IDENTIFICATION OF GENES INVOLVED IN PHOSPHATE METABOLISM IN ARBUSCULAR MYCORRHIZAL FUNGI
Alessandra Benedetto, L. Lanfranco, P. Bonfante
Dipartimento di Biologia Vegetale, Università di Torino
Arbuscular mycorrhizal (AM) fungi form symbiotic associations with most land plants and promote the growth of the host through enhanced uptake of phosphorus. Once the association is established, the fungi take up inorganic phosphate (Pi) from the soil through extraradical hyphae, accumulate Pi into vacuoles as polyphosphates (polyP) and translocate polyP to intraradical hyphae. This is assumed to be followed by hydrolysis of polyP and subsequent release of Pi into the plant-fungus interfacial apoplast. The molecular basis of these processes are not completely known as, up to now, just one Pi transporter gene has been isolated from an AM fungus (M. Harrison & van Buuren 1995, Nature). In this work we used different approaches to identify genes involved in P metabolism in AM fungi. Degenerate primers were designed on conserved amino acid domains of Pi transporter and used in PCR experiments to amplify genomic DNA from AM spores. A fragment of 846 bp showing high similarity with Pi transporters was identified from the fungus Glomus mosseae (BEG12). In parallel experiments, a screening of a genomic library from Gigaspora margarita (BEG34) with a heterologous probe led to the isolation of a positive plaque which is currently under analysis. In addition, a clone showing high similarity with polyphosphate synthases was identified from an EST collection from germinated spores of Gigaspora margarita (BEG34).
2
HIGH LEVELS OF d1-PYRROLINE-5-CARBOXYLATE DEHYDROGENASE SEEM REQUIRED FOR ARGININE UTILIZATION IN CULTURED CELLS OF SOME SOLANACEAE
Alberto Campani*, Giulia Corona+, Erik Nielsen+ and Giuseppe Forlani*
*Dept of Biology, UNI-FE; +Dept of Genetics and Microbiology, UNI-PV
Plants do not normally utilize exogenous sources of organic nitrogen. However, they synthesize several compounds in specific tissues as endogenous sources of nitrogen for growth, among which the amino acid arginine shows a favourable N:C ratio. Arg was found to be dissimilated into ornithine, ammonia and bicarbonate in both Arabidopsis leaf chloroplasts and soybean seedling cotyledons. However, when cultured cells of three Solanaceae (namely Lycopersicon esculentum, Solanum tuberosum and Nicotiana plumbaginifolia) were grown in a modified MS medium containing Arg as the only nitrogen source, only in the latter case a significant growth rate could be detected. Free amino acid contents were quantified in cell suspension cultures at increasing time after the inoculum in the modified MS medium containing Arg as the only N source, or in MS medium supplemented with Arg. Results showed that in no case ornithine was accumulated, and suggested the conversion of ornithine to glutamate as the critical point in Arg utilization. This conversion takes place through two steps, catalyzed by ornithine-d-aminotransferase and pyrroline-5-carboxylate dehydrogenase (P5C-DH, EC 1.5.1.12). When P5C-DH was quantified in extracts from cultures cells of the above species by means of Western blot analysis, remarkably higher levels were evident in tobacco than in potato or in tomato. These data also strengthen the possibility that two P5C-DH isozymes involved in the oxidation of either Pro-deriving or Arg-deriving P5C may indeed exist.
3
POLYAMINE OXIDASE IS REGULATED BY LIGHT AND AUXIN IN THE MAIZE MESOCOTYL
Alessandra Cona, Rodolfo Federico, Paolo Mariottini, Manuela Cervelli, Francesco Cenci, Sandra Moreno, Daniela Cesare and Riccardo Angelini.
Dipartimento di Biologia, Università "ROMA TRE", Rome, Italy
H2O2 is a mediator in important physiological processes such as programmed cell death, lignification, wall stiffening and cellular defense. It has been recently suggested that polyamine oxidase (PAO) behaves as a H2O2 delivering system in the above-mentioned events in maize tissues. In particular, it has been hypothesized that PAO may have a role in wall stiffening of epidermal cells occurring during the light-mediated inhibition of maize mesocotyl growth (Laurenzi et al., 1999, Planta, 208: 146-154). In the latter event a strong reduction of free IAA level was observed in the mesocotyl epidermis (Barker-Bridgers et al., 1998, Planta, 204: 207-211). Recent studies pointed out that maize PAO is regulated by light at the transcriptional level and PAO promoter sequence analysis revealed the occurrence of cis-acting light- and auxin-responsive elements. In this work we report the evidence that exogenously supplied auxin inhibits, in vivo, light-induced increase of PAO activity in the outer tissues of maize mesocotyl. PAO protein levels analyzed by western immunoblotting revealed a similar pattern as compared to that of enzyme activity. Ethylene did not affect maize PAO activity, thus excluding an effect of auxin by increasing ethylene biosynthesis. The auxin transport inhibitor naphthylphtalamic acid (NPA), caused a further increase of PAO activity in outer tissues under light. The slow increase of PAO activity, normally occurring in the mesocotyl epidermis during plant development in the dark, was inhibited by auxin as well and stimulated by NPA, although to a lesser extent as compared under light condition, thus suggesting a complex regulation of PAO expression by light and auxin.
4
TRANSGLUTAMINASES AND THEIR PLASTIDIAL SUBSTRATES IN ZEA MAYS
M Della Mea, S Del Duca, A Di Sandro e D Serafini Fracassini.
Dip. di Biologia e. s, Università degli Studi di Bologna., via Irnerio 42 - 40126 Bologna, Italia.
Transglutaminases (TGases) are an enzyme family which catalyses the covalent linkage between a glutaminyl and lisyl residues or other molecules with primary amino-groups like polyamines. In plants, TGases are also involved in post-translational modifications of plastidial proteins. In fact, in H. tuberosus and Z. mays chloroplasts, apoproteins of antenna-complexes and Rubisco act as substrates. Among the factors influencing the plastidial activity, the most interesting one is the light which enhances the enzimatic incorporation of polyamines. On the basis of light-induced events of plastidial proteins phosphorylation, we have studied the TGase activity under phosphorylating and dephosphorylating conditions. The phosphorylation seems not to be involved in these mechanisms. The activity measurement, performed on purified thylakoids and stroma, shows a very low incorporation if compared to that of whole chloroplast. The addition of the Guinea pig liver TGase (SIGMA) is able to enhance the activity in both fractions, but it causes the disappearance of light/dark differences. On this basis, we have hypotesized that either stromal or thylakoidal enzymes and substrates are functionally interacting. Besides light acts directly on plastidial TGases rather than on substrates. Afterwards, we tested the maize LHCII as substrate of TGase. Obtained data are in accord with this hypotesis and, probably due to SIGMA TGase or to polyamines used as substrates, we have not registered a clear variation of activity depending on light/dark conditions. Till now we do not have good evidences on the physiological meaning of plastidial transglutaminase activity.
5
DIFFERENT REGULATION OF CHLOROPLASTIC AND PLASTIDIC GLUCOSE-6P DEHYDROGENASE ISOFORM IN BARLEY
Sergio Esposito1, Graziella Massaro 1, Vincenza Vona1, Vittoria Di Martino Rigano2, Simona Carfagna 1, Carmelo Rigano 1
1 - Dipart. di Biologia Vegetale - Università di Napoli "Federico II" Via Foria, 223 - 80139 Naples - ITALY. 2 - Dipart. Farmacologia Sperimentale - Università di Napoli "Federico II"- Via Montesano - 80131 Naples - ITALY Tel.(39)-081-2538514 . Email: esposito@cds.unina.it
The main regulated enzyme of the oxidative pentose phosphate pathway (OPPP) is glucose-6P dehydrogenase (G6PDH - EC 1.1.1.49), which catalyses the first reaction of the cycle. The occurrence of two different G6PDHs in plants, located in the cytosol and in the plastids, has been proved both in green (Schnarrenberger et al. 1973) and in non-photosynthetic tissues (Hong and Copeland 1991; Esposito et al., 2001); different genes encoding for cytosolic, chloroplastic and plastidic isoforms have been recently sequenced in potato (Wendt et al. 2000). Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterised from ammonium-fed roots, exhibiting different pH optima and Km values for glucose 6P; particularly, G6PDH 2 showed similar kinetic parameters to the unique G6PDH present in roots of barley grown without any nitrogen source. Western blot analysis confirmed the existence of two G6PDH in barley roots under ammonium feeding conditions, while only G6PDH 2 could be detected in roots grown without any nitrogen source. G6PDH extracted from isolated chloroplasts shows kinetic and regulatory characteristics and subunit molecular weights different with respect to the root plastidic G6PDH. The results suggest differences in genes encoding for particulate enzyme between eterotrophic and photosynthetic tissues (Wendt et al. 2000). References: Emes and Fowler (1983) Planta 158: 97-102 Esposito et al. (2001) Planta 212: 627-634 Hong and Copeland (1991) Plant Physiol 96: 862-867 Schnarrenberger et al. (1973) Arch Biochem Biophys 154: 438-448. Wendt et al. (2000) Plant J 23: 723-733
6
TRIDIMENSIONAL STRUCTURE OF THE NON-REGULATORY A4 ISOFORM OF SPINACH CHLOROPLAST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
Fermani S1, Ripamonti A1, Sabatino P1, Zanotti G2, Scagliarini S3, Sparla F3, Trost P3, Pupillo P3
1Dept. Chemistry "G. Ciamician", UNI Bologna; 2Dept. Organic Chemistry, UNI Padova;3 Dept. Biology, UNI Bologna
Photosynthetic glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) of higher plants are made up of either one type (A) or two types (A and B) of subunits, whereas glycolytic GAPDHs are tetrameric enzymes formed by C-type subunits. Here we report the first crystal structure of a photosynthetic GAPDH complexed with NADP. The enzyme purified from spinach chloroplasts, is constituted by a single type of subunits A arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference toward NADP. The structure has been solved to 3.0 Ċ of resolution by molecular replacement. The protein belongs to the Rossman fold family of dehydrogenases and reveals a large structural homology with known glycolytic GAPDHs. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt bridge with any positively charged residue in its surroundings, being instead set in place only by hydrogen bonds. Although the overall structure of the A4-GAPDH is similar to the homologous cytosolic GAPDH from bacteria and eukaryotes, the spinach chloroplast tetramer is peculiar in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys203 residues. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.
7
CHARACTERIZATION OF ARABIDOPSIS THALIANA AND YEAST HEXOKINASES OVEREXPRESSED IN DIFFERENT TRANSGENIC LINES
Silvia Gonzali *, Amedeo Alpi *, Federica Blando§, Luigi De Bellis +
* Dip. di Biologia delle Piante Agrarie, Pisa, §IRBA- CNR, Lecce + Dip. di Scienze e Tecnologie Biologiche ed Ambientali, Lecce
Three different Arabidopsis thaliana transgenic lines overexpressing AtHXK1, AtHXK2 and Saccharomyces cerevisiae HXK2 genes, respectively, were analyzed. The catalytic properties of the overexpressed HXKs were studied by non-denaturing electrophoresis followed by activity-staining. Three distinct hexokinase enzymatic activities were detected and named, respectively, HXK1, HXK2 and YHXK2. In our experimental conditions, these enzymes shared most of their catalytic behavior but some differences were revealed. Arabidopsis HXK1 and HXK2 showed high affinity for glucose and mannose, and a lower specificity for fructose. In comparison, YHXK2 exhibited a higher affinity for both mannose and fructose. Concerning the phosphate donor substrate, HXK1 and HXK2 employed ATP with the maximum efficiency, but could also perform glucose phosphorylation in the presence of other nucleoside triphosphates, although with a lower efficiency. The effect of various metabolites was analyzed. ADP proved to act as a strong inhibitor of the enzymes, while AMP determined a lower inhibition. Galactose, 3-O-methyl glucose and 6-deoxyglucose (which are not HXK substrates) showed no influence on hexokinase activity on glucose, while glucosamine and 2-deoxyglucose (both phosphorylable by HXK) produced a clear inhibition. As a whole, these results provide useful information for a discussion concerning the sugar response of transgenic plants overexpressing AtHXK or YHXK2 genes.
8
SEQUENCING OF THE CDNA ENCODING PROTOCHLORO- PHYLLIDE OXIDOREDUCTASE IN MAIZE (ZEA MAIS L.)
Isabella Moro, Luisa Dalla Valle, Nicoletta La Rocca e Nicoletta Rascio
Dip. di Biologia, UNI Padova
NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the photoreduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Two distinct isozymes of POR, PORA and PORB were identified, and respectively cDNAs were also cloned in some higher plants (Holtorf et al., P.N.A.S., 92:3254, 1995). In our study we sequenced the maize POR and analyzed the gene expression. To investigate the POR expression in maize, isolation of total RNA was carried out on 9-day-old dark-grown seedlings and on 9-days-old dark-grown seedlings illuminated with continuous light for various lengths of time. The RNA was subjected to RT-PCR analysis with oligonucleotide pairs designed on highly conserved regions of both PORA and PORB cDNA of barley, available in GeneBank. After 3'- and 5'- RACE analyses, only one 1.3 kb of maize POR cDNA was sequenced. Northern hydridization analyses showed that in maize a single transcript of POR occurred. Moreover, the gene expression level, valued by the transcript quantity, reached the maximum at 8 hours of illumination, decreasing successively, with a trend similar to that noticed in cucumber (Fusada et al., Photosynth. Res., 64:147, 2000), another plant in which only one gene encoding the POR enzyme was found.
9
A MUTATION AFFECTING PHYTINE SYNTHESIS AND INCREASING FREE PHOSPHATE CONTENT IN MAIZE SEED
E. Nielsen^ and S. R. Pilu*
^Dip. di Genetica e Microbiologia, Univ. di Pavia; *Dip di Produzione Vegetale, Univ. di Milano.
Phytine (IP6), is the major storage compound of phosphorous in plants, accumulating predominantly in seeds (up to 4-5% of dry weight) and pollen. In cereals, mature seeds contain high phytine-phosphate (up to 80%) and low free phosphate levels. However, so far, many questions regarding the biochemical arrangement of phytine synthesis are still unanswered. In order to isolate mutations that may be useful for the charachterization of phytine pathway, we carried out a screening for high level of free phosphate in the seed, a quick and inherently sensitive assay for the rescue of lpa (low phytic acid) genotype. A population of EMS (ethyl methanesulfonate)-induced maize mutants was generated using the pollen-treatment method, and approximately 600 M2 families were screened. The first-level screening was carried out by titrating free phosphate by the molybdate staining assay. Putative mutants were then challenged by a TLC method allowing the simultaneous detection of free phosphate and phytine. Most of the mutations isolated this way turned out to perturb germ or aleurone development. However, one monogenic recessive mutation (named lpa 3-1), even causing an about ten fold increase in the amount of free phosphate titratable in -/- homozygous seeds (and a simultaneous decrease in phytine level), did not affect normal germination or seedling growth. Moreover, also in +/- heterozygous seeds, free phosphate content was 2-3 fold increased compared to wild type. Genetic analysis of this mutation, as well as its further biochemical characterization, are under way.
10
IDENTIFICATION OF GLUTATHIONE S TRANSFERASE IN ARABIDOPSIS GENOME, CLONING AND SCREENING OF GST MUTANTS IN ARABIDOPSIS T-DNA TAGGED LINES.
E. Nutricati, P. Rampino, M. Lanzilotti, L. De Bellis
Dip. di Scienze e Tecnologie Biologiche e Ambientali, UNI Lecce
Increasing plant yield potential is a realistic goal in plant breeding. The understanding of the mechanism of plant resistance or tolerance to some stresses is fundamental information to develop a biotechnological approach toward yield increase and quality improvement. Glutathione S-transferases (GST) are enzymes that catalyse the conjugation of the tripeptide glutathione (GSH) to a variety of hydrophobic, electrophilic and cytotoxic substrates. The role of GSTs in plant metabolism is unclear, although their complex regulation by environmental stimuli implies that they have important protective function. In fact, plant GSTs seem to play a role in numerous stress responses, including those arising from pathogen attack, oxidative stress, cold stress and heavy-metal toxicity. The glutathione S-transferase exists as a large gene family in plant. We have identified 33 GSTs in Arabidopsis genome and a phylogenetic analysis revealed the presence of three GST sub-families. Four full-length GST cDNAs, with at least one member for every sub-family, were cloned and overexpressing experiments are in progress. Couples of primers were developed to search and identify GST mutants in Arabidopsis T-DNA tagged lines and study the expression of the different GSTs in plant development. Mutant plants will undoubtedly help to clarify the role of the huge GST gene family. Reference: Edwards R. et al., 2000. Trends in Plant Science, 5: 193-198.
11
CLONING AND EXPRESSION OF ARABIDOPSIS PUTATIVE ACYL-COA SYNTHETASE
P. Rampino*, M. Lanzilotti*, E. Nutricati*, P. Giuntini+, L. De Bellis*
*Dip. di Scienze e Tecnologie Biologiche e Ambientali, Università di Lecce
+ Dip. di Biologia delle Piante Agrarie, Pisa
One of major goals of modern plant biotechnology is to manipulate lipid metabolism in oilseed crops to produce new and improved edible and industrial vegetable oils. Lipids constitute the structural components of cellular membranes and act as source of energy for the germinating seed and are therefore essential plant cell function. Towards this goal is important to characterize the plant acyl-CoA synthetases (ACSs) which play an important role in both de novo synthesis and modification of existing lipids. The acyl-CoA synthetase exists as a large gene family in plants, as it appears from the presence of twelve putative ACSs in Arabidopsis genome. A cDNA clone encoding Arabidopsis thaliana acyl-CoA synthetase (clone RZ114c11 obtained from the Kazusa DNA Research Institute, Kirazasu, Japan) was cloned in frame into pET24 (Novagen, Madison, USA). The construct was utilized to transform the E. coli strain BL21(DE3) which allowed the expression of high level of ACS in inclusion body. Finally, couples of primers were developed to evaluate the expression of the different ACSs during Arabidopsis development. Reference: Ohlrogge J, 1999. Current Opinion in Plant Biology, 2:121-122.
12
OVEREXPRESSION OF PEA DIAMINE OXIDASE AND MAIZE POLYAMINE OXIDASE IN TRANSGENIC PLANTS OF N. TABACUM
Rea G., Gobbi V., Pironi L., Angelini R., Federico R., Tavladoraki P.
Dipartimento di Biologia, Università 'ROMA TRE', Viale G. Marconi 446, Rome 00146, Italy
In plants, polyamines have been implicated in several processes such as cellular proliferation, adaptation to environmental stresses and defense mechanisms. Diamine oxidase (CuAO) and polyamine oxidase (PAO) are copper- and flavin-containing enzymes, respectively, involved in polyamine catabolism. The production of H2O2 raised upon polyamine oxidation has been correlated with oxidative burst, cell-death, lignification and suberisation occurring during ontogenesis and defense responses. To study the physiological role of pea CuAO and maize PAO, we have obtained transgenic plants of N. tabacum over-expressing these enzymes in the cell wall. Analysis of R0 and R1 generations revealed high level of CuAO or PAO expression and purification of the engineered proteins demonstrated unmodified catalytic properties. Furthermore, evaluation of the free polyamine content showed a 50% decrease in the CuAO or PAO transformed plants in respect to the wild type plants. It has been also determined that, supplying exogenous polyamines, the transgenic plants producd higher H2O2 levels than the untransformed plants, suggesting that polyamine secretion in the cell wall is a rate-limiting event in tobacco plants. Challenging experiments with P. syringae pv phaseolicola and pv tabaci indicated that CuAO or PAO over-expression do not interfere with bacterial growth. Experiments are in progress to study the hypersensitive response of the transgenic plants.
13
NITROGEN METABOLISM IN ALGAL CELLS
C. Rigano*, V. Di Martino Rigano**, V. Vona*, G. Massaro*, S. Carfagna*, and S. Esposito*
*Dipartimento di Biologia Vegetale, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy. **Dipartimento di Farmacologia Sperimentale, Università di Napoli Federico II, via Montesano 49 I-80131 Napoli, Italy.
Cells of C. sorokiniana cultivated under light conditions in a complete medium showed a continuous growth. They assimilated NH4+ at a constant rate, and contained constant level of starch. When transferred to darkness these cells cease immediately to use NH4+ and to grow; they did not show appreciable decrease in the content of starch, denoting a drastic falling down in energy and carbon requirement. Cells starved of N for 24 h showed a rate of dark respiratory oxygen consumption which was only 70% that of N-sufficient cells, and exhibited a considerable higher content of starch. They showed a photosynthetic capacity (Pc) which was only 30% that of N-sufficient cells. The uptake of re-supplied NH4+ by N-starved cells was immediate both in light and darkness; there was a concomitant increase in the intracellular concentration of glutamine, and a rapid decrease in concentrations of 2-ketoglutarate, glutamate and PEP; there occurred also a 3-fold stimulation of respiration. Then, concentrations of PEP and 2-ketoglutarate raised to concentrations similar, or slightly higher (in light) than those of the starting cells, and there was also increase in the concentrations of pyruvate (in the light) and alanine (either in light and dark). There occurred a rapid starch mobilisation either in illuminated or darkened cells. The starch mobilisation in illuminated cells, supports that the very low Pc of N-starved cells was unable alone to furnish sufficient carbon substrates and energy as a full assimilation of NH4+ would require. By summing, however, carbon compounds derived from photosynthetic activity to those derived from starch mobilisation, then it results that illuminated cells had an higher carbon availability than darkened cells, which could explain why, after NH4+ addition, the former cells showed a more active recovery from N starvation than the latter cells, and exhibited also resumption of growth.
14
Chimeric mini-protease inhibitors derived from the oil rape proteinase inhibitor type III
Maurizio Trovato1, Bruno Maras2, Elena Caroli Casavola1, Paolo Ascenzi3, Paolo Costantino1
1Dip. di Gen. e Biol. Molecolare; 2Dip. di Sc. Biochimiche, UNI Roma1; 3 Dip. di Biologia, UNI Roma3.
Protease inhibitors are widespread in the plant kingdom and are involved in a number of developmental stages as well as in the protection against phytopathogen organisms. Their capability of inhibiting the proteolitic enzymes of parasites and insects has been exploited to generate transgenic plants expressing proteinase inhibitor genes. Although successful expression of proteinase inhibitor genes have been reported in transgenic plants, this strategy is generally hampered by the pest capability to develop resistance against proteinase inhibitors. Reduced mini-inhibitors, capable to resist insect proteolitic digestion while still endowed of catalitic activity and multi-functional protease inhibitors are currently being developed to overcome these problems. We report the successful design of a chimeric protein composed by the minimized reactive site domain of the rape trypsin inhibitor from Brassica napus (var oleifera) and the murine dihydrofolate reductase (DHFR). The DHFR-mini-RTI-III chimeric protein was expressed in E.coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin is similar to that determined for native RTI-III and witness the feasibility of using small active domains in the development of multi-domain chimeric inhibitors. Mutant variants as well as smaller chimeric proteinase inhibitors are currently being studyied in our laboratory and will be described.
15
LEAF ISOFORMS OF SAPORIN: LOCALIZATION, PURIFICATION AND ESI-MS ANALYSIS
A.Tucci, A.Di Tullio*, F.De Angelis*, A.Poma, A.M. Ragnelli, L.Spanò
Dip. di Biologia di Base e Applicata, * Dip. di Chimica, Ingegneria Chimica e Materiali, Univeristà dell'Aquila, 67010 Coppito AQ
The immunocytochemical analysis of Saponaria officinalis leaves reveals the presence of saporin, one of the most studied type 1 ribosome-inactivating protein, both in the extra-cellular (apoplastic) and the intra-cellular (vacuolar) compartment of the mesophyll cells; the toxin isoforms were purified by means of cation exchange chromatography and HPLC. N-terminal sequence analysis, in vitro and in vivo activity tests, as well as results from ESI-MS, allows the identification of the apoplastic and vacuolar enzymes as belonging to the seed-type and, respectively, leaf type saporins.
ADATTAMENTO DEL CLOROPLASTO DI KOLIELLA ANTARCTICA ANDREOLI ET AL. SOTTOPOSTA A SIMULATA NOTTE AUSTRALE: STUDIO MICROSPETTROFLUORIMETRICO ED ULTRASTRUTTURALE.
Costanza Baldisserotto*, Lorenzo Ferroni*, Nicoletta La Rocca°, Carlo Andreoli° e Simonetta Pancaldi*.
*Dipartimento di Biologia - Sezione Botanica, Università di Ferrara, C.so Porta Mare, 2 - 44100 Ferrara °Dipartimento di Biologia, Università di Padova, Via Ugo Bassi, 58/b - 35131 Padova
Koliella antarctica Andreoli et al. (Trebouxiophyceae, Chlorophyta) è stata isolata nell'estate australe 1989/90, alla profondità di 3 m sotto il pack-ice, presso la Baia di Terra Nova (Antartide). Trasferita presso il Dip. di Biologia dell'Università di PD, è stata mantenuta in condizioni riconducibili a quelle del sito di origine (T = 4°C, salinità = 34, irradianza = 20 µmol m-2 s-1). Essa possiede un unico cloroplasto parietale/cellula con lunghi tilacoidi agranali e privo di pirenoide. Al fine di valutare i cambiamenti adattativi del plastidio a simulate condizioni di notte australe, colture liquide cresciute alla luce sono state poste al buio per 2 mesi, senza variare gli altri parametri di coltura. L'esame ultrastrutturale, l'analisi microspettrofluorimetrica dell'assetto del PSII, l'analisi spettrofotometrica dei pigmenti fotosintetici hanno mostrato che l'alga nel periodo di buio attua un rimodellamento del suo plastidio. Questo comprende: 1. una specifica sequenza di degradazione di costituenti non necessari per la vita al buio ed il loro immagazzinamento in strutture intraplastidiali specializzate; 2. il mantenimento di buona parte delle membrane tilacoidali, strettamente appressate in fascetti alla periferia dell'organulo; 3. la comparsa di emissioni di fluorescenza proprie di protoclorofille associabili a strutture simili a corpi prolamellari poco evoluti. Tali cambiamenti appaiono come risultato di due momenti di adattamento, uno che si realizza nei primi 6 giorni di buio, e l'altro, più lento, che si instuara nelle settimana successive.
17
THE MAJOR ANTENNA COMPLEX OF PHOTOSYSTEM II (LHCII) HAS A XANTHOPHYLL BINDING SITE NOT INVOLVED IN LIGHT HARVESTING.
Stefano Caffarri (1) , Roberta Croce (1) , Jacques Breton (2) and Roberto Bassi (1)
(1) Dipartimento Scientifico e Tecnologico, Università di Verona. Strada Le Grazie 15, 37134, Verona, Italy. (2) Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, Bât. 532, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France
We have characterised a xanthophyll binding site, called V1, in the major light harvesting complex of Photosystem II, distinct from the three tightly binding sites previously described as L1, L2 and N1. Xanthophyll binding to the V1 site can be preserved upon solubilisation of the chloroplast membranes with the mild detergent dodecyl-a-D-maltoside while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2 and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesised zeaxanthin which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.
18
DIFFERENTIAL ACCUMULATION OF Lhcb GENE PRODUCTS IN THYLAKOID MEMBRANES OF ZEA MAYS PLANTS GROWN UNDER DIFFERENT LIGHT AND TEMPERATURE CONDITIONS.
Stefano Caffarri and Roberto Bassi
Dipartimento Scientifico e Tecnologico. Facoltà di Scienze MM.FF.NN. Università di Verona. Italy.
It is well know that at many different genes encode Lhcb proteins in higher plants. Evolutionary conservation of this genomic complexity suggests that individual gene products play different roles in light-harvesting and photoprotection depending on environmental conditions. We have tested the hypothesis that the expression/accumulation level of the individual proteins is regulated in order to adapt plants to different growing conditions. Zea mays plants were grown in different temperature (13°C vs 24°C) and light (high light vs low light) conditions. The thylakoid membranes were isolated and fractionated by sucrose gradient and the protein content of the different bands was analysed by SDS-PAGE. Significant differences were found in the accumulation of both the major LHCII complex and the minor antenna proteins CP29, CP26 and CP24. In particular the temperature seems to play a major role in driving the expression/accumulation of the different proteins: the LHCII/minor antenna ratio increases with decreasing temperature. The pigment composition and the spectroscopic properties of LHCII complex isolated from low temperature grown plants is significantly different from those of LHCII from high temperature grown plants. The functional properties of the different LHCII preparations are under investigation.
19
Studies on the role of tyrosine phosphorylation on the 14-3-3-mediated activation of the plasma membrane H+-ATPase
L. Camoni1, M. Vecchi1, M. Marra2 and P. Aducci1
1Università di Roma "Tor Vergata", 2Università del Sannio-Benevento
The H+-ATPase is the major ion pump in the plasma membrane of higher plants. This enzyme generates an electrochemical gradient which provides the driving force for a number of key physiological processes, such as stomata opening, phloem loading and root ion uptake. In the last years, some light has been shed about the molecular mechanism of regulation of H+-ATPase. In fact, it has been demonstrated that the activity of the enzyme is stimulated by a phosphorylation dependent association of 14-3-3 proteins to the C-terminal autoinhibitory domain of the proton pump (Camoni et al., J. Biol. Chem 2000). The 14-3-3 binding site has been identified at the extreme end of the enzyme and contains a phosphorylated threonine residue. Recently, it has been observed that in vivo treatment with phenylarsine oxide (PAO), a tyrosine phosphatase inhibitor, strongly inhibits the fusicoccin-induced activation of the proton pump (Olivari et al., Plant Physiology 2000). This result suggests that a phosphorylation event on a tyrosine residue inhibits the association of 14-3-3 proteins to the H+-ATPase, thus representing a further regulatory mechanism of the enzyme activity. In the present study, we investigated the possible role of tyrosine phosphorylation on 14-3-3 proteins on their ability to activate the proton pump. To this purpose, in vivo treatment of maize roots with PAO was carried out, and cytosolic 14-3-3 proteins were purified and analyzed. Furthermore, the capability of 14-3-3 proteins purified from PAO-treated roots to activate the H+-ATPase was studied.
20
PYROPHOSPHATE IN PLANT MITOCHONDRIA
Valentino Casolo, Stefano Micolini, Francesco Macrì e Angelo Vianello
Dip. di Biologia e Economia Agro-industriale, Sezione di Biologia Vegetale, Università di Udine
The matrix level of pyrophosphate (PPi) in mitochondria from etiolated pea (Pisum sativum L., cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 mM. This can derive from cytoplasmic PPi entering the mitochondria via adenine nucleotide translocase (ANT), because F- and Ca2+ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied by PPi. This result was also confirmed by measuring oxygen consumption and the electrical potential (DY in succinate-energized mitochondria). In a medium free of phosphate (Pi), the addition of PPi before the substrate, rendered possible an ADP-stimulated oxygen consumption that was inhibited by F- or Ca2+. In a similar experiment, ADP induced the dissipation of DY whether it was added after the succinate-generated DY had reached a steady-state and, again, F- inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H+-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H+-PPiase (EC 3.6.1.1), acting as a synthase. This evidence, arises from the observation that Pi stimulated oxygen consumption with a respiratory control ratio of 1.7 and that this stimulation was inhibited by F- or Ca2+. The physiological role of the mitochondrial H+-PPiase is discussed in the light of the consideration that this enzyme can catalyze a readily reversible reaction.
21
IN VITRO RECONSTITUTION OF THE PHOTOSYSTEM I LIGHT-HARVESTING COMPLEXES (Lhca1-4)
Simona Castelletti1,2, , Tomas Morosinotto1, Bruno Robert 2, Roberto Bassi 1 and Roberta Croce1
1 Dipartimento Scientifico e Tecnologico, Università di Verona, Italy. 2 Départément de Biologie Moléculaire et Cellulaire, CEA Saclay, France.
Lhca1-4 are protein-pigment complexes of higher plants Photosystem I and are responsible for the long wavelength fluorescence emission of the chloroplasts. These unusual fluorescence properties are due to the presence of long wavelength absorption forms while the reasons of such a shift are still unknown. Molecular analysis of LHCI is needed in order to understand the basis of the peculiar spectroscopic properties of this pigment-protein complex. Unfortunately, within the Lhc family these proteins are among the less characterised due to the difficulty of their purification. In particular the characteristics of the individual Lhca proteins are not well known since they form stable heterodimeric complexes whose dissociation leads to denaturation of the pigment-protein complexes. To overcome these difficulties, we have produced recombinant Lhca proteins by overexpression of the apoproteins in E.coli and in vitro reconstitution of the holoproteins. In this work we describe in vitro reconstitution of PSI antenna complex Lhca1-4 using the apoproteins of Lhca1, Lhca2, Lhca3 and Lhca4 of Arabidopsis thaliana overexpressed in E.coli and a mix of pigments (chlorophylls, xanthophylls and b-carotene) in appropriate ratios.
22
MODULATION OF K+ATP CHANNEL AND CYTOCHROME c RELEASE IN PLANT MITOCHONDRIA
Elisa Chiandussi, Elisa Petrussa, Enrico Braidot, Francesco Macrì e Angelo Vianello
Dip. di Biologia ed Economia Agro-industriale, Sezione di Biologia Vegetale, Università di Udine
In this work, we present evidence on the modulation of the K+ATP channel of pea stem mitochondria by NO and H2O2, and on its possible involvement in cytochrome c release. Pea stem mitochondria, resuspended in a KCl medium (de-energized mitochondria), underwent a swelling, as a consequence of K+ entry, that was inhibited by ATP. This inhibition was partially restored by GTP and diazoxide (K+ channel openers). In addition, glyburide and 5-hydroxydecanoate (K+ATP channel blockers) induced an inhibition of the GTP-stimulated swelling. Mitochondrial swelling was inhibited by H2O2 (generated by glucose-glucose oxidase), but stimulated by NO (released by potassium nitroprusside). The same results were also obtained in succinate-energized mitochondria. When the succinate-dependent electrical potential had reached a steady-state, the addition of KCl induced a dissipation that was favoured by NO and prevented by the NO scavenger, carboxy-PTIO. Hydrogen peroxide prevented the stimulation by NO of the KCl-induced dissipation of the electrical potential. The aperture of this channel was linked to the partial rupture of the outer membrane and the latter effect led to a release of cytochrome c, but not of adenylate kinase. These results show that the plant mitochondrial K+ channel resembles that present in mammalian mitochondria. This channel can be modulated by NO and H2O2 and be involved in the release of cytochrome c, occurring during the manifestation of programmed cell death.
23
TUNGSTATE AS A TOOL TO STUDY NITRATE UPTAKE: EVIDENCES FOR A PROTON-SYMPORT MECHANISM I MAIZE ROOTS.
Luca Espen, Fabio Nocito e Marizio Cocucci
Dip. di Produzione Vegetale, UNI Milano
It has been proposed that NO3- uptake occurs through a H+ symport operating with a > 1 H+/NO3- stoichiometric ratio. Although this mechanism would lead to an acidification of the cytoplasmic pH (pHCyt), experiments conducted using intracellular electrodes showed an alkalization of pHCyt during NO3- influx. This result was attributed to a high NO3- assimilation activity, which is a proton-consuming process. In order to study the effect of nitrate uptake on pHCyt, we used tungstate as inhibitor of nitrate reductase (NR) activity. Tungstate inhibited NR activity, as well as the 15NO3- assimilation. The in vivo 31P-NMR analysis allowed to exclude a generalised toxic action effect of tungstate on cell metabolism. In the same conditions, NO3- uptake was not affected. In tungstate-treated roots, the addition of 0.1 mM Ca(NO3)2 induced an acidification of pHcyt. Differently, an alkalization was observed in the control. Transmembrane potential differences of root cortical cells (Em) was transiently depolarised under all conditions analysed. However, in plants treated with tungstate, the initial depolarisation was followed by a gradual hyperpolarisation of Em. Neverthelessb, the treatment with tungstate strongly reduced the depolarising effect of 0,2 mM KNO3. The activity of plasma membrane H+-ATPase resulted higher in tungstate-treated than in control roots. These results suggest that NO3- uptake in maize roots involves a transport system potentially able to induce acidification of pHcyt, according to the hypothesis of a H+/NO3- symport mechanism.
24
Effect of phosphorylation on the Viral K+ Channel KCV
SABRINA GAZZARRINI*, PAOLA VALBUZZI*, MARIA ANNA SEVERINO*, GERHARD THIEL^, ANNA MORONI*
*Dip. di Biologia, UNI Milano; ^UNI Darmstadt
Kcv is a K+ channel gene identified in the genome of PBCV-1, a DNA virus which infects chlorella-like green algae. Previous data have shown that, when heterologously expressed in Xenopus laevis oocytes, Kcv forms a K+-selective ion channel displaying several properties of the structurally more complex mammalian and plant channels. Kcv sequence analysis revealed two putative CKII (casein kinase II) phosphorylation sites (T9 and S49). We have therefore explored the possibility that Kcv is regulated by phosphorylation by testing the effect of several kinase inhibitors (DRB, A3, H89) on Kcv currents. We have also performed site specific mutations (T9A and S49A) in order to neutralize the phosphorylation sites and check the effect of the kinase inhibitors on the mutated proteins currents.
25
Non-photochemical Quenching in KOLIELLA ANTARCTICA, AN NEW ANTARCTIC GREEN ALGAE ISOLATED IN THE ROSS SEA
GM. Giacometti, N. La Rocca, S. Zanetti, C. Andreoli
Dip. di Biologia, Università di Padova, Via Ugo Bassi 58 B, 35121 Padova, Italia
PSII fluorescence emission in a cell suspension of Koliella antarctica Andreoli, was characterised by an unusually high Fo and low Fv. This indicates that even when all the PSII reaction centers are open only a small fraction of excitation absorbed by antenna is converted into charge separation. When actinic light was turned on, a maximum fluorescence level Fp rapidly attained and a non-photochemical quenching quickly built up bringing the fluorescence emission to a steady state level Fs well below the Fo level. When the actinic light was turned off, after a fast decrease to a Fo' level, the fluorescence increased back exponentially approaching to Fo. Generation of described quenching is abolished or significantly reduced by addition of DCMU or of uncoupling agents, showing that the observed QNP is dependent on membrane energization in agree with its relaxation time. It is tempting to interpret this behaviour as an adaptation strategy of this algae which utilizes the light energy absorbed part for photosynthetic work and part to increase the local temperature to make biosynthetic activity faster at the temperature of his natural habitat.
26
EFFECTS OF CAROTENOGENESIS INTERRUPTION BY CPTA ON BARLEY ETIOPLASTS
Nicoletta La Rocca , Isabella Moro e Nicoletta Rascio
Dip. Di Biologia, UNI Padova
The altered organization of etioplast membranes and the deregulation of protochlorophyllide (Pchlide) synthesis were noticed in etiolated barley plants treated with amitrole (La Rocca et al., Planta, 213:201, 2001), a herbicide which inhibits carotenoid biosynthesis leading to accumulation of hydrocarbon precursors. It has been hypothesized that free hydrocarbon carotenoids could destabilize the thylakoid membranes by inserting themselves in the hydrophobic matrix environment (Havaux, T. Plant Sci.., 3:147, 1998). This led us to suppose that also in etioplasts, anomalous quantities of carotenoid precursors could account for the membrane disorders and that this latter might interfere with the regulation of Pchlide synthesis. In order to verify this idea, etiolated barley plants were treated with CPTA (N,N-diethyl-N-[-2-(4-chlorophenylthio)ethyl]amine), which interrupts carotenogenesis by specifically inhibiting the lycopene cyclase (Fedtke et al., Pest Manag. Sci., 57:278, 2001). The analyses confirmed that also this treatment, leading to the accumulation of relevant quantities of lycopene gave rise to a disordered aggregation of etioplast membranes. This ultrastructural alteration was related to Pchlide levels 3-4 times higher than those of control plants, showing the deregulation of the pigment biosynthesis. Moreover the fluorescence emission spectra evidenced that the prevalent Pchlide form was the non-phototransformable one.This demonstrated the difficulty in forming the ternary complex Pchlide-Pchlide oxidoreductase-NADPH in the altered membranes of the orgenelles.
27
Progress on the identification of the protein kinase involved in the 14-3-3-mediated activation of the plasma membrane H+-ATPase. Lalle M., Visconti S. and Aducci P.
Department of Biology, University of Rome "Tor Vergata"
In eukaryotic cells, protein phosphorylation is a central mechanism in post translational regulation of proteins activity. A variety of signalling molecules that regulate proteins by binding to short motifs containing phosphoserine or phosphotreonine have been identified as 14-3-3, a family of dimeric a-helical proteins. In plants 14-3-3s are involved in the regulation of many enzymes such as nitrate reductase and sucrose phosphate synthase. Also the plant plasma membrane H+-ATPase is regulated by 14-3-3 proteins interaction. H+-ATPase generates an electrochemical gradient across plant cell plasma membrane that provides the driving force for secondary active transport and for cell turgor maintenance. The activity of H+-ATPase is regulated by its C-terminal region that functions as an autoinhibitory domain. 14-3-3s are able to activate the proton pump by interaction with the last three amino acids (YpTV that is a non-canonical 14-3-3 binding site) of C-terminus of the enzyme. The binding is promoted by phosphorylation of threonine. At present there are some indirect evidences that the involved protein kinase that phosphorylates the penultimate threonine is associated with the plasma membrane. Using two different approaches we are attempting to identify this protein kinase. The first is a molecular strategy based on a functional screening of a lgt11 corn cDNA expression library. The second is a biochemical analysis of plant extracts of various tissues from corn and spinach tested for specific protein kinase activity.
28
WATER RELATIONS OF NtAQP1-AQUAPORIN-ANTISENSE TOBACCO PLANTS
Claudio Lovisolo +*, Franka Siefritz*, Andrea Schubert +, Ralf Kaldenhoff *.
+DCA UNI Torino; *Universität Würzburg
The significance of the plasma membrane intrinsic aquaporin NtAQP1 in whole plant water relations was studied comparing anti-sense plants with wild types, after a one-week moderate water stress. TOBACCO PLANTS, IMPAIRED IN NTAQP1 EXPRESSION, WERE GENERATED BY LEAF DISC TRANSFORMATION OF AN ANTI-SENSE NTAQP1 CONSTRUCT, DRIVEN BY THE STRONG CONSTITUTIVE 35S CAMV PROMOTER TOGETHER WITH A KANAMYCIN RESISTANCE MARKER GENE (NPT II). REGENERATED PLANTS WERE SELF-POLLINATED AND THE RESULTING T2 GENERATIONS OF 4 INDEPENDENTLY TRANSFORMED LINES WERE ANALYZED. At the end of the drought period, soil water potential was not significantly different among AS-plants and controls. Stem water potential was higher in WT with respect to AS-plants; this caused a reduced water potential gradient (=energy necessary to absorb water from the soil) into WT plants than into AS-plants. In AS-plants stomatal conductance and plant transpiration were lower than in controls. Root hydraulic conductivity (kh) was higher in controls than in AS-plants both in vivo and on excised plants in lab. Reduction of root kh had profound effects on plant physiology: AS-plants reduced stomatal conductance in order to compensate for lower root water uptake, and this in turn reduced transpiration and water potential. MOREOVER, BREAKING DOWN THE SYMPLASTIC RESISTANCE BY INACTIVATING MEMBRANES WITH FREEZE-THAW CYCLES INCREASED ROOT KH MUCH MORE IN AS- THAN IN CONTROL PLANTS, AS EXPECTED IF SYMPLASTIC RESISTANCE WERE HIGHER IN THE AS- THAN IN CONTROL PLANTS.
29
Regolazione dell'attività della Ca2+-ATPasi del plasmalemma da fosfolipidi acidi
Laura Luoni, M.Cristina Bonza e M.Ida De Michelis
Dip. di Biologia, UNI Milano
La Ca2+-ATPasi del plasmalemma (PM) delle cellule vegetali catalizza l'estrusione di Ca2+ dal citoplasma verso l'apoplasto contro un elevato gradiente elettrochimico; è una ATPasi di tipo P caratterizzata dalla presenza di un dominio autoinibitorio che lega la calmodulina (CaM). La CaM è il regolatore dell'attività della Ca2+-ATPasi del PM meglio noto; il legame della CaM al sito autoinibitorio determina l'attivazione dell'enzima. L'attività delle Ca2+-ATPasi del PM delle cellule animali è regolata anche dai fosfolipidi acidi (APL) che attivano l'enzima attraverso un meccanismo complesso solo in parte sovrapponibile a quello della CaM. L'effetto degli APL sull'attività della Ca2+-ATPasi del PM delle cellule vegetali è stato studiato in PM isolato da semi germinanti di ravanello dopo rimozione della CaM endogena con EDTA. Gli APL stimolano l'attività basale della Ca2+-ATPasi nel seguente ordine di efficienza: fosfatidilinositolo4,5-difosfato > fosfatidilinisitolo 4-monofosfato > fosfatidilinositolo > fosfatidilserina > acido fosfatidico. L'analisi della dipendenza da concentrazione di Ca2+ libero dell'attività della Ca2+-ATPasi mostra che gli APL determinano un aumento di Vmax e una diminuzione della Km apparente per il Ca2+ libero a valori inferiori rispetto a quelli misurati in seguito al taglio proteolitico del dominio di legame della CaM. Gli APL quindi attivano anche la Ca2+-ATPasi del PM delle cellule vegetali attraverso un meccanismo solo in parte sovrapponibile a quello della CaM, implicando l'esistenza nella Ca2+-ATPasi del PM delle cellule vegetali di un sito di legame per gli APL diverso da quello per la CaM.
30
(-)-MENTHOL-INDUCED MEMBRANE DEPOLARIZATION PARALLELS INTRACELLULAR CALCIUM VARIATIONS IN CUCUMBER ROOT CELLS
Massimo Maffei e Wanda Camusso
Dip. Biologia Vegetale, Università di Torino, V. Mattioli, 25 10125 Torino.
Monoterpenes inhibit seed germination and plant growth in the chemical interaction among plants known as allelopathy. Peppermint (Mentha piperita L.) essential oils affect root and mitochondrial respiration as well as membrane permeability and ion uptake of cucumber (Cucumis sativus L.) root cells. In this communication we report the effect of the monoterpene (-)-menthol on membrane depolarization and variation in intracellular calcium concentration [Ca2+]i in cortical root cells of cucumber. The transmembrane potential (Vm) was determined in the root elongating zone before and during perfusion with 300 ppm (-)-menthol with glass micropipettes. To evaluate [Ca2+]i as a response to (-)-menthol perfusion, two [Ca2+] indicators, Fura-2 and Fluo-3 AM, were used using a Nikon Eclipse E-400 Epifluorescence Microscope and a Zeiss Axiovert 100M connected to a Zeiss LSM 510 Laser Scanning Confocal Microscope. Ca2+ channel inhibitors as well as G-Protein pathway inhibitors were also used. The results obtained show a rapid Vm depolarization which is maintained until (-)-menthol is perfused. Washing out (-)-menthol restores the initial Vm. The increase in [Ca2+]i parallels Vm depolarization, even though the [Ca2+]i increase is transient. After a peak around 300 s. (corresponding to maximum depolarization), [Ca2+] drops down to initial values, while Vm remains depolarized. The physiological, biochemical and allelopathic effects of (-)-menthol on cucumber root system are discussed along with general considerations on the (-)-menthol signal transduction pathway.
31
PPI1, UNA NUOVA PROTEINA CAPACE DI INTERAGIRE CON IL DOMINIO C-TERMINALE DELL' H+-ATPASI DEL PLASMALEMMA
Piero Morandini, Cristina Albumi, Marco Valera, Maria Cristina Bonza, Irene Murgia, Carlo Soave e Maria Ida De Michelis
Dipartimento di Biologia, Università di Milano
Il dominio autoinibitorio C-terminale dell' H+-ATPasi del plasmalemma costituisce il principale sito di regolazione dell'enzima: la sua rimozione proteolitica, così come il legame di proteine 14-3-3 ad una sequenza contenuta in questa regione, causano un aumento dell'attività enzimatica. Utilizzando il metodo del doppio ibrido abbiamo identificato una proteina di 612 aa (Ppi1, proton pump interactor), la cui porzione N-terminale (88aa) interagisce con il dominio C-terminale dell'H+-ATPasi, isoforma AHA1. Diversi geni di Arabidopsis thaliana e alcuni EST di diverse specie vegetali mostrano una omologia di sequenza significativa (50-70% in porzioni di 200-600 aa) con Ppi1. La regione N-terminale di Ppi1 è stata espressa in E. coli come proteina di fusione con la GST o con una coda di istidine (His6-Ppi). In esperimenti di overlay, entrambe le proteine di fusione si legano all'H+-ATPasi immunoprecipitata da una frazione di plasmalemma purificata da cellule in coltura di A. thaliana His6-Ppi stimola l'attività dell'H+-ATPasi; lo stimolo è maggiore a pH 6.4 (massimo stimolo a 20 mM His-Ppi) che a pH 7.3, dove lo stimolo non è saturato neppure alla concentrazione di 60 mM. L'attivazione indotta da Ppi1 è sinergica con quella indotta da fusicoccina e da tripsina; inoltre His6-Ppi si lega all'H+-ATPasi trattata con tripsina. Il legame dell'attivatore avviene perciò ad un sito diverso da quello delle 14-3-3 e a monte del sito di taglio della tripsina.
32
The origin of the red absorption forms of PSI-200: mutational analysis of Lhca1
Tomas Morosinotto, Simona Castelletti , Roberto Bassi and Roberta Croce
Dipartimento scientifico e tecnologico, Università degli studi di Verona, Italy
Lhca1 is one of the four antenna complexes of Photosystem I, which are characterised by absorption forms at low energies. Lhca1 from Arabidopsis thaliana was expressed in E.coli and reconstituted in vitro adding the pigments. The reconstituted complex shows a low energy absorption form, with large FWHM, at 687 nm, and emission at 694 nm. To identify the pigment responsible for this absorption, site selected mutagenesis on the putative Chls binding sites of Lhca1 was performed. The data indicate that the interactions between pigments play a major role in determining the spectroscopic properties of the complex, in contrast to what observed for the light-harvesting complexes of Photosystem II. In particular it was observed that the red absorption form originate by the excitonic interaction between the Chls in sites A5 and B5 with the involvement of the carotenoid located in L2 site. Biochemical and spectroscopic characterisations of Lhca1-WT complex and all mutants are presented.
33
ION CHANNELS IN PROTOPLASTS FROM THE MARINE PLANT POSIDONIA OCEANICA
A. Paganetto, A. Naso, E. Pesce, A. Carpaneto e F. Gambale
Istituto di Cibernetica e Biofisica, CNR, Via De Marini 6, 16149-Genova
Posidonia oceanica is a marine plant that grows exclusively in the Mediterranean Sea, where it is the dominating species at depths from 2 to 30 m. Given its importance for the ecosystem, the ecological role of this plant is being extensively studied. However, little is known about the correlation between marine physico-chemical parameters (such as high salinity, light levels, hydrostatic pressure, wave action and slow diffusion of inorganic carbon species) and the functional characteristics of Posidonia nutrient transport systems. To investigate in particular the transport occurring through ion channels we enzymatically isolated protoplasts from the basal part of P. oceanica leaves. By applying the patch-clamp technique to these isolated protoplasts, we were able to record different ion channels showing properties similar to those of the corresponding channels from terrestrial plants. Among the various identified channels, we studied the biophysical characteristics of an outward channel open at voltages >-40 mV. This channel is selective for potassium (sodium does not permeate) and sensitive to the external K+ concentration. We have also started the cloning of Posidonia channels. Functional and structural characterisation of channels cloned from P. oceanica is an important step to clarify the physiological processes that allow this plant to grow, survive and play a major role in the ecology of the Meditteranean Sea.
34
A HISTIDINE RESIDUE IN THE S5-S6 LINKER OF THE KDC1 CHANNEL ALTERS SENSITIVITY TO ZINC IONS.
Cristiana Picco+, Monica Bregante+, Andrea Paganetto+, Alessia Naso+, Paola Gavazzo+, Patrick Downey*, Fiorella Lo Schiavo*, Rainer Hedrich# e Franco Gambale+
+Istituto di Cibernetica e Biofisica, CNR, Genova; *Dipartimento di Biologia, Universita' di Padova, Padova; #Julius-von-Sachs Institut fur Biowissenschaften, Lehrstuhl fur Molekulare Pflanzen Physiologie und Biophysik, Würzburg, Germany
KDC1 is a potassium channel gene recently cloned from carrot roots. It has been shown that this channel is involved in the modulation of ionic currents when co-injected with other potassium channels in Xenopus laevis oocytes. In particular, when KDC1 is co-expressed with KAT1 (a potassium channel cloned from Arabidopsis thaliana) ionic currents display much slower activation kinetics and their sensitivity to heavy metal ions is also significantly modified. The current of homomeric KAT1 channel is blocked by Zn2+, while in the heteromeric KDC1:KAT1 channel, the current is enhanced by zinc. One of the hypotheses made to explain this behaviour is that the current enhancement is due to the presence of some extraplasmatic histidines in the aminoacidic sequence of KDC1 which are absent in the KAT1 channel. To investigate which histidines are involved in this process, point mutations have been introduced in the KDC1 channel; in particular, two histidines (H224, H225) in the S5-S6 linker to the external face of the membrane of KDC1 have been mutated. mRNA containing either the H224A or the H225A mutation of the KDC1 channel has been co-injected with that of KAT1 (ratio 1:1) in oocytes. Ionic currents have been measured using the voltage-clamp technique. In the case of the H224A mutation, the current enhancement previously observed in the heteromeric wild-type channel (KDC1:KAT1) does not occur: the presence of Zn2+ inhibits ionic current and the heteromeric channel behaves like the homomeric one (KAT1) i.e the channel is blocked by external zinc. Conversely, the channel containing the H225A mutation shows the same current enhancement as the wild-type channel. These results have also been confirmed by the co-injection of KAT1 with the double mutated (H224A-H225A) KDC1 channel. Zn2+ has been found to inhibit the potassium current in this experiment as well. Therefore, we suggest that the residue H224 located in the S5-S6 linker of the KDC1 channel may play a key role in the zinc enhancement of the heteromeric KDC1:KAT1 current.
35
ISOLATION OF SEQUENCE AND EXPRESSION ANALYSIS OF AQUAPORIN IN VITIS VINIFERA L.
Riccomagno Nadia e Schubert Andrea
Dip. Colture Arboree, UNI Torino.
Aquaporins are integral membrane proteins of vacuolar and plasma membranes that facilitate the passage of water through these membranes. These channels are encoded by a family of genes characterized by conserved sequences. The aim of this work was to isolate new Vitis vinifera aquaporin cDNAs. Using a PIP1b probe from A.thaliana, by Southern hybridization we obtained four different DNA bands representative of four putative genes. RT-PCR with primers designed on conserved NPA motifs of the same gene showed expression of only two aquaporin genes. We cloned one of these cDNAs, VvAQP1. The VvAQP1 sequence is 600 nucleotide long and is highly similar to aquaporin sequences of Vitis berlandieri´Vitis rupestris, Vitis vinifera cv Pinot Noir, Arabidopsis and tobacco. Northern analysis gave expression of VvAQP1 in root and not in leaves and berries. The tissue-specific expression of this gene and the apparent redundancy of aquaporin can be explain by a specialized function of each aquaporin.
36
INDUCTION OF NITRATE UPTAKE AND PLASMA MEMBRANE H+-ATPase GENE EXPRESSION IN MAIZE ROOTS
Simonetta Santi, Rossella Monte, Geraldine Locci, Roberto Pinton, Zeno Varanini
Dip. di Produzione Vegetale e Tecnologie Agrarie, UNI Udine
We have previously shown that root plasma membrane (PM) H+-ATPase activity increases when maize (Zea mays L.) seedlings are treated for 24 h with NO3- (Santi et al., Plant Physiology 1995). To analyse in further detail the response of the enzyme, etiolated maize seedlings were induced for NO3- uptake, and the changes in activity, quantity, and gene expression of root PM H+-ATPase were monitored throughout the first hours of contact with the anion. The results show that there is a close link between the process of NO3- uptake and the activity and quantity of the enzyme. Furthermore, Northern analyses were carried out using a cDNA sequence from a conserved region of MHA2 gene of maize (PM) H+-ATPase, results indicate that NO3- treatment enhances PM H+-ATPase gene expression with kinetics similar to those observed for other NO3--inducible genes thus suggesting that the anion may act as a signal triggering the expression of PM H+-ATPase. With the aim to show if specific (PM) H+-ATPase isoforms are involved in this phenomenon, four partial cDNAs coding for the (PM) H+-ATPase have been isolated by reverse transcription-coupled Rapid Amplification of cDNA Ends (RACE) from total RNA of maize roots. Furthermore, a partial cDNA sequence for a putative high affinity NO3- transporter (NRT2) has been identified by using degenerate primers in RACE experiments. Studies of gene expression by northern analysis are in progress.
Supported by Italian MURST-COFIN 2000 to Z.V.
37
PROTEOMICS OF THE PHOTOSYSTEM II ANTENNA COMPLEX FROM DIFFERENT SPECIES: ANALYSIS AND COMPARISON
A.M. TIMPERIO+, S. RINALDUCCI+, S. TROIANI+*, M BIANCHETTI+, C. G. HUBER*, AND L. ZOLLA
+ Department of Environmental Science, University of Tuscia, 01100 Viterbo, Italy and * Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, 6020 Innsbruck, Austria
A comprehensive overview on the molecular masses of the major (Lhcb1, Lhcb2, Lhcb3) and minor (CP24, CP26, CP29) light-harvesting protein components of photosystem II (PSII) in a variety of different plant species has been performed by RP-HPLC-ESI-MS. The measured molecular masses were in excellent agreement with the molecular masses of the individual components where the amino acid sequence is known, and in good agreement with the values calculated on the basis of their nucleotide-derived amino acid sequences, allowing to asses that the apoprotein heterogeneity is from the expression of distinct genes. In all species examined Lhcb1 was present in several isoforms with molecular masses ranging from 24,680 to 25,014. Thus, Lhcb1 proteins seem to be highly conserved among different species, such that they belong to a single gene group which has several different gene family members. Lhcb2 was present in only one form with a molecular mass ranging from 24,600 to 24,800, whereas the masses of Lhcb3 were close to 24,300. Two isoforms of Lhcb3 were found in Petunia and tomato (Lycopersicon esculentum). The minor antenna protein, CP24, was the most divergent of all LHC sequences, with molecular masses ranging from 22,600 to 22,800, and the greatest number of isoforms were found in Petunia, tobacco (Nicotiana tabacum), tomato and rice ( Oryza sativa). In contrast, CP26 and CP29 were only present in one form with molecular masses that ranged from 26,500 to 27,500 and from 28,100 to 28,600, respectively. Genes from some species which have been proposed to code for CP26 and CP29 have been reviewed and newly assigned.
38
Expression analysis of a K+ channel in Daucus carota L.
Zambon M*., Costa A*., Downey P*. Lo Schiavo F*. e Varotto S°.,
*Dipartimento di Biologia Università degli Studi di Padova Via Colombo, 3 35121 °Dipartimento di Agronomia Ambientale e produzioni vegetali Università degli Studi di Padova via Romea,16 35020 Legnaro (PD)
Plants have developed highly specific mechanism to take up potassium from the soil: these include the expression of K+ transporters and potassium channels in the root. KDC1 is a novel plant potassium channel isolated from carrot root RNA. Whole mount hybridization performed on carrot roots showed that the transcript of this gene is present in the root hair cells. In order to determine the precise cellular localisation of the channel, mRNA "in situ" hybridisation experiments were carried out in young seedling root sections. The hybridisation signal was observed in root epidermis and in some vessel elements. The expression pattern of the channel was also analysed in carrot embryos during somatic embriogenesis. RT-PCR experiments showed that the channel is present in early stages of embriogenesis (globular stages). "In situ" hybridisation on somatic embryos sections showed a detectable hybridisation signal in some cells of the embryo at the early torpedo stage. These data were confirmed by Western analysis performed with a specific antibody. Electro physiological analysis of carrot root hair protoplasts showed that the K+ channel is involved in mechanism of resistance to heavy metals. In order to confirm these results, Arabidopsis thaliana plants are going to be transformed and analysed. Moreover for detecting the physiological induction probably exerted by auxin on the channel, "in situ" hybridisation experiments will be carried out on sections of young seedlings germinated in a auxin-enriched medium.
39
REGOLAZIONE DA ABA DEI CANALI Kin DI COLEOTTILI DI MAIS
Luisa Zingarelli*, Barbara Locatelli*, Gerhard Thiel° e Raffaella Cerana^
*Dip. Biologia UNI Milano, °UNI Darmstadt, ^DISAT UNI Milano-Bicocca
Precedenti studi (Zingarelli et al., XXXIX Congresso SIFV), condotti con la tecnica del patch-clamp in configurazione whole-cell, avevano evidenziato che l'acido abscissico (ABA) 20 mM inibiva l'attività dei canali per il K+ rettificatori entranti (canali Kin) di protoplasti isolati da coleottili di mais. La massima inibizione (ca. 50%) si raggiungeva in 15 min e appariva irreversibile e indipendente dalla concentrazione citoplasmatica di Ca2+ e H+. In questo lavoro abbiamo analizzato l'effetto dell'ABA sulle proprietà biofisiche del canale e, dato che mutanti di mais insensibili all' ABA sono difettivi per proteine fosfatasi (Armstrong et al.1995), abbiamo studiato l'effetto dell'ATP sull'inibizione da ABA dell'attività di questi canali. I risultati mostrano che: i) l'ABA non modifica né la cinetica d'attivazione né la voltaggio-dipendenza del canale, suggerendo che l'effetto sia sulla conduttanza; ii) l' inibizione da ABA dell'attività dei canali cambia se nel lato citoplasmatico viene aggiunto ATP: in particolare sono necessarie concentrazioni più elevate (200 mM vs. 20 mM) di ABA per osservare l'effetto inibente; iii) la staurosporina, inibitore aspecifico delle proteine chinasi, inibisce l'attività dei canali. Questi risultati suggeriscono che l'attività dei canali da noi studiati è regolata dal loro stato di fosforilazione o da quello di proteine regolatrici. L'ABA, agendo su tale stato, ne inibisce l'attività e ciò potrebbe contribuire alla diminuzione dell'ingresso di K+ e, quindi, all'inibizione della crescita per distensione del coleottile indotta dall'ormone.
POLYAMINE PATTERN AND GENE EXPRESSION IN FLORAL ORGANS OF NORMAL AND PARTHENOCARPIC TOMATO PLANTS
F. Antognoni+, F. Ghetti+, M. Franceschetti+, A. Mazzucato* e N. Bagni+
+Dipartimento di Biologia evoluzionistica sperimentale, Università di Bologna; *Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Viterbo, Italia.
POLYAMINES ARE WIDELY DISTRIBUTED IN PLANTS AND APPEAR TO PLAY AN IMPORTANT ROLE IN SEVERAL GROWTH AND DEVELOPMENTAL PROCESSES. AMONG OTHERS, POLYAMINES SEEM TO BE INVOLVED IN THE FLOWERING PROCESS, I.E.THE FLORAL INDUCTION AND THE DIFFERENTIATION OF FLORAL ORGANS, ALTHOUGH THEIR PRECISE ROLE IN THIS PROCESS IS STILL POORLY UNDERSTOOD. THE USE OF FLORAL MUTANTS WITH SPECIFIC DEFECTS IN ORGAN DEVELOPMENT HAS BEEN AN USEFUL TOOLS IN ANALYZING THE ROLE OF THESE POLYCATIONS IN FLOWER DEVELOPMENT. Parthenocarpy expression in tomato can be affected by environmental conditions and genetic background. Among the different sources of genetic parthenocarpy, the mutation referred to as parthenocarpic fruit (pat) is caused by a single recessive mutation with pleiotropic effects (Philouze and Pecaut, 1986). Among them, the pat gene is responsible for aberrations that affect the androecia development and the female fertility. In this paper we report on polyamine profile and biosynthetic enzyme activities in floral organs of normal and pat mutant tomato plants during flower development. Transcript levels of arginine-, ornithine-, and S-adenosylmethionine decarboxylases were also examined in ovaries and anthers and the possible regulation of their genes throughout flower development is discussed.
41
OLIGOGALACTURONIDES AFFECT SOMATIC EMBRYOGENESIS IN Daucus carota L.
Baldan Barbara, Berto Michela, Bertoldo Alessandro and Mariani Paola
Dipartimento di Biologia , UNI Padova
Oligogalacturonides (OGs) are pectic fragment of the plant cell wall. They have been shown to elicit several defence responses and to induce the transcriptional activation of defence-related genes. Furthermore OGs, at micromolar levels, can regulate several events of plant growth and development. We analysed in D.carota the effect of OGs on somatic embryogenesis, a very complex morphogenetic process regulated by exogenous and endogenous factors. The obtained results point out an impaired embryogenesis following the OG treatment. The embryogenic efficiency is lower and the embryo development is severely delayed with respect to the control. Early embryos regularly acquire the shoot-radicle axis polarity. In older embryos the radicle is stimulated to develop, whereas shoot is deeply disorganized: random cell proliferations occur and the embryonal axis is lacking. Seedlings obtained from altered embryos exhibit a multiple shoot and a well-developed root apparatus.
42
Requirement for topo Ia or topo Ib expression for plant cell viability revealed by antisense RNA strategy
Balestrazzi A., Guidi M. and Carbonera D.
Dip. Di Genetica e Microbiologia "A: Buzzati-Traverso",Università di Pavia
Although many studies link eukaryotic topoisomerase I to important cellular functions related to transcription and then gene expression regulation, this enzyme is dispensable in yeast, but is essential during the embryogenesis of Drosophila melanogaster, mouse and Caenorhabditis elegans. Differently from previous evidence that top1 gene was present in single copy in eukaryotes, two distinct top1 loci (a and b) in the nuclear genome of Daucus carota cells has been documented for the first time by Balestrazzi et al. (1). Both genes are transcribed in cycling cells and both proteins are located in carrot nuclei. The presence of two top1 genes has also been recently confirmed in the pufferfish Fugus rubripes (2). In order to define the requirement of topo Ia or topo Ib expression for plant cell viability an antisense RNA strategy was used. This approach offers the opportunity of producing carrot cell lines having down-regulated top1 genes. Results from the molecular analysis and response to the specific drug camptothecin of these cell lines will be discussed with the aim to highlight the contribution of topoisomerases a and b on carrot cell growth. (1) Balestrazzi A., Chini A., Bernacchia G., Bracci A., Luccarini G., Cella R. and Carbonera D. (2000) J. Exp. Bot., 51:1979-90. (2) Smith S.F., Metcalfe J.A. and Elgar G. (2001) Gene, 265: 195-204.
43
DNA METHYLATION AND CARROT SOMATIC EMBRYOGENESIS
Giovanni Bernacchia, Simona Marinello, Laura Ciarrocchi and Rino Cella#
Dip. Biologia, Sez. Biologia Evolutiva, Uni FE, Via Borsari 46, 44100 Ferrara; # Dip. Genetica, Uni PV, Pavia.
Our research work concentrates on the relationships between DNA methylation and plant development. We study the interactions between carrot somatic embryogenesis and DNA-methyltransferases-encoding genes as a model system. We have cloned and characterized two carrot DNA METase genes differentially expressed in plant tissues and embryos. Transgenic carrot cell lines, bearing either antisense and sense constructs, revealed that the embryogenetic progress is highly sensitive to DNA methylation variations. Similar conclusions have been obtained by treating carrot proembryogenic masses with recently identified inhibiting molecules purified from carrot cells (Pedrali-Noy, Bernacchia and Cella). Even at low concentration, these molecules, which can affect DNA methylation in vivo, are able to block in a reversible way the somatic embryogenesis. To better define the role of DNA METase genes, we are isolating the promoter regions of the two genes and testing their structure by protoplast transient expression and carrot transgenic calli. An inducible MET antisense construct has been obtained which can be activated by treatment with the glucocorticoid hormone (Zuo and Chua, 2000, Curr. Op. Biotecn. 11, 146-151). Transgenic carrot cell lines bearing the inducible vector will be described.
44
THE BALANCE BETWEEN METHYL JASMONATE AND HORMONES INTERFERES WITH POLYAMINE CONJUGATION IN TOBACCO LEAF DISKS
Stefania Biondi*, Valeria Scoccianti+, Sonia Scaramagli*, Vanina Ziosi* e Patrizia Torrigiani*
*Dip. di Biologia e.s., Università di Bologna; +Ist. di Botanica, Università di Urbino.
Jasmonic acid (JA) and its methyl ester (MJ) are ubiquitous cyclopentanone compounds which are claimed to represent a new class of plant growth regulators. They exert numerous biological effects ranging from promotion of leaf senescence and tuberization to germination of non-dormant seeds. It has been shown that treatment with jasmonates induces the over-accumulation of secondary metabolites, in particular the conjugates polyamine-hydroxycinnamic acids (HCAs). These compounds, abundant in Solanaceae, accumulate dramatically in relation to biotic and abiotic stress. In some cases altered levels of free polyamines, and of their biosynthetic gene expression have been reported to occur in response to treatment with jasmonates. Nevertheless, a complete picture of the effects of jasmonates on polyamine metabolism is still lacking and a few data about their relation with phytohormones are available. In this work the interactions between auxin/cytokinin and MJ and their effects on polyamine conjugation in tobacco leaf disks were investigated. Preliminary results show that MJ alone or in the presence of auxin and cytokinin (but not of cytokinin alone) dramatically enhances soluble and especially insoluble HCA titers. Northern analysis confirms that transcript levels of S-adenosylmethionine decarboxylase (SAMDC), which leads to spermidine synthesis, are increased in leaf disks under the same experimental conditions.
45
ROLE OF ATHB-2 IN ROOT DEVELOPMENT
Carabelli Monica1, Steindler Corinna1, Mittempergher Francesca1, Sessa Giovanna1, Morelli Giorgio2, and Ruberti Ida1.
1Centro Acidi Nucleici, C.N.R., Rome, Italy, 2Unità Nutrizione Sperimentale, INRAN, Rome, Italy.
ATHB-2 encodes for a homeodomain-leucine zipper-class II (HD-ZIP II) transcription factor in Arabidopsis. Previous studies have shown that this gene acts in the shade avoidance response, which comprises a set of morphological changes that take place when the plant grows in a light environment with a low red:far red ratio (R:FR). Recently, we have shown that at least some of the morphological changes observed either upon low R:FR light treatment or upon overexpression of ATHB-2 require an intact auxin pathway, leading to the hypothesis of a crosstalk between light and auxin pathways. On the basis of these findings, we postulate a model in which low R:FR light, via ATHB-2, induces a re-direction of auxin flux from the vasculature to external cell layers, resulting in a reduced auxin flux in the root. It has been recently shown that formation and maintenance of the root merystem structure in Arabidopsis depends on the presence of an auxin maximum in specific meristematic cells. Therefore, to test our hypothesis, we are currently analysing the cellular structure of the root apex in wild type plants upon low R:FR light treatment, in plants overexpressing ATHB-2 and in athb-2 mutants. Moreover, we are also testing the relative auxin levels in the root merystem of the same plants, by the use of the DR5::GUS transgenic line.
46
STUDIES ON PLANT INHIBITORS OF PECTIN MODIFYING ENZYMES: POLYGALACTURONASE-INHIBITING PROTEIN (PGIP) AND PECTIN METHYLESTERASE INHIBITOR (PMEI)
C. Caprari1, B. Mattei1, A. Raiola1, L. Federici2, G. Salvi, D. Bellincampi1, G. De Lorenzo1, F.Cervone1, A. Giovane3, L. Camardella4
1Dipartimento di Biologia Vegetale e 2Dipartimento di Scienza Biochimiche , Università di Roma "La Sapienza", 3Dipartimento di Biochimica e Biofisica, II Università di Napoli, 4Istituto di Biochimica delle proteine ed Enzimologia, C.N.R., Napoli
Pectin of the plant cell wall is either degraded during microbial attacks or remodelled during the various phases of growth and development. We are studying two protein inhibitors of pectic enzymes: polygalacturonase-inhibiting protein (PGIP) and pectin methylesterase inhibitor (PMEI). PGIP is specific for fungal polygalacturonases (PGs) and is involved in defence against phytopathogenic fungi by allowing the accumulation of elicitor active oligogalacturonides (OGs). PMEI is active against plant pectin methylesterase (PME) and likely involved in the regulation of the degree of methylation of the cell wall pectin during plant growth and development. PME and PG inhibitors have been purified to homogeneity in our laboratories and biochemically characterised. Both PMEI and PGIP are competitive inhibitors. Detailed kinetic analyses of enzyme-inhibitor interactions using Surface Plasmon Resonance (SPR) will be presented.
47
PAL activity in skins of sugar incubated grape berries
A. Carra, S. Guidoni and A. Schubert
Dipartimento di Colture Arboree, Università di Torino
Berries of Vitis vinifera L. 'Nebbiolo' were harvested at three dates during the growing season 2000 and incubated in sucrose, glucose and fructose 0,3 M, while incubations in H2O and in mannitol 0,3 M were used as controls. Glucose, fructose and sucrose content in the skins of the incubated berries was measured to verify the extent of sugar uptake after the treatments. Total proteins from the berry skins were extracted and PAL activity was measured using a HPLC-based method. Glucose and fructose content increased in the glucose and fructose - incubated berries in comparison to controls, indicating that these sugars are transported into the cells of the skins during treatments. Sucrose content in the skins of the incubated berries was undetectable, probably because cleaved to glucose and fructose by invertase. PAL activity was significantly higher in skins of berries incubated in glucose in comparison to that incubated in other sugars and to controls. When berries were incubated at an early ripening stage, when no coloration was visible, PAL activity was nearly undetectable in all the treatments. When treatments were performed on berries sampled at full ripening, a similar level of PAL activity was detected in sugar incubated berries and in the controls. These results suggest that developmentally regulated transcriptional activation and internal stimuli provided by hexose accumulation are integrated in the regulation of an early flavonoid biosynthetic gene in grape berries.
48
Microalgae of Euganean Thermae: scientific and practical aspects.
Cristina Ceschi Berrini+, Francesca De Appolonia+, Gabriele Marcolongo* and Carlo Andreoli+
+ Dip. di Biologia, UNI Padova; * Dip. di Chimica Fisica, UNI Padova
Since ancient time, in the Euganean area, mud therapy has represented an effective treatment which even today is not well known. Thermal mud is a mixture of water, clay and organic compounds coming from microalgae living in this system. In this "extreme" environment, microalgae are able to survive and to grow reaching sometimes very high cell densities. Before therapeutic use, the mud is undergone to a "maturation" process during which thermophile microorganisms colonize not only the surface but even the deeper layers of mud. As the consequence of this physico-chemical and biochemical characteristics change and the mud become therapeutically effective. To understand the complex dynamics of the maturation process, in the last few years the Centro Studi Termali "Pietro d'Abano" has been supporting a research on the thermal microalgae living in the Euganean area. The goals of this research are the study of the different species living in this environment and the isolation and purification of those species involved in the maturation process. From these unialgal cultures, it has just begun the extraction and characterization of lipidic substances that could have pharmacological properties. These substances could be proposed as bio-markers for a more rational and controlled management of the maturation process.
49
STORAGE PROCESSES AND REDOX SYSTEMS DURING MATURATION OF WHEAT KERNELS
Laura De Gara1,, Maria C de Pinto1, VM Cristiana Moliterni2, Maria G. D'Egidio2
1Dip.di Biologia e Patologia Vegetale, UNI Bari, 2Ist. Sperimentale di Cerealicoltura, Roma
Seed development and maturation is a multi-step process during which embryos are formed and supplied with the carbohydrates, proteins and lipids needed for the subsequent germination. Many data are available on the capability of cereal kernels of accumulating storage compounds and acquiring desiccation tolerance. The behaviour of some parameters characterizing the development of wheat kernels, including storage of carbohydrates and accumulation and maturation of proteins was studied. In addition, attention has been directed to ascorbate (ASC) and glutathione (GSH) metabolisms, two redox pairs involved both in plant developmental processes as well as in the protection against reactive oxygen species (ROS). In particular the changes in the redox state of ASC and GSH, as well as in the activities of the enzymes responsible for the recycling of their oxidised forms and for detoxification or utilisation of hydrogen peroxide (ascorbate peroxidase, catalase and secretory peroxidases) were analysed. The results indicate that both the activities of the ASC and GSH redox enzymes and those of catalase and peroxidