SESSION I - GENE REGULATION

ORAL COMMUNICATION

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S1a

THE LONG AND SHORT OF RNA SILENCING

D. BAULCOMBE

RNA silencing is a eukaryotic genome defence system that involves processing of double strand (ds) RNA into 21-26nt short RNAs. The short RNA mediates sequence specific suppression of genes corresponding to the dsRNA. In plants RNA silencing is primarily an antiviral defense system. However, during evolution, it seems that there has been duplication and functional differentiation of the genes required for silencing. Consequently there are now different versions of the silencing mechanism operating in the nucleus and cytoplasm and that are targeted at transcription, RNA stability, translation and possibly other processes required for gene expression. I will describe our progress to unravelling the complexity of RNA silencing through the analysis of gene families and different types of short RNA. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S1b

HOW TO KILL TWO BIRDS WITH ONE STONE: ACTIVATION OF THE MYBLEU-REGULATED PATHWAY ALLOWS RICE TO TOLERATE AND ESCAPE ANOXIA
C. VANNINI*, M. BRACALE*, M. MARSONI*, F. LOCATELLI**, S. MANCUSO***, F. FAORO****, A. GENGA**, I. CORAGGIO**

*) Dipt di Biologia Strutturale e Funzionale, Università dell’Insubria, via J.H. Dunant 3, 21100 Varese, Italy
**) Istituto di Biologia e Biotecnologia Agraria, C.N.R., via E. Bassini 15, 20133, Milano, Italy
***) Dipt. Ortoflorofrutticoltura-UniFI, viale delle Idee 30, 50090 Sesto Fiorentino (FI)
****) Istituto di Virologia Vegetale, CNR, via Celoria 2, 20133 Milano, Italy
Mybleu is a natural incomplete transcription factor of rice, consisting of a partial Myb repeat followed by a short leucin zipper. In rice, its presence is restricted to the apical region of roots and coleoptiles. We previously demonstrated its ability to dimerize with canonical leucin zipper and selectively activate transcription. To identify the biological function of Mybleu, we constitutively expressed the Mybleu coding sequence under the control of the constitutive CaMV35S promoter in transformed A. thaliana plants. The localization in the apical region and the expression in both aerobic and anaerobic conditions in rice coleoptiles, and only in aerobic conditions in roots, suggested a Mybleu involvement in cellular differentiation and/or elongation. According to its putative role in cell elongation in the differentiation zone, its constitutive expression induces a change in the root apex morphology. Mybleu expression in A. thaliana, dramatically increases hypoxia tolerance, measured both as survival and ability to germinate. One of the most toxic effects of anoxia/hypoxia depends on the lowering of the cellular pH; on the other hand cell elongation is mediated by H+ extrusion in the cellular wall. According, we supposed a Mybleu role in the proton pump function at low oxygen concentration. Actually, measuring the ionic fluxes at several O2 concentrations, we demonstrated the ability of Mybleu expressing plant to preserve pump functionality (K+ and H+) at low oxygen pressure. We also demonstrated the ability of the Mybleu expressing transgenic plants, in contrast with the wild type, to elongate under hypoxia. Finally we assayed the energetic charge in wt and transformed plants in aerobic and hypoxic conditions.

Mybleu is the first and up to now the only gene able to confer tolerance to low oxygen condition. Its ability to confer both hypoxia tolerance and ability to growth in limiting oxygen environments suggests that it is a key component in the rice coleoptile peculiarity to survive and grow in submerged soil up to the air.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication - S1c

TRANSCRIPTIONAL ACTIVATION OF RETROTRANSPOSONS INDUCES DESICCATION TOLERANCE IN CALLUS TISSUE OF CRATEROSTIGMA PLANTAGINEUM

A. FURINI, L. BORGATO, S. VAROTTO, F. SALAMINI, D. BARTELS

Dip. Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona

Drought tolerance in the resurrection plant Craterostigma plantagineum is restricted to fully developed plants. In callus this phenomenon can be induced by treatment with abscisic acid. 

Based on T-DNA activation tagging, an element (CDT-1) was isolated whose induced transcription resulted in the formation of a desiccation-tolerant callus in the absence of exogenously applied abscisic acid. Craterostigma genes normally induced by desiccation are constitutively induced in the transgenic callus. Hybridization analysis with the cDNA probe revealed that the CDT-1 is a member of a gene family; the analysis of cDNA and genomic clones indicates that the isolated element do not resemble canonical members of so far known plant signalling pathways, since it codes for unusual primary transcript with no large ORFs for proteins. Transcribed regions of the CDT-1 are flanked by short direct sequence repeats. A sequence alignment of these direct repeats shows that they are of different lengths, but carry common core sequences. This feature of repeats suggests a close analogy to sequence duplications flanking the integration target sites of eukaryotic retrotransposons. 

Further experiments demonstrated that the polyadenylated transcripts of CDT-1 act as regulatory RNAs and the transcripts are present only in particular cell types of Craterostigma tissue.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S1d

Molecular dissection of TYLCSV Rep-derived resistance mechanisms Illustrates the complexities of VIRUS-HOST interactions

A. Lucioli*, E. Noris**, A. Brunetti*, R. Tavazza*, V. Ruzza*, A. Castillo***, E. Bejarano***, A. Fammartino**, G.P. Accotto**, M. Tavazza*

*) ENEA CR Casaccia, Biotec, Rome, Italy
**) IVV, CNR, Torino, Italy
***) Universidad de Malaga, Spain

dominant negative mutants, silencing, PTGS

Transgenic expression of pathogen-derived sequences has been extensively used to obtain virus-resistant plants. However, not always the predicted molecular interference mechanisms were those actually operating in resistant transgenic plants. 

The replication-associated protein (Rep) of geminiviruses is the only viral protein that is absolutely required for viral replication. Rep is involved in several biological processes brought about owing to the presence of distinct functional domains and different states of protein aggregation. We previously exploited the multifunctional character of Rep to develop an interference strategy to impair viral infection. In this respect, we showed that transgenic expression of N-terminal 210 amino acids of Tomato yellow leaf curl Sardinia virus Rep (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication (Brunetti et al. 2001 Journal of Virology).

In the present study, using both biochemical and transgenic approaches, we carry out an extensive investigation of the molecular resistance mechanisms operating in Rep-210 transgenic plants. We show that Rep transcriptional repression does not require the oligomerization domain and that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by a tight inhibition of viral Rep gene transcription, while that to heterologous virus (Tomato yellow leaf curl virus, Portugal isolate) is due to the interacting property of the Rep-210 oligomerization domain. Moreover, we show that in Rep-210 expressing plants the duration of resistance is related to the unexpected ability of the challenging virus to shut off transgene expression by a post-transcriptional homology-dependent gene silencing mechanism and to spread in virus-silenced plants. According to the ability of TYLCSV to spread in virus-silenced transgenic plants, we show that TYLCSV is able to infect transgenic plants accumulating, before the inoculation, PTGS-generated Rep-210 transgene-specific 21-25 nt small interfering RNAs.

We propose a model in which the resistant or susceptible phenotype can be regarded as the result of a battle where the transgenic plant uses the Rep-210 to repress viral transcription and to assemble dysfunctional oligomers, while the virus induces silencing of the transgene. The impact of TYLCSV-mediated transgene silencing on the development of geminivirus-derived resistances will be also discussed.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S1e

DETECTION OF CIS-ACTING REGULATORY VARIATION THAT AFFECTS MAIZE GENE EXPRESSION 

F. CATTONARO, S. AMADEO, M. MORGANTE

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università degli Studi di Udine, Via delle Scienze 208, 33100 Udine, Italy

maize, gene expression, SNPs, RT-PCR, heterosis 

Variation in coding sequence is classified as synonymous and non-synonymous, reflecting nucleotide variants with and without an effect on phenotype at the level of protein primary structure and can be recognized directly from DNA sequence. Variation in non-coding cis-regulatory DNA sequences, which affects gene expression levels, has also been proposed as a major component of the genetic basis for phenotypic evolution. In humans it has been proposed to influence disease susceptibility. In contrast to the former, the latter type of variation can not be recognised from nucleotide sequences alone. 

A method to detect changes in transcript levels, due to cis-acting sequence differences, without the necessity to recognize specific regulatory variants (which can be hundreds or even thousands of bases upstream from the transcription unit), has been developed in maize, based upon recent papers about methods for measurement of allelic levels of gene expression in humans and mouse (Yan et al., Science, 2002; Cowles et al., Nature Genetics, 2002). 

The method involves the study of two alleles of a gene under identical circumstances and the comparison of the transcript expression associated with each. This is done by comparing the expression of alleles from two maize inbred lines (B73 and H99) in the F1 hybrid lines (B73XH99 and H99XB73). To distinguish between the transcripts derived from each of the two parental alleles we used SNP markers (Single Nucleotide polymorphisms) in the transcript itself. 

The SNP assay we developed, involves RT-PCR amplification of the region surrounding the SNP marker, Single Base Extension (SBE) of a primer adjacent to the variant base in the presence of fluorescently labeled nucleotides and detection on a capillary DNA sequencer. The ratio of the two alleles is inferred by comparison with known mixtures of B73 and H99 genomic DNA used as reference standard, which, after PCR amplification, are subjected to the same SNP assay.  

Preliminary results on genes expressed at medium and high levels in seedlings revealed abundant cis-acting variation in maize: 3 of 5 (60%) genes tested showed greater than 1.5 fold (range: 2.1-4.0) differences in expression among alleles, with no difference among reciprocal hybrids. This is in contrast with what was found in mouse genes where 4 out of 69 genes (6%) showed allelic differences in gene expression (Cowles et al., Nature Genetics, 2002) 
The high levels of cis-acting variation in maize genes we are finding agree with recent discoveries about very high levels of sequence diversity in maize transcribed and promoter regions (Ching et al., BMC Genetics, 2002), lack of sharing of repeats/intergenic regions and genes in maize inbred lines (Fu and Dooner, PNAS, 2002) and influence of (retro)transposons insertion/deletions on expression of flanking genes in maize and wheat (Selinger and Chandler, PNAS, 1999; Selinger and Chandler Plant Physiology, 2001; Kashkush et al., Nature Genetics, 2003). Beside representing an important source of phenotypic and quantitative variation, regulatory variation may also provide a possible molecular explanation of the overdominance genetic hypotesis, which at least partly explains the hybrid vigor (heterosis) phenomenon in maize. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S1f

MADS-BOX TRANSCRIPTION FACTORS CONTROLLING THE FLORAL TRANSITION IN RICE

M.M. KATER*, F. FORNARA**, N. PELUCCHI**, V. GREGIS**, R. RIZZARDI*, G. FALASCA***, M. ALTAMURA***, L. COLOMBO**

*) Dipartimento di Scienze Biomolecolari e Biotecnologie
**) Dipartimento di Biologia, Università, degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
***) Dipartimento di Biologia Vegetale, Università La Sapienza, P.le Aldo Moro 5, 00185 Roma

MADS-box, flowering, development

MADS-box transcription factors have shown to be key regulators of plant developmental processes. The best studied MADS-box genes are those involved in flower development. In Arabidopsis several of them have been shown to control the switch from vegetative to reproductive development, which include AP1, FUL, SVP and AGL24. We have isolated 3 MADS-box genes from rice, OsMADS18, OsMADS22 and OsMADS47 that are homologous to these genes. 

OsMADS18 and OsMADS22 are expressed in all tissues analyzed, whereas OsMADS47 is expressed only in leaves. We are analyzing the function of these genes to understand whether these rice MADS-box genes play similar roles in rice. For these analyses we use overexpression and RNA interference strategies.

Overexpression of OsMADS18 induces early flowering. Detailed histological analyses of these transgenic plants showed that this early flowering phenotype is probably the result of an acceleration of the maturation of these transgenic plants. 

To study the conservation of the function of these MADS-box genes between monocot and dicot plants we have used the rice genes to complement the Arabidopsis mutants. 

POSTER

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.01

CHARACTERIZATION OF THE NF-Y SUBUNITS IN ARABIDOPSIS THALIANA

V. CALVENZANI, M. LAPILLA, T. SALA, K. PETRONI, C. TONELLI 

Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria 26, 20133 Milano

transcription factor, CAAT binding proteins, NF-Y, Arabidopsis

NF-Y is a CCAAT-specific binding factor composed of three dinstinct subunit. In vertebrates and fungi all three subunit are encoded by evolutionary conserved single copy genes. In a statistical analysis of over 500 eukariotic promoters, the CCAAT box was one of the most ubiquitous elements, being present in 30% of them (1).

Most of the information about the CCAAT binding protein is provided from studies in yeast and mammals, but our knowledge of the biology of NF-Y genes in plants is still rudimentary.

In our laboratory all the members of the NF-Y family in Arabidopsis thaliana have been cloned. This family is composed of 29 subunits genes: 10 NF-YAs, 10 NF-YBs, 9 NF-YCs. Gene specific probes were used for the preliminary characterization of the different members by semi-quantitative RT-PCR on RNA samples obtained from several tissues and developmental stages. In general, the emerging picture is extremely complex, with all genes expressed at least in one stage of flower development and a maximum of 20 genes induced in senescent flowers, 3 days after fertilisation. The phylogenetic analysis showed the existence of different subgroups of sequences inside each family, with a good correlation between the belonging to a certain subgroup and the corresponding expression patterns (2, 3). 

In order to identify the NF-Y subunits of Arabidopsis which interact with other transcription factors, 25 out of 29 Two-Hybrid  constructs have been prepared. In particular, the complete ORF of 24 subunits (7 NF-YA, 8 NF-YB e 9 NF-YC) has been fused in frame to the activation domain of Gal4 (AD vectors) or to the DNA binding domain of Gal4 (BD vectors). The constructs have been sequenced to verify the frame and have been introduced in yeast to check them for autoactivation. The constructs prepared were used to test, in a Two-Hybrid screening, the possible interactions with different transcription factors from Arabidopsis prepared together with other laboratories within an European project. The positive results will be confirmed and these genes will be functionally characterized by searching of insertional mutants in Arabidopsis T-DNA and transposon populations. Moreover the phenotypes of the double mutants between genes  belonging to the same subgroup will be further characterised.

(1) Mantovani (1998), Nucleic Acid Res. 26:1135-1143
(2) Gusmaroli et al. (2001), Gene 264:173-185
(3) Gusmaroli et al. (2002), Gene 283:41-48

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.02

ARABIDOPSIS MBD PROTEINS SHOW DIFFERENT BINDING SPECIFICITIES AND NUCLEAR LOCALIZATION

F. SCEBBA*, G. BERNACCHIA**, M. DE BASTIANI**, A. ANDREUCCI***, M.T. LOCCI****, L. PITTO*

*) Laboratory of Gene and   Molecular Terapy, Institute of Clinical Fisiology, CNR, Pisa, Italy
**) Department of Biological Sciences University of Ferrara, Italy
***) Department of Botanical Sciences, University of Pisa, Italy
****) Department of Human Morphology and Applied Biology, University of Pisa, Italy

DNA-binding proteins, symmetric and asymmetric methylation, GFP tagged proteins 

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control (Rice and Allis, 2001, Nature,414:258-61).  In plants DNA methylation affects both symmetrical and asymmetrical cytosines by means of different DNA-methyltransferases. In vertebrates it is known that these modifications are interpreted by a group of proteins (methylated DNA binding domain proteins, MBDs) able to specifically bind methylated CpG (Hendrich and Bird, 1998, Mol Cell Biol. 18:6538-47). All MBDs, except MBD4, a thymine glycosylase that forms a complex with the DNA mismatch-repair protein MLH1 (Bellacosa et al, 1999, Proc Natl Acad Sci U S A. 96:3969-74) form complexes  with histone deacetylases (HDAC) which they recruit to methyl CpG enriched regions to repress transcription (Ballestar and Wolffe,2001. Eur J Biochem. 268:1-6.). In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is yet to be completed. Here we present results concerning (six) different MBDs from Arabidopsis chosen on the basis of differences in their expression profiles as judged by semi-quantitative RT-PCR. All the corresponding polypeptides, expressed in E. coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only three of them (namely AtMBD5, AtMBD6 and AtMBD11) were able to bind methylated oligonucleotide duplexes. Moreover we were able to differentiate AtMBD5 and AtMBD6, despite their high homology, for their specific affinities: AtMBD5 is able to specifically bind cytosine methylated in symmetrical and asymmetrical contexts while AtMBD6 efficiently complexes only sequences methylated in the canonical CpG site. AtMBD11 differs from the other tested AtMBD proteins for its capacity to strongly bind methylated and unmethylated DNA. The binding  specificity of these proteins was tested not only on arbitrarily chosen probes but also on Arabidopsis E2F-recognition sequence containing two CpG sites. Our data indicate that methylation can trasform a sequence target for the members of the AtE2F transcription factors family in a binding site for AtMBD5 and AtMBD6. Transient expression experiments of AtMBD5, AtMBD6 and AtMBD11 tagged with green fluorescent protein in Arabidopsis protoplasts showed that AtMBD5, AtMBD6 and AtMBD11 are nuclear protein. Moreover our data indicate that AtMBD5 and AtMBD6 localize in heterochromatin and this localization is affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificities and distribution, therefore they might have different roles in methylation-mediated transcriptional silencing. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.03

THE AtRHA1 GENE OF ARABIDOPSIS: STRUCTURE AND POSSIBLE FUNCTIONS

S. PICONESE, C. ROSI, F. MIGLIACCIO 

Institute of  Agroenvironmental and Forest Biology; Consiglio Nazionale delle Ricerche, Via Salaria Km 29.300, 00016 Monterotondo (Roma), Italy

Arabidopsis, gravitropism, auxin, HSFs

The mutant Atrha1 shows in the roots reduced gravitropic response, reduced slanting toward the right-hand, and increased resistance  to the auxinic hormones, their inhibitors, and ethylene. Taking advantage of a T-DNA tag inserted in AtRHA1, the gene was cloned through the TAIL-PCR technique.  The gene appears to be a new heat shock factor (HSF) of Arabidopsis, made up of three EXONS, two of which translated, and two INTRONS. It maps on chromosome 5, close and above the RFLP marker mi61. Through RT-PCR it was confirmed that the gene is expressed in the wild-type, but not in the mutant. Complementation of the Atrha1 mutants  with the wild type gene abolished the mutated phenotype.

AtRHA1 shows notable homology in the DNA binding motif, at the level of the aminoacid sequence, with other HSFs from plants (Arabidopsis, Licopersicum peruvianum, maize), Coenorhabdites, Drosophila, mouse, yeast, and humans. It belongs to the A group of plant HSFs. We suggest that AtRHA1, apart its function in the activation of Heat Shock Proteins, could be involved in the transduction of signals coming from gravity and auxin.. Possibly, as shown for the HSF2 from humans (Hong and  Sargent, 1999  J. Biol. Chem. 274, 12967- 70), it could be involved in the regulation of the PP2A phosphatase, which has been demonstrated to regulate the transport of auxin in Arabidopsis (Garbers et al. 1996 EMBO J. 15, 2115-24). 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.04

CHARACTERIZATION OF PUTATIVE UPSTREAM REGULATORS OF BKN3, THE BARLEY HOMEOBOX GENE RESPONSIBLE FOR HOODED MUTATION 

M.R. STILE*, L. SANTI**, L. ROSSINI***, C. POZZI****, M. OSNATO***, Y. LIU**, E. FILIPPONE*, F. SALAMINI**

*) Department of Soil, Plant and Environmental Sciences–School of Biotechnological Sciences, University of Naples, Portici, Italy
**) Max-Planck-Institute for Plant Breeding Research, Carl-von-Linnè-weg 10, 50827 Colo gne, Germany
***) University of Milan, Dipartimento di Produzione Vegetale, Via Celoria 2, 20133 Milan, Italy
****) Fondazione Parco Tecnologico Padano, V. Haussmann 11, 26900 Lodi, Italy

barley mutant, Shoot Apical Meristem, Knox genes

Molecular and genetic analyses have revealed a subfamily of homeobox-containing genes, Knox (Knotted1-like homeobox) genes that play a vital role in meristem function. Knox  activity has been proposed to maintain an indeterminate state in the cells of the shoot apical meristem (SAM). In agreement with this model, Knox genes are specifically expressed in the central region of the SAM and are repressed in lateral organ primordia.

Knox gene function may require the cooperation of, or be inhibited by, other factors that are expressed in a spatially or temporally restricted pattern during development. Loci that regulate Knox expression have been isolated and characterized in both dicot and monocot species, e.g. phantastica (phan) in Antirrhinum (Waites e Hudson, 1995) and rough-sheath2 (rs2) in maize (Schneeberger et al., 1998). The phenotypes of phan and rs2 recessive mutants resemble those conditioned by gain-of-function mutations in Knox genes, suggesting that phan and rs2 act as repressors of Knox gene expression. So far regulators of Knox genes have been characterized by mutant analysis, but no molecular evidence has been reported for the direct role of these proteins in the control of Knox gene expression.

In barley, the dominant Hooded phenotype is caused by the duplication of a 305bp element in the IV intron of Bkn3, a Knox gene. This element has been used as a bait in a yeast one hybrid screen aimed at identifying putative DNA-binding proteins involved in Bkn3 regulation. This approach led to the isolation of four barley cDNAs, B42, B65, B89, and BBR (Barley B Recombinant, Santi et al., 2003).

In order to gather insight into how these proteins interact with the 305bp intron sequence, we have undertaken the molecular characterization of these barley genes. Genomic and full-length cDNA sequences have been determined and all four genes have been positioned on the established AFLP/RFLP/ISTR linkage map (Castiglioni et al., 1998). Transcript levels for the four genes were compared in wild-type and Hooded barley tissues. Yeast two hybrid assays, conducted with partial and full length cDNA sequences, revealed selective interactions between the proteins. RFP fusion constructs of all four proteins exhibited nuclear localization in tobacco protoplasts, consistent with their putative role in the transcriptional regulation of Bkn3.

References:
Castiglioni et al., 1998, Genetics, 149:2039-2056
Santi et al., 2003, Plant Journal, 34:813-826
Schneeberger et al., 1998, Development, 125:2857-2865
Waites e Hudson, 1995, Development,  121:2143-2154.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.05

EXPRESSION PATTERN AND CELLULAR LOCALISATION OF ZEA MAYS RPD3-TYPE HISTONE DEACETYLASES DURING PLANT DEVELOPMENT

S. VAROTTO*, S. LOCATELLI**, S. CANOVA*, A. PIPAL***, M. MOTTO**, V. ROSSI**

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università di Padova, viale dell’Università 16, I-35020 Legnaro (PD), Italy 
**) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, via Stezzano 24, I-24126 Bergamo, Italy
***) Department of Molecular Biology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria

chromatin, expression pattern,  HDACs, histone modifications, protein interaction

In chromatin, eukaryotic DNA is tightly wrapped around octamers of histones proteins, restricting its accessibility to factors  involved in DNA replication and transcription. Local or extended structural chromatin changes play an important role in the control of gene expression and are controlled by complexes that remodel chromatin and by enzymes that posttranslationally modify histones (Verdin et al. 2003 Trends Genet 19: 286-293). Histone acetylation is the best-characterized type of histone modifications (Wu and Grunstein 2000 Trends Biochem Sci 25: 619-623). The enzymes responsible for maintaining the steady-state balance of histone acetylation are the histone acetyltransferases (HATs) and histone deacetylases (HDACs). Many of the recently identified HATs and HDACs have been found to be transcriptional co-activators and co-repressors, thus establishing a direct link between histone acetylation and regulation of gene transcription.

Different HDAC genes have been identified in plants and their classification into three distinct gene families has been proposed by The Plant Chromatin Initiative (http://chromdb.biosci.arizona.edu). The first family, the HDA gene family, is the Rpd3/Hda1 super-family, which contains members related to the yeast sequences Rpd3. The expression pattern and cellular localization of the maize Rpd3-type histone deacetylases ZmRpd3/101, ZmRpd3/102, and ZmRpd3/108 showed that their transcripts are expressed in all the organs and cellular domains analyzed but that their amount changed during development. A similar expression pattern and a nucleus/cytoplasmic localization was observed for ZmRpd3 proteins. GST pull-down assays showed that ZmRpd3 proteins can interact with the maize retinoblastoma-related (ZmRBR1) and retinoblastoma-associated (ZmRbAp1) proteins and that the three ZmRpd3s did not compete with each other in the binding. These results indicate a global role of ZmRpd3 genes in plant cell cycle and development, probably by affecting several metabolic pathways and suggest mechanisms regulating their transcription and protein accumulation. The important role played by HDAC in maize development is also emerging from the characterisation of antisense and over-expressing mutants that is in progress in our labs. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.06

MOLECULAR CHARACTERIZATION OF THE gl1 GENE OF MAIZE

M. STURARO, H. HARTINGS, R. VELASCO, M. GATTUSO, M. MOTTO 

Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo 

epicuticular waxes, glossy mutants, drought adaptation, transposon tagging

Epicuticular waxes, a complex mixture of long chain lipidic compounds, cover the aerial organs of terrestrial plants and represent a preformed defense against several biotic and abiotic stresses. Many mutants impaired in their biosynthesis have been isolated in different species, including maize and Arabidopsis, helping the elucidation of some steps of the poorly defined pathways leading to wax synthesis and deposition.

In maize, two different pools of waxes are produced from two distinct biosynthetic pathways, the former throughout the entire plant life cycle, while the latter is restricted to the juvenile developmental phase (up to 5 to 6 leaf stage). At least 18 different glossy (gl) loci have been defined by mutations affecting maize juvenile wax biosynthesis but only a few of them have been characterized with molecular or biochemical approaches.

The glossy 1 mutant shows a strong reduction of total juvenile waxes suggesting a specific block at an early step of the corresponding biosynthetic pathway or a role of the gene product in the translocation of  wax components onto the plant surface.

In an attempt to characterize this mutant at the molecular level, we performed a transposon tagging  with the En/Spm element which led to the isolation of the  glossy 1 gene, and we cloned the corresponding cDNA by RT-PCR. These genomic and cDNA sequences differ from the putative gl1 gene and transcript previously identified by others. The protein encoded by gl1 shows significant homology with the entire sequence of the CER1 gene product, a putative aldehyde decarbonylase involved in cuticular wax production in Arabidopsis. Additional insights into Gl1 activity were obtained from the analysis of a collection of 20 stable independent mutations. The gl1 gene is actively transcribed in response to water stress.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.07

MOLECULAR, FUNCTIONAL AND CELLULAR ANALYSES OF WILD-TYPE AND MUTANT ALLELES OF THE OPAQUE-2 REGULATORY LOCUS

B. LAZZARI, C. COSENTINO, F. GAVAZZI, S. DOLFINI, A. VIOTTI 

Istituto Biologia e Biotecnologia Agraria, CNR, Milano, Dipt. Genetica e Microorganismi, Università, Milano

opaque-2 alleles, o2-polypeptydes, cellular location 

The Opaque2 b-ZIP transcriptional activator is involved in the transcriptional regulation of several genes in the subaleurone layers of the maize endosperm cells. Three wild-type and five mutant alleles (o2T, o2It, o2-676 o2-52, and o2R) were considered in this study. The wild-type alleles encode polypeptides with highly conserved amino acid sequence having only a short hypervariable region within the amino terminal part of the protein. The o2R allele is a null o2-transcript allele and thus represents a suitable control in these analyses. The o2T allele is characterised after 645 bases from the ATG by a 25 nucleotide deletion that introduces a stop codon thereby producing a truncated polypeptide (o2T) missing both the basic and the L-Zipper motifs. The o2-52 allele contains an insertion of 4 base pairs 796 bases downstream from the start codon, which causes a frame shift, giving rise to another truncated polypeptide that lacks all the carboxy terminal part downstream from the second leucine of the L-Zipper. Conversely, the o2It allele encodes for two different polypeptides, o2It-S and o2It-L. The o2-676 polypeptide contains both the basic and the L-Zipper motifs but show an amino acid substitution within the basic region leading to a non-functional protein. Western analyses with specific amino terminal and carboxy terminal O2-antisera confirm the presence of differentially shortened o2 polypeptides. Biochemical analyses of native wild-type and mutant polypeptides followed by IPG-IEF fractionation show that the native proteins are highly heterogeneous by charge, and this was associated to a different extent of protein phosphorylation. In the wild-type and in the o2It-L, o2-676 and o2.52 proteins six to nine isoforms were detected with the non- and hypo-phosphorylated forms more abundant in respect to the hyper-phosphorylated ones. The o2T and o2It-S polypeptides were, however, fractionated into three to four isoforms.  

In order to investigate the functional meaning of the-phosphorylation process and domain region we performed southwestern experiments on both wild-type and mutant O2s before and after phosphatase treatment and in addition immuno-detection to localise these polypeptides at the subcellular level.  The functional analysis reveals that only the hypo-phosphorylated forms of the wild-types are able to bind the O2 target sequence, while the hyper-phosphorylated forms acquire DNA binding activity only after phosphatase treatment.  Localisation studies reveal that more than 75% of the wild-type O2 is present in the nucleus. The o2It-L, o2-52 and o2-676 mutants are similarly localised into the nucleus while the o2T remains confined into the cytoplasm. In conclusion, wild-type and mutant polypeptides reveal functional and cellular behaviours mediated by their structural and biochemical features.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.08

FUNCTIONAL ANALYSIS OF RICE GSTs

G. DE TOMA, M. SARI GORLA, C. FROVA 

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano

Glutathione transferases (GSTs), transcriptional profiling, structure-function relationship

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes ubiquitously distributed in all aerobic organisms. These proteins have evolved multiple functions in the metabolism of endogenous as well as foreign toxic compounds, and have important roles in cellular protection against a wide range of biotic and abiotic stresses. In plants they detoxify herbicides, organic pollutants and natural toxins and are involved in counteracting oxidative injury from various sources, including heavy metals. Most plant species harbour tens of GST genes: the estimates in Arabidopsis, maize and soybean are 48, 42 and 25 members respectively. In rice, we have isolated and cloned 58 putative GST genes, belonging to the four major plant classes: Phi (F), Tau (U), Zeta (Z) and Theta (T). 

However, so far little is known about the specific function of individual members. With the aim to functionally characterise rice GSTs, their expression patterns were analysed by 1) systematic screening of rice EST collections and 2) DNA macroarray analysis. By comparing whole genome data with ESTs, about 55% of rice GST genes appear to be expressed. Inspection of EST libraries indicates that 67% of the individual cDNAs correspond to Tau class GSTs, 26% to Phi, 5% to Zeta and 1.4% to Theta. Individual rate of expression, estimated by the number of EST hits, shows that 19% are poorly expressed (1-3 hits), but 10% are very highly expressed (15 or more hits). Analysis in selected tissues indicates the lowest expression in leaves and the highest in callus.

As for macroarray analysis, we previously reported (SIGA 2002) that several rice GST genes are transcriptionally induced by either H2O2 or Cadmium or both. Interestingly, 2 Tau class genes (U9 and U3), which share a very high sequence identity, showed different transcriptional profiles in response to the two stresses. We thus further investigated these two genes also at the protein level: close sequence inspection shows that non-synonimous aminoacid substitutions are clustered in the co-substrate binding domain (an  helix made up by a ten aminoacid stretch), likely changing the two GSTs specificities. This hypothesis is supported by simulations performed through the SWISS-MODEL 3D program, which show that in GSTU3 this  helix structure is lost due to the specific aminoacid substitutions.

Macroarray transcriptional profiling of GST genes in response to two herbicides (the chloroacetanilide Pretilachlor and the thiocarbammate Molinate) and the safener Fenchlorim is currently under way.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.09

CHARACTERIZATION OF THE WRKY GENE FAMILY IN ORYZA SATIVA

S. BERRI, M. KATER, E. PÈ 

Department of Biomolecuar Science and Biotechnology, University of Milan

WRKY, expression profiling, abiotic stress

WRKY genes encode a family of transcription factors unique to plants characterized by the presence of one or two WRKYGQK motifs within a wider 60 aa consensus domain. Very little is known about this gene family, although some studies (mainly in Arabidopsis thaliana) showed their involvement in a variety of processes like the response to biotic and abiotic stresses, trichome development, senescence and biosynthesis of secondary metabolites. Recently, expression profiling of all 72 Arabidopsis WRKY genes demonstrated that many members of this family are actually transcriptionally regulated by pathogen infection and salicylic acid treatment. (Jixin et al., 2003)

Annotation of the rice genome sequence (which is still incomplete) resulted in the identification of 53 WRKY genes. The total predicted number is around 80 genes. 

With the aim to characterise this gene family in rice, we compared the protein sequences of the 53 WRKY genes and produced a tree using the C-terminal consensus sequence. By including also the Arabidopsis sequences, we showed that similarity among homologous genes of different species is higher than among genes of the same species. Furthermore, this analysis showed that the Arabidopsis classification of WRKY genes (Eulgem et al., 2000) can be applied to rice genes as well.

To study the expression of the rice WRKY genes with regards to their expression pattern under oxidative stresses (H2O2), temperature stresses (cold) or salicylic acid induction, gene specific probes were spotted on filters and hybridised with cDNA probes derived from RNA extracted from the stressed plants.

For the functional analysis of WRKY genes we are analysing insertion (knockout) mutants. Four Tos17 mutants were identified in the rice insertion mutant database (http://tos.nias.affrc.go.jp/~miyao/pub/tos17/index.html.en). The analysis of these lines is in progress.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.10

CHARACTERISATION OF THREE FUNCTIONAL HIGH AFFINITY AMMONIUM TRANSPORTERS IN LOTUS JAPONICUS WITH DIFFERENTIAL TRANSCRIPTIONAL REGULATION AND SPATIAL EXPRESSION

A. ROGATO, E. D’APUZZO, U. SIMON-ROSIN, H. EL ALAOUI, A. COSTA, A. BARBULOVA, A.-M. MARINI, K. M. UDVARDI, F. LO SCHIAVO, M. CHIURAZZI

Institute of Genetics and Biophysics “A. Buzzati Traverso”, Via Marconi 12, 80125, Napoli, Italy

N metabolism, symbiosis, nodule organogenesis

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can be additionally obtained by the nodule infected cells as result of the symbiotic nitrogen fixation. NH4+ is not only the final product of symbiosis but also a main regulator of the early as well as late symbiotic interaction steps and hence ammonium transporters are likely to play important roles in the control of nodule formation and functioning. PCR techniques were used to isolate two new full-length clones of L. japonicus homologue of LjAMT1;1 (LjAMT1;2 and LjAMT1;3) and the 5’ regulatory sequences of the three members. The full-length cDNAs when cloned into the yeast expression vector pMET426 were able to complement a yeast mutant unable to grow on media with NH4+ as the sole nitrogen source. A measurement of the accumulation of [14C]methylamine in the yeast strains expressing the three LjAMT1 genes permitted their biochemical characterization and differentiation. The observed biochemical features were correlated with the transcripts abundance observed in mature plants grown in hydroponics conditions in presence of different N regimes. The expression patterns of the three LjAMT1 members were examined in different organs and a 60% decreased expression of LjAMT1;1 was observed in leaves of plants growing under high CO2 conditions. Furthermore, we report a map of the pattern of expression of the Lotus AMT1 members obtained by promoter-gusA fusions analysis, mRNA in situ and GFP-fusion localizations.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.11

SENESCENCE IN CELL SUSPENSION CULTURES IN MEDICAGO TRUNCATULA AND ARABIDOPSIS THALIANA

E. BARIZZA, R. DE MICHELE, F. CARIMI, M.GROTTO, M. TERZI 

Dipartimento di Biologia, Università di Padova

Arabidopsis thaliana cell culture, Medicago truncatula cell culture, senescence, PCD

High concentrations of cytokinin block cell proliferation and induce cell death in Arabidopsis cells.

Cell death is demonstrated to be of the programmed type by various parameters including chromatin condensation and oligonucleosomal DNA degradation (laddering). PCD is preceded by senescence, an active process requiring active synthesis, identified by specific markers, the most typical being SAG12 a cystein-protease whose expression was found originally in leaves and now also in cultured cells.

We are now moving to Medicago truncatula, a model plant for leguminous species, whose cells can be cultivated in vitro. On Medicago truncatula cells we are reproducing prematurely induced senescence and associating it with markers which, on a preliminary bioinformatic analysis, show homology with SAG12 and other senescence markers whose function has been identified in Arabidopsis.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.12

VIRUS INDUCED GENE SILENCING TO STUDY GENE FUNCTION IN PEACH (PRUNUS PERSICA L. BATSCH)

E. CONDELLO, D. GIANNINO, C. NICOLODI, G. TESTONE, A. TARTARINI, D. MARIOTTI 

Istituto di Biologia e Biotecnologie Agrarie del Consiglio Nazionale delle Ricerche, Sezione Territoriale di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma)

gene silencing, tobravirus based vectors, peach

Peach plants are relatively recalcitrant to the standard leaf disk transformation mediated by Agrobacterium tumefaciens, due to the low efficiency of shoot regeneration. Consequently, the study of gene function and manipulation of this species have been hampered and delayed. The virus induced gene silencing (VIGS) is a manifestation of an RNA-mediated defence mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. The gene silencing vectors based on tobacco rattle virus (TRV) have been excellent tools to study gene function in crops such as tobacco and tomato. In fact, TRV vectors carrying plant host genes are able to induce silencing of the corresponding genes in infected plants. These vectors are engineered in binary vectors and subsequently transferred into host leaves via syringe inoculation of A. tumefaciens strains. They are also able to target host RNAs in plant meristems and the consequent VIGS for a particular gene mimics the phenotype of loss of function mutant. Though the species of Rosaceae family are not usual hosts of tobraviruses, a TRV-VIGS apporach has been applied to peach by infecting plants, at different stages of growth, with control vectors and TRV carrying gene markers, full length or partial cDNAs derived from genes previously cloned in peach. Preliminary molecular analyses indicated that the transgene insertion and expression occurred in all the inoculated plants, that the vector itself was transmitted to the basal and apical parts of infected plants and did not induce visual symptoms. Putative VIGS mutant phenotypes are being under analysis and will be presented in relation to the silenced gene.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.13

ARABIDOPSIS GENE EXPRESSION CHANGES DURING AND AFTER OZONE STRESS

N. TOSTI*, S. PASQUALINI**, E. FALISTOCCO**, G. DELLA TORRE**,***, L. EDERLI**, F. PAOLOCCI*

*) Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, sezione di Perugia
**) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia
***) Dipartimento di Arboricoltura e Protezione delle Piante, Università degli Studi di Perugia

GeneChip microarray, Arabidopsis, ozone

The concentration of tropospheric ozone (O3) has increased during the past decades due to human activities, and it has been estimated that in year 2100, 50% of global forest will be exposed to potentially phytotoxic O3 concentrations. Plants respond to high ozone levels showing both sensitivity, manifested as the appearance of rapid lesion formation, or tolerance. A. thaliana ecotype Col-0 is reported to be particularly tolerant to ozone and severe ozone fumigation did not induce any significant visual modification on rosette leaves and this makes this plant species of extreme interest to study genes involved into stress adaptive mechanisms. Therefore, to dissect the effects of ozone on plant gene expression profile, a microarray analysis was performed by means of GeneChip probe array developed by Affymetrix. The GeneChip contains more than 22,500 A. thaliana  oligonucleotide probe sets, representing approximately 24,000 genes.

9-week A. thaliana old plants were exposed to a single dose of 300 ppb ozone for 6 h. Entire plant rosettes were harvested before (0h) and after 3 h (3h) of ozone treatment as well as after 6 h from the end of the treatment (12h). Plants after fumigation were left in growth chamber to recover.

Total leaf RNA was isolated from of 4 bulked plants for each of time points and used as probe for microarray. As control, time point 0h and 3h microarray analyses were carried out in duplicate using as probe newly isolated  RNA.   

The hybridising arrays, over a background level, ranged from 55-60 % for the control, to 62 % for 3h and 12h plants of the total probes. After 3 hours of treatment 12.28 % and 7.66 % of the total probes were up and down regulated, respectively. These percentages decreased down to 4.25 and 2.8 when only probes showing a two-fold difference with respect to the control were considered. The gene-related probes belonged to several gene families and many pathways resulted to be involved in response to ozone stress, while in some cases genes belonging to the same biosynthetic pathway were up and down regulated. 

The log ratio of the expression values for each probe set between the control (0h) and treated samples (3h and 12h) was calculated. For each comparison, the probe sets that showed greater than two-fold difference from the control in at least one sample were selected. The expression values of the selected samples were subjected to a log2 transformation then to a Self Organising Tree Algorhytm (SOTA). We found 29 gene clusters that were subdivided in four principal groups. An analysis of the SOTA dendrogram showed that in some cases genes belonging to the same gene family were clustered together. Moreover, the study of the promoter region of the up regulated genes showed the presence of over-represented motifs that were statistically related to some clusters of the SOTA dendrogram.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.14

DIFFERENTIAL GENE EXPRESSION INDUCED BY OZONE STRESS IN SENSITIVE AND TOLERANT POPULUS SPP. 

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

*) 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, Populus, suppression subtractive hybridisation

Although definitive proof of a link between ozone and forest damage exist only in California, large number of data supports a role for this tropospheric pollutant in forest decline in eastern USA and Europe in combination with other biotic and abiotic factors. 

Plant subjected to elevated (but often close to reality) concentrations of tropospheric ozone undergo several biochemical changes, before any morphological damage can be detected, that are the direct result of a qualitative and quantitative modification of gene expression.

A lot of regulating events, responsible for specific physiological reactions, often depend on the activation or inactivation of particular genes: identification of differentially expressed genes after ozone treatment can help to understand molecular mechanisms that are at the basis of the response to this kind of stress. 

In order to identify differentially expressed genes after the exposition to an acute ozone dose (a single pulse of 150 ppb for five hours) we have used suppression subtractive hybridisation, SSH in two poplar species characterised by a different sensibility to this kind of pollutant: Populus deltoides x maximowiczii, Eridano clone, e Populus x euoramericana, I-214 clone, sensitive and tolerant to O3 respectively. 

This method allows to obtain cDNA library enriched of transcript only present in one of the two compared samples (in our case treated sample and control sample).

The main feature of this technique is the peculiar effectiveness in isolating rarely expressed transcripts. This minor fraction is particularly of interest, since transcript for many regulatory proteins fall into this category.

By using SSH, four cDNA libraries were obtained, two libraries made of products of genes activated by stress treatment in sensitive and tolerant plants separately (FS and FT libraries), and two libraries made of genes whose transcription is suppressed or diminished under ozone stress, once again in sensitive and tolerant plants separately (RS and RT libraries).

A primary screening for all libraries was performed, and several differentially expressed cDNA clones were selected. From FT library e.g. we have isolated phenyalanine ammonia-lyase (PAL), ascorbate peroxidase (APX), an important antioxidant enzyme that helps plants to survive oxidative stress and a cDNA sequence that presents a conserved domain typical of transcription factors belonging to the WRKY superfamily that include members involved in the regulation of various physiological program as pathogens defense.

The expression of some of the most interesting isolated clones has been analysed furthermore.
 
Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.15

ANALYSIS OF THERMOTOLERANCE IN DURUM WHEAT: A MOLECULAR CHARACTERISATION

M. CORRADI*, P. RAMPINO**, M. CIAVARRELLA***, M. GULLÌ*, N. DI FONZO***, N. MARMIROLI*, C. PERROTTA**

*) Department of Environmental Science, University of Parma, Italy
**) Department of Biological and Environmental Sciences and Technologies, University of Lecce, Italy
***) Experimental Institute of Crop Research, S.S 16 km 675 - 71100 Foggia (Italy).

thermotolerance, triticae, molecular markers, differential display

High temperature represents a major constraint affecting organisms’ adaptability and survival. Plants exhibit a very different capacity to respond to this environmental stress, and inter and intra specific variations are mostly due to genetic variability. Different wheat genotypes were evaluated for their cellular thermotolerance, by cell membrane stability (CMS) assay. We tested cultivated and wild durum wheat and some wild wheat relatives (Aegilops spp.) originating from semi-arid areas. Some genotypes selected on the basis of their thermotolerance as the most heat resistant were used to study the molecular basis of thermotolerance. The level of induction of several HSP genes was analysed in both tolerant and sensitive genotypes in order to identify differences in the induction of these genes that have been suggested to be correlated to tolerance. DDRT-PCR was performed on one of the most tolerant Aegilops to identify sequences differentially expressed after a temperature shift from 23 to 42°C. About 20 bands specific to the stress response were identified and cloned. Moreover genetic variation in traits associated with thermotolerance was studied using AP-PCR and LP-PCR markers. Different patterns were identified, associated with inter and intra species differences, specific bands were isolated and sequenced.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.16

IDENTIFICATION OF SEQUENCES RELATED TO DROUGHT STRESS IN DURUM WHEAT

P. RAMPINO*, M. MALATRASI**, M. GULLÌ**, N. MARMIROLI**, P. PERROTTA*

*) Department of Biological and Environmental Sciences and Technologies, University of Lecce
**) Department of Environmental Science, University of Parma

drought stress, wheat, differential display

Drought is one of the adverse environmental factors causing severe yield loss in crop plants. Plants have developed different strategies to cope with this stress, involving changes in the pattern of gene expression due to activation of stress-specific genes and to a general re-programming of genetic activity in the cell. Ten day-old seedlings of two cultivars of durum wheat sensitive to drought to varying degrees (cv Adamello, more sensitive and cv Ofanto less sensitive) were subjected to dehydration for 2, 4 and 6 hours. Total RNA extracted from these samples was used for PCR analysis using different primer sets. The first set of primers was designed on dehydrin conserved sequences, selected from drought-specific sequences found in databases. For the second set of primers, the EST database was used to select sequences obtained from drought-specific libraries. A third set was designed on the sequences of some DDRT clones corresponding to mRNAs expressed in barley after cold or drought treatments. The difference in the induction of the transcripts corresponding to the selected primers was analysed by RT-PCR, after the different stress conditions. RT-PCR analysis demonstrated that the level of transcripts increased under drought stress but at a different rate in the two cultivars.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.17

CLONING OF A WRKY TRANSCRIPTION ACTIVATOR INVOLVED IN COLD AND DROUGHT RESPONSE IN BARLEY

E. MAZZUCOTELLI, C. MARÈ, C. CROSATTI, G. DALFINO, D. GUERRA, L. CATTIVELLI 

Experimental Institute for Cereal Research, Fiorenzuola d’Arda

barley, cold acclimation, transcription factor

In the past years a number of cold-responsive (cor) genes expressed during acclimation to low temperature have been isolated and characterised in many crop species. Nevertheless only the CBF/DREB1 gene family of Arabidopsis thaliana codes for transcription activators which have been shown to control many cold and drought induced genes. CBF/DREB1 genes are themselves cold-regulated and their transcripts begin to accumulate within 15 min of transfer to low temperature, suggesting that differential screening techniques could be suitable for the isolation of new transcription factors.

By differential display RT-PCR technique we have isolated, from barley leaves, a cDNA induced after 3h of low temperature exposure. This transcript was characterised by an early and rapid expression in comparison with other known barley cor genes usually detectable only after 24 h of cold treatment. By using the differentially expressed cDNA the corresponding full length clone was recovered from a cDNA library. Sequence analysis of isolated cDNA revealed that this gene encodes for a 38kDa protein homologous to Avena fatua ABF2 a member of the large WRKY (pronounced “worky”) family of plant transcription factors. The barley gene, named corWRKY38, is characterised by the WRKY domain together with a zinc-finger-like motif and a leucine zipper sequence. The accumulation of corWRKY38 transcripts was investigated by northern blot analysis. During hardening at +2°C the corWRKY38 expression is rapid and transient and achieves its highest level after 12 hours in the leaves and 5 hours in the roots. Since the corWRKY38 mRNAs disappear after 24h at +2°C, we have examined if a further temperature decrease promotes a new induction of corWRKY38. Barley plants were hardened at +2°C for one week, then submitted to –3°C or –8°C for 48 hours. The temperature steps down induced a rapid and stable accumulation of corWRKY38 mRNA providing that frozen temperatures still promote a molecular response. corWRKY38 mRNA accumulation is also promoted by drought treatment, but not affected by ABA. Gel mobility shift assay experiments demonstrated that an in vitro synthesised WRKY38 protein binds oligonucleotides containing two W-box motifs suggesting that corWRKY38 can potentially act as transcription factors also in vivo.

The WRKY proteins, defined by the conserved amino acid sequence WRKYGOK, are involved in the regulation of varied physiological processes: pathogen defence, senescence and trichome development. This work provides the first evidence of involvement of WRKY transcription factors in cold-stress response.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.18

A CLUSTER OF CBF GENES ON CHROMOSOME 5H REGULATES THE ACCUMULATION OF THE COR14b AND TMC-Ap3 COLD-INDUCED PROTEINS AND FROST TOLERANCE IN BARLEY

E. FRANCIA*, D. BARABASCHI*, A. APRILE*, A. TONDELLI*, C. CROSATTI*, A.M. STANCA*, G. GALIBA**, P.M. HAYES***, N. PECCHIONI****

*) Istituto Sperimentale per la Cerealicoltura, I-29017 Fiorenzuola d’Arda, Italy
**) Agricultural Research Institute of the Hungarian Academy of Sciences, H-2462 Martonvásár, Hungary
***) Department of Crop and Soil Science and Department of Horticulture, Oregon State University 97331 Corvallis, Oregon USA
****) Università di Modena e Reggio, Facoltà di Agraria, Via Kennedy 17, I-42100 Reggio Emilia, Italy

barley, CBF, COR protein, Low temperature tolerance, QTL

Low temperature stresses limit crop productivity. A better understanding of low temperature tolerance mechanisms could significantly improve the yield of fall-sown cereals. Barley (Hordeum vulgare subsp. vulgare) is an economically important diploid model for the Triticeae. We developed a new resource for genetic analysis of winter hardiness-related traits, the ‘Nure’ x ‘Tremois’ linkage map, based on a doubled-haploid population that is segregating for low-temperature tolerance and vernalization requirement. Three measures of low-temperature tolerance and one measure of vernalization requirement were used and, for all traits, QTLs were mapped to the long arm of chromosome 5H. QTLs for three measures of low temperature tolerance are coincident, and we report for the first time segregation of two low temperature QTLs on a group 5 chromosome of the Triticeae.

Using Composite Interval Mapping, a first, distal set of coincident QTLs for low temperature tolerance and vernalization requirement was identified at the VrnH1/Fr1 region. A second, proximal set, of coincident QTLs for low temperature tolerance was identified and was coincident with the QTLs regulating the accumulation of two different and well characterized COR proteins (COR14b and TMC-Ap3). Several barley genes with the CBF transcription factor signature have been mapped in cluster in this region, and are the candidate genes underlying this QTL. A CRT/DRE recognition site in the promoter of the cor14b gene with which a CBF protein could interact has been found. These results support the hypothesis that highly conserved regulatory factors, such as members of the CBF gene family, may regulate the stress responses of a wide range of plant species. Moreover, these findings will be of immediate utility to researchers interested in marker-assisted selection for low temperature tolerance and in the genetic dissection of low temperature regulons.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.19

MOLECULAR ANALYSIS OF THE RELATIONSHIP BETWEEN PLANT DEVELOPMENT AND COLD ACCLIMATION IN BARLEY

C. CROSATTI, D. PAGANI, C. CAMPOLI, F. RIZZA

Experimental Institute for Cereal Research , Section of Fiorenzuola d’Arda, Via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC), Italy

frost tolerance, barley, cold-regulated genes, cor14b, stage of development

Agronomic studies indicated that frost tolerance of barley and wheat, besides genetic factors, depends on plant developmental stage. The maximum level of frost tolerance is reached when plants undergo to freezing at three-four leaf stage suggesting a possible interaction between plant development and cold acclimation capacity. In this work we have investigated the interaction between plant development and cold acclimation in a field experiment through the analysis of cor14b messenger and protein accumulation . cor14b was used as a molecular tool to monitor the development of cold acclimation since the expression of this gene was found associated with the acquisition of frost tolerance. Our results demonstrate that the amount of COR14b protein was related to both growth habit (winter cultivars accumulate more COR14b than spring ones) and stage of development. We found that plants accumulate  more COR14b and are indeed more frost tolerant at the third leaf stage than in the first one. The comparison of protein accumulation in the fields vs. growth chamber suggested that the higher amount of COR14b in earlier sowed plants is mainly because they spend more days under hardening conditions.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.20

A SMALL HEAT SHOCK GENE IS POSITIVELY REGULATED BY ECTOPIC EXPRESSION OF A MAIZE GENE CODING FOR A bHLH TRANSCRIPTION FACTOR

F. DAMIANI, N. TOSTI, M. RAGANO-CARACCIOLO, F. PAOLOCCI

C.N.R. Istituto Genetica Vegetale sez. di Perugia

condensed tannins, cDNA-AFLP, real-time PCR, gene expression, Lotus corniculatus

The production of bloat-safe alfalfa is recognised as one of the main goal of breeding of such species. In fact the most of proteic fraction of such crop is wasted for degradative processes occurring either in the rumen or during silage.

The presence of moderate amount of condensed tannins is claimed as the solution of such problem. However, notwithstanding the remarkable progresses of last years (Devic et al., 1999; Xié et al., 2003; Tanner et al., 2003) the process leading to the synthesis of such compounds is not completely described and some genes necessary for their synthesis need to be identified, yet.

At this purpose several approaches have been attempted. In our laboratory we produced, through genetic transformation with Sn, a maize gene coding for a bHLH transcription factor, some high contrasting phenotypes (Paolocci et al., 1999; Robbins et al., 2003) of Lotus corniculatus, where tannins level in leaves was highly enhanced (E) or strongly depleted (D). With such materials a strategy for isolating the genes involved in tannin synthesis consisted on the comparison of cDNA–AFLP pattern of two enhanced plants (E), two suppressed plants (D) and a control plant (C).

216 primer combinations out of the 256 possible (two selective bases for each primer) were carried out. 

A total of 201 putative differentially expressed fragments were recovered from the gel and 77 of them were sequenced. Only 43 (55.8%) sequenced bands matched with known genes. In this paper we report the results of the study on a gene coding for a small Heat Shock Protein (HSP), that gene resulted highly expressed in plants rich of tannin while almost absent in the control plant and in the suppressed plants.

The differential expression of such gene was confirmed through northern reverse and RT-PCR analysis. Full messenger and promoter region of the gene were recovered trough Genome Walking.

Real Time PCR was performed on the selected gene utilising ABI PRISM 5700 SDS on the different cDNA of the same plants and compared with that of dfr and Sn. The first gene is strongly up-regulated in the E plants and Sn is expressed in the E plants only. A silencing mechanism has been proposed to explain the production of D plants (Damiani et al., 1999).

HSP resulted up-regulated (8.9 and 17.2 fold, for the two enhanced tannin plants) with respect to the control plant (C) The D plants had a different trend between them: one showed a decrease level of the HSP gene level (0.59), while the other maintain substantially the level of the C plant (1.3). The level of the up regulation was greater then those of dfr in both the E plants. As expected Sn expression was detected only in E plants.

From this study comes out that Sn that transactivates dfr, can also affect, directly or indirectly, HSP; however, while dfr over-expression is consistent with the increase of tannin level, it seems hard to find a direct correlation between HSP expression and tannin increase. Experiments of transformation with chimeric HSP constructs are going to be performed to clarify this point. However, as general comment, we can hypothetise that the bHLH gene enhances generic mechanisms of resistance to stresses among which tannins and heat shock proteins represent different outcomes.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.21

INVOLVEMENT OF AtMYB41 GENE IN OSMOTIC STRESS RESPONSE IN ARABIDOPSIS THALIANA

E. COMINELLI, D. CALVI, T. SALA, C. TONELLI 

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano

R2R3MYB, drought, salt stress, abi mutants

In the last years it has been demonstrated that overexpression of some transcription factors in transgenic plants activates many stress tolerance genes and improves plant tolerance to osmotic stresses. This shows that characterization of transcription factors that control the coordinate expression of multiple genes involved in stress response is very important in the perspective of improving plants tolerance.
 
Analysis of the expression of dehydration-inducible genes in Arabidopsis have indicated that at least four independent signal pathways function in the induction of these genes. Two are abscisic acid (ABA) -dependent and two are ABA-independent.

In this study we analyzed the expression pattern of AtMYB41 gene, belonging to the R2R3 MYB family of Arabidopsis, in response to different osmotic stresses in wild type and abi mutants plants. Then we analyzed molecularly and phenotipically plants overexpressing this gene.

AtMYB41 is not expressed in any organs and at any developmental stages in wild type plants grown in normal conditions, while its transcript is accumulated in a specific manner in response to drought, PEG and NaCl treatments, but not after ABA supply. Expression pattern of AtMYB41 gene in response to ABA treatment in wild type plants has been compared to that obtained in ABA-insensitive mutants abi1-1 and abi2-1. In this cases AtMYB41 m-RNA is accumulated during the treatment, suggesting a role for ABI1 and ABI2 phosphatases in the regulation of this gene activity. 

The overexpression of AtMYB41 in transgenic plants (35S::AtMYB41) results in severe growth retardation under control conditions. A phenotypic characterization of 35S::AtMYB41 plants is in progress to understand if reduced plants dimensions are a consequence of problems in cell expansion or in  cell division.

The expression patterns of some stress induced genes were compared between wild type and 35S::AtMYB41 plants in normal growth conditions and in response to PEG and NaCl treatments. Since so far no differences have been detected, we decided to do a large scale analysis through the hybridization of macroarrays containing 8000 Arabidopsis ESTs.

35S::AtMYB41 plants did not show improved tolerance in response to drought and salt stresses. Probably AtMYB41 constitutive expression is too dangerous for the plants. Transgenic plants harbouring AtMYB41 fused downstream the drought inducible promoter of RD29A gene, are now under analysis.
Transgenic plants for a construct harbouring a 1kb sequence of the putative AtMYB41 promoter fused upstream the GUS reporter gene are also under analysis. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.22

THE ISOLATION OF EARLY-COR GENES IN DURUM WHEAT REVEALS THE PRODUCTION OF ALTERNATIVELY SPLICED TRANSCRIPTS IN RESPONSE TO LOW TEMPERATURE 

A.M. MASTRANGELO*, S. BELLONI**, S. BARILLI**, N. DI FONZO*, L CATTIVELLI**

*) Experimental Institute Cereal Research, Foggia (Italy)
**) Experimental Institute Cereal Research, Fiorenzuola d’Arda (Italy)

cold, drought, PCR-select, alternative splicing

The exposure of plant to abiotic stress determines a physiological and molecular reaction that depends, in initial phases, on a complex cascade of stress signal transduction in which few regulatory genes control many downstream genes that contribute to stress tolerance as a whole. Most of known cold-related genes in cereals belong to the group of downstream genes, beginning to be expressed after 20-24 hours of cold stress, and only few early induced genes with a regulatory role have been described. To elucidate the early molecular events in stress sensing and signal transduction, a suppression-subtractive library of control and cold stressed (6 hrs at 3°C) durum wheat samples has been constructed by using the Clontech PCR-select cDNA Subtraction Kit.

This work yielded twelve different clones reconstructed through RACE experiments. Three clones gave positive matches in database with genes already known to be involved in plant stress response (actin-binding protein gene, S-adenosylmethionine decarboxilase gene and a sequence coding for a farnesylated protein). Four clones showed similarity with genes never studied in relation to stress tolerance, a putative ribokinase, a putative ARM repeat containing protein, a putative transmembrane protein  and a rice protein of unknown function. Three clones found matches only with bread wheat ESTs, and for the last two clones any significant similarity has been found in database. All these clones have been defined e-cor (early cold-regulated) genes, being over expressed or induced by cold within the first 6 hours of exposition to low temperature.

For some genes isolated in this work, the cold stress showed to influence not only, quantitatively, the abundance of transcripts but also the type of transcript present: a putative post transcriptional regulation mechanism based on alternative splicing has been found for two of isolated clones. 

The gene coding for the putative ribokinase (7H8), characterised by a complex coding region containing 8 exons and 7 introns, revealed the production of different alternative transcripts, on the basis of the presence/absence of some introns, following the cold treatment. The majority of these transcripts appeared in a transient manner between four and eight hours of low temperature exposure. This mechanism has been found also in other cereals, bread wheat and barley, but not in homologous gene of the dicot Arabidopsis. The 6G2 clone, similar to a rice hypothetical protein, showed the production of two alternative transcripts, around four hours of cold treatment, containing or not a 100bp intron in the 3’ UTR region of the messenger.

The presence of the intron could influence some properties of the transcript like its stability or binding capacity to regulatory proteins or to the ribosome. Several examples are described in literature, in which multiple transcript forms are synthesised from a single pre-mRNA in response to biotic and abiotic stresses. In this cases the alternative splicing has a role in the regulation of stress tolerance, and it is not a simple target site for stress damage. So further studies are in progress in order to clarify the meaning of the production of 7H8 and 6G2 alternative transcripts as a putative mechanism for the post transcriptional regulation of gene expression in response to cold stress.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.23

A BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE ISOLATED FROM HORDEUM VULGARE IS DIFFERENTIALLY REGULATED BY ABIOTIC STRESS

M. MALATRASI*,**, M. GULLI’*, M. CORRADI*, T.J. CLOSE**, N. MARMIROLI*

*) Dipartimento di Scienze Ambientali, Sez. Genetica e Biotecnologie Ambientali, Parco Area delle Scienze, 43100 Parma
**) Department of Botany and Plant Sciences, University of California, Riverside, USA

branched-chain amino acid aminotransferase, drought stress, barley, differential display, RT-PCR

Plants have developed several adaptive strategies to cope with environmental stress. Among the abiotic factors water availability is probably the most important in determining plant surviving and the most common to experience. The plant reacts to osmotic stress by a complex of responses leading to enhance the stress tolerance. These begin with stress perception, which initiates signal transduction pathways that cause changes at the cellular, physiological and developmental levels (involving hormones balance, modification of metabolites and repression or induction of many regulated genes). For genetic improvement programs the characterisation of new genes involved in stress response is fundamental. In fact many of the water-deficit induced genes encode for specific stress proteins with possible protective function, even if the only expression during stress does not guarantee that a gene product promote the ability of the plant to survive stress. In this research changes in the patterns of gene expression were analysed in drought, cold and ABA-treated seedlings of barley (Hordeum vulgare cv. Georgie) by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Here we report the characterisation of the gene HvBCAT-1, corresponding to the clone DD12 isolated by DDRT-PCR, which belongs to the branched-chain amino acid aminotransferase (BCAT) gene family. BCAT catalyze the final transamination step in the pathway that produces essential amino acids leucine, isoleucine, and valine; these enzymes are also involved in the degradation of these three amino acids. BCAT activity has been detected in a wide range of eukariotic systems, including maize, spinach, peas, barley, tomato fruit, soybean, yeast, rats and human. So far identification and cloning of plant BACT genes has been successful in potato, where the two genes analyzed were demonstrated to be differentially expressed (Campbell et al., 2001) and in Arabidopsis where a family of seven members with different subcellular location was described (Diebold et al., 2002). Through the screening of a barley BAC library the genomic clone corresponding to DD12 has been isolated, and the structure of the gene has been elucidated. A comparative analysis with the corresponding genes from rice and Arabidopsis has been performed in order to identify conserved structural patterns. HvBCAT-1 has been mapped on chromosome 4H. The expression analysis of HvBCAT-1 transcript was analysed by both qualitative RT-PCR and Northern hybridisation and several conditions were tested. Through the RT-PCR analysis it was revealed the presence of the transcripts in all the analysed conditions, but Northern blot analysis demonstrated that the level of transcript increased under drought stress and ABA treatments as compared with control condition. 

This is the first example of the identification of a BCAT gene in barley regulated by drought condition; this observation may suggest a role for BCAA metabolism in stress response that could be explained by the need for precursors for stress inducible secondary metabolites derived from these amino acids.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.24

A NEW NAC-PROTEIN GENE OF ARABIDOPSIS INDUCED IN RESPONSE TO NITRIC OXIDE AND PATHOGENS 

G. MALACARNE, E. MICA, M. PEZZOTTI, M. DELLEDONNE, A. POLVERARI

Dip.Scientifico e Tecnologico, Università degli Studi di Verona

NAC transcription factors, disease resistance, hypersensitive reaction

Transcript profiling by cDNA-AFLP of Arabidopsis thaliana following treatment with the nitric oxide (NO) donor sodium nitroprusside (SNP) allowed the identification of a homologue to the ATAF2 protein (ATAF2-like), which is induced 1 hour after the treatment. The gene product belongs to the NAC protein family of Arabidopsis, which contains a number of transcription factors unique to plants, associated with the response to various stresses. Given the well-known involvement of NO in the plant response to pathogens, we used the Real-time RT-PCR to analyse the expression level of ATAF2-like in the interactions between A. thaliana Col-0 and Pseudomonas syringae pv. tomato, both virulent and avirulent strains, and between A. thaliana and Turnip Crinkle Virus (TCV) in both compatible (ecotype Col-0) and incompatible (ecotype Dijon 17) interactions. Both the incompatibilities result in a hypersensitive reaction in Arabidopsis, that is a programmed cell death at the infection site that contributes to pathogen limitation and is often associated to an efficient race-specific resistance. Induction by NO was also confirmed.

ATAF2-like transcript abundance showed a 10-12 fold increases following nitric oxide treatment and appeared to increase in the compatible and incompatible interactions with P. s. pv. tomato, 4-fold and 8-fold respectively, 10 hours post-inoculation. TCV infection does not seem to affect ATAF2-like expression in the two ecotypes, at least within the first 24 hours.

Arabidopsis Col-0 insertional mutants carrying a single, homozygous T-DNA insertion in the coding region of ATAF2-like have been obtained and research is in progress to further characterise the functional role of this gene in the hypersensitive reaction. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.25

EXPRESSION OF SELF-COMPLEMENTARY HAIRPIN RNA UNDER THE CONTROL OF THE ROLC PROMOTER CONFERS SYSTEMIC DISEASE RESISTANCE TO PLUM POX VIRUS WITHOUT PREVENTING LOCAL INFECTION

B. MOLESINI, T. PANDOLFINI, L. AVESANI, A. SPENA, A. POLVERARI 

Dipartimento Scientifico-Tecnologico, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

PTGS, virus resistance, sharka, hairpin construct

Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection.

In the ihprolC-PP197 gene (intron hairpin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter. N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants is resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation.

The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.26

PLUM POX VIRUS (PPV) RESISTANCE: PRODUCTION OF TRANSGENIC PLANTS EXPRESSING RECOMBINANT INTRON-SPLICED PPV HAIRPIN RNAs 

E. DI NICOLA-NEGRI, A. BRUNETTI, V. INNOCENZI, V. ILARDI 

Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Rome, Italy, Fax +39.06.86802296
fisiopatologia@ispave.it
http://www.ispave.it/

PPV, transgenic plants, RNA silencing

Sharka is the most important disease of stone fruits in terms of agronomic impact and economic importance. The disease is caused by Plum pox potyvirus (PPV) a single-stranded RNA virus. There are five PPV strains but the two major and agronomically important are D and M. Until now no useful natural resistance has been found against this pathogen. 

Transgenic expression of pathogen-derived sequences encoding self-complementary “hairpin” RNA that undergo to an efficient and predictable posttranscriptional silencing (Smith et al., 2000. Nature, 407:319-320) is a new and agricultural sustainable strategy to obtain virus-resistant plants avoiding the production of transgenic viral proteins. 

Four PPV genomic sequence regions, highly homologous between D and M strains, were selected by in silico analysis as potential silencing targets. One of the four sequences starts at the 5’ end of PPV genome and includes a part of the P1 gene, the other three partially cover the HC-Pro gene that encodes the viral suppressor of RNA silencing. Each predicted-region was amplified by RT-PCR from a PPV M Italian isolate and arranged to form an intron-spliced RNA hairpin structure. The four sequences were introduced by Agrobacterium transformation into Nicotiana benthamiana plants under the transcriptional control of a cauliflower mosaic virus 35S promoter. Virus resistance experiments to test the ability of the different constructs to interfere with PPV replication are in progress. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.27

REGULATION OF PATHOGENESIS-RELATED GENES IN TRANSGENIC PLANTS WITH CONSTITUTIVELY ACTIVATED DEFENCE SYSTEMS

F. FIOCCHETTI*, M. TUCCI**, C. CARUSO***, L. BERTINI***, F. SACCARDO*

*) Dipartimento di Produzione vegetale, Università della Tuscia, Viterbo
**) CNR – IGV Istituto di Genetica vegetale, sez. Portici (NA)
***) Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, Viterbo

PR proteins, PR genes, fungal pathogens, genetic transformation, signal transduction pathways

Plants respond to pathogen challenge by activating defence strategies which include synthesis of “pathogenesis-related” (PR) proteins. Most PR proteins show strong antifungal activity in in vitro assays and possibly exert protective roles in vivo.

In recent years, extensive research has been devoted to elucidating the signal transduction pathways leading to activation of plant defences upon pathogen attack. These studies have been mainly focused on specific host-pathogen interactions, involving the presence of a specific avirulence (Avr) gene in the pathogen and a specific resistance (R) gene in the host. Neverthless, the complex signalling mechanisms finally leading to the activation of the whole array of plant responses to pathogens have been only partially unravelled.

The elucidation of the molecular signals activating PR gene expression could greatly contribute to the general understanding of the signal transduction pathways involved in the plant-pathogen interaction.

To this aim, we have studied the differential expression of PR genes in wild type and transgenic tomato plants constitutively expressing tobacco osmotin, a PR5 defence protein, upon treatments with known inducers of defence genes. Namely, expression of PR1, PR4 and PR5 genes was monitored through RT-PCR at different time points after treatments with salicilyc acid (SA) and its functional analogue acibenzolar-S-methyl (benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester) (BTH).

PR1 expression was induced by BTH at late time points after the treatment (15 days) in transgenic seedlings, while SA did not trigger the expression of PR1 in seedlings of both the transgenic and the wild type at 7, 10 or 15 days after the treatment. Upon SA treatment of detached leaves, expression of PR1 increased in the wild type already after 4 hours, while it was constitutively elevated in the transgenic line.

PR4 expression was induced by BTH but not by SA at 10 and 15 days after the treatment in transgenic and wild type seedlings, while SA triggered PR4 expression at 4 and 24 hours in detached leaves of the same genotypes.

The steady-state levels of PR5 RNA did not change upon treatments with either SA and BTH in transgenic and wild type seedlings at long time points, while they increased in detached leaves within 24 hours from the treatment with SA in both the transgenic and the wild type. As expected, PR5 expression was very high in the transgenic line, since the primers used for the RT-PCR detect also the high constitutive expression of the ectopic PR5 gene.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.28

DIFFERENTIAL EXPRESSION OF ISOFLAVONOID GENES DURING CICER-ASCOCHYTA INTERACTION BY REAL TIME PCR

C. ROSATI

ENEA, C. R. Trisaia, S.S. 106, km 419+500, 75026 Rotondella (MT), Italy

isoflavonoid pathway, plant-pathogen interaction, leguminous plants, Real Time PCR

Chickpea (Cicer arietinum) is a leguminous crop extensively cultivated in the Mediterranean area. Ascochyta blight, caused by the fungus Ascochyta rabiei, is the most serious fungal disease affecting chickpea, with yield losses higher than 50% in susceptible varieties. To investigate of view the early steps of Cicer-Ascochyta interaction leading to isoflavonoid phytoalexins biosynthesis from the molecular point, the expression of flavonoid and specific isoflavonoid genes was studied in the varieties Principe and Sultano, respectively susceptible and resistant to Ascochyta. The chickpea sequences of genes coding for chalcone synthase (CHS), chalcone isomerase (CHI), chalcone reductase (CHR), isoflavone synthase (IFS) and isoflavone reductase (IFR), known to be expressed during Cicer-Ascochyta interaction, were used to design specific primers and probes for Real Time PCR. The first two genes belong to the general flavonoid pathway, while the latter three encode proteins specifically involved in the synthesis of isoflavonoid phytoalexins, accumulated at different levels upon Ascochyta infection. The expression of such genes was monitored at 1, 3, 9 and 24 hours after inoculation with Ascochyta inoculum and blank solution, and compared with expression at time zero. Gene expression data were further normalized with respect to the expression of EF-1alpha gene. For chr, chs and ifr, the accumulation of transcript peaked 3 hours after inoculation. Qualitative and quantitative differences in gene expression patterns were found between the two varieties. CHS, the key enzyme in phenylpropanoid-flavonoid pathways, was constitutively expressed at high levels, though its transcript abundance was not affected by inoculation. Moreover, ifr was more expressed in the resistant variety Sultano than in Principe, and chi was showed a peak 3 hours after inoculation only in Sultano. On the other hand, chr was expressed at higher levels in the susceptible variety Principe than in Sultano, pointing out a negative correlation with mechanisms of resistance. A cross population Principe x Sultano is being bred to extend analyses to segregating progenies and validate the presented preliminary data.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.29

DIFFERENTIALLY EXPRESSED GENES IN THE APPLE CULTIVAR FLORINA AFTER SCAB INOCULATION

C. Toller*, c. Gessler**, A. patocchi**, E. Zini*, P. Baldi*, s. passerotti*, M. Komjanc*

*) Istituto Agrario di San Michele all’Adige, via E. Mach n°1, 38010 San Michele all’Adige (TN), Italy
**) Swiss Federal Institute of Technology Zurich (ETH) Rämistrasse 101, 8006 Zurich - CH

apple, PCR-Select, differentially expressed genes, apple scab

For years the Agricultural Institute of  San Michele all’Adige has been interested in the study of the plant - pathogen interaction in apple and in 2000 it has started a project called “Advanced Biology Applied to Grape, Apple and Salmons” financed by the Caritro foundation.

Our contribution in this project is 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), a method already known from literature to be very useful for the identification and isolation of rare and differentially expressed genes.

The current study is turned to the molecular analysis in order to identify defence and signal transduction genes after scab inoculation and salicylic acid treatment (a second messenger associated with SAR -  Systemic Acquired Resistance) in Florina apple leaf tissue.

In this way we will be able to find out the differences and similarities between the two signal transduction pathways.

We have isolated 1152 colonies; among them we have selected 280 positive clones by Southern analysis and 203 of them have been sequenced. 

The results revealed 10 most frequent sequences which showed a very high homology ( > 85%) with known sequences in blast analysis. 

All these genes have been studied by Northern analysis.

The two most representative genes were a 14 -3 - 3 protein and a Mal d 1, with a frequency respectively of  22 % and 8 % on the total of the sequenced clones. Both of them were strongly up-regulated after 48 h of treatment.

We have then isolated two different sequences of chitinase (class II and class V) that by Northern analysis revealed a differential expression, the first one was up-regulated after the treatment with the fungal pathogen while the second one was down-regulated. 

This result is consistent with two papers in which it has been demonstrated how some isoforms of chitinase could be up-regulated while other down-regulated after the same treatment.

Our next steps will be to obtain the full length of these genes and then to study their expression considering different times after scab inoculation to detect the pattern of activation of each gene in the plant.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.30

Addressing Heterosis in Maize by Molecular and Quantitative Genetics Approaches

G. PEA*, E. Frascaroli**, F. Cattonaro***, L. Gianfranceschi*, P. Landi**, M. Morgante***, M. Villa*, M. A. Canè**, M. E. PÉ*

*) Dip. Scienze Biomolecolari e Biotecnologie, via Celoria 26, 20133 Milano
**) Dip. Scienze e Tecnologie Agroambientali, via Fanin 40, 40127 Bologna
***) Dip. Produzione Vegetale e Tecnologia Agraria, via delle Scienze 208, 33100 Udine

heterosis, maize, QTLs, gene expression

The exploitation of heterosis in plant breeding is considered one of the most revolutionary advancement in plant improvement. However, despite a long story of successes, there is still a striking gap between the extensive agricultural practice of hybrid vigour utilization and our understanding of the cause of such phenomenon. An integrated approach comprising both molecular and quantitative analyses is being applied to gain some understanding of the heterotic phenomenon in maize: combination and integration of large scale transcriptional profiling data and modern quantitative genetics is being performed, in order to assess the importance of gene expression in determining heterosis and to shed light on the genetic and molecular mechanisms underlying it.

Appropriate plant material was produced, to provide a common set of genotypes for each experimental approach and to allow data integration. The material meets two contrasting needs: sufficient complexity in order to allow an accurate genetic interpretation of the phenotypic values, and simple genetic design to ease molecular biology analysis. In particular a population of 142 recombinant inbred lines (RILs), obtained from the original cross B73 x H99 was used for the production of three pseudo-back cross (PBC) populations by crossing each of the 142 RIL with both the original parental lines (PBC-P1 and -P2) and their F1 hybrid (PBC-F1). In addition, a tester population (TC) was also produced, by crossing each of the 142 RIL with tester line Mo17. Owing to the mating design adopted, the mapping population of reference is the B73 x H99 RILs. This population has been already characterized for more than 200 molecular markers, mainly Simple Sequence Repeats and AFLPs.

All the populations described (i.e. RILs per se, PBC-P1, PBC-2, PBC-F1 and TC, for a total of 716 genotypes) were grown in three locations (2 reps per location). Data for components of vegetative growth, plant cycle and yield were collected and analyzed. To identify the underlying QTLs, Composite Interval Mapping was employed (LOD > 2) by using PLABQTL (Utz, H.F. & Melchinger, A.E. 1996). Analyses of their effects and relationships across all populations allowed us to place emphasis on heterotic effect evaluation and thus to chose from 6 to 9 relevant QTLs. Results suggest that there are loci actually involved in heterosis and that dominance plays a role in it. For those QTLs, Near Isogenic Lines (NILs) are going to be produced for further genetic dissection and molecular analysis. Results concerning plant vigour and yield are discussed.

A comparison of the expression profiles of sets of genes in the hybrid and in the corresponding inbred parents is being used to identify the components of the genetic circuits that control heterosis. DNA microarrays on glass slides (University of Arizona), spotted with thousands of maize ESTs, were hybridized with fluorescently labelled cDNA obtained from 15 dap kernels and young leaves (endosperm and Unigene1 microarrays, respectively). Preliminary results of Significance Analysis of Microarray (SAM) are presented. Allele-specific expression levels in inbreds and hybrids will also be determined. The approach is based on the detection of Single Nucleotide Polymorphisms (SNPs) located within the transcript by single base extension methods followed by capillary electrophoresis separation using RNA as a template.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.31

ISOLATION AND CHARACTERIZATION OF CELL CYCLE GENES IN NICOTIANA TABACUM

M. ATALLAH, M. L. SAVO SARDARO, E. PORCEDDU

Dipartimento di Agrobiologia e Agrochimica, Università degli studi della Tuscia

cell cycle, cDNA-AFLP, Cdc20, d123

Cell division is a basic characteristic of all living organisms. 

A cDNA-AFLP approach was used to study gene expression in synchronized cell culture of Nicotiana tabacum TBY2. 20 fragments showing cell cycle regulated expression were excised from the gel, cloned and sequenced. Database comparison of these sequences identified high similarity to genes directly involved in cell cycle regulation.

The Cdc20 (the homolog of Fizzy in yeast and vertebrates) is a WD proteins involved in the M Phase, a key factors for function and regulation of the APC/C complex. APC-Cdc20 activity is present during mitosis and initiates the metaphase-anaphase transition. Cdc20 is regulated by the spindle assembly checkpoint monitoring the attachment of each chromosome to the mitotic spindle.

D123 is a novel 44-kDa gene product, which has been proposed as necessary for S-phase entry of the cell cycle, A point mutation of D123 resulted in a temperature-sensitive arrest in G1-phase in mammalian system. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in alpha-helix.   

For molecular characterization of these two gene we have made several constructs for gene overexpression, gene silencing and GFP fusion. This constructs have been introduced into Nicotiana tabacum plant and inTBY2 cell suspension.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.32

MOB-LIKE PROTEINS ARE INVOLVED IN BOTH CELL CYCLE PROGRESSION AND PROGRAMMED CELL DEATH IN ALFALFA

G. Barcaccia*, S. Varotto*, S. Citterio**, S. Sgorbati**, M. Lucchin*, P. Parrini*

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università degli Studi di Padova, Agripolis, Via dell’Università 16, 35020 Legnaro, Padova
**) Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, 20126 Milano

Medicago sativa L., reproductive mutants, gene expression, cytokinesis, apoptosis

Reproductive mutants of Medicago spp. that form 2n eggs by restitutional apomeiosis (FDR) and failure of cytokinesis (SDR) have been discovered and cytologically characterized. The movement of chromosomes and cytokinesis are accomplished by cytoskeletal structures, the spindle and the phragmoplast, respectively. The formation of 2n eggs may depend upon alterations of the cytoskeleton, especially defects in proteins which affect the function and integrity of spindle and phragmoplast tubules. A differential display of mRNAs allowed us to select a flower-specific EST with structural homology to MOB (Mps-one-binder) gene family members. The full-length cDNA clone of the Mob-like gene of alfalfa (GenBank acc. AJ319713) showed a significant amino acid similarity with several Mob1s from a variety of eukaryote organisms. Several Mob1 homologues have been recently cloned in Arabidopsis, chickpea, rice and maize, but the function of this gene in plants remains unexplored. Alfalfa genomic clones amplified by UTR-specific and ORF-specific primers enabled to find out two introns of 394 and 105 pb in the Mob1 gene. RT-PCRs with Mob1-specific primers followed by sequencing analysis revealed a messenger from the apomeiotic mutants bearing two adjacent stop codons and an insertion of 66 nt never found in wild-types. Transcripts of this member were detected only in flower buds at the mega-sporogenesis stage confirming the organ-specificity revealed by Northern blot hybridization, while roots, stems, leaves and pods revealed constitutive transcripts of a distinct Mob1 member. In situ localization of mRNAs with DIG-labeled Mob1 riboprobes in ovaries at different developmental stages revealed two different antisense hybridization patterns: in ovules undergoing a regular meiosis the signal was detected in apoptotic megaspores, whereas in apomeiotic ovules the signal was visualized in either MMCs and embryo sacs. The hybridization signal was also detected in anther tapetal cells when PCD takes place to allow pollen grain dispersal and in degenerating ovules with non-viable embryo sacs. Interestingly protein database searches revealed the existence of ovary-specific Mob1 gene products in human and mouse tumor tissues. It has been shown that the Phocein-MOB domain (pfam 03637) can be combined (e.g. in Arabidopsis and rice) with the NB-ARC domain (pfam 00931), a signaling motif shared by animal cell death gene regulators as well as with Leucine Rich Repeats (pfam 00560), short sequence motifs involved in protein-protein interactions. The potential link between Mob1 expression and apoptosis in alfalfa reproductive tissues was investigated in flower bud sections by TUNEL: apoptotic MMCs of the apomeiotic type were never observed and DNA fragmentation was observed solely in degenerating meiotic megaspores. Polyclonal antibodies against the Mob1 proteins were produced and tested for their specificity of recognition by immunoblotting and immunocytochemical assays. Western blotting of alfalfa proteins isolated from 3-day old plantlet roots and cotyledons revealed two distinct doublets of about 28 KDa and 55 KDa providing that Mob1 may be a component of multi-domain proteins. Along with a cortical localization in synchronized root tip cells, the Mob1 proteins were visualized at the cell plate site during cytokinesis by marking the progressive formation of the division septum. Further experiments are in progress to better understand the Mob1 protein function during mitotic and meiotic spindle assembling and activity, and to check whether in alfalfa mutants Mob1 expression is correlated to either an apomeiotic or apoptotic fate of the ovule cells.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.33

GENETIC ANALYSIS OF NEW VIVIPAROUS MUTANTS OF MAIZE

A. GIULINI*, G. CONSONNI*, F. DALLA VECCHIA**, N. RASCIO**, N. LA ROCCA**, R. PILU*, S. STEFANELLI***, R. TUBEROSA***, G. GAVAZZI*
*) Department of Crop Production, Via Celoria 2, 20133 Milano, Italy 
**) Department of Biology, Via Colombo 3, 35121 Padova, Italy 
***) Department of Agroenvironmental Sciences and Technology, Via Farin 44, 40127 Bologna, Italy

abscisic acid, maize, viviparous mutants

Abscissic acid (ABA) regulates many agronomically important aspects of plant development, including synthesis of seed storage proteins and lipids, promotion of seed desiccation tolerance and dormancy. In addition, ABA mediates some physiological responses to environmental stresses such as stomatal closure, or induction of tolerance to water. Mutants, altered in ABA metabolism, provide a good opportunity to identify which functions at the molecular, biochemical, cellular and/or whole plant level are affected by impaired ABA biosynthesis or sensitivity.

We selected out of a large sample of vp mutants, originated in our lab or received from other scientists, four mutants yielding green seedlings. The majority of the vp mutants in maize are associated to white or pale yellow endosperm and seedling because they are due to a mutational event occurring in the first part of the biosynthetic route that is shared by ABA biosynthesis and carotenogenesis.

The mutants thus selected and provisionally symbolized vp374*, vp390*, vp105*, vp D*, affect in downstream steps of the biosynthesis or they represent regulatory genes involved in the perception and/or transduction of ABA signal.

They were intercrossed to establish their allelism relationship and crossed to vp1, a regulatory gene of the ABA signal and to vp13, a mutant specific of the ABA biosynthesis.

Double mutants vp1/vpD* and vp1/vp105* were also tested. We will present data on their response to exogenous ABA, on their endogenous ABA content, on their pattern of ABA inducible genes during embryo maturation.

Their analysis will be extended to vegetative tissues to establish how the mutation affects water loss and stomatal aperture with the aim to establish whether the genes under study are specifically expressed during embryogenesis or if they are also important during vegetative growth.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.34

Cloning S-locus sequences related to sporophytic self-incompatibility in Cichorium intybus L.

G. Barcaccia, S. Varotto, M. Soattin, M. Lucchin, P. Parrini

Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Facoltà di Agraria,
Università di Padova, Agripolis, Via Romea 16, 35020 Legnaro, Padova, Italy

chicory, S-locus receptor kinase (SRK), S-locus glycoprotein (SLG), gene expression

In chicory (Cichorium intybus L.) inbreeding is hampered by a sporophytic incompatibility mechanism that prevents both selfing and crossing between plants showing identical phenotype at the multiallelic S-locus. Little information is available on the incompatibility system of this species and a deeper knowledge on the genetics of this reproductive barrier is needed for its exploitation in breeding programs. In chicory the incompatibility reaction occurs soon after pollination: trinucleate pollen grains cannot adhere (self-pollen) or germinate (allo-pollen) on the dry stigma. Cytological features indicate that the incompatibility reaction is partially similar to the pollen rejection observed in Brassica species, where the sporophytic incompatibility mechanism has been partly elucidated at molecular level. The products of two closely related genes, an S-locus receptor-like kinase (SRK) and an S-locus secreted glycoprotein (SLG) with multiple alleles organized in distinct haplotypes, cooperate to function as molecules for the recognition and rejection of the incompatible pollen on the stigmatic surface. SRK and SLG share a common domain called the S domain. A number of S domain-related sequences, unlinked to the S locus, have been found not only in Brassica, but also in several genomes of both dicots and monocots. In spite of the numerous attempts made for identifying S-gene products using RT-PCR and degenerated primers constructed on the mostly conserved regions of the S-gene products of Brassica, no related sequences were identified in chicory. The cDNA-AFLP method allowed us to obtain information on gene expression level and on its changes during the incompatibility reaction: although transcripts differentially expressed between self-compatible (SC) and self-incompatible (SI) plants of a C. intybus x C. endivia hybrid progeny were identified, no S-locus related sequences were found. Primers designed by Nasrallah et al. (1987, Nature 326:617-9) on the coding sequence of S-locus alleles of B. oleracea were used to screen by PCR different cDNA samples obtained through reverse transcription of total RNA isolated from SI and SC plants of C. intybus. Primer combination A1-forward and D1-reverse revealed a 1200 pb product that was cloned into a plasmid and sequenced. Homologies for the cDNA clone were searched in public databases to compare nucleotide and translated sequences. The sequence of chicory (AJ551426) showed a 99% similarity degree with an S-locus glycoprotein of B. oleracea (CAA34254), while the alignment with the SLG conserved domain scored a 8e-59 value. Moreover, sequencing analysis of genomic AP-PCR fragments obtained with the M13 universal primer revealed by chance a 430 nt long polymorphic amplicon of C. intybus partially homologous to an S-locus gene of Raphanus sativus (AY052578). Structural homologies were searched in protein databases to compare its deduced amino acid sequence with known proteins. The most significant amino acid sequences were used for pair-wise alignments to point out shared regions with respect to the translated sequence of chicory adopted as query. The highest similarity estimates were found with S-locus receptor-like kinases of Brassica. Semi-quantitative PCRs with internal primers using ds-cDNA templates isolated from SC and SI plants of a cross between genotypes contrasting for the incompatibility reaction (FB4 of C. intybus and SA12 of the self-compatible C. endivia) confirmed the differential expression of the putative SRK transcript. Research is in progress and 30-mer primers specific of the chicory SRK-like clone will be designed to obtain the full-length by performing both the 5' and 3' RACE. Both the SLG member and the SRK-related marker will be assigned to linkage groups of a functional map of C. intybus.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.35

AN INTEGRATED APPROACH FOR THE STUDY OF SEXUAL DIFFERENTIATION OF CANNABIS SATIVA L.

V.M.C. MOLITERNI*, L. CATTIVELLI**, G. MANDOLINO*, P. RANALLI*

*) Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40128 Bologna
**) Istituto Sperimentale per la Cerealicoltura, strada S. Protaso 302, 29017  Fiorenzuola d’Arda (PC) 

Cannabis sativa, dioecy, sexual differentiation, sex linked markers, cDNA AFLP

Cannabis sativa is a dioecious species with unisexual flowers and sexual dimorphism occurring in a late stage of plant development only. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (2n = 18+XY) and female is the homogametic one (2n = 18+XX). Despite of its chromosomal determination, the sexual phenotype of Cannabis often shows some flexibility. Anomalies in fact may occur in floral development, leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). For some genotypes it is also possible the complete sexual reversion by treatment with activators or inhibitors of the ethylene biosynthetic pathway.

The microscopic analysis of male and female apices at different stages of development, revealed that the earliest step in their sexual differentiation occurs as the leaves of the fourth node emerge. The genetic expression of male and female apices at this stage has been compared by cDNA AFLP. In order to discriminate the two sexes before sampling, a rapid method for sex determination has been developed, based on the PCR amplification of a 391 bp long, male-specific SCAR marker, directly from a leaf  tissue fragment. 

Seven of the several cDNA AFLP polymorphic fragments, have been identified to be differentially expressed in males and females apices at the fourth node; cloning and sequencing of the sub-clones, revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. The results of BLAST analysis for this clones led to the identification of their putative coding function and to their possible roles in this earliest step of sexual differentiation. Four of the induced clones belong respectively to the mRNA for a putative permease, for a putative kinesin, an SMT3 like protein and a RAC-GTP binding protein like . 

The induction of these clones in female apices at this stage may suggest that they are involved in the early phase of the female sex differentiation; on the other hand, their under-expression in male apices could be correlated to the repression of female characteristics at this precocious stage of male apex sexual differentiation .

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.36

CLONING OF CANDIDATE GENES FOR APOMIXIS IN KENTUCKY BLUEGRASS (P. PRATENSIS L.)

E. ALBERTINI, G. MARCONI, M. FALCINELLI

Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy

apomixis, cDNA-AFLP, differential display, Poa pratensis 

Poa pratensis L., one of the most important forage and turf grasses in temperate climates, has a versatile and dynamic mode of reproduction that ranges naturally from nearly obligate apomixis to complete sexuality. In this species apomixis is functionally the result of two processes: apospory and parthenogenesis. Despite extensive efforts aimed at understanding the inheritance of apomixis, the identity of the gene/s controlling the reproductive process is still obscure as well as it is not known whether the combination of apomictic features (apospory with parthenogenesis) derives from the expression of one single gene or a few linked genes. Our research project deals with the elucidation of the genetic control of apomixis and with the cloning of candidate genes for both apomixis as a whole and its elements (i.e. apospory and parthenogenesis). A gene expression study was carried out by using the cDNA-AFLP technique in order to select and isolate differentially expressed mRNAs for apospory and parthenogenesis. Within an F1 population of P. pratensis segregating for the mode of reproduction an highly apomictic (aposporic and parthenogenetic), a completely sexual (non-aposporic and non-parthenogenetic), and also a recombinant (aposporic and non-parthenogenetic) genotypes were selected on the basis of auxin test and cytohistological investigations and used for mRNA profiling by AFLP markers. The approach enabled us to select 172 mRNAs either apomictic- or sexual-specific and to detect transcriptional changes potentially related to apospory and parthenogenesis. About 30% of clones showed no similarity with genes of interest whereas the other 70% showed similarity with genes involved with meiotic or embryo development pathways. Interestingly a group of genes differentially expressed between apomictic and sexual genotypes showed an high similarity with a group of genes tightly linked in Arabidopsis thaliana. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.37

ANALYSIS OF THE ROLE OF C4-3, A TOBACCO GENE EXPRESSED DURING FLOWER GROWTH, IN THE DEVELOPMENT OF ANTHERS

V. CECCHETTI*, M. POMPONI*, S. D'ANGELI*, M.M. ALTAMURA *, G.B. TORNIELLI **, M. PEZZOTTI***, P. COSTANTINO*, M. CARDARELLI***

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

auxin, rolB, anther development, C4-3

Auxin has a substantial role in floral organ development. We demonstrated an effect of auxin in the process of anther development and dehiscence by means of the localised expression of the Agrobacterium rhizogenes rolB oncogene that induces a cell-autonomous state of auxin hypersensitivity. Tobacco pDMC1:rolB plants, that express  the rolB oncogene under the control of  the meiosis specific promoter AtDMC1, are male sterile due to alterations in meiosis causing a reduced number of pollen grains as well as a severe delay in anther dehiscence that causes pollen release when the stigma is no longer receptive to impollination. Moreover these plants display reduced stamen growth due to shorter filaments as compared to controls. Real-time RT-PCR analysis of mRNA extracted from male sterile pDMC1:rolB anthers, at meiotic and posmeiotic stages, shows an increase in the transcript level of C4-3, a c-DNA overexpressed in tobacco protoplasts expressing rolB. Remarkably, tobacco plants transformed with an antisense C4-3 c-DNA under the control of the CaMV35S promoter show an increase in stamen lenght, suggesting a role for C4-3 in filament elongation.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.38

ARE CLASS B HOMEOTIC GENES INVOLVED IN THE EXPRESSION OF PARTHENOCARPY IN TOMATO?

I. OLIMPIERI, F. SILIGATO, R. CACCIA, A. MAZZUCATO

Dipartimento di Agrobiologia e Agrochimica, Sezione di Genetica, Università degli Studi della Tuscia, Via S.C. de Lellis snc, 01100 Viterbo, Italy

floral mutants, homeotic genes, parthenocarpy, tomato

In flowering plants, the ovary develops into a fruit only after pollination and fertilization, when the requirements for seed development are thoroughly fulfilled. The genetic and molecular mechanisms that negatively control ovary development before pollination are not well understood. A recently proposed theory postulates that stamens act as regulatory factors towards the fourth floral whorl, by repressing the development of the ovary until fertilization has taken place. Accordingly to this theory, the silencing of a class B homeotic gene has been shown to be the cause of parthenocarpy in apple and a correlation between stamen disruption and parthenocarpy has been reported in transgenic tomatoes.

In tomato, natural or induced parthenocarpic mutants offer a useful method to regulate fruit production and a suitable experimental system to study ovary and fruit development. Among them, the parthenocarpic fruit (pat) mutation deserves particular interest because of its strong expressivity and the pleiotropic effects that affect anther and ovule phenotype. In such mutant in fact, the anthers show homeotic transformation into carpel-like structures, seldom bearing external ovules on the adaxial surface and ovules show arrested integument growth with absence of meiosis and consequent unviability. To date, no research has tested the hypothesis that stamen aberrations and parthenocarpy are driven by cues coming from the altered expression of class B genes in this genotype.

In this research, we address this possibility by constructing double mutants between pat and tomato mutations putatively involved in the B function, such as stamenless-2 (sl-2, that shows homeotic conversion of stamens into carpels) and pistillate-2 (pi-2, that shows strong stamen and subtle petal aberrations). In addition, the expression patterns of tomato class B (LeAP3 and TM6) and class B-related (TM8 and TM29) genes have been studied in time courses of tomato WT and pat mutant ovaries.

F1 hybrids between the pat mutant and sl-2 or pi-2 showed wild-type (WT) phenotype, indicating that the mutations affect different loci. In F2, whereas pat and sl-2 segregated independently and numerous double mutants could be recovered, for pat and pi-2 no double mutant could be unambiguously recognized out of 200 progeny plants, thus suggesting tight linkage. pat and sl-2 showed mainly additive effects in double mutant segregants, while some phenotypes of the pat mutation were counteracted by a single dose of the sl-2 allele. These results indicated that pat and sl-2 mutants might share only partially overlapping genetic determinants.

RT-PCR based expression analysis revealed that all the four assayed genes were down-regulated in the pat mutant ovary or fruit in comparison with the WT. For those transcripts characterized as being specifically transcribed in ovules (AP3), such lower expression in the mutant could be merely explained by the presence of very few normal ovules inside the pat ovary. However, the down-regulation of LeAP3 might also be directly involved in the parthenocarpic phenotype, because this pattern was also observed in the pat-2 mutant, a different tomato mutation for parthenocarpy that shows normal ovule development. Similarly, the lower expression of TM29 in the pat mutant can be directly related to parthenocarpy, because this gene is normally expressed in the young ovary pericarp.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.39

SUMO REGULATES FLOWERING IN ARABIDOPSIS

G. MURTAS*, P. H. REEVES*,**, Y.-F. FU*,**, I. BANCROFT*, C. DEAN*, G. COUPLAND*,**

*) John Innes Centre, Colney Lane Norwich NR4 7UH UK
**) Max Planck Institute for Plant Breeding, Carl von Linne Weg, 10, D-50829 Cologne, Germany

flowering, development, SUMO

Flowering is controlled by a network of genetic pathways in Arabidopsis thaliana. We have characterised an extreme early flowering mutant early in short days 4 (esd4) that dramatically accelerates the transition from vegetative growth to flowering in Arabidopsis. In addition to its flowering time phenotype, esd4 has effects on plant development, which include broadening of the silique, alterations in phylotaxy and a reduction in stature.

Genetic analysis indicates that ESD4 is most closely associated with the autonomous pathway. The MADS box transcription factor FLOWERING LOCUS C (FLC) plays a central role within this pathway as a potent repressor of flowering and the flowering phenotype of esd4 is partly caused by reduced abundance of FLC mRNA, as well as an FLC-independent mechanism. 

By map-based cloning we have isolated ESD4 and this gene encodes a nuclear protein containing a segment of over 200 amino acids at the carboxy-terminus with strong similarity to yeast and animal proteases that are specific for the protein modifier Small Ubiquitin-related Modifier (SUMO). We demonstrate that ESD4 has a similar protease function by showing that in vitro it processes the precursor of Arabidopsis SUMO (AtSUMO). Moreover, in esd4 mutants the abundance of free AtSUMO is reduced, suggesting that in vivo the ESD4 recycles AtSUMO from conjugates.

The effects of mutations in SUMO metabolism have not been described in a multicellular organism and this is the first demonstration of the importance of SUMO in plant development. ESD4 defines important roles for protein modification by AtSUMO within the genetic network that regulates flowering and in other aspects of plant development.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.40

CHARACTERIZATION OF cDNA SEQUENCES DIFFERENTIALLY EXPRESSED IN WHEAT (TRITICUM AESTIVUM L.) SPIKELETS

M. CIAFFI, A.R. PAOLACCI, E. d’ALOISIO, O.A. TANZARELLA, E. PORCEDDU

Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo
ciaffi@unitus.it

wheat, flower development, heading stage, differential analysis, gene expression

The past decade has provided a wealth of information concerning the molecular and genetic control of flowering in higher plants. Numerous studies have led to the insight that inflorescence and flower development in higher eudicotyledonous species, such as the predominant model system Arabidopsis, are controlled by a hierarchy of interacting regulatory genes, most of them encoding transcription factors. Comparatively little is understood on genetic and molecular aspects of flowering in monocots. The elucidation of the molecular basis of grass flower development can be considered just at its beginning, since most of the genes involved in floral transition (flowering time genes), specification of floral meristem (meristem identity genes) and regulation of size and number of floral organs (floral meristem size genes and floral organ pattern genes) remain to be cloned and characterised. Moreover, most “floral organ identity genes” have yet to be detected in wheat, as only three MADS-box genes belonging to the AP1, AP3 and AG groups had been cloned. The aim of the present study was the isolation of cDNA sequences differentially expressed in wheat spikelets at the heading stage. 

RNA was extracted from fully emerged spikes and flag leaves of Triticum aestivum cv. Chinese Spring.  Purified mRNAs were analysed by a modified version of the Simple Differential Display Method (SDDM), which consists of the first-strand cDNA synthesis primed by random hexamers, followed by PCR amplification using random 10-mer oligonucleotides and then size separation of cDNA fragments by denaturing polyacrilamide gels. Out of 60 analyzed decamers, 38 detected 142 differential amplification products. Each differential band was extracted from the gel and amplified with the same primers used for the previous amplification. On the basis of northern hybridisation signals on RNA from spikelets and leaves, the 142 clones were assigned to four groups: group A included 40 clones which hybridised exclusively to the spikelet RNA; group B consisted of 25 clones whose hybridisation signals were higher in RNA from spikelets than in that from leaves; group C comprised 42 clones that showed similar hybridisation signals in both tissues; group D, finally, included 35 clones which did not detected any hybridisation signal in RNA from both spikelets and leaves. A total of 100 clones of the groups A, B and D were wholly sequenced. Group D clones were included because the lack of hybridisation signals in both analysed tissues could be due to very low expression level, which is peculiar of transcription factors and regulatory genes. Specific primers were designed for each of the 35 clones of group D and their expression in spikelets and leaves was checked by RT-PCR analysis. Differential expression in the spikelets was detected for 20 of the 35 clones of D group, whereas 8 clones were expressed in both spikelets and leaves and 7 primer combinations did not show any amplification product in both tissues. The search of nucleotide and deduced amino acid sequences of 85 clones, whose preferential expression in the spike had been previously shown either by Northern or RT-PCR analyses, in several databases allowed their assignment, on the basis of their putative functions, to four main functional groups: I) sequences involved in the morphogenesis of floral organs; II) sequences expressed in anther tissues and/or pollen; III) transcription factors; IV) kinases and LRR proteins. Some sequences probably related to the intense cell proliferation which takes places within the floral organs, such as cyclins, microtubule and centromeric proteins, or in the protein trafficking within the secretory pathway; were also cloned. The expression patterns of several cloned sequences have been studied by RT-PCR analysis of RNAs extracted from different plant tissues and floral organs of Triticum aestivum cv. Chinese Spring.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.41

ISOLATION OF DNA FRACTIONS DIFFERENTIALLY EXPRESSED IN CITRUS DURING RIPENING PROCESS

D. SALUZZI, L. MILELLA, G. MARTELLI, I. GRECO

Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Università degli Studi della Basilicata, Contrada Macchia Romana, 85100 Potenza

citrus, ripening process, differentially DNA fractions expression

Development, maturation and ripening of fruits has received considerable experimental attention, primarily due to the uniqueness of such processes to plant species and the importance of fruit as a significant aspect of human dietary intake and nutrition. Molecular and genetic analysis of fruit development, and especially ripening of fleshy fruits, has resulted in significant gains in knowledge over recent years especially considering metabolic pathway characterizing commercial and nutritional fruit  quality (Canel et al., 1996; Moriguchi T., et al 2002).

Ripening has an impact on fibre content and composition, lipid metabolism, and the levels of vitamins and various antioxidants (Ronen et al., 1999). The ability to understand and manipulate, through breeding or biotechnology, key control points or regulatory points of specific ripening process such as carotenoid, flavonoid, vitamin, and flavour volatiles, will allow the control of nutrition and quality characteristics linked to ripening. 

The present work was pointed to isolate DNA fractions differentially expressed in Citrus during ripening process. 

To investigate this process fruits of four orange genotypes, were collected at two different ripening stages: green and ripe. The fruit were frozen in liquid nitrogen and stored at -80°C. Totally RNA and mRNA were isolated from different fruit tissue and cDNA was obtained in order to perform several experiments of differential display and cDNA-AFLP. Specific and random primers were also utilized in order to isolate sequences with high level of similarity with gene involved in ripening process. 

Using that techniques, 150 differentially expressed fraction were isolated: 48 using cDNA-AFLP experiment and the others with random and specific primers. All fragments differentially expressed among the ripening stages and coming from the different genotypes utilized were  sequenced. The sequences obtained were compared against public databases for similarity with genes yet isolated. 

Twenty sequences shown high homology with genes encoding protein with known activity  as epimerase, carotene desaturase and Zinc methabolism enzymes.

The results obtained by the use of specific primers inspire to improve the analyses of the ripening process with the aim of isolate key control points of specific metabolic pathway.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.42

MUTANT GENES RELATED WITH ETHYLENE PERCEPTION AND SYNTHESIS (Nr, rin, nor) MODULATE THE DEGREE OF AUTONECROSES IN THE TOMATO (LYCOPERSICON ESCULENTUM MILL.) "LESION MIMIC" MUTANT V20368 

V. FONZO*, P. MOSCONI*, R. CACCIA*, E. SANTANGELO**, G.P. SORESSI*

*) University of Tuscia, DABAC, via S.C. de Lellis s.n.c., 01100 Viterbo
**) Consorzio Agrital Ricerche, viale dell'Industria 24, 00057 Maccarese (Roma)

ethylene, lesion mimic, PCD, tomato

A distinctive trait of plant-pathogen incompatible reaction is the hypersensitive reaction (HR), a mechanism of programmed cell death (PCD) used by the plant in order to limit the pathogen spreading. The ethylene hormone is one of the putative regulators of PCD in some physiological processes (He et al., 1996; Orzaez and Granell, 1997; Young and Gallie, 2000; De Jong et al., 2002) and its role as signal molecule (with salicilic and jasmonic acid) is well known (Lund et al., 1998; O’Donnell et al., 2001; Gu et al., 2000).

For the comprehension of mechanisms activated during the PCD, an important tool is the study of the “lesion-mimic” mutants, mimicking forms of cell death resembling those triggered during the plant-pathogen interaction. In tomato an "autonecrotic" mutant was described first by Rick and Butler (1956) and recently studied by Kruger et al. (2002). The necrotic lesions are triggered by high temperature and luminosity on 6 week old plants with an acropetal direction and are caused by the interaction between the Cf-2 resistance gene (introduced from L. pimpinellifolium) and a second gene (Rcr3esc) belonging to L. esculentum and encoding for a cystein protease.

In a new tomato autonecrotic mutant (line V20368), previously characterised from a genetic, biochemical and molecular point of view, the role of ethylene in the modulation of the necroses formation was investigated, by crossing the autonecrotic line with the tomato mutants for perception (Nr) and synthesis (nor, rin) of ethylene. 

Parental lines (V20368, Nr, rin and nor) and the F1 hybrids between V20368 and the ethylene mutants were grown in tunnel during environmental conditions leading to the autonecroses. The phenotypic manifestation of necroses was evaluated and the ethylene (by gas chromatograph) and cell integrity (by ion leakage) measured on the basal, central and apical leaves when the necrotic spots appeared on the basal ones.

From the data emerged differences in terms of time of appearing, number and diameter of lesions, in relation to the presence of Nr, rin and nor genes. The necrose evolution seem affected by the ethylene reduction due to the nor and rin genes, but not by its perception due to the Nr gene. In fact, the F1 between V20368 and rin or nor appeared less necrotic, while the F1 with Nr exhibited earlier and more severe necrotic lesions. A maternal effect was also observed.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.43

DIFFERENTIAL GENE EXPRESSION IN CITRANGE TROYER OVEREXPRESSING ARABIDOPSIS PHYTOCHROME B GENE, AS DETERMINED BY REAL TIME PCR

P. LATINI*, G. DISTEFANO**, A. CONTINELLA**, S. LA MALFA**, M. C. INTRIERI*, D. ZINIU**, E. RUGINI*, A. GENTILE**, R. MULEO*

*) Dipartimento di Produzione Vegetale, Università della Tuscia, Viterbo, Italy
**) Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari sez. Arboricoltura, Università degli Studi di Catania, Italy

PhyB, Quantitative RT-PCR, Light qualities, gene expression

Light is an important environmental cue, which regulates plant development from seed germination up to flowering and fruit formation. Many genes are under control of light qualities, through the signal transduction of some classes of photoreceptors like the family of Phytochrome, Cryptochrome, UV-receptor and Phototropins. Among them, phytochrome family is the best characterized receptor. In particular, phytochrome B (PHYB) is the most important activated phytochrome under light conditions. In some plants the overexpression of alien phyB gene induces strong modification in gene expression and in morphology, i.e. decrease in stem elongation and in chlorophyll content, increase in branching and delay in senescence.

In this study, genetically transformed citrange Troyer [Citrus sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.9] overexpressing Arabidopsis phyB gene have been used to investigate the regolative role on the transcription of target genes linked to photosynthetic activity and light signal transduction itself: cab, rbcs, psbA, phyB. Leaves were sampled for molecular analyses from wild type and overeexpressing plants after their exposition to red light. Real Time quantitative RT-PCR (LightCycler, Roche), using SYBR Green I dye method, was performed to quantify the amount of transcripted target genes. Ribosomal 18s genes (18s) were used as an internal control (housekeeping gene) for the normalization of the results.

Results showed a strong reduction in the amount of transcripts of rbcs and cab genes (ranging for 40 to 70%) in plants overexpressing phyB as compared to wild type plants. The other genes studied did not show significant variation of expression levels, excepted for phyB gene. Transgenic plants showed a higher CO2 exchange rate (CER) compared to that of wild type ones, especially during midday hours. 

Moreover, morphological traits seem also to be affected from the transferred DNA, probably, by the alteration of the phyB signal transduction regulating morphogenic events. We speculated that overexpressed phyB may alter the signalling cascade playing a pivotal role in the interaction between PHYB and G-protein and /or PIF3.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.44

PROFILING GRAPE GENE EXPRESSION DURING LEAF DEVELOPMENT AND SENESCENCE BY cDNA ARRAYS

M. PINDO*, C. MOSER*, P. GATTO*, C. SEGALA*, P. FONTANA*, H.A. BECKER***, E. BLANZIERI**, R. VELASCO*

*) Area Biologia Avanzata, Istituto Agrario S. Michele a/Adige, S. Michele a/Adige, Trento
**) Dip. di Informatica e Telecomunicazioni, Università di Trento
***) Max Plank Institut für Züchtungsforschung, Koeln, Germany

grape, gene expression, ESTs, arrays, leaf development

High-throughput sequencing of ESTs  and array technologies are important tools for genome-wide studies of essential biological processes like leaf development and senescence. At our institute, we are interested in understanding the process of grape leaf senescence from a physiological and molecular point of view. Early symptoms of senescence in grapevines leaves can indeed severely affect the quality of the grape harvest. 

In order to obtain a collection of grape expressed sequences we constructed several standard cDNA libraries from bud, leaf, berry, root (cultivar 'Pinot noir'), sprout and inflorescence (cultivar 'Regent', in collaboration with the Institut fuer Rebenzuechtung, Geiweilerhof, Germany). In addition three subtracted cDNA libraries were constructed by coupled subtractive hybridizations of leaf, berry and root ('Pinot noir'). A subset of these cDNA clones (from leaf, bud, sprout and inflorescence tissues) were sequenced and amplified by PCR and then orderly arrayed on nylon membranes for gene expression studies. The high-density nylon filters contain 4010 amplified cDNAs double spotted corresponding to about 2300 clusters (unigenes). Positive and negative controls were also included. Radioactively-labeled RNAs from 'Pinot noir' leaves at 5 different development stages were used to hybridize the high-density filters to profile gene-expression during development and senescence of grape leaves. 6 independent measurements for each condition were collected and the data were then normalized making the assumption that global expression does not change among filters and conditions. Analysis of gene expression data has been carried out by looking at genes involved in specific metabolic functions and by clustering methods. Gene clusters will contain those transcripts whose expression is similar in the 5 tested conditions. Preliminary results, to be further validated by RT-PCR, high-lighted genes that are up-regulated during the senescent phase.

This work was supported by the project "Advanced Biology" funded by the Fondazione delle Casse di Risparmio di Trento e Rovereto (Trento-Italy).

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.45

EXPRESSIONS LEVELS OF CHS, DFR AND ANS AND ANTHOCYANIN CONTENT IN 19 GENOTYPES OF CITRUS L.

M. P. RUSSO*, P. S. COTRONEO*, A. R. LO PIERO**, A. CONSOLI**, I. PUGLISI**, G. PETRONE**, T. CORALLO*, G. REFORGIATO RECUPERO*

*) Istituto Sperimentale per l’Agrumicoltura, Acireale (CT)
**) DACPA, Facoltà di Agraria, Università di Catania

anthocyanins, orange flesh, Real Time RT-PCR, Citrus spp. L.

Italian orange fruit production is characterized by the presence of anthocyanins, an important class of flavonoids, representing, due to their antioxydant healthy value, the mean of promotion for the marketing of this product. Making use of clonal selection, inside this group, have been differentiated many early and late selections, characterized by anthocyanin content, diversely expressed in flesh and rind. Anthocyanin content has varying expression levels and is strictly dependent on climate conditions.

The aim of this study is to characterise the relationships between the expression of biosynthetic genes CHS (chalcone synthase), DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase) and the anthocyanin content. Evaluation of expression levels of the enzymes was tested by Real Time RT-PCR, in 16 blood oranges selections, in Cadenera, a blond orange cultivar, and in AMOA 8 and OTA 9, two hybrids obtained crossing respectively: Moro orange per Avana mandarin, and Oroval clementine per Tarocco, the elongation factor (EF), present in Citrus at constant level, was used as internal reference.

Anthocyanin buildup in the flesh of fruits picked in mid march was notably varying, from a maximum of 56.4 mg/g tissue in the AMOA 8 hybrid to a minimum of 0.17 mg/g in Tarocco Sciara. On the basis of their content 3 groups were detectable: the first varying from 56.4 to 10.8 mg/g, the second from 6.2 to 1.4 mg/g and the third from 0.67 to 0.17 mg/g. Anthocyanin content was positively related with the expression of biosynthetic genes. CHS, DFR e ANS levels were basically related among them in the different selections.

Real Time RT-PCR results suggested that the expression levels of CHS, DFR and ANS chiefly corresponded to the amount of anthocyanins. The presence of variability mostly marked in anthocyanin content is due to its accretion. This is confirmed by data from Tarocco Messina, a late Tarocco orange selection, which begins later than the others its pigmentation process, having a very low content of anthocyanins, although  showing intermediate levels of  biosynthetic genes expression. In Cadenera orange no presence of the pigment was detected, even though  CHS and ANS expression levels were similar to the ones observed for the group showing the lowest levels of anthocyanins. The low expression of the biosinthetic genes in the blond orange Cadenera suggests that there are other factors controlling red coloration. Recently Kobayashi et al., 2001 suggested that in white cultivars of grape and in their sports with red skin the phenotipic change is thought to be the result of a mutation in some regulatory gene controlling the expression of UDP-glucose, flavonoid 3-O- glucosyltransferase (UFGT).

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.46

ISOLATION AND FUNCTIONAL DISSECTION OF THREE CHS PROMOTERS IN VITIS VINIFERA

A. SCIALPI, A. ROSATI, M. MASIERI, G. MERSI, M. BUIATTI, P. BOGANI

Dipartimento di Biologia animale e Genetica “Leo Pardi”, Università di Firenze

Vitis vinifera, Chs, promoters, cis-acting elements, gene regulation, functional markers

Chalcone synthase (CHS) is the key enzyme in the first step of the biosynthetic pathway of anthocyanin and flavonoid compounds (Holton and Cornish, 1955). Genes encoding CHS form a multigene family the copy number of which varies among different species, from the single copy in Arabidopsis (Koch et al., 2001), to eigtheen gene isoforms characterized in Asteraceae (Yang et al., 2002).

In grapevine, up to the present Chss genes have been estimated at three to four (Sparvoli et al., 1994) and three have been sequenced and deposited in the GenBank DNA database (Goto-Yamamoto et al., 2002). Expression analysis, based on RT-PCR, suggested the genes to be under a different transcriptional control. 

As to our knowledge, Chs grapevine promoters, have not been yet investigated, in this work we report the isolation of upstream regions of Chs1, Chs2 and Chs3 coding sequences, with the aim to identify conserved cis-acting elements conferring functional patterns of gene regulation. For this purpose, based on the Chs coding sequences, reverse primers were designed which annealed to the first exon, while forward ones were supplied by “Genome walker kit” (Clontech). PCR amplification products from DNA of Vitis vinifera cv Cabernet Sauvignon, were then cloned and sequenced by the dideoxy chain termination method (Sanger et al., 1977) with the DYEnamic™ ET Terminator cycle sequencing kit (Amersham Biosciences), using the ABI Prism 310 automated sequencer (Applied Biosystem). The obtained sequences were successively analyzed with PLACE (Higo et al., 1999), PlantCARE (Lescot et al., 2002), TRANSFAC (Wingender et al., 2000) softwares for the search of putatively conserved motifs related to the control of specific gene regulation. Moreover, we analyzed the amplified regions with the “STRETCHES” algorithm, developed in our laboratories for the search of hypervariable homogeneous stretches in coding and non coding regulatory regions and the detection of polymorphisms, to be used as functional markers in the assessment of genetic variability.

References
Goto-Yamamoto, N., Wan, G.H., Masaki, K., Kobayashi, S. (2002). Plant Science, 162: 867-872.
Higo, K., Ugawa, Y., Iwamoto, M., Korenaga, T. (1999). NAR, 27: 297-300. 
Holton, T.A., Cornish, E.C. (1995). Plant Cell, 7: 1071-1083.
Koch, M.A., Weishaar, B., Kroymann, J., Haubold, B., Mitchell-Olds, T. (2001). Mol. Biol. Evol. 18: 1882-1891.
Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P., Rombauts, S. (2002). NAR, 30:325-327
Sanger., F, Nicklen, S, Coulson, A.R. (1977) PNAS., 74:5463-5467.
Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C. (1994). Plant Mol Biol, 24:743-755.
Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys, V., Meinhardt, T., Prüß, M., Reuter, I. and Schacherer, F.(2000). Nucleic Acids Res. 28, 316-319.
Yang, J., Huang, J., Gu, H., Zhong, Y., Yang, Z. (2002). Mol. Biol. Evol. 19: 1752-1759.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.47

USING FUNCTIONAL GENOMICS TOOLS TO EXPLOIT STILBENS AND FLAVONOIDS PATHWAYS OF GRAPEVINE

P. GATTO*,**, J. MUTH**, D. PRUEFER**, L. ZULINI*, C. MOSER*, M. S. GRANDO*, F. MATTIVI*, R. VELASCO*

*) Istituto Agrario San Michele all’Adige (IASMA), 38010 San Michele all’Adige, Italy
**) Fraunhofer Institut (IME) fuer Molekularbiologie und angewandte Oekologie, 52074 Aachen, Germany

grapevine, resveratrol, phenolic compounds, subtracted libraries, gene expression analysis

Grapevine contains many phenolic substances, most of which originate in the berry. These compounds, which include the phenolics acids, trihydroxy stilbenes and flavonoids, have a number of important oenological functions: they affect the tastes of bitterness and astringency of the wine, determine its colour, especially of the red wine, and they are the key wine preservative and the basis of long aging. In the last years a new interest has surfaced regarding to the natural antioxidant properties of phenols which seem to have beneficial effects against cancer and protective effects on the cardiovascular system. Among these phenolic substances, a hydroxylated stilben, known as resveratrol, has attracted much interest because relatively high quantities are found in grapes and wine, which are considered to be the most important dietary sources of this compounds. Besides the beneficial health effects, resveratrol is known to present an important antifungal feature which may be of interest in wine production technology: resveratrol is a phytoalexin produced by grape in response to microbial attack by the grapevine pathogen Botrytis cinerea. In order to get new information about the factor regulating the synthesis of phenolic compounds in grape, with particular attention to resveratrol, gene expression analysis will be carried out with array technologies. Starting point was the isolation of high quality total RNA from three different grapevine tissues (Vitis vinifera): leaf, root (cv. Pinot noir) and berry (a mix of four cultivars characterized by different resveratrol content: cv. Pinot noir, Teroldego, Friburg, Moscato bianco). The corresponding cDNAs were subjected to cDNA subtraction in order to be enriched for tissue specific differentially expressed genes. The subtracted cDNA pools were then cloned and about 10000 clones of each library picked into 384-well microtiterplates to generate three tissue-specific libraries. The inserts of 4000 clones of each library were then amplified. Next step will be the arraying of the PCR products on Nylon filters, which will be used first to investigate the expression of genes involved in the grape defense mechanisms against grey mould (B. cinerea). For this aim, the cDNA arrays will be hybridized with cDNA probes from healthy and infected leaves, collected at different time points, of two grape cultivars, Friburg and Teroldego, which are resistant and susceptible to the disease. Further hybridizations aiming to the identification of genes regulating the synthesis of phenolic compounds under different conditions such as in developing grape berries, will be performed. The expression profiles derived by array hybridisations will be compared with biochemical analysis of resveratrol and other phenolic compounds content in selected cultivars under different agronomic conditions. The coupling of genomic analysis and biochemical approaches will lead to a better knowledge of the biosynthetic pathways of grape phenols, which could be exploited for commercial applications.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.48

Cloning and functional characterization of the promoter for a stilbene synthase gene from Cissus juttae

F. GEUNA*, C. MAITTI*, F. LOCATELLI**, A. SCIENZA*

*) Dipartimento di Produzione Vegetale, Università di Milano, Via Celoria 2, I-20133 Milano
**) Istituto di Biologia e Biotecnologia Agraria, CNR, Via Bassini 15, I-20133 Milano

resveratrol, stilbene, promoter, transient expression, Vitis

Resveratrol is a natural compound renowned for its role as an antifungal agent in the species where it has been studied. Beside its properties in plant pathogen protection, it is now widely recognised also as a strong antioxidant and antitumor agent for human health. From the agronomic point of view, active research is underway worldwide to improve plant resistance to major fungal pathogens by transgenic expression of the gene coding stilbene synthase, the key enzyme for its biosynthesis. Genes for stilbene synthase have been cloned from Vitis vinifera and other species belonging to its family, as well as from Arachys hypogea and some conifers. In this study we report the cloning and preliminary characterization of the promoter belonging to one member of the stilbene synthase gene family from a Vitacea producing resveratrol and other related compounds.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.49

PARADOX EFFECTS OF CAROTENOID METABOLIC ENGINEERING IN tomato (Lycopersicon esculentum L.) FRUITS

G. Diretto*, C. Rosati**, P. Pallara**, B. Camara***, G. Giuliano*

*) Ente per le Nuove tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Casaccia Research Centre, PO Box 2400, Roma 00100AD, Italy
**) Ente per le Nuove tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Trisaia Research Centre, 75026 Rotondella (MT), Italy
***) CNRS, Plant Molecular Biology Institute, 67084 Strasbourg Cedex, France

tomato, beta-carotene 

Plant carotenoids are 40-carbon isoprenoid compounds involved in many physiological and biochemical functions, such as light harvesting and photoprotection from excess light energy; additionally, they colour many flowers and fruits, probably to attract animals and to protect reproductive structures by high light intensity. Carotenoids also play a crucial role in human health by improving normal vision and by preventing degenerative diseases like cancer. Animals do not produce carotenoids and therefore they must get them by a nutrient supplementation, as for Beta-carotene, the major dietary precursor of vitamin A. In this context we began a metabolic engineering approach on tomato (Lycopersicon esculentum L.) fruits, which, in their wild-type form, contain almost pure lycopene. In this work, the pepper Beta-carotene hydroxylase cDNA, cloned in sense orientation under the control of tomato fruit-specific PDS promoter, was introduced, via Agrobacterium tumefaciens, in tomato plants (cv MoneyMaker) and transformed plants were checked by PCR assay. Phenotypic analyses revealed that, contrary to expectations, the colour of the ripe fruits varied from the complete red of wild-type cv Moneymaker to red-orange to complete orange of several transgenic lines. Further studies are in progress in order to characterize transgenic fruits at the biochemical (by HPLC analyses) and molecular (by Real Time PCR and DNA microarray) levels.

References

•	Giuliano, G., Aquilani, R. and Dharmapuri, S. (2000) Metabolic engineering of plant carotenoids. Trends Plant Sci 5 (10), 406-409
•	Rosati, C., Aquilani, R., Dharmapuri, S., Pallara, P., Marusic, C., Tavazza, R., Bouvier, F., Camara, B. and Giuliano, G. (2000) Metabolic engineering of beta -carotene and lycopene content in tomato fruit. Plant J 24 (3), 413-419
•	Hirschberg, J. (2001) Carotenoid biosynthesis in flowering plants. Curr Opin in Plant Biol 4, 210-218
•	Dharmapuri S., Rosati C., Pallara P., Aquilani R., Bouvier F., Camara B. and Giuliano G. (2002) Metabolic engineering of xanthophyll content in tomato fruits. FEBS Lett 519, 30-34.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.50

Metabolic engineering of CAROTENOID CONTENT in POTATO (solanum tuberosum) TUBERS

D. PIZZICHINI*, F. MOURGUES**, R. WELSCH***, P. BEYER***, V. PAPACCHIOLI*, R. TAVAZZA*, G. Giuliano*

*) Ente per le Nuove tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Casaccia Research Centre, PO Box 2400, Roma 00100AD, Italy
**) Ente per le Nuove tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Trisaia Research Centre, 75026 Rotondella (MT), Italy
***) Albert-Ludwigs-Universitat, Institute for Biologie II, 79014 Freiburg, Germany

potato, beta-carotene, antisense

Carotenoids are a class of isoprenoid compounds involved in pigmentation of plants and animal tissues, in light harvesting, and in photoprotection of photosyntetic organisms. In animals, carotenoids are dietary precursors of retinoids, a class of molecules associated with many physiological processes, such as vision (trans-retinal), functionality of mucosal epithelia (vitamin A), morphogenesis (retinoic acid). Carotenoids have antioxidant activities: for this reason they are also thought to prevent some types of cancer .

In order to increase beta-carotene (pro-vitamin A) content in potato tubers we followed two metabolic engineering approaches: a) a push approach, in which early genes of the carotenoid pathway (phytoene synthase, phytoene desaturase) were introduced in sense orientation; b) a stop approach, employing antisense fragments of late genes (lycopene epsilon-cyclase and beta-carotene hydroxylase); both types of transgenes were cloned between the tuber-specific Patatin promoter and the Nos polyadenylation signal. Tubers from from approach b) were characterised through HPLC analysis, which revealed in different lines, an increase of 2-9 fold of ß carotene and important modifications of xanthophyll content. Real time PCR experiments revealed levels of gene silencing ranging from 30% to 70%. Further studies will focus on the characterisation of global transcriptional profiles through DNA microarray analyses.

References

•	Giuliano, G., Aquilani, R. and Dharmapuri, S. (2000) Metabolic engineering of plant carotenoids. Trends Plant Sci 5 (10), 406-409.
•	Hirschberg, J. (2001) Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 4, 210-218.
•	Romer, S., Lubeck, J., Kauder, F., Steiger, S., Adomat, C., and Sandmann, G. (2002) Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-suppression of carotenoid epoxidation. Metab Eng 4, 263-272.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.51

FROM SEED GERMINATION TO FLOWERING, CRYPTOCHROMES AFFECT MULTIPLE ASPECTS OF TOMATO PLANT DEVELOPMENT

L. GILIBERTO*, M. TAVAZZA*, P. PALLARA**, F. GIORNO**, G. PERROTTA**, G. GIULIANO*

*) Ente per le Nuove Tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Casaccia Research Centre, PO Box 2400, Roma 00100AD, Italy
**) Ente per le Nuove Tecnologie, l'Energia e l'Ambiente (ENEA), Biotechnology Unit, Trisaia Research Centre, 75026 Rotondella (MT), Italy

tomato, photoperiodism, de-etiolation

Cryptochromes are flavin-type blue light photoreceptors, widespread in plants and animals. We have started the functional characterisation of the tomato cryptochrome gene family. Tomato contains 2 Cry1 genes, 1 Cry2 gene and 1 putative CryDash gene. Homologous, ectopic expression of the Cry2 gene produces some phenotypes similar to what already observed in Arabidopsis and tobacco (hypocotyl and internode shortening, anthocyanin accumulation), while other phenotypes are novel: modification of leaf chlorophyll and carotenoid content, of seed germination, and of flowering time. Some of the phenotypes are reversible through posttranscriptional gene silencing with a viral vector carrying a fragment of the Cry2 gene. Experiments conducted under controlled conditions show that tomato, (cv. Moneymaker), like Arabidopsis, flowers earlier under long day conditions and requires light for seed germination. Ectopic expression of Cry2 affects seed germination, and retards flowering. Analysis of cry1 mutant plants confirms that tomato cryptochromes control tomato seed germination and flowering in a fashion radically different from Arabidopsis.

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 Journal 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 Molecular Biology 42, 765-773.
Perrotta, G., Yahoubyan, G., Nebuloso, E., Renzi, L., and Giuliano, G. (2001). Tomato and barley contain duplicated copies of cryptochrome 1. Plant Cell and Environment 24, 991-997.
Weller, J.L., Perrotta, G., Schreuder, M.E., Van Tuinen, A., Koornneef, M., Giuliano, G., and Kendrick, R.E. (2001). Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2. Plant Journal 25, 427-440.
Lin, C. (2002). Blue light receptors and signal transduction. Plant Cell 14, S207-225.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.52

EXPRESSION ANALYSIS OF G1-1, A GENE ISOLATED IN POTATO TUBER DURING TRANSITION FROM DORMANCY TO SPROUTING

R. BIANCHI, C. AGRIMONTI, G. VISIOLI, N. MARMIROLI

Dipartimento di Scienze Ambientali, Università di Parma

sink-source transition, dormancy, sprouting, Real-Time RT- PCR

In potato tubers the transition from sink to source function is interrupted by a period of dormancy in which sprouts growth is completely inhibited. To investigate some of the molecular events of this process, an analysis by means of the differential display technique was previously undertaken, identifying the cDNA sequence G1-1, whose expression is induced in potato tubers at the end of the dormancy period and in the early phases of tuber sprouting.

The short ORF of G1-1 (226bp) shares 81 and 75% of nucleotide identity with two ESTs of tomato and soya respectively, isolated in different tissues having in common an intense mitotic activity. Northern analysis using mRNA from whole potato tubers had shown very low levels of expression, with a small increase in transition from the dormancy to the sprouting phase. In the present study, we have examined G1-1 expression at a more thorough level, in the tissues of tuber eyes, parenchyma, whole tuber, sprouts and leaves in different physiological stages, using the highly sensitive quantitative real time RT-PCR technique (TaqMan method). 

The highest expression levels were found in actively growing tissues, such as sprouts and leaves  collected during the first stage of their development, when they are still in the sink phase. The expression of G1-1 decreases, even though it remains quite high, in mature leaves, after their transition  in the source phase. G1-1 is weakly expressed in parenchyma and whole tuber tissues, but an increasing expression has been observed in tuber eyes during the breakage of dormancy.

Thus, these results may suggest a connection between the expression of G1-1 and actively dividing tissues of sink organs.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.53

"STAY GREEN" MUTANTS OF DURUM WHEAT: ANALYSIS AT MOLECULAR LEVEL

P. RAMPINO*, G. SPANO**,***, P.R. SHEWRY****, J.A. NAPIER****, C. PERROTTA*, N. DI FONZO***

*) DiSTeBA, Università di Lecce
**) Dip. Scienze Alimentari, Università di Foggia
***) Ist. Sper. Cerealicoltura-Foggia
****) Long Ashton Res. Station, Dept. of Agricultural Sciences, University of Bristol

stay green, durum wheat, molecular analysis, differential display

Examples of the ‘stay green’ phenotype have been identified in a number of crop species (Thomas and Howart, 2000) and, although this phenotype is superficially similar in all species the genetics and physiological routee to the trait may differ. To elucidate the molecular mechanisms responsible for the ‘stay green’ phenotype is relevant not only to understand the senescence phenomenon itself but also for practical purposes, such as yield improvement. Four mutants with delayed leaf senescence were obtained by ethylmethanesulfonate (EMS) mutagenesis of durum wheat (cv Trinakria). All the photosynthetic parameters seem to be affected in these mutants (Spano et al, 2003), that have the characteristics of functional ‘stay green’ according to Thomas and Howarth (2000). This type of ‘stay green’ mutants may arise by alteration of genes involved in the timing of the initiation of senescence, or in the regulation of its rate of progress (Smart, 1994). In order to identify genes whose expression is up or down-regulated in our mutants, a comparative analysis between mutant and control plants was performed by differential display of mRNAs. Total RNA was extracted from the flag leaves of plants grown in a controlled glasshouse, and different primer combinations were used. About 20 fragments were confirmed as differentially expressed in leaves of mutant plants. These fragments were cloned and subsequently sequenced. BLAST analysis of the sequences obtained was performed.

References
Spano et al, 2003, J. Exp. Bot. 54: 1415-1420 
Thomas and Howart, 2000, J. Exp. Bot. 51: 329-337
Thomas and Smart, 1993, Ann. Appl. Biol. 123:193-203

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.54

STRAWBERRY cDNA MICROARRAYS AS A TOOL FOR STUDYING FRUIT QUALITY TRAITS AND COMPARING ELITE GENOTYPES

C. ROSATI, F. MOURGUES, F. GIORNO, F. CARBONE, M.A. CARBONI, G. PERROTTA 

ENEA, C. R. Trisaia - S.S. 106, km 419+500, 75026 Rotondella (MT), Italy

strawberry, microarrays, fruit quality, ripening, functional genomics

Cultivated octoploid strawberry (Fragaria x ananassa) is a fruit crop with hundreds of varieties grown over a wide range of latitudes. To assess fruit quality and correlate gene expression data with those from biochemical analyses and panel tests, we amplified 4512 clones of a cDNA library obtained from RNA extracted from ripening fruits and analyzed the 3061 expressed sequence tags (ESTs) which produced a sequence larger than 250 bp. Two successive runs of contig assembly reduced sequence complexity and grouped the ESTs into 1353 sequences (580 contigs and 773 singletons), 632 of which shared similarity with plant gene sequences. The highest number of BLAST hits were assigned to the Rosaceae family (652 out of 3061 processed ESTs, 21.3%), with 476 sequences (15.6% of total, 73% of Rosaceae) belonging to the genus Fragaria (both octoploid F. x ananassa and diploid F. vesca). The 50 largest contigs related to plant genes with known functions comprised 725 ESTs (23.7% of total), including genes associated with ripening (cell wall metabolism, sugar and acid synthesis, pigment formation, vitamin synthesis and allergenic properties), as well as with plant-pathogen interactions, abiotic stresses and housekeeping functions. Moreover, 125 consensus sequences were similar to mRNAs or putatively encoded proteins not yet characterized and 721 did not give any significant BLAST match. These 846 “unknown” sequences represent an invaluable resource for finding genes with new functions. Representative subsets of ESTs were used to produce cDNA microarrays and carry out experiments on genotypes with different fruit quality. In the frame of the Italian FISR Project “QUALIFRAPE”, our investigations will point out the genes whose expression level is most involved in fruit quality and post-harvest traits. Comparison of molecular, biochemical and panel test information are underway to correlate analytical data with overall consumer’s preference.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.55

GENE EXPRESSION ANALYSIS IN GRAPES: THE CASE OF BERRY RIPENING

A. FERRARINI*, M. CRESPAN**, M. PEZZOTTI*, A. COSTACURTA**, C. LORENZONI***, M. DELLEDONNE*

*) Università degli Studi di Verona, Dipartimento Scientifico e Tecnologico, Strada Le Grazie 15, 37134 Verona 
**) Istituto Sperimentale per la Viticoltura, Via 28 Aprile 26, 31015 Conegliano Veneto, Treviso
***) Istituto di Botanica e Genetica Vegetale, Università Cattolica S.C., Via Emilia Parmense 84, 29100 Piacenza

grape, ripening, AFLP-TP

Grape cultivation and wine production are the most remunerative sectors of Italian agriculture. Wine quality is strictly connected to grape quality and it is the result of a complex interplay of physiological, genetic and environmental factors. Recent development of molecular biology has given us the tools to examine them at a molecular level. Among these tools AFLP-TP is a very powerful expression profiles analysis technique based on PCR, characterized by high sensitivity and response linearity. These features permit to detect also poorly represented transcripts and little expression differences.

We are using AFLP-TP to analyse transcript profiles during the ripening of berry in two varieties of Vitis vinifera: “Manzoni bianco” (white) and “Refosco” (red). The aim of this work is to identify genes differentially expressed during berry ripening and genes that are specificly activated in each variety during the ripening process. Berry samples were collected from preveraison to the completion of ripening of the berry. Skin and flesh from berry are analysed separately and transcription profiles from different developmental stages of the two varietis are compared to each other. Fragments corresponding to the transcripts identified as differentially expressed are sequenced and/or cloned and genes corresponding to the sequenced fragments are identified by homology searches on public databases.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 1.56

NONSYMBIOTIC PLANT HAEMOGLOBINS AND  REGULATION OF NITRIC OXIDE SIGNALING

E. ZAGO*, M. PERAZZOLLI**,*, C. ANZI*, A. MAROCCO**, P. DOMINICI*, M. DELLEDONNE*

*) Università degli Studi di Verona, Dipartimento Scientifico e Tecnologico, Strada Le Grazie 15, 37134 Verona
**) Istituto di Botanica e Genetica Vegetale, Università Cattolica S.C., Via Emilia Parmense 84, 29100 Piacenza

nonsymbiotic plants haemoglobin, nitric oxide,  plant defence response 

Nitric oxide (NO) is an important signaling molecule regulating gene expression during plant defence responses and in other important physiological processes like seed germination, de-etiolation, senescence and hypoxia. Recent studies demonstrated that NO production is based on nitric oxide synthase (NOS) and nitrate reductase (NR) activities. The mechanisms for removal of this signal are however still unknown.

In different organisms, from bacteria to mammals, haemoglobins (Hb) control NO homeostasis by converting this molecule to nitrate. Also in plants Hb genes have been identified and have been classified into two groups, symbiotic and nonsymbiotic haemoglobins (nsHb).  The function of ubiquitous nsHb has not been clearly identified.  

Arabidopsis thaliana possesses two nsHb, AHb1 and AHb2 with different expression pattern and oxygen binding properties. AHb1 is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHb1 has an high oxygen affinity. AHb2 is expressed at a low level in rosette leaves and is low temperature-inducible. AHb2 has a lower affinity for oxygen than AHb1. AHb1 has been purified to analyse the interaction between NO and nsHb, and a detoxification model has been proposed. AHb1 shows a dioxygenase activity that convert NO to nitrate with a catalytic cycle in presence of NADPH.  To investigate this function in vivo, AHb1 was cloned in the constitutive expression vector pBI-121 and Arabidopsis thaliana transgenic plants, with an increased level of AHb1, were generated. Northern and western analysis allowed selection of lines with higher level of AHb1. These transgenic plants with elevated NO detoxification system give the possibility to analyse the involvement of NO in several different physiological condition in order to get deeper inside the NO-dependent expression pattern.