

finding, the STRSs re-localize to the nucleoplasm in response to various abiotic stresses. These

results suggest either that at least one STRS function takes place in the nucleolus and that this

function is inactivated during abiotic stress, or that the STRSs are sequestered in the nucleolus

and function in the nucleoplasm.

Adaptation to Abiotic Stresses of Wild Cruciferae Species from Different

Habitats

Gil Eshel1, Yana Kazachkova1, Vered Chalifa-Caspi2, Michal Gordon2, Ruth Shaked1, Anna Amtmann3, Yitzhak Gutterman1, Shirli Bar-David1, Aaron Fait1 and Simon Barak1

1French Assoc. Inst. for Agriculture and Biotechnology of Drylands, Jacob Blaustein Insts. for Desert Research, Ben-Gurion University of the Negev, Midreshet, Ben-Gurion, Israel; 2Bioinformatics Core

Facility, National Inst. for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beersheva, Israel; 3Inst. of Molecular, Cellular and Systems Biology, University of Glasgow, Glasgow, UK.

Abiotic stresses reduce average crop yields by more than 50%. In addition, most new land with

the potential for agriculture is situated in harsh environments. Consequently, understanding the

genetic basis of stress tolerance is vital for improvement of crop yields under stress. We are

investigating the adaptation mechanisms of the naturally stress-tolerant Arabidopsis relatives

(Cruciferae) Anastatica heirochuntica (salt-, heat-, low N-tolerant crucifer from the Negev

Desert) and Thellungella salsuginea (salt-, cold-, low N-tolerant crucifer from saline coasts in

China [Shandong ecotype]). Preliminary transcriptome sequencing of Anastatica shoots under

control conditions using Arabidopsis thaliana as a reference transcriptome revealed over 800

genes that have a role in the Arabidopsis stress response. Many of these genes are induced by

stress in Arabidopsis, yet were highly expressed in unstressed Anastatica plants suggesting that

Anastatica may be primed for stress. Principal component analysis of metabolic profiles of

primary carbon and nitrogen metabolites in Arabidopsis, Thellungiella and Anastatica under 0,

50, 100 and 200 mM NaCl demonstrated that the first component (51% of variance) separated

metabolite abundance by species while the second component (15% of variance) separated the

metabolites according to salt treatment. Hierarchical clustering analysis showed clear separation

of the three species based on their metabolic strategies and salt tolerance. Arabidopsis (salt-

sensitive glycophyte) was present on a separate clade from the two extremophile species, which

were themselves separated on sub-clades. These results suggest that the two extremophiles have

developed both common salt tolerance mechanisms even though they are from very different

habitats as well as species-specific mechanisms.

Stress-induced dedifferentiation revealed by ANAC2 promoter activation and pericentric chromatin decondensation

Assa Florentin and Gideon Grafi

French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.

Dedifferentiation signifies the withdrawal of cells from a given differentiated state into a stem cell-

like state, a process preceding switch in cell fate including reentry into the cell cycle and death.

Open chromatin conformation has emerged as a fundamental feature characterizing dedifferentiating

cells as well as stem cells both in plants and animals. Recent data suggest that dedifferentiation is

often induced following exposure to extreme stress such as pathogen infection, protoplasting and

long exposure to dark. To further substantiate the linkage between stress response and

dedifferentiation we generated a tool based on the activity of a putative dedifferentiation-specific

promoter derived from the ANAC2 (At1g01720) gene that controls the expression of a reporter

nuclear protein AtMBD6 fused to GFP coupled with the analysis of pericentric chromatin

conformation. We found that ANAC2 promoter is essentially inactive in mature leaves but highly

active in the shoot and the root apical meristems as well as in flowers and siliques at early stages of

their development. In the shoot apical meristem, ANAC2 promoter was active not only in the central

zone of the meristem but also in the peripheral zone including leaf primordia. Activation of ANAC2

was observed in leaves following exposure to various extreme stress conditions including

protoplasting, wounding, heat and long exposure to dark and was associated with pericentric

chromatin decondensation. These results showed that plant cells respond to extreme stress

conditions by undergoing dedifferentiation and acquisition of stem cell like state, which is

characterized by ANAC2 promoter activation associated with pericentric chromatin decondensation.

Reduced epigenetic constraints evolved in the Zygophyllacea family

Gila Granot and Gideon Grafi

French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, 84990, Israel.

The desert plant Zygophyllum dumosum displays unique epigenetic constraints, not found in

other perennial desert plants, namely, it possosses mono- but not di- and trimethylated histone

H3 at lysine 9. Here we investigated the proposal that lack of H3K9 dimethyl is not restricted to

Z. dumosum but a feature uniquely evolved in the Zygophyllaceae family. To this end, we

analyzed H3K9 methylation status in Zygophyllaceae species including Z. simplex (annual),

Peganum harmala (hemicryptophyte) and Balanites aegyptiaca (tree) from the Negev Desert

(Israel) and Larrea tridentata (Creosote bush) a prominent species in the Mojave, Sonoran, and

Chihuahuan Deserts of western North America. All plants examined showed methylation of H3

at K4 but no detectable levels of dimethylated H3K9. Furthermore, Nitraria retusa that was

recently separated from the Zygophyllaceae family does possess dimethylated H3K9, further

supporting its partition into a distinct family (Nitrariaceae). Surprisingly, however, two species

within the Oxalidaceae, namely, Oxalis conriculata and O. pes-caprae were deficient in

dimethylated H3K9 similarly to Zygophyllaceae species, while the analysis of Krameria

cistoidea of the Kramericeae family, which is listed under the Zygophyllales showed the

presence of dimethylated H3K9. Considering that most species in the Zygophyllaceae family

inhabit dry and semidry regions of the world, it is suggested that lessening epigenetic constraints

might have an adaptive value in variable desert regions enabling plants to quickly respond to

environmental cues.

Ref:

Granot G, Sikron-Persi N, Gaspan O, Florentin A, Talwara S, Paul LK, Morgenstern Y, Granot Y, Grafi G (2009) Histone modifications associated with drought tolerance in the desert plant Zygophyllum dumosum Boiss. Planta 231:27-34

Protoplasting induces dedifferentiation and genomic variation

Yaakov Morgenstern and Gideon Grafi

French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.

The ability of some organisms to regenerate organs is mostly based on the capability of somatic

cells to undergo dedifferentiation, that is, acquire stem cell like state prior to reentry into the cell

cycle. Dedifferentiation characterizes the transition from leaf cells to protoplasts and is

accompanied by widespread chromatin decondensation. We examined the possibility that

dedifferentiation event might induced genetic variation by means of activation of transposable

elements. To test this possibility I have generated a transposition construct in which the Tag1

element was mutated to generate a non-autonomous dTag1 element introduced between the 35S

promoter and a reporter AtMBD5-GFP. Only transposition followed by faithful end joining

could reconstitute an active construct. Indeed, I found that in transient assays introduction of this

35S--dTag1--AtMBD6-GFP construct into Arabidopsis protoplasts resulted in activation of

AtMBD6-GFP in at least 5% of transformed cells. Using immunoblotting analysis I found that

H2AX become phosphorylated at serine 139 in protoplasts but not in leaves, thus indicating the

occurrence of double strand DNA breaks (DSBs) in dedifferentiating protoplasts. Finally, I have

generated a tool for following foci formation, which is indicative for DSBs, whereby H2AX is

fused to GFP under the control of the 35S promoter. Introduction of this construct into

protoplasts resulted in dotted distribution inside the nucleus. Thus this work suggest that

stressing cells by removal of cell walls to produce protoplasts triggers multiple nuclear reactions

that could bring about genetic variation and genome instability.

Natural variability in the metabolic responses of tomato seeds to salinity

Rosental Leah1, Perelman Adi2, Saranga Yehoshua2 and Fait Aaron1

1French Associates Institute for Agriculture and Biotechnology of Drylands, the Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel. 2R. H. Smith Faculty of

Agriculture, Food and Environment, the Hebrew University of Jerusalem, Israel.

A rising need to conserve fresh water has brought to widespread use of saline water in

agriculture. However, few works addressed the effect of saline irrigation on developing seeds. In

the present study, we used a large-scale field experiment of 76 different introgression lines (ILs)

and their control M82, grown under fresh water (EC=1.5) or saline water (EC=6) irrigation

regimes during two consecutive seasons. Dry seeds were subject to metabolic profiling using Gas

Chromatograph Mass Spectrometry (GC-MS). Metabolites (n=61) where annotated in both

seasons. M82 showed a relatively mild metabolic response to stress, which may testify to

breeding for stability. Several ILs showed many metabolic changes significantly different than

M82 under saline irrigation, providing promising regions for salt stress response QTLs. Over all

the population, the metabolites that showed the most significant changes in the saline seed

development conditions were: glycerol-3-phosphate, arginine, phenylalanine, methionine, urea

and glucose. Glycerol-3-phosphate and polyamine precursors, arginine and methionine, are

related to seed germination under stress conditions (Quettier, 2008 and Urano, 2004). The lower

levels of glucose in the seeds under salinity were associated with higher BRIX degree in the fruit

of most significantly differing lines, implying a competition on C-resources between fruit and

seed. Changes in urea content can be indicative for protein degradation related to salt stress. The

above results together with data of germination under saline conditions, will lead to better

understanding of the salt stress response in tomato seeds and putative regulating QTLs.

Study and identification of haploid Hylocereus lines (vine cacti) using morphological traits, cytological tools and SSR markers

Arroyave M.F. and Tel-Zur N.

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel

The formation of haploid plants is possible via anther or ovule culture. Anti-mitotic

reagents, as colchicine, are used to induce chromosome doubling in the haploid lines

leading to the production of homozygous (pure) lines from heterozygous parents in a single-

step. Anther and ovule culture techniques were successfully applied in two vine cacti

species, the diploid Hylocereus monacanthus and the tetraploid H. megalanthus. Currently,

about 77 putative haploid lines are under study. The ploidy level of the putative haploid

lines was evaluated by flow cytometric analysis. Morphological and cytological traits were

carried out to compare these lines with the donor plants. Flow cytometric analysis showed

that 25% of the lines obtained from the tetraploid H. megalanthus are dihaploid, as

expected, and the others are tetraploid, as the donor plant. The dihaploids lines exhibited

abnormal flowers, low pollen viability and lower fruit weight in comparison with the donor

plant and the other tetraploid lines. Flow cytometric analysis of lines obtained from the

diploid H. monocanthus showed that those plants were also diploids. These plants never

flower and their vegetative growth is very slow. Simple sequence repeat (SSR) marker

analysis was conducted to verify the origin of these diploid lines, proving their microspore

origin and homozygous nature. The obtained plant material will be further used for breeding

programs.

Metabolic Response and Mechanism Associated with Drought Adaption and

Recovery Strategy in Seedlings of Two Emmer Wheat Genotypes Differing in

Drought Response

Lydia Quansah1, Tamar Krugman2, Aaron Fait1

1French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel; 2Department of evolutionary and

environmental Biology, Institute of Evolution, Faculty of Natural Sciences, University of Haifa Mt. Carmel, Haifa 31905, Israel.

Environmental stresses greatly affect crop plants establishments and these can duly affect their growth, metabolism and yield. Thus crops must adapt and evolve strategies to increase their survival and tolerance to their changing environment. The degree of plants tolerance to environmental stress varies not only between different species but also between different genotypes of the same plant species. A detailed understanding of the morphological, physiological and metabolic base for such differences in stress tolerance could be used in selecting crops for breeding improvement under environmental stress conditions. Two natural emmer wheat genotypes; Amirim and Yehudiyya reported to be sensitive and tolerant to drought respectively were used in this study by subjecting them to osmotic stress using 200 mM mannitol concentration. Seedlings were exposed to three days stress and three days recovery. Sampling was done 1 and 3 days during stress imposition and recovery. The sensitive genotype was observed to have decreased its water content by almost 50% three days after stress imposition while the tolerant genotype maintained a high water status.

Metabolic profiling done using GC-MS revealed the tolerant genotype having higher levels of drought related metabolites as well as maintain some of these metabolites during recovery period than the sensitive genotype. Principal component analysis performed for the two time points taken each during stress and recovery showed separation between the two genotypes. The major effect that contributed to the separation was genotypic effect followed by treatment both during stress and recovery. The two time points during drought of the tolerant genotype grouped together while a more separation occurred for the sensitive genotype. During recovery, the tolerant genotype at each time point recovered to the control level where the control samples of the sensitive genotype grouped together with separation of the recovered samples far from each other. The study helped to understand the mechanism behind recovery strategy in the tolerant genotype that is lacking in the sensitive genotype.

Genetic relationships among species of the vine cactus Hylocereus as revealed

by AFLP markers

Aroldo Cisneros and Noemi Tel-Zur

French Associates Institute for Agriculture and Biotechnology of Drylands. The Jacob Blaustein Institutes for Desert Research (BIDR), Ben-Gurion University of the Negev (BGU) Israel

Phone: (972) 8-656 3462 (office); (972) 8-659 6700 (lab) / Fax: (972) 8- 659 6742; e-mail: cisneros@post.bgu.ac.il and telzur@bgu.ac.il / http://www.bgu.ac.il/

Species of the vine cactus Hylocereus are increasingly being grown in Israel as new exotic fruit crops that are particularly suited to cultivation in semi-arid and arid lands. Hylocereus species are characterized by high genetic variability; in the current study, we investigated genetic similarity and distance, genetic relationships, allele frequency and polymorphic information content (PIC) of 15 accessions belonging to four commercially important Hylocereus species. As a first step in defining the above genetic characteristics of the investigated accessions, five amplified fragment length polymorphism (AFLP) primer combinations were used. These five sets of primer combinations amplified different subsets of restriction fragments. For each primer, 59 to 91 bands were identified that independently revealed similar patterns for relationships among the species under analysis. About 94.5% of the total number of bands developed were polymorphic, with the majority of the bands being small in size, i.e., between 50 and 250 bp. The highest similarity was found between accessions 96–664 and 96–680, and the lowest, between accessions 88–023 and 96–664, with all four accessions belonging to the species H. megalanthus. PIC varied between primers from 0.27 to 0.37 and between species from 0.40 to 0.50, revealing a high level of heterozygosity. Grouping of the accessions showing high similarity produced four main clusters. Two points of interest emerged: (i) The H. undatus accession 70–02–04 belonged to the cluster of H. megalanthus accessions, which suggested that this H. undatus accession is a putative natural hybrid, and (ii) H. undatus accession 89–024 and H. megalanthus accession 88–023 were clustered in the same node, showing a close association between them, with more than 50% of similarity. From the genetic distance analyses three clusters were obtained; one cluster including three H. monacanthus accessions, one H. costaricensis accession, and two H. undatus accessions (but not 89–024 and 70–02–04); one cluster comprising accession 89–024 (H. undatus) and accession 88–023 (H. megalanthus); and one cluster comprising six H. megalanthus accessions and H. undatus accession 70–02–04. The AFLP analyses confirmed the high genetic variability among Hylocereus species and the technique provide an efficient tool for assessing genetic relationships. The study also confirms the excellent prospects for conserving and domesticating these exotic fruit crop species.

Chilling requirement and the effect of low temperature on flowering time in

Ziziphus jujube

Meir M.1, Zaccai M.2., and Tel-Zur N.1

1French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel. 2Department of Life Science. Ben-

Gurion University of the Negev (BGU). Bergmann Campus 84105, Israel. E-mail: meir.michal@gmail.com; telzur@bgu.ac.il

Ziziphus jujube is a deciduous fruit crop and a promising candidate for the agriculture in the Negev. The goal of this research is to assess its chilling requirement and effect of exposure to low temperatures on flowering and vegetative growth. The research was performed during two consecutive years in campus Bergman, Beer Sheva. During the first year, beginning of winter, dormant trees and isolated branches were exposed to 240, 480 and 720 hours (h) at 10°C or 4°C. During the second year the treatments were 240, 480, 720, 960 and 1200h at 4°C. After cold treatment, the trees and isolated branches were transferred to controlled conditions, i.e. 26/21°C (day /night) cycle and 14h/10h day/night photoperiod. The controls constituted of trees and isolated branches not exposed to cold during the winter as well as under natural conditions. Dormancy release was followed together with flowering time, flowering intensity and vegetative growth. In both years, exposure of trees to cold temperatures induced dormancy release. Exposure to 4°C for 720h preceded dormancy release up to 82 days (about three months) earlier in comparison to natural conditions and 72 days earlier in comparison to control. Exposure to 4ºC for 720, 960 and 1200h showed the highest effect on dormancy release. All trees flowered between 27 to 42 days following the dormancy release, similar to control. The effect of low temperature on the time of flowering was indirect, preceding dormancy was followed by the preceding of flowering. No statistically significant differences were observed on flower bud number or on flowering intensity as well as on vegetative growth among the treatments. All branches broke dormancy significantly earlier than branches under natural conditions. In the first year, no statistical differences were found between treatments and control, but in the second year all branches exposed to 4°C broke dormancy significantly earlier than branches under control conditions. Under the same conditions all branches tend to break dormancy earlier than trees, suggesting that the detaching of the branches from the tree had also an effect on dormancy release.The results of this study show that both time of exposure and temperature have an effect on dormancy release in Ziziphus jujube resulting in an indirect effect on flowering time.

Metabolic and cytological changes associated with autopolyploidy in species of

vine cacti (Cactaceae)

Hagai Cohen, Aaron Fait and Noemi Tel-Zur

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel email: Hagai@bgu.ac.il

Genome duplication is one of the main mechanisms whereby plants acquire additional numbers

of gene copies, thereby facilitating genetic and evolutionary novelty in organisms. Polyploidy

has been estimated as a very frequent phenomenon in angiosperms evolution, with 30 to 70% of

plant species having at least one polyploidy event in their lineages. The processes of

polyploidization often have multi-level effects on the plant, but very little is known of its

metabolic implications. Therefore, the research aim was to study the metabolic changes

associated with autopolyploidization in the fruits and seeds of the exotic hemiepiphytes

Hylocereus species, along with extensive cytological and morphological observations. Fruits,

seeds, and embryos were examined for morphological traits, and flow cytometric analysis and

microscopic observations assessed the increase in genome size and the associated cytological

changes. Fruit pulp and seeds were subjected to metabolite profiling using GC-MS and UPLC-

QTOF-MS/MS, respectively.

The results indicated that the autopolyploid lines produced larger pollen grains with lower

viability, larger stomata cells with lower density, smaller fruits with lower seed set, and less

normal tetrads in PMCs. Profiling of primary metabolites revealed a decrease in the abundance

of sugars in fruit and seed samples of both autopolyploid lines accompanied by an increase in the

metabolism of amino acids, TCA cycle intermediates, and organic acids. Fruit pulp secondary

metabolism analysis revealed a significant decrease in betacyanins content accompanied with an

increase in several flavonoids and hydroxycinnamates. Less indicative changes occurred in the

seeds secondary metabolism.

Taken together, our results indicate that autopolyploidization have significant (negative)

effects on the plant at the level of fruit and seed quality. This study shows a clear impact of

autopolyploidization on carbon partitioning in fruits and seeds alike, and provides a first

description of the metabolic changes associated with autopolyploidy.

Multi component solute transport in drainage lysimeters

Iael Raij and Naftali Lazarovitch

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel

Excess irrigation with saline water is widespread in arid and semi-arid environments.

This type of irrigation can lead to inefficient use of water and fertilizers resulting in economic

losses and groundwater pollution. Proper irrigation and fertilization scheduling based on in-situ

water and solute balances could help to minimize these consequences.

Lysimeters are an accurate research method for water and solute balances and are being

used successfully for agricultural research. However, lysimeters are expensive to produce, buy

and install, and complicated to operate and maintain. Developing a low cost drainage field

lysimeters that practitioners could use for irrigation and fertilization decision making could

optimize water and fertilizers inputs. The main obstacles for such low cost lysimeter include size

determination (depth and area) of the lysimeter itself and temporal resolution for the water and

solute balances. In this study, field and numerical experiments will be conducted with the aim of

finding a representative elementary volume and time for establishing water and solute balances

under field conditions. In addition, field and numerical experiments will explore the effect of the

lower boundary condition and volume of the lysimeter on transport of major ions.

The numerical experiments used the UNSATCHEM code that allows a numerical

solution for water flow and solute transport in unsaturated conditions considering complex ion

chemistry. This solution takes into consideration the complex interactions between the soil

matrix and the major ions in the soil including equilibrium and kinetic non-equilibrium

chemistry. The lysimeters are designed in a 3D-axisimetrical domain, with one dripper source in

the middle. The preliminary results from the numerical experiments of different lysimeter

volumes show differences in the breakthrough curves of the various ions.

Organ-Specific and Developmental Regulation of Sulfite Reductase Promoter

in Plants Dmitry Yarmolinsky and Moshe Sagi

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University, Israel

Sulfite reductase (SiR) is an essential part of sulfur assimilation pathway and catalyzes

the reduction of sulfite to sulfide. The expression pattern of SiR in Arabidopsis plants was

studied. A 2.2-kb fragment containing SiR promoter was cloned and fused with Dronpa (a GFP-

like protein) and GUS genes to direct expression of the reporter genes. Homozygous transgenic

plants of Arabidopsis thaliana (ecotype Col-0) were selected after Agrobacterium-mediated

transformation and expression of the reporter genes was studied. Organ-specific expression of

SiR was demonstrated by Western blotting with specific antibodies. The SiR promoter was

active in all organs, albeit not equally expressed, starting from the early stages of plant

development. Thus, high level of Dronpa and GUS was observed in root tip and developing

siliques. Also the elevated activity of SiR promoter was associated with bundles located in

flowering stem and leaf vessels. The expression of the reporter genes was found to be highly

increased in phloem zone and S-cells of stem bundles. Our data indicate a semi-constitutive,

organ-specific profile of SiR expression that probably associating with demand in sulfur

assimilation.

Determination of Sulfite in tomato leaves

Galina Brychkova and Moshe Sagi

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben-Gurion University, Sede Boqer Campus 84990, Israel

Sulfite detection in plant tissue is a very complicated procedure, due to the up to 70% sulfite

losses upon extraction and derivatization with monobromobimane [1]. A sensitive enzymatic

assay for sulfite determination in wild type and sulfite oxidase compromised tomato plants was

employed using chicken sulfite oxidase. The level of the detected sulfite was further supported

by a modified fuschin-based detection method and by additionally employing both, purified plant

SiR and OAS-TL to generate cysteine. The detected sulfite varied only by 2 to 9% among the

methods. Importantly, the sulfite calibration curves determined in the presence or absent of plant

sulfite, were linear for all three detection methods, exhibiting correlation coefficients higher than

0.998 [2].

[1] Tsakraklides G. et al, 2002. Plant J 32:879-889.

[2] Brychkova G et al, 2012. Plant Science 190:123-130.

Photochemistry in near isohydric and anisohydric grapevine (Vitis vinifera)

cultivars

Hochberg Uri, Degau Asfaw, Fait Aaron, Rachmilevitch Shimon

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben-Gurion University, Sede Boqer Campus 84990, Israel

Drought stress is known to limit photosynthesis rates and to inflict photo-oxidative damage in

grapevines. Grapevines, which are considered drought-tolerant plants, are characterized by

diverse hydraulic and photosynthetic behaviors, depending on the cultivar. This research

compared the photosynthesis and the photorespiration of Cabernet Sauvignon (Cs) (isohydric)

and Shiraz (anisohydric) in an attempt to acquire a wider perspective on the iso/anisohydric

phenomenon and its implications.

Shiraz and Cs were subjected to terminal drought in the greenhouse. Soil water content

(θ), leaf water potential (Ψl) and stomata conductance (gs) were measured to determine the

cultivars’ hydraulic behavior. Gas exchange and fluorometry measurements were taken at 21 and

2% O2 to acquire photosynthesis and photorespiration characteristics. Cs was found to behave in

a near isohydric manner whereas Shiraz behaved in a near anisohydric manner. Compared to

Shiraz, the reduced stomata conductance values of Cs were accompanied by higher water use

efficiency and photorespiration rates, as well as photosystem II photochemical potential (Fv/Fm).

As compared with Shiraz, Cs compensated for lower stomata conductance by higher

photosynthesis and photorespiration. These two processes contributed to higher electron flow

rates that might have a role in photoinhibition avoidance, which was observed in the stability of

Fv/Fm under drought stress.

