Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5a

MAPK CASCADES IN HORMONE, STRESS AND DEFENSE SIGNALING

SHEEN J.

Department of Molecular Biology, Massachusetts General Hospital
Department of Genetics, Harvard Medical School
Boston, MA 02114, USA

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling modules with essential regulatory functions in eukaryotes, including yeasts, worms, flies, frogs, mammals, and plants. Numerous studies have shown that plant MAPKs are activated by abiotic stresses, pathogens and pathogen-derived elicitors, and plant hormones. The Arabidopsis genome and EST sequencing projects have revealed large gene families encoding MAPKs and their immediate upstream regulators, MAPKKs and MAPKKKs. However, little is known about the constitution of plant MAPK cascades and the specific roles that particular MAPK cascade genes play in particular plant signal transduction pathways. We have developed a comprehensive approach based on genomic information, transient expression assays, and transgenic and genetic analyses to determine the function of all Arabidopsis MAPK cascade genes involved in essential plant signaling pathways. The transient nature of the protoplast systems allows direct functional analysis of plant genes at an unprecedented high throughput rate. The experimental approaches are especially powerful in unraveling the functions of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. The involvement of MAPK cascades in hormonal, stress and innate immune responses will be presented. Since the functions of MAPK cascades in plant signal transduction pathways are likely conserved, our studies using the Arabidopsis genome resources will have broad implications and applications in other plant species. The elucidation and manipulation of MAPK cascades in plants has revealed fundamentally important intracellular signaling processes and provided new tools for crop improvement in stress tolerance, disease resistance, and yield enhancement.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5b

FUNCTIONAL ANALYSIS OF THE MYB-LIKE TRANCRIPTION FACTORS IN ARABIDOPSIS

Galbiati M., Cominelli E., Conti L., Tonelli C.

Dipartimento di Genetica e Biologia dei Microrganismi, Via Celoria 26, 20133 Milano, Italy

transcription factors, MYB-like genes, Arabidopsis, trichomes, anthocyanin

In the attempt of elucidating the functional role of the MYB-LIKE Trancription Factor-S (TFM-S) we have analyzed the expression profile of the wild type allele in different plant tissues by RT-PCR and in-situ hybridization. Data indicate that TFM-S is expressed in the epidermal and sub-epidermal layers of leaves, sepals and carpels.
To further characterize the expression of TFM-S we have cloned a 900bp upstream region in front of the reporter gene GFP. Transgenic plant expressing the fusion construct exhibit a strong GFP expression in the guard cell of the stomata present in cotyledons, rosette leaves, stems, sepals and carpels. 
Recently, we have identified, by a PCR-based screening approach, a null allele of TFM-S, in the Yale University T-DNA insertion lines. Plants homozygous for the insertion closely resemble the phenotype of ttg1 and ttg2 mutants . Mutant TFM-S individuals exhibit a glabrous phenotype in the rosette leaves, stems and sepals. Abnormal trichomes are exceptionally found only at the leaf margin. Moreover, mutant seeds lack the characteristic purple anthocyanin pigment in the endothelium layer and do not produce mucilage upon germination. 
All together, these preliminary observations, suggest a role for the wild type TFM-S gene product in controlling different metabolic pathways confined to the epidermal and sub-epidermal layers of Arabidopsis organs.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5c

THE TOMATO CRYPTOCHROME GENE FAMILY: MOLECULAR AND FUNCTIONAL CHARACTERISATION

GILIBERTO L.*, TAVAZZA M.*, PERROTTA G.**, GIORNO F.**, CHIESA M.**, WELLER J.***, GIULIANO G.

*) ENEA, Casaccia Res. Ctr., Roma, Italy
**) ENEA, Trisaia Res. Ctr., Matera, Italy
***) University of Tasmania, Hobart, Australia

Cryptochromes are blue light photoreceptors controlling plant growth and photoperiodism, showing similarity to DNA photolyases. Our laboratory is characterising the cryptochrome gene family of tomato. We will report on the following:

•	The existence in tomato of 3 cryptochrome genes (unlike Arabidopsis, which contains 2 genes)
•	The characterisation of Cry1 function, through transgenic / classical genetic approaches
•	The characterisation of genes under the control of Cry1, using microarray profiling in a Cry1 mutant
•	The attempts to characterise Cry2 and Cry3 function through overexpression / virus-induced gene silencing.

References

•	Ninu, L., Ahmad, M., Miarelli, C., Cashmore, A.R. and Giuliano, G.  (1999) Cryptochrome 1 controls tomato development in response to blue light. Plant J 18, 551-556.
•	Perrotta, G., Ninu, L., Flamma, F., Weller, J.L., Kendrick, R.E., Nebuloso, E. and Giuliano, G. (2000) Tomato contains homologues of Arabidopsis  cryptochromes 1 and 2. Plant Mol Biol 42, 765-773.
•	Weller, J.L, Perrotta, G., Schreuder, M.E.L., van Tuinen, A., Koornneef, M., Giuliano, G. and Kendrick, R.E. (2001) Genetic dissection of blue-light sensing in tomato using mutants deficient in phytochromes A, B1 and B2 and cryptochrome 1. Plant J 25(4), 427-440.
•	Perrotta, G., Yahoubyan, G., Nebuloso, E., Renzi, L., and Giuliano, G. (2001) Tomato and barley contain duplicated copies of cryptochrome 1. Plant Cell Env 24, 991-997.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5d

ANALYSIS OF THE ROLE OF AUXIN IN THE DEVELOPMENT OF ANTHERS  AND PISTIL

POMPONI M.*, MARSILIO S.**, ALTAMURA M.M.*, CECCHETTI V.*, TORNIELLI G.B.***, COSTANTINO P.*, CARDARELLI M.**

*) Università La Sapienza Roma
**) CNR Roma
***) Università degli Studi Verona

auxin, rolB, anther dehiscence, meiosis, pistil development

Auxin plays a crucial but as yet not fully comprehended role in floral organ development.To study the effect of auxin in the process of anther development and dehiscence, we utilised a strategy based on the localised expression of the Agrobacterium rhizogenes rolB oncogene that induces a cell-autonomous state of auxin hypersensitivity. Tobacco plants were transformed with two different transcriptional fusions of the rolB coding region with the promoter of the Arabidopsis gene DMC1, specifically active during meiosis, and, respectively, with the promoter of the Petunia gene FBP7 involved in ovule development. 

DMC1::rolB plants are male sterile due to 1) a delay in anther dehiscence probably preventing successful pollination and 2) alterations in meiosis causing a reduced number of pollen grains.

Delayed anther dehiscence is also observed in FBP7::rolB plants. However, these plants are not male-sterile as delayed pollen release takes place when the stigma is still receptive due to a corresponding delay in pistil development.. Furthermore, in these plants no alterations were observed in pollen grain number and morphology.

These data suggest that auxin might play a role in the meiotic process and in anther dehiscence, as well as in pistil development.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5e

NITRIC OXIDE-MEDIATED TRANSCRIPTIONAL CHANGES IN ARABIDOPSIS THALIANA 

MOLESINI B.*, DELLEDONNE M.*, BUONAURIO R.**, MALACARNE G.*, MICA E.*, POLVERARI A.* 

*) Dipartimento Scientifico Tecnologico, Università degli Studi di Verona, Ca’ Vignal 1, Strada Le Grazie 15, 37134 Verona
**) Dipartimento di Arboricoltura e Protezione delle Piante, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia

transcript profiling, cDNA AFLP, cell death, hypersensitive reaction

Nitric oxide (NO) production in plants has been shown to be an essential step in several processes related to development and stress response, with many important parallels with its role in animal cells. In particular, NO is the essential molecule that mediates the hypersensitive response of plants to pathogens, in synergy with reactive oxygen species (ROS). However, the molecular events downstream of NO still have largely to be elucidated. Hypersensitive cell death and related resistance responses are active phenomena, requiring de novo protein synthesis and transcriptional changes of a large array of genes are likely to have an essential role in the process. By applying a new cDNA-AFLP technique, we investigated the expression profile of Arabidopsis thaliana at different times after leaf infiltration with the NO donor sodium nitroprusside (SNP), which results in an HR-like cell death. The transcription profiles of about 3.000 genes have been examined, 120 of which showed an altered expression pattern in comparison to the control water-infiltrated plants. The large majority (about 98%) of the genes showed a transcriptional induction, often very early after the treatment (10’). Comparison of 75 selected sequences with genes reported in genomic and EST databases revealed homologies with genes involved in a wide range of functions (basic metabolism, signal transduction, disease resistance, photosynthesis, etc.), or with predicted proteins of unknown function. Functional genomic approaches are now undertaken to characterize the role of a few selected genes, which might have a regulatory role downstream of NO. Our findings indicate a wide and fast effect of NO on plant transcriptional activity, and confirm gel-based differential display techniques as an essential tool in new gene discovery.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5f

UBC10, A UBIQUITIN CONJUGASE INVOLVED IN STRESS-RELATED HORMONE SIGNAL TRANSDUCTION

FRUGIS G.*, PUANGSOMLEE P.**, MATHIAS ZEIDLER M.**, CHUA N.-H.**

*) Istituto di Biologia Agroambientale e Forestale (IBAF), CNR, Area della Ricerca di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma), Italia
**) Laboratory of Plant Molecular Biology, Rockefeller University, 1260 York Avenue, 10021 New York, NY, USA

ubiquitin conjugase, Arabidopsis thaliana, stress response, NACs

Ubiquitin-proteasome pathway (UPP) of  protein degradation is an emerging theme in cell biology. Evidences for a key role in the control of many different cell functions such as cell cycle progression, endocytosis, protein sorting, regulation of transcription and receptors activity has been widely provided both in yeast and in animal field. 

Ubiquitin is conjugated to target proteins through sequential reactions that involve activating (E1), conjugating (E2) and ligase (E3) enzymes.

We isolated a ubiquitin conjugase of Arabidopsis thaliana, UBC10, that interacted with members of the NAC family, in two-hybrid assays.  NACs are transcription factors unique to plants that are involved in different aspects of plant development and hormone signal transduction.

In particular, the transcripts of the two NAC genes, that encode for the UBC10-interacting proteins, are modulated in response to wounding, methyl jasmonate (MeJa), abscisic acid (ABA) and ethylene. We found that UBC10 is expressed ubiquitously, downregulated in the dark and increased around two-fold in response to MeJa, ABA, SA and ethylene.
 
In order to investigate the role of UBC10 in the stress processes mediated by these hormones,  a reverse genetics approach was used. Transgenic plants that overexpressed the UBC10 antisense or the UBC10 protein, either wild-type or  mutated in the cystein required for ubiquitin-thiolester formation (dominant negative), were obtained. 

Here we present the isolation of UBC10 and the characterization of transgenic plants that misexpress UBC10, with particular attention to stress-response and involvement in specific hormone signal transduction pathways. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5g

FUNCTIONAL CHARACTERIZATION OF RPD3-LIKE HISTONE DEACETYLASES IN MAIZE

LOCATELLI S., MOTTO M., ROSSI V.

Istituto Sperimentale per la Cerealicoltura - Bergamo

Zea mays, histone deacetylases, epigenetic factors

Gene expression can be stably modified by epigenetic components that can either silence or superactive selected DNA templates. Among these components are factors affecting chromatin structure by covalent modification of histones. It has been shown that histone modifications define epigenetic marks within the genome, thus establishing an “epigenetic code” superimposed to the DNA-code based control of gene activity.
Histone acetyltransferases (HATs) and deacetylases (HDACs) are the best characterized enzymes involved in the control of histone modification; in particular, a positive, although not general, correlation between histone acetylation and gene activity has been proposed. HATs and HDACs have been identified and characterized in plant systems and it has been shown that the basic features of histone acetylation in plants resemble those of other eukaryotes, although plant-specific features has also been reported.
It has been shown that HDACs are involved in several pathways for transcriptional repression This is best illustrated by the product of the retinoblastoma tumor suppressor gene (pRb) that can recruit mammalian Rpd3-type class I HDACs to repress gene transcription. Specifically, the binding of the Rpd3/pRb complex to members of the E2F family of transcriptional activators results in the formation of a transcriptionally incompetent chromatin environment, thus repressing the expression of E2F-target genes and, consequently, prevents cell cycle progression at the G1/S transition. Since several components of the pRb/E2F pathway and the basic features of histone acetylation are conserved in metazoans and plants we investigated whether this mechanism might also be relevant in plants. Our study demonstrate that ZmRpd3I, a maize class I HDAC, can associate with ZmRBR1, a maize retinoblastoma-related protein, and that the two proteins cooperate in repressing gene transcription. This provides the first evidence that plant HDACs can be recruited by a regulator of important biological processes to control gene transcription and found both conserved and distinct features.
The monocot maize (Zea mays) is a model organism for the detailed biochemical, enzymatic, and molecular characterization of different HAT and HDAC types. Three different maize Rpd3-like genes have been identified so far. To understand the biological role of the maize Rpd3-type HDACs we have analyzed the expression and localization profile of Rpd3 genes. In addition, Mu-tagged mutants and transgenic plants with an altered level of ZmRpd3I HDAC activity have been characterized. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5h

THE EFFECT OF ABIOTIC STRESS ON NATURAL POPULATIONS STRUCTURE: ANALYSIS USING MOLECULAR MARKERS TARGETED TO STRESS-INDUCIBLE GENES

JONES-EVANS E., MAESTRI E., MARMIROLI N.

Division of Genetics and Environmental Biotechnology, Department of Environmental Sciences, University of Parma, Italy

AP-PCR, abiotic stress, tolerance, antimony 

The presence of heavy metals in soils is a recurring problem, resulting both from anthropogenic industrial activities and as a natural phenomenon. The toxic conditions found on these soils influence on the biomass production and reproductive capacity of plants in a negative way, as well as exerting strong selective pressures. A number of species however have evolved mechanisms to tolerate these extreme conditions and have colonised contaminated soils. Molecular markers generated by the AP-PCR (Arbitrary Primed-Polymerase Chain Reaction) technique were employed to asses the genetic variability within and among populations of three plant species, Achillea ageratum, Plantago lanceolata and Inula viscosa, growing around a disused antimony mine, in order to gain an insight into how natural populations behave under stressful conditions. Primers (19-21nt) were designed on conserved coding sequences of stress responsive genes such as metallothioneins groups 1 and 2, phytochelatin synthase using Arabidopsis and tomato gene sequences for primer design. As these molecular markers are gene-targeted, they will be subjected to selection and hence valuable information on the genetic structure of populations subjected to abiotic stress can be gleaned. Results show that in each species, a small but significant proportion of genetic variation was attributed to differences among populations, in A. ageratum in particular. In I. viscosa and P. lanceolata, populations sampled from the most contaminated site show significantly less genetic variability with respect to the control population (from an area of low contamination) indicating an effect due to the contaminant. Principal Coordinates Analysis of a matrix of genetic distance values among pairs of individuals showed that individuals from the same population clustered together. Moreover a significant correlation was seen between environmental parameters and specific loci.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Oral Communication Abstract - S5i

SIGNAL NETWORKS IN PLANT DISEASE RESISTANCE

LAMB C.

John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK
Tel: +44 1603 450000; Fax: +44 1603 450016
chris.lamb@bbsrc.ac.uk

Attempted infection of plants by an avirulent pathogen elicits a battery of defenses often accompanied by the collapse of challenged host cells.  This hypersensitive cell death results in a restricted lesion clearly delimited from surrounding healthy tissue, and is thought to contribute to the restriction of pathogen growth and spread.  One of the earliest events in this hypersensitive response (HR) is the rapid generation of superoxide (O2) and subsequent accumulation of hydrogen peroxide (H2O2) in an oxidative burst, reminiscent of that leading t to the production of such reactive oxygen intermediates (ROI) in activated macrophages during the mammalian innate immune response. Activation of the oxidative burst in the plant HR is a central component of a highly amplified and integrated signal system, which also involves salicylic acid and perturbations of cytosolic calcium, to trigger the expression of disease resistance mechanisms, and to mediate a systemic signal network in the establishment of plant immunity.  The oxidative burst is necessary but not sufficient to trigger localised host cell death, and recent data indicate that nitric oxide (NO) co-operates with ROI in the activation of hypersensitive cell death.  NO also functions independently of ROI in the induction of various defence genes including pathogenesis-related proteins and enzymes of phenylpropanoid metabolism involved in the production of lignin, antibiotics and the secondary signal salicylic acid.  Recent data indicate that while plants and animals use a similar repertoire of signals in disease resistance, ROI and NO are deployed in strikingly different ways to trigger host cell death.

Local attack by a nectrotising pathogens also induces systemic acquired resistance (SAR) to a broad range of normally virulent pathogens.  A genetic screen was carried out using avriulent Pseudomonas syringae pv. tomato (Pst) as infecting organism to obtain SAR-defective mutants throughout the SAR pathway in Arabidopsis thaliana.  The T-DNA tagged dir1-1 (defective in induced resistance) mutant responds like wild-type plants to primary infections with both avirulent and virulent Pst.  The DIR1 gene was cloned and encodes a putative non-specific lipid transfer protein.  Initiation of SAR by inoculation with avirulent Pst was unaffected in dir1-1, but the ability to produce or perceive the phloem-mobile SAR signal was defective.  Studies with phloem sap exudates from petioles suggested that the mobile SAR signal was absent in dir1-1 plants.  We suggest that the DIR1 protein may either be the SAR mobile signal, or may transport the SAR signal to the phloem for export to distant tissue.  This is the first report that genetically assigns a biological function to a lipid transfer protein and DIR1 is the first gene identified whose product is involved in SAR mobile signal production and or transport.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.01

CLONING A PLANT (GA/TC) BINDING FACTOR-SUPPORTS AN INTRON MEDIATED MODEL FOR PLANT HOMEOBOX GENE REGULATION

Santi L.*, Stile M.R.**, wang Y.*, MULLER K.*, roig C.*, MULLER J:*, POZZI C., RODHE W.*, Filippone E.**, Salamini F.*

*) Max-Planck-Institute fur Zuchtungsforschung, Carl-von-Linnè-Weg 10, 50829 Koeln, Germany
**) Department of Soil, Plant and Environmental Sciences, School of Biotechnology Science, Via Università 100, 80055 Portici (Italy)
***) Parco Tecnologico Padano, Via Hausmann c/o DIPROVE-UNIMI, Via Celoria, Milano, Italy

barley, homeotic genes, transcriptional factor, development mutans

The Hooded mutation, which causes the formation of an extra flower of inverse polarity, is governed by the dominant genetic locus K that maps on the short arm of chromosome 4. This mutation has been associated with Bkn3, which belongs to the plant Knox gene family. The mutation is caused by direct duplication of 305 bp in the large intron IV of the gene resulting in anectopic expression of Bkn3 in the lemma of the barley mutant flower. In order to understand the mechanism of this regulation, the 305 bp has been investigated: it can enhance, in a tissue specific fashion, the expression of a GUS reporter gene driven by a 35S minimal promoter in transgenic tobacco. Using this 305 bp element in a one-hybrid screen we identified a protein interactor named BBR. This protein shows homology with 3 proteins, of unknown function, present in Arabidopsis. BBR carries the DNA binding domain at the C-terminus and has a clear Q rich region at the N-terminus portion of the protein. Using in vitro binding assays we identified a stretch of (TC) 8 repeats on the element where the protein binds. Interestingly Trithorax like of Drosophila, one of the few GAGA factor cloned, is involved, together with polycomb genes, in the regulation of homeotic mutations. According to this analogy, we are investigating the role of this novel plant GAGA factor in controlling plant leaf development.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.02

cloning of early-stress induced gene in durum wheat via PCr-select

Mastrangelo A.M.*,**, mazzucotelli E.*,*****, Marè C.***, barilli S.***, ruperti B.****, grossi M.***, di fonzo N.*

*) Experimental Institute for Cereal Research, I-71100, Foggia, Italy
**) Department Genetics and Microbiology, University of Milano, 20133 Milano, Italy
***) Experimental Institute for Cereal Research, I-29017, Fiorenzuola d’Arda (PC), Italy
****) Department of Environmental Agronomy and Crop Science, Univ of Padova I-35020 Legnaro (PD)
*****) Department of Biology, University of Ferrara, 44100 Ferrara, Italy

cold, drought, transcription factor, PCR-select

In a general view the cellular response to the stress event consists of three steps, the first one being the sensing of the stress presence at the cellular membrane level; this information is then transferred to the nucleus by means of a phosphorilation cascade, and here, regulatory proteins direct the expression of downstream genes whose products mediate stress tolerance. The use of cloning techniques has led to the isolation of a large number of genes whose expression is up-regulated by the stress event, but the majority of them belong to the group of downstream genes, and only few regulatory genes have been studied, so that the topic is now to understand the early molecular events in stress sensing and signal transduction.

To this aim suppression subtractive libraries of control and cold stressed durum wheat samples have been constructed by using the Clontech PCR-select cDNA Subtraction Kit. This is a unique method based on selective amplification of differentially expressed sequences, which overcomes technical limitations of traditional subtraction methods. The experiment has been carried out on seven days-old plants, exposed to low temperature (3°C) for six hours.

From a first screening, about fifty putatively positives clones were individuated and subjected to further analysis. By Northern experiments, fourteen clones showed to be really cold-induced or up-regulated.

These clones have been sequenced and analysed using the FASTA, BLASTX and BLASTN programmes. Five clones gave positive matches in database with known genes, representing two putative transcription factors, a putative farnesylated protein (a class of plasmalemma interacting protein  often involved in signal transduction) and three downstream stress-related genes. Four clones found matches with bread wheat ESTs, and five represent completely new sequences since they did not find matches, not even in dbEST. The putative transcriptional factors belong to MYB and AP2 families. In particular, the second one is a new stress-induced transcription factor which has the AP2 domain, but does not belong to the CBF/DREB family.

The mRNA accumulation of selected clones has been analysed in a time course from 6h to 10 days of low temperature treatment. Based on their expression profile the clones can be divided in two groups: some of them showed a transient mRNA accumulation, from 6h to 18h or 2 days of cold exposition, while other clones correspond to mRNA accumulated for all cold treatment duration.

The transcript accumulation of the isolated sequences has been studied also under dehydration stress: 7 days-old plants were dehydrated for 0.5, 1.5 and 3h. Some clones showed to be positive also to dehydration, while others are specific for cold-induced response.

This work shows as the method used is very useful in the isolation of cold-induced genes involved in early phases of the stress response. In this work genes whose products are probably involved in stress signal transduction and in the regulation of stress-induced gene expression have been identified. Some of these transcripts showed a very low expression level, suggesting that this method can be useful also in the identification of rare transcripts. The finding of clones that have never been previously isolated, not even as EST, opens new possibilities for the isolation of novel genes, also with respect to advanced techniques like cDNA microarrays.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.03

OZONE INDUCED GENE RESPONSES IN TOLERANT AND SENSITIVE PLANTS 

RIZZO M.*, SALVINI M.*,**, TADDEI S.*, BERNARDI R.*, NALI C.***, LORENZINI G.***, DURANTE M.*

*) Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti 1/B, 56124 Pisa
mrizzo@agr.unipi.it, rbernard@agr.unipi.it, mdurante@agr.unipi.it
**) Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126 Pisa 
msalvini@agr.unipi.it
***) Dipartimento di Coltivazione e Difesa delle Specie Legnose "G. Scaramuzzi" Via del Borghetto 80, 56124 Pisa 
cnali@agr.unipi.it, glorenz@agr.unipi.it

ozone stress, Phaseolus vulgaris,  Populus , suppression subtractive hybridisation 

Among the major gaseous pollutants, tropospheric ozone surely possesses the higher phyto-toxic potential: ozone has been demonstrated to alter basic metabolic processes of plants, and the alterations can affect crop productivity and contribute to forest decline.

Environmental concentrations of ozone increased from 1% to 2% per year during the past 20 years in Europe and United States, and in the next decades the critical threshold concentration of 40 nl l-1 will be reached more often in rural regions than in urban areas.

The response of plant to ozone exposure includes a number of physiological and biochemical changes that are the direct result of selective increase or decreases in gene expression. Although the physiological and morphological responses of plant to ozone have been well characterised, little is known about the molecular basis for these responses.

To elucidate the molecular mechanisms of ozone responses in arboreous and herbaceous species, we have chosen hybrid poplars (Populus deltoides x maximowiczii Eridano clone, O3-sensitive, e P. x euramericana I-214 clone, O3-tolerant) and bean (Phaseolus vulgaris var. Groffi, O3-tolerant, e Phaseolus vulgaris var. Pinto, O3-sensitive). 

With the aim to characterise the gene expression induced by ozone exposure and the molecular basis of ozone tolerance we have applied an improved techniques for finding differentially regulated genes, such as Suppressive Subtractive Hybridisation (SSH): this methods is based on the construction of subtracted cDNA libraries that allow the identification and isolation of differentially expressed transcript.

At present we have isolated some interesting clones. The sequences, coming from automated sequencing of the clones, were analysed using the FASTA, BLAST, ProDom, BLOCK  software for their identification. For example, in the cDNA library obtained from  Phaseolus vulgaris var. Pinto, O3-sensitive,  proteins were recognised that are required in the defence response for different type of biotic or abiotic stress (components of 26S proteasome; proteins that catalyse the covalent attachment of ubiquitin to other proteins; glutathione S-transferases that participate in the detoxification of reactive electrophilic compounds by catalysing their conjugation to glutathione). This may suggest that the mechanisms involving ozone-induced proteins are quite different in sensitive or tolerant varieties and that the roles of the above mentioned proteins are not sufficient in conferring tolerance.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.04

Genomic approaches to dissect cold and drought Signal transductions in cereals

Crosatti C.*, Marè C.*, mazzucotelli E.**,******, Govoni C.*, Mastrangelo A.**,*******, Galiba G.***, Dubkovski J.****, Pecchioni N.*, Bassi R.*****, Cattivelli L.*

*) Experimental Institute for Cereal Research, 29017, Fiorenzuola d’Arda, Italy
**) Experimental Institute for Cereal Research, 71100, Foggia, Italy
***) Agricultural Institute of the Hungarian Academy of Sciences, Martonvásár, Hungary
****) Department of Agronomy & Range Science, University of California, Davis, CA 95616, USA
*****) University of Verona, Facolty of Science, 37134, Verona, Italy
******) Department of Biology, University of Ferrara, 44100 Ferrara, Italy
*******) Department Genetics and Microbiology, University of Milano, 20133 Milano, Italy

cold, drought, mutant, transcription factor, QTL

The molecular dissection of cold and drought stress response in cereal has revealed a complex situation, where the co-ordinated expression of a series of stress-related genes is associated with increased levels of resistance. According to their expression patterns the cereal stress related genes isolated in our lab can be classified as cold-induced, cold and drought-induced or cold drought and ABA-induced, suggesting the existence of different stress-related signal transduction pathways.
In the recent years we have followed three genomic strategies to identify components involved in the regulation of the stress response.
Identification of mutants with impaired stress related gene expression
We have found that the expression of several stress related genes induced by low temperature only is impaired in plants carrying albino or xantha mutations suggesting that a chloroplast factor is involved in the cold-dependent, but not in other stress-related signal transduction pathways.
Identification of cis-elements and cloning of stress induced transcription factors
Deletion and mutation analysis were used to identify a novel cold-responsive cis-element in the promoter of the cold induced genes cor14b. To prove the activity of this motif several chimeric cor14b-uidA constructs were produced and assayed by transient expression after biolistic particle bombardment into leaves of wild type and albino barley plants. 
Since other known stress related transcription factors are themselves stress-regulated and their transcripts begin to accumulate during the early events of stress response, we used differential display RT-PCR and suppression subtractive library (developed using Clonthech PCR-select subtraction kit) to identify early stress related genes. Among about 15 different early induced stress-related sequence three shared a clear homology with MYB, AP2 and WRKY (pronounced “worky”) transcription factor gene families. They expression was equally promoted both by cold and drought stress. While stress induced MYB and AP2 sequences were already described in other species the involvement of a WRKY sequence in stress response represent a novel finding.
Mapping of QTL for cold-induced gene expression and co-segregation with candidate genes
We have shown by genetic analysis that the expression of the some cold-induced genes is positively correlated with the level of frost tolerance in barley and wheat. Studies made with chromosome substitution lines in wheat as well as with segregant populations in barley and in T. monococcum, showed that the expression of cor14b is controlled by the same genomic region where the QTL’s for frost resistance have been located. Current experiments aim to map all available stress related transcription factors in order to verify their association with frost resistance.
The results obtained from the approaches described above, led to the identification of several signal transduction components homologous to those found in arabidopsis as well as of novel features not described in other species.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.05

ISOLATION OF GENES RESPONSIVE TO WATER STRESS IN POTATO CELL CULTURES

COSTA A.*, DI GIACOMO M.*, MASSARELLI I.*, LEONE A.**, GRILLO S.*

*) Research Institute for Vegetables and Ornamental Plant Breeding - CNR-IMOF, Portici, Italy
**) Department of Pharmaceutical Science, University of Salerno, Italy

water stress, gene isolation, cDNA-AFLP, potato

Drought is one of major stress limiting factors for crop productivity. This stress condition affects almost all plant functions, including growth and development. Plants respond to osmotic stresses by activating mechanisms of repair and protection of cellular metabolism as a result of the action of different genes that operate in a complex and coordinate network. In order to identify key genes involved in response to drought, we studied the differential expression of potato cells upon PEG-mediated osmotic stress of different intensity (5-20% PEG) and duration (15min, 1h, 24h). The systematic comparison, by cDNA-AFLP, of changes in gene expression during gradual adaptation to low water potential with those induced by abrupt exposure to water stress, allowed to identify cDNA fragments specific for each response. As first screening, about 60 cDNAs fragments were identified as to be differentially expressed. Fourteen cDNA clones were isolated and their pattern of expression confirmed a clear up-regulation upon stress as determined by RT-PCR. Many of these AFLP tags match typical stress responsive genes belonging to different functional classes, i.e. protein synthesis (elongation factor 1, ribosomal proteins), chaperone activity and protein degradation (hsp20, RER), ROS scavenging pathway (phenylalanine ammonia-lyase, peroxidase). Sequence homology search revealed that the remaining six cDNA clones encode hypothetical or unknown proteins, never described as to be stress-inducible before. Three cDNAs are highly homologous to potato or tomato EST, one homologous to a cDNA expressed during arabidopsis seed development. When potato cDNA sequences were converted into arabidopsis hortologs, conserved domains were identified, such as plant DNA binding homodomain (clone #96) or transmembrane domains (clones #102 and 103). No hits were found in current databases for fragments #114 and 131. Further characterization of the most interesting clones is in progress for the isolation of the corresponding full-length cDNAs and the functional analysis of the genes in arabidopsis mutants.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.06

MONITORING THE EXPRESSION PATTERN INDUCED BY THE OSMYB4 OVEREXPRESSION BY USING ARABIDOPSIS THALIANA MICROARRAY ANALYSIS 

mizzi L.*, Bracale M.**, Vannini C.**, Locatelli F.***, Magnani E.***, Coraggio I.***

*) Dipartimento di Genetica e di Biologia dei Microorganismi, Università dell’Insubria, Via J.H. Dunant 3, 21100 Varese, Italy
**) Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Milano, Via Caloria 26, 20133 Milano, Italy
***) Istituto Biologia e Biotecnologia Agraria, CNR, Via E. Bassini 15, 20133, Milano, Italy

microarray, cold tolerance, Myb transcriptional factor, Oryza sativa, transgenic plants

Osmyb4 is a rice gene, encoding for a myb transcriptional factor involved in cold acclimation. The Osmyb4 expression, detected at low level in rice coleoptiles grown for three days at 29°C, is strongly induced by treatments at 4°C. Its expression is induced in rice seedlings subjected to sub-lethal temperatures (10 and 15 °C) and not by other stresses or ABA treatment. We previously demonstrated the Myb4 transcriptional activity in transient expression on several cold-induced promoters (PAL2, ScD9 SAD and COR15a). We also investigated the Osmyb4 function in vivo over-expressing its cDNA in Arabidopsis thaliana plants under the control of the constitutive CaMV35S promoter. The Myb4 over-expressing plants showed a significant increased cold and freezing tolerance, both as membrane and PSII stability and as whole plant tolerance. Myb4 is the first Myb factor involved in cold acclimation characterized up to now and therefore, there is not any information in literature on the pathway regulated by this transcriptional factor. In this study, we applied the microarray analysis, using 15000 ESTs of A. thaliana, to identify Myb4-regulated genes. Fluorescently labelled cDNAs were prepared from mRNAs isolated from wild-type and Osmyb4 transgenic plants. In total, almost 800 Myb4-inducible and 500 Myb4-repressed genes were identified by this analysis. In particular, it is of particular interest the induction of genes involved in: signal transduction (as transcriptional factors, kinases and phosphatases), stress and pathogen response (as PAL2, -xylosidase, etc.), lipid and amino acid metabolism (as lipase , lipid transporter and asparagines synthase), translation machinery (as IF2, and ribosomal proteins). The  exaustive analysis of microarray data will permit to identify the role of Myb4 in the cold induced pathway also in relation to already described pathway branches (such as CBF and SCOF1 regulated ones).

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.07

TOWARDS THE FUNCTIONAL CHARACTERISATION OF RICE GSTs

Frova C., De Toma G., Soranzo N., Sari Gorla M.

Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano

GSTs, rice, macroarrays, cadmium, hydrogen peroxide

Glutathione S-transferases (GSTs) are a super-family of multifunctional enzymes ubiquitously distributed in bacteria, fungi, plants and animals. These proteins have evolved multiple functions in the metabolism of endogenous compounds as well as foreign toxic compounds and have important roles in cellular protection against a wide range of biotic and abiotic stresses. Recent studies in maize, soybean and Arabidopsis have shown that the family encompasses 42, 25 and 47 members respectively. With the aim of extensively characterise the Gst gene family in rice,  we have previously isolated in silico 58 putative Gst genes and 3 pseudogenes, belonging to the four major plant phylogenetic classes: phi (16), tau (39), zeta (3), theta (2), plus 1 gene for a putative microsomal GST.  
As a first step towards the functional characterisation of the diverse members of this super-family in rice, 29 cDNAs representing all GST classes were cloned by RT- PCR and used to study OsGST expression in response to treatment with oxidative stress (H2O2), heavy metals (Cadmium) and a panel of herbicides. The analysis was performed by probing specifically prepared macroarray filters with mRNA from roots of rice seedlings hydroponically grown in control conditions and in presence of the above stress factors. 
The first results indicate that some tau and zeta class GSTs are transcriptionally induced by oxidative stress. The response of phi GSTs is more heterogeneous, at least one being apparently induced and one repressed by H2O2 treatment. Analysis of the response to cadmium is currently under way.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.08

EXPRESSION IN TOBACCO OF TOMATO PROSYSTEMIN ACTIVATES GENES INVOLVED IN  DIRECT AND INDIRECT PLANT DEFENCE RESPONSE

TORTIGLIONE C.*, MAZZITELLI L.**, GUERRIERI E.***, RAO R.**

*) Istituto per il Miglioramento Genetico delle Piante da Orto e da Fiore, CNR, Portici,  
**) Dipartimento di Scienze del Suolo, della pianta e dell’ Ambiente, Università di Napoli, Federico II, Portici 
***) Istituto per la Protezione delle Piante, CNR, Portici

lipoxygenase,  proteinase inhibitor, attractiveness, A. ervi

Plants respond to herbivore attack with a dramatic functional reorganization that involves the activation of direct and indirect defences. These defences, include the local and systemic production of molecules targeting directly gut membranes or inhibiting gut proteases and the production of volatiles attracting insects and mites that prey upon or parasitize herbivores. In tomato the molecule responsible of the wound induced signalling transduction pathway is systemin, a18-amino acid long peptide. This peptide induces the activation of genes directly involved in the plant defence towards herbivorous insects through the jasmonate dependent-pathway. Jasmonates are active both inside and outside the plants. In fact volatile jasmonates influence insect behaviour playing a role in defence by attracting herbivore predators and parasitoids.
Genes coding for systemin have been identified in potato, nightshade and pepper, but not in tobacco, a more distantly related solanacea. Tobacco plants were found unable to responde to exogenously supplied tomato systemin, supporting the existance of a different polypeptide involved in the wound signalling. Recently, in this species two molecules involved in the systemic wound signal transduction have been isolated and they resulted structurally different from other systemins and sharing no homologies to them.  Herein, we report that tomato prosystemin driven by a constitutive promoter when stably expressed in tobacco plants is able to induce constitutively the synthesis of lipoxygenase (LOX), the first enzyme of the octadecanoid pathway, and proteinase inhibitor (PI-II), a downstream gene directly involved in insect defence response. Furthermore transgenic tobacco plants exert attraction on A. ervi, an insect parassitoid, thus supporting evidence that also the indirect defence response is activated by the expression of prosystemin gene. These data suggest the presence in tobacco of the protease activities necessary to properly process the tomato prosystemin precursor and  activate wound signal transduction pathway.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.09

CHARACTERIZATION OF A NOVEL ARABIDOPSIS THALIANA PROTEIN THAT INTERACTS WITH A METHYL-JASMONATE INDUCIBLE NAC TRANSCRIPTION FACTOR

DI GIACOMO E., FRUGIS G., MELE G., MARIOTTI D.

Istituto di Biologia Agroambientale e Forestale (IBAF), CNR, Area della Ricerca di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma), Italy

Arabidopsis thaliana, methyl jasmonate, NAC, signal transduction

Jasmonates (JAs), including jasmonic acid (JA) and its methyl-ester methyl jasmonate (MeJa), are synthesized from linolenic acid via an inducible octadecanoic pathway. They act as plant hormones by regulating developmental processes and responses to environmental cues that include root growth, pollen development, abscission, senescence and responses to wounding and pathogens infection.
NACs are a family of genes specific to plants that play a role in a diverse set of developmental processes. The encoded proteins are characterized by highly conserved domains in the N-terminal region, whereas their C-terminal sequences are divergent in both length and amino acid composition. As a family, NACs show no definitive sequence homology to any other proteins and do not present any known functional motif. 
Previous studies suggested an involvment of some Arabidopsis thaliana NAC genes in different hormone signal transduction pathway. As a matter of fact at least two NAC genes (NAC4 and NAC5) have been shown to be upregulated in response to MeJa treatment. 
Two-hybrid assays using NAC5 protein as a bait, identified two putative NAC5 interacting proteins (NAC5-ip). 
Here we present the characterization of ProtX, a NAC5-ip, that showed no homology with any other known protein. The full length cDNA of ProtX was isolated and placed either in sense or antisense direction, under the control of both 35S promoter and XVE glucocorticoid inducible system. These constructs were used to obtain Arabidopsis thaliana transgenic plants. We will present the phenotypical and molecular characterization of plants that misexpress ProtX gene in response to methyl jasmonate as well as other stress-related hormones. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.10

BOTRYTIS CINEREA INFECTION ACTIVATES ETHYLENE AND JASMONIC ACID-RELATED GENE EXPRESSION IN HARVESTED TOMATO FRUITS

De Martinis D.

ENEA, Ente Nazionale per le Nuove Tecnologie, Energia e Ambiente, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione di Genetica e Genomica Vegetale, C.R. Casccia, Via Anguillarese 301, 00060, Roma, Italy

plant-pathogen interactions, tomato, fruit, Botrytis cinerea, ethylene, jasmonic acid, gene expression

The aim of our study is to identify (new) genes related to (new) metabolic pathways or regulatory pathways that affect fruit spoilage, nutritional quality and marketability.
We are studying the mechanism of fruit spoilage derived from Botrytis cinerea (Botrytis) mould in tomato berry fruit. Botrytis is a plant pathogenic fungus that infects over 200 different plant species and provokes significant crop losses, particularly after harvest. Infection results in volatile compounds release from the tomato berry fruit; ethylene, ethanol, short chain aldehydes (hexenals) and carbon dioxide, that may be useful markers to monitor the physiological status of the infected fruits, as early signals of disease progression. We used a RT-PCR approach to monitor fruit (tomato) and fungus (Botrytis) gene expression related to the monitored volatile compounds in fruit in standard and infected conditions. We monitored gene related expression of ACCsynthase (ACS) and ACCoxidase (ACO) for ethylene biosynthesis, lipoxygenase (LOX), allene-oxide synthase (AOS) hydroperoxide lyase (HPL) for the biosynthetic pathway of potential natural pesticides (hexenals and jasmonic acid). Preliminary analysis in infected tomato fruits  provided the expression profile of this gene pool during disease progression, clearly showing the activation of ACO and AOS genes and the Jasmonate/ethylene-dependent PDF1.2 gene. Our analysis based on the monitoring of specific metabolic compounds related to fruit ripening/spoilage/pathogenesis will indicate “key” time-point of physiological changes of the fruit. Those key points will be evaluated for cDNA library construction that will be used to study massive differential gene expression by serial analysis of gene expression (SAGE) and by cDNA-microarray technology. The expression profiling will enable the analysis of thousand of genes, thus enabling the isolation of cDNA clones whose expression is related to spoilage/pathogenesis maybe unravelling new metabolic pathway or differential regulation of described ones. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.11

GALA APPLE TRANSFORMED FOR SCAB (VENTURIA INAEQUALIS) RESISTANCE WITH CLONED VF GENE REGION CONSTRUCT

BELFANTI E.*, BARBIERI M.*, TARTARINI S.*, VINATZER B.*, SANSAVINI S.*,
DILWORTH E.**, GIANFRANCESCHI L.**, HERMANN D.**, PATOCCHI A.**, GESSLER C.**

*) Dipartimento di Colture Arboree (DCA), University of Bologna, Italy
**) Plant Pathology, Institute of Plant Science, ETH Zurich, Switzerland

Venturia inaequalis, Malus  domestica, Vf-resistance gene cloning, genetic transformation, GM-apples

Scab is the most serious disease of apple in many districts throughout the world and can require more than 15-20 treatments per year. The monogenic resistance conferred by the Vf gene against Venturia inaequalis has been employed in traditional breeding programs for over fifty years. Despite the many cultivars that have been released from these efforts, the problem has not yet been resolved because the resulting fruit quality is less than satisfactory and because this kind of vertical resistance has been overcome by V. inaequalis races 6 and 7 in several European countries. Eight years ago, Bologna’s DCA and Zurich’s ETH undertook the cloning of the Vf gene starting from a saturated map; the markers AL07 SCAR and M18 CAPS proved to be closely linked to the Vf gene.

The BAC library derived from cv. Florina extended more than five times the size of the apple haploid genome itself. Chromosome walking was than used to screen this library with Vf marker and yielded a contig of the Vf locus of 550 kb. Recombinant analysis of the seedling population enable the restriction of the contig to 350 kb made up of 5 BAC clones, including the Vf region. The next step was the construction of a cDNA library from Florina leaves elicited with V. inaequalis conidia ; this library’s subsequent screening yielded more than one hundred hybridizing cDNAs. A cluster of receptor-like genes homologous to Cf Cladosporium fulvum resistance gene family of tomato was identified, sequenced, isolated from the Vf region and named HcrVf sequences.

A plasmid construct of the HcrVf2 sequence (about 2,000 base pairs), selected among several of the same cluster , was introgressed mediated by Agrobacterium tumefaciens into Gala leaf and internode explants using the binary vector pCambia2301 (containing two nptII genes and a GUS reporter gene with its promotor CaMV35s). PCR assays on about 50 regenerated plantlets after 15 months on selection medium showed that almost 90% had HcrVf2 and the control genes. The acquired resistance was corroborated by repeated in vitro infection with V. inaequalis conidia: on the transformed plants V. inaequalis conidia germinated and formed appressoria, however on most leaflets of particular transformant lines no stroma was observed, contrary on untransformed controls extended stroma was observed in most leaflets. A particular transformant line did not express resistance and V. inaequalis was able to form stroma similar to the control plants. The definitive demonstration of acquired resistance is to be conducted in vivo with artificially and naturally inoculated plants.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.12

MOLECULAR CLONING OF A TRYPSIN INHIBITOR CDNA FROM SNAIL MEDIC (MEDICAGO SCUTELLATA) SEEDS

BALESTRAZZI A.*, CONFALONIERI M.**, TAVA A.**, ODOARDI M.**, CARBONERA D.*

*)Dipartimento di Genetica e Microbiologia “A. Buzzati-Traverso”, Università di Pavia
**) Istituto Sperimentale Colture Foraggere - MiPAF, Lodi

Medicago scutellata, proteinase inhibitors

Proteinase inhibitors from plants have been intensively investigated in order to define their chemical structure and biological function. They act as defence proteins in plants against pathogen attack, regulators of endogenous protease activity and as storage proteins. In a previous study the expression pattern of MsTI gene from Medicago scutellata, an annual pasture legume, has been evaluated by northern blot analysis, using an heterologous probe from Medicago sativa. Following exposure to several biotic and abiotic stresses, both local and systemic accumulation of MsTI mRNA was observed. Moreover, the presence of a multigene family has been demonstrated. We have recently isolated a small (180 bp) cDNA fragment, encoding the MsTI protein. The MsTI cDNA was obtained by RT-PCR performend on poly(A)+ mRNA purified from immature seeds using degenerated oligonucleotides. The nucleotide sequence showed an homology of 75% compared with the corresponding sequence of the ATI inhibitor from M. sativa. Isolation of both 5’ and 3’ cDNA ends of the MsTI cDNA will be performed using a RACE-based approach. The availability of the full-lenght MsTI cDNA will allow performing structural studies on the corresponding recombinant protein as well as production of transgenic plants for insect pest resistance. Identification and functional characterization of the wound-inducible MsTI promoter will be also relevant for biotechnological applications.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.13

ATCYS FROM ARABIDOPSIS THALIANA ENCODES A CYSTEINE-PROTEASE INHIBITOR THAT FUNCTIONS AS A NEGATIVE REGULATOR OF HYPERSENSITIVE CELL DEATH

BELENGHI B., PERAZZOLLI M., DELLEDONNE M.

Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy
 massimo.delledonne@univr.it

cysteine protease inhibitors, nitric oxide, hypersensitive cell death, plant-pathogen interaction

In plants, cysteine protease inhibitors (cystatins) are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. The genome of Arabidopsis most likely contains no more than two cystatins. We have identified and characterized one member of this family (Atcys) which encodes a protein of 102 amino acids and contains the conserved motif of the cystatin superfamily (gln-val-val-ala-gly). Atcys was constitutively expressed in roots and in developing siliques. In leaves, it was strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). Overexpression of Atcys blocked cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both Arabidopsis suspension cultured cells and in transgenic tobacco plants. However, the drastic inhibition of hypersensitive cell death following infection with the avirulent pathogen did not reduce the host capacity to control the spread of pathogenic infection, suggesting that Atcys has a role in plant defense. Furthermore, the observed inhibition of NO-mediated cell death in Atcys overexpressors indicated that NO is not cytotoxic for the plant and functions as cell death trigger through the stimulation of an active process, in which cysteine proteases appear to play a crucial role.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.14

A TOBACCO MAP KINASE MODULE INVOLVED IN CYTOKINESIS

CALDERINI O.*, HEBERLE-BORS E.**, Wilson C.**

*) IRMGPF-CNR, Perugia, Italy
**) Institute of Microbiology and Genetics, University of Vienna, Austria

MAP kinase, cytokinesis, protein-protein interaction

Numerous plant molecules  belonging to a typical MAP kinase signalling module (MAP kinase, MAP kinase kinase, and MAP kinase kinase kinase) have been identified. Recent studies have started to link some of these molecules into a functionally significant interacting partnership. In particular  three tobacco MAP kinase-pathway members and a kinesin have been involved in a module that operates during cell division.

In synchronised cell suspension cultures the activation of the tobacco p43Ntf6 MAP kinase is regulated in a cell cycle-dependent manner, occurring during anaphase/telophase. The p43Ntf6 protein localises at the phragmoplast equator (1). Similarly the tobacco MAP kinase kinase kinase NPK1 and the kinesin NACK1also localise at the equatorial zone of the phragmoplast and expression of dominant-negative mutants of both NPK1and NACK1 results in defects in cytokinesis (2,3). A novel tobacco MEK, NtMEK1, was identified in a yeast two-hybrid screen using p43Ntf6 as bait. NtMEK1 is a MAP kinase kinase that can interact with and activate p43Ntf6 (4). NtMEK1 is reported to interact with NPK1 (2), therefore a MAP kinase module composed of NPK1, NtMEK1, and p43Ntf6 might have a role during cytokinesis in tobacco cells. Characterization of novel members of the pathway is in progress to further clarify the mechanism of cell plate formation in plants.

1. Calderini, O., Bögre, L., Vicente, O., Binarova, P., Heberle-Bors, E., Wilson, C. (1998) J. Cell Sci. 111, 3091-3100.
2. Nishihama, R., Ishikawa, M., Araki, S, Soyano, T., Asada, T., Machida, Y. (2001) Genes Dev. 15, 352-363.
3. Nishihama R, Soyano T,Ishikawa M,Araki S, Tanaka H, Asada T, Irie K, Ito M, Terada M, Banno H (2002) Cell 109: 87-99.
4. Calderini, O., Glab, N., Bergounioux, C., Heberle-Bors, E., Wilson C. (2001) J. Biol. Chem. 276, 18139-18145.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.15

The albino nd-1 mutant of sunflower is impaired at THE -carotene desaturase step in carotenoid biosynthesis*

CONTI A.*, PUGLIESI C.*,****, FAMBRINI M.*, MICHELOTTI V.*, PANCALDI S.**, BONORA A.**, SALVINI M.*,***

*) Università di Pisa, Dip. Biol. Piante Agr. Sez. Genetica, Via Matteotti 1/B 56124 Pisa
**) Università di Ferrara, Dip. delle Risorse Naturali e Culturali, Sez. Sez. Botanica, C.rso Ercole I D'Este 32 44100 Ferrara
***) Scuola Normale Superiore, Piazza dei Cavalieri 7 56126 Pisa

Helianthus annuus, carotenoids, mutant, -carotene

Carotenoids are isoprenoids essential for all photosynthetic organisms with interesting applications for human health and many industrial products as feed additives, cosmetics and pharmaceuticals. In fungi, bacteria and higher plants the cloning of several carotenogenic genes makes it possible the biotechnological alteration of pathways. Recently, a new carotenoid biosynthesis has been identified (1-Deoxyxylulose-5-phosphate pathway) in bacteria and plants which is responsible for Isopentenyl diphosphate synthesis (IPP), a precursor of Geranylgeranyl diphosphate (GGPP). The conversion of GGPP to phytoene is the first committed step in carotenoid biosynthesis. In plants, phytoene and -carotene desaturase are required to produce lycopene. It can undergo cyclization and hydroxylation to yield xanthophylls. Several biochemical and molecular informations have been reported for phytoene desaturase but about -carotene desaturase, the data are more incomplete. This carotenogenic enzyme catalyzes the two desaturation steps to lycopene by introducing two double bounds at position 7,8 of -carotene and 7’,8’ of neurosporene. To date, the better characterization of the -carotene desaturase (Zds) gene has been conducted in Capsicum annuum and the vp9 of maize is the first mutant recovered for -carotene accumulation. Here we report the analysis by HPLC of the carotenoid content in the viviparous nd-1 sunflower mutant (Fambrini et al., 1993), demonstrating that under very dim light, cotyledons and leaves accumulate a high level of -carotene. The Zds gene of Helianthus annuus has been isolated and we present results on the molecular characterization of nd-1.

Fambrini M., Pugliesi C.., Vernieri P., Giuliano G., Baroncelli S.  Theor. Appl. Genet. 87: 65-69, 1993.
* This work was supported by Grant of Scuola Normale Superiore.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.16

Monitoring the expression pattern of 624 tomato genes under white/blue light in both wild type and mutants deficient in cryptochrome 1

Giorno F.*, Giliberto L.**, Maugliani A.*, Chiesa M.*, Perrotta G.*

*) ENEA Trisaia Research Center, 75026 Rotondella, Matera
**)ENEA Casaccia Research Center, PO Box 2400, 00100AD Roma, Italy

blue light photoreceptors, expression profiling, microarrays

Cryptochromes (cry) are blue light photoreceptors that mediate many photomorphological responses in land plants such as hypocotyl shortening, cotyledon expansion, induction of anthocyanin synthesis and the control of flowering time. Though Arabidopsis codes for two cryptochromes (Cry1 and Cry2), in angiosperms the CRY gene family seems to span from 2 to 3 elements. Several evidences indicate that Cry1 is the major photoreceptor mediating most of the blue light induced responses under high irradiances, while Cry2 enhances the effect of Cry1 at lower irradiances and  promotes flowering.
Despite the relevance of the physiological changes induced by blue light perception, the  molecular and functional characterization of cryptochromes has been partially elucidated only in Arabidopsis and tomato. Besides, very little is known about the signalling mechanism and the possible molecular targets of the cryptochromes.
To get further insight the functional interactions between cryptochromes and other molecular species possibly involved in the blue-light responses, we have compared the expression profiling of both tomato wild type and a cry1 deficient mutant probing a tomato cDNA microarray.
In order to characterize the tomato coding sequences for the microarray construction, we have sequenced 1904 clones from a fruit ripening cDNA library. Sequences have been blasted against GenBank, assembled and clustered using the software tools available in GCG Wisconsin Package.  As a result 561 non redundant clones representing as many tomato expressed genes has been selected, PCR amplified and spotted over the microarray. The information stored over the microarray has been then further implemented by spotting 63 RT-PCR products corresponding to genes known to be involved in light transduction signals in tomato or in other plant species.
For profiling experiments, tomato seedlings from wild type and cry1 lines have been grown under dim white light for 2 weeks and then exposed to intense blue light for 2hr and 6 hr. At each step part of the plant material has been harvested and RNA extracted. Later, RNA from tomato wild type and cry1, grown under 2hr and 6 hr of blue light, respectively has been used for synthesis of fluorescent cDNA and compared to the corresponding cDNA from tomato grown in dim white light, by microarray hybridisation. In a second set of experiments the labelled cDNA from wild type has been compared to that of cry1 in conditions of dim white light, 2 hr and 6 hr of blue light treatment, respectively.  
Data analysis is currently in progress in order to select genes which possibly show appreciable differences in their expression levels and therefore are candidates to play a role in the mechanism of action of tomato cryptochrome 1. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.17

LIGHT-INDUCED EXPRESSION OF A DEHYDRIN-ENCODING GENE OF SUNFLOWER

GIORDANI T., NATALI L., MAESTRINI P., MARTINELLI F., VERNIERI P., LERCARI B., CAVALLINI A.

Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti 1/B, 56124 Pisa

dehydrin, gene expression, light, promoter, sunflower

Dehydrins are an immunologically distinct family of proteins, produced by plants in the late stages of embryo development, but also following any environmental stimulus involving dehydration, such as drought or cold stress and salinity, as key components of dehydration tolerance. A dehydrin cDNA, HaDhn1, induced by drought stress, has been isolated and sequenced in sunflower, whose accumulation has been found to correlate to drought tolerance; this gene is expressed in the latest stages of Helianthus annuus embryogenesis, depending on abscisic acid accumulation; HaDhn1 transcripts accumulated after drought stress even in ABA-deficient sunflower mutants. Moreover, HaDhn1 is expressed also in plantlets exposed to low temperatures.
We have tested the effects of light quality on the expression of this gene. Plantlets of sunflower were germinated in the dark and, after 5 days, treated with different lights for 3, 6, 12, and 24 hours. Exposure to white light determined HaDhn1 transcript accumulation in the cotyledons, compared to plantlets grown in the dark. No differences in abscisic acid production were observed between light- and dark-grown plantlets. HaDhn1 transcripts increased also treating plantlets with monochromatic lights, either red and blue. However, red light induced higher transcripts levels than blue light, indicating phytochrome involvement in light signal transduction. Further experiments, performed saturating active phytochrome by yellow light, in combination or not with blue light, showed that also cryptochrome is involved in the HaDhn1 transcripts accumulation after exposure to light.
To verify possible mechanisms of induction of gene expression we have isolated and studied the putative promoter sequence of the HaDhn1 gene. Sequence analysis showed that different putative cis-elements recognizable by transcription factors occur in the isolated sequence: beside the putative TATA box (at -76 bp), one ABA-responsive element, one dehydration responsive element (DRE), two MYB- and one MYC- related elements were found. Moreover, a putative light-responsive G-box was found.
We are now performing Western blot analysis to verify if HaDhn1 RNA synthesis is followed by dehydrin-protein accumulation or not. If not, light-induced HaDhn1 gene expression might be considered as a preliminary response to possible drought stress conditions that can occur during the day in association to high light intensity.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.18

MADS-BOX GENES CONTROLLING THE FLORAL TRANSITION IN RICE

KATER M.M.*, FORNARA F.**, PELUCCHI N.**, MONTI G.**, GREGIS V.**, COLOMBO L.**

*) Dipartimento di Genetica e Biologia dei Microrganismi 
**) Dipartimento di Biologia, Università di Milano, Via Celoria 26, 20133 Milano, Italy

MADS-box transcription factor, protein interactions, flowering, rice

In plants MADS-box genes are involved in various steps of vegetative and reproductive development. In particular, their role is essential in the regulation of the floral transition and in the determination of identity of the inflorescence, and floral meristems and flower organs.
Recently, we have isolated three rice MADS-box genes putatively involved in the transition to flowering: OsMADS18, OsMADS22, OsMADS47. Phylogenetic analysis places OsMADS18 in the SQUA sub-family, closely linked to AP1 and FUL from Arabidopsis. The predicted OsMADS18 amino acid sequence is also highly homologous to PFG, a Petunia gene which promotes flowering. OsMADS22 and OsMADS47 belong to the StMADS11 clade together with SHORT VEGETATIVE PHASE (SVP) from Arabidopsis, a MADS-box gene involved in flowering repression.
In situ expression analysis showed that OsMADS18 and OsMADS22 transcripts can be detected in all plant tissues at different developmental stages and in particular in meristematic tissues. As revealed by northern blot analysis, OsMADS47 expression is restricted to leaves. 
We have started with a transgenic approach to study the function of these genes by overexpression and by down-regulation using RNAinterference. Transgenic rice plants in which OsMADS18 was overexpressed have been obtained and analysed. These plants show a reduction in growth and flower earlier in respect to wild-type plants. Expression analysis showed that OsMADS47 is down-regulated in OsMADS18 overexpressing plants. Based on these data together with protein-protein interaction data a working-model to explain the involvement of MADS-box factors in the floral transition will be discussed.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.19

AUXIN MEDIATES A DISACCHARIDE SIGNALING IN ARABIDOPSIS THALIANA

Loreti E.*, Novi G.**, Poggi A.**, Gonzali S.**, Paolicchi F.**, Alpi A.**, Perata P.***

*) Institute of Agricultural Biology and Biotechnology, CNR, Pisa
**) Dept. of Crop Plant Biology, University of Pisa
***) Dept. of Agricultural Sciences, University of Modena and Reggio Emilia, ITALY

auxin, sugar-sensing, cross-talk

We used sucrose analogs to study a sucrose-specific signaling pathway in Arabidopsis thaliana. Addition of sucrose analogs during the post-germinative  phase results in an altered phenotype of the seedlings, showing reduced hypocotyl and root growth. The effects of sucrose analogs on gene expression was evaluated using DNA microarrays. Mutants insensitive to the analogs used were identified. Overall the results suggest that sucrose analogs mimic the effects of sucrose as a  signaling molecule, and that the long-term effects observed in seedlings treated with the sucrose analogs are due to auxin overproduction as a consequence of  sucrose sensing. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.20

Oligogalacturonides Inhibit the induction of Late but not of Early Auxin-Responsive Genes Involved in Differentiation

MAURO M.L.*, DE LORENZO G.**, BELLINCAMPI D.**

*) Dipartimento di Genetica e Biologia Molecolare
**) Dipartimento di Biologia Vegetale 
Università "La Sapienza"di Roma, P.le Aldo Moro 5, 00185 Roma

auxin-regulated genes, oligogalacturonides, differentiation, tobacco

From recent studies in animals and plants the notion has emerged that disease resistance mechanisms and developmental processes may share regulatory signals .Oligogacturonides (OG), well known elicitors of plant defence responses, exhibit various regulatory effects on plant growth and differentiation.
OGs are generally considered localised active signals and the effects induced by OGs in plant defence and development are considered to be receptor-mediated.
The analysis of different responses produced by OGs suggests the existence of different pathways leading to the activation of defence responses or the regulation of developmental processes. In many of these latter effects OGs seem to act as antagonists to auxin.
To investigate on the mechanism of the antagonism of OGs to auxin, we analysed the effect of different auxin-responsive promoters of genes likely to be involved in developmental and growth processes.
Auxin-responsive genes are classified as early or late-responsive on the basis of the kinetics of their response to the hormone. In this work, we examined genes belonging  to both classes. 
Analysis by GUS reporter gene shows that submicromolar concentrations of OGs with a DP of 9 to 18 inhibit the late auxin-regulated activation of the promoters of the genes pNt114 from tobacco, prolB and prolD from Agrobacterium rhizogenes in tobacco seedlings. In contrast, OG concentrations as high as 100 g/ml, do not affect the early activation of pGm-GH3 from soybean, indicating  that this promoter  responds to auxin via an OG-insensitive signal  transduction pathway. 
To confirm our results, we analysed by RNA gel blot the effect of OGs on two additional early auxin-responsive tobacco genes: Nt-gh3, a GH3 related gene and Nt-iaa 2.3/Nt-iaa 2.5, belonging to the Aux/IAA large multigene family. We demonstrate that, in addition to pGm-GH3 these early responsive genes are insensitive to OGs. Moreover histochemically analysis shows that the inhibitory effect exerted by OGs was not tissue-specific. The OG effect is not correlated with  the auxin concentration necessary to activate the genes. These results taken toghether with a comparative analysis of promoter sequences suggest the possibility that OG-triggered transduction pathway(s) intersect(s) those induced by auxin far downstream of the perception event. A model of OG as auxin antagonist will be presented. The signalling element(s) at the intersection(s) between the OG and IAA transduction networks remains to be identified.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.21

Mapping and dissection of AtMYB11 promoter from Arabidopsis

Petroni K., Calvenzani V., Allegra D., Tonelli C.

Dipartimento di Genetica e Biologia dei Microrganismi, Via Celoria 26, 20133 Milano, Italy

transcription factors, MYB-like genes, Arabidopsis, meristem, promoter

In vertebrates MYB proteins form a small family with a central role in controlling cellular proliferation, cell cycle progression and differentiation, while higher plants contain a large number of MYB genes with mainly unknown functions. Despite single- and three-repeat MYB proteins have been found, the R2R3-MYB subfamily remains predominant in plants. In Arabidopsis, more than 125 R2R3-MYB genes have been identified, representing one of the widest family of plant transcription factors described. The information available on the function of a few plant MYB proteins suggest an important role of this family in various processes like: the regulation of metabolic pathways, the control of cell division and of plant morphogenesis, the response of plants to different stresses and in hormone signal transduction.

One of the MYB genes under study in our laboratory is expressed throughout flower development and in 4 days-old seedlings, where the activation is mediated by light. Furthermore, in 4 days-old seedlings the transcript is mainly present in the vegetative meristem and in cotyledons, while during flower development the transcript is present in the inflorescence meristem and floral apex, in all flower bud except sepals, in ovule primordia, mature ovules, ovary wall and in the epidermis of embryos. Aim of the present work is the exact mapping of the full-length promoter and the identification of the cis-regulatory element which drives the expression of this gene in a tissue-specific and developmental-specific manner within the embryo epidermis and /or the flower.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.22

STUDY OF DIFFERENTIALLY EXPRESSED GENES IN APPLE

Toller C., Zini E., Baldi P., Komjanc M.