The unique photosynthesis in the desert plant Anastatica hierochuntica (Rose

of Jericho), in different growth environments

Amir Eppel, Ruth Shaked, Simon Barak, Shimon Rachmilevitch

French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben-Gurion University, Sede Boqer Campus 84990, Israel

Annual desert plants have to complete their life cycle in a short period of time during

which they are exposed to high light intensities and limited water. In our study, we have found

that the C3 annual desert plant, Anastatica hierochuntica (Rose of Jericho) has a unique

photosynthetic response, in which NPQ induction was significantly lower and electron transport

rate was significantly higher under high light intensities, in comparison to other plant species

such as Arabidopsis thaliana, Thellungiella salsuginea and Helianthus annuus (sunflower). The

A. hierochuntica carbon assimilation rates were higher than those of Arabidopsis and

Thellungiella and similar to those of sunflower. In order to find how photosynthesis in A.

hierochuntica plants is affected by growth in different light intensities, and whether this unique

photosynthesis occurs under natural conditions, plants were grown in different conditions and

light intensities: low, moderate (both in a growth chamber) and high (outside in the field). The

NPQ response to high light intensity was similar and therefore was not affected by growth

conditions; however carbon assimilation and electron transport rate were much higher in the

plants grown under moderate light intensities. In conclusion A. hierochuntica has a unique

photosynthetic response that may be beneficial for annual plants growing in desert environment

under high light intensities.

Bassia indica for salt phytoremediation in constructed wetlands

Oren Shelef1, Amit Gross2, Shimon Rachmilevitch1

1French Associates Institute for Agriculture & Biotechnology of Drylands,

The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel; 2Zuckerberg Institute for Water Research, the Jacob Blaustein Institutes for Desert Research,

Ben-Gurion University of the Negev, Israel

The treatment and reuse of wastewater in constructed wetlands offers a low-cost,

environmentally-friendly alternative for common engineered systems. Salinity in treated

wastewater is often increased, especially in arid and semi-arid areas, and may harm crops

irrigated from wetlands. We have strong evidence that halophyte plants are able to reduce the

salinity of wastewater by accumulating salts in their tissues. Bassia indica is an annual halophyte

with unique adaptations for salt tolerance. We performed three experiments to evaluate the

capability of B. indica for salt phytoremediation as follows: a hydroponic system with mixed salt

solutions, a recirculated vertical flow constructed wetland (RVFCW) with domestic wastewater,

and a vertical flow constructed wetland (VFCW) for treating goat farm effluents. B. Indica plants

developed successfully in all three systems and reduced the effluent salinity by 20-60% in

comparison with unplanted systems or systems planted with other wetland plants. Salinity

reduction was attributed to the accumulation of salts, mainly Na and K, in the leaves. Our

experiments were carried out on an operative scale, suggesting a novel treatment for green

desalination in constructed wetlands by salt phytoremediation in desert regions and other

ecosystems.

Prof. Michael Evenari (1904-1989) By Prof. (Emeritus) Yitzchak Gutterman Prof. Michael Evenari was borne in Metz, France, in 1904. He received his Ph.D. from Frankfort University in 1926, and taught until he left for the Hebrew University of Jerusalem in 1933. In 1951 he was nominated to be a full Professor, and became the head of the Department of Botany in the Hebrew University of Jerusalem, which he was for 40 years, until his retirement in 1973.

In 1953 Prof. Michael Evenari was elected Vise President of the Hebrew University of Jerusalem, for 7 years. Prof. Michael Evenari is well known in the world, for his achievements in his research on the environmental factors affecting seed germination and on the survival mechanisms of plants under extreme desert conditions. In 1959, Prof. Michael Evenari reconstructed the ancient farm near the ancient city Avdat, and developed it into an international desert research center. Prof. Michael Evenari also reconstructed the ancient farms of Wadi Mashash and Shivta, using “run off water” for agriculture. Prof. Michael Evenari received The Israel Prize and the international Balsam Prize. In 1974, the Government of Israel decided to establish The Desert Research Institute as a campus of Ben-Gurion University near Sede Boqer. When Prof. Amos Richmond was nominated head of this Institute, Prof. Michael Evenari was one of the founders that used his connections to help Prof. Amos Richmond in the first steps of the Institute. In 1978, Prof. Evenari established the “Unit of Desert Ecology” in this Institute and was the head of this unit for some years. I was lucky to be his student and later his colleague from 1962 to 1989. Prof. Michael Evenari died in 1989 and since then we organize the Annual Prof. Evenari Symposium in his memory.