Istituto Agrario di San Michele all’Adige- via E. Mach n°1-38010 San Michele all’Adige (TN)

apple, PCR-select, differentially expressed genes

For years the Agricultural Institute of San Michele all’Adige carried out the study of the plant - pathogen interaction in apple and recently has started a project called “Advanced  Biology Applied to Grape, Apple and Salmons” financed by the Caritro foundation.

In the project the apple research is developed on three principal lines: MAS (marker assisted selection) for scab and oidium resistance; identification of molecular markers related to Va scab resistance gene and isolation and cloning of resistance genes analogous and differentially expressed sequences related to fungal infection resistance and salicylic acid treatment.  

Our contribution is focused on the isolation and characterisation of differentially expressed genes and sequences associated to signal transduction pathways involved in apple defence mechanisms.

In this study the PCR-Select technique is used (kit “PCR-Select cDNA Subtraction” by Clontech); method already used by E.Wang et al. (Nature Biotechnology 2001) for the identification and isolation of rare and differentially expressed genes.

Literature data say that such method is more efficient and effective in identifying rare genes than the differential display and  cDNA AFLP.

The current study is turned to the molecular analysis in order to identify defence and signal transduction genes during salicylic acid treatment and SAR (systemical applied resistance) response. 

At present 347 clones have been isolated and 121 of these sequenced. 

The results revealed 8 most frequent sequences; the two most representative genes were a proline synthetase and a beta-1,3-glucanase (with a frequency respectively of  21.5 % and 17.3 % on the total of the sequenced clones). 

It’s interesting to note also the presence of a sequence coding for a hypothetical protein highly homologue (89% of identity) to the 9 multispanning membrane protein family. 

All these genes are known to be induced by salicylic acid treatment and/or fungal infection, but have never been studied in apple.

Our next step will be to confirm, by Northen analysis, the real expression of these genes in the leaf  tissue.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.23

POLYAMINE APPLICATION DELAYS PEACH (Prunus persica L.) FRUIT RIPENING INDEPENDENTLY OF INHIBITION OF ETHYLENE BIOSYNTHESIS 

ZIOSI V.*, SCARAMAGLI S.*, BREGOLI A.M.**, RASORI A.***, BIONDI S.*, COSTA G.**, TORRIGIANI P.*

*) Dip. Biologia evoluzionistica sperimentale 
**) Dip. Colture Arboree, Università di Bologna
***) Dip. Agronomia Ambientale e Produzioni Vegetali, Agripolis, Università di Padova

polyamines, ACC synthase, ACC oxidase, ethylene biosynthesis, peach fruit ripening

Polyamines (PAs) are ubiquitous plant growth regulators, e.g. tobacco mutants overproducing PAs display vegetative and floral abnormalities; moreover, which can affect molecular events in signal transduction. The interaction between PAs and ethylene is much studied since they share the common precursor SAM, a possible metabolic switch, depending upon the physiological conditions (Biondi et al. 1998 Protoplasma 202, 134-144). During fruit development a relationship between increases in PA content and cell division has been demonstrated (Biasi et al. 1991 Plant Physiol Biochem 29, 497-506). In fact, PA titres generally decline during peach fruit development and ripening (stages S1–S4) (Kushad 1988 J Am Soc Hort Sci 123, 950-955). Previous results show that exogenous PAs affected ethylene synthesis and flesh firmness in ripening Redhaven peaches modestly altering the endogenous levels (Bregoli et al. 2002 Physiol Plant 114, 472-481).

In the present work, several PAs and aminoethoxyvinylglycine (AVG), the ACS activity inhibitor, concentrations were applied on peach trees (Prunus persica L. Batch cv. Stark Red Gold) in middle S3 (87 dAFB) and in S4 (107 dAFB) fruit growth stages. Control and treated nectarines were selected for uniform size and sampled at weekly intervals while (commercial) harvest was performed at 115 dAFB. The double sigmoidal fruit growth curve was established, quality traits analysed and ethylene measured. PA titresjj were evaluated by HPLC; a peach SAMDC gene fragment was obtained by PCR; expression studies were performed by northern analysis on SAMDC transcript levels as well as on those of the ethylene biosynthetic genes ACS and ACO. 

Results show that PA treatment delays flesh softening and affects other parameters earlier than inhibition of ethylene emission while reduction of fruit drop was achieved by earlier PA applications. Moreover, no residual polyamines were detected in addition to the endogenous complement in the epicarp or mesocarp one month following treatment. Transcript levels of ethylene biosynthetic enzymes were repressed or not affected. Equally, expression of polyamine biosynthetic genes was repressed or not affected depending upon concentration. AVG application led to similar effects. These results lend support to the contention that PAs delay ripening independently of ethylene. 

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract -5.24

ANALYSIS OF EMBRYO-SPECIFIC MUTANTS IN MAIZE REVEALS THAT EARLY ARREST IN MAIZE EMBRYOGENESIS IS ASSOCIATED WITH IRREGULAR CELL DIVISIONS 

CONSONNI G., BARBANTE A., BRETTSCHNEIDER R., ASPESI C., DOLFINI S., GIULIANI C., GIULINI A., HANSEN S., PILU R., GAVAZZI G.

Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano

emb mutants, embryogenesis, morphogenesis, program cell death, Zea mays

The process that, following embryogenesis, leads to embryo formation appears to follow a defined pattern whose sequential developmental steps under strict genetic control can be analyzed through the analysis of mutants affecting embryogenesis. In this paper we present the analysis of four embryo specific (emb) mutants of maize arrested at the proembryo or early transition stage and defining five separate genes on the basis of their chromosomal location and complementation pattern.
A common feature emerging from their histological analysis is that suppression of morphogenesis is accompanied by an uncontrolled pattern of cell division, an indication that cell division and differentiation are interconnected events. The block in embryo development is associated with an abnormal suspensor proliferation possibly due to the absence of a signal elaborated by the embryo proper and required for suspensor cell identity maintenance.
Mutant endosperm development is not impaired as shown by the formation of the expected domains, i.e. the aleurone, the starchy endosperm, the embryo surrounding region and the basal endosperm transfer layer.
The program of cell death appears impaired in the mutants, as expected if this process is essential in determining the shape and morphology of the developing organs.
An unexpected result is obtained when mutant embryos rescue is attempted. Immature embryos in fact transferred to a basal medium germinate yielding small but otherwise normal seedlings, an observation not consistent with the histological evidence of a complete absence of morphogenetic potential.
The analysis of emb mutants appears a promising tool to elucidate crucial points of embryo development such as the coupling of cell division with morphogenesis, the cell to cell interactions, the relationship between embryo and endosperm development and the interaction between embryo proper and suspensor.

Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1

Poster Abstract - 5.25

PROJECT EURICE: ANTIFUNGAL GENES IN RICE AS A STRATEGY FOR CROP PROTECTION – SECOND YEAR ACTIVITIES

LUPOTTO E. - (coordinator) on behalf of the EURICE (QLK5-CT1999-1484) Consortium

Istituto Sperimentale per la Cerealicoltura, via Stezzano 24, 24126 Bergamo (Italy)

biotechnology, crop protection, fungal pathogens, defence genes

The Project EURICE aims to improve and evaluate the use of genes involved in defence against pathogenic fungi in the control of rice blast (Magnaporthe grisea) and other fungal diseases, as major pests for rice in Europe. This approach aims to create new rice genotypes enhanced for resistance and addresses to the obtention of modified cultivars protected against massive attacks of the pathogens, as well as to an evaluation of the genetically modified genotypes in the environment. Three genes of different origin, shown to be implicated in the process of fungal infection, were chosen as potential source of resistance because of their different characteristics and modality of action: the maize gene b32, the fungal gene AFP, and the insect gene cecA. They were initially cloned under the strong monocot promoter ubi1 of maize and introduced into chosen European rice varieties (Oryza sativa L. ssp. japonica). The rice genotypes chosen are representative of the main rice cultivating regions in Europe: Italy, Spain, Greece and France. First events of transformation were subjected to molecular and genetic analyses; expression of the transgenes in leaf tissues at various stages of plant development was confirmed, and seed amplified for the first field trial, which is being carried out in Italy, summer season 2002. Meantime, the most susceptile old Italian variety Maratelli was transformed with single and combination of genes in order to assess the “gene pyramiding” strategy. To date, in vitro assays and pathogenicity tests on primary reganerants and T1 plants confirmed protection against leaf blast for at least two of the three genes under study. During the second year, a potential inducible promoter was also identified and confirmed to be suitable for the project’s purposes: the maize ZmPR4 promoter cloned from maize. Expression vector were constructed with the antifungal genes driven by the short version of ZmPR4 and plants are being raised. The conventional field trials carried out in Greece within the project, established an efficient protocol for the evaluation of leaf and neck blast disease in the field, by means of the Honey Comb design, and pathogenicity assays in greehouse conditions supported by confocal microscopy analyses allowed to define parameters for a detailed study of the plant/pathogen interaction. Objectives and detailed results of the second year activities are reported in the poster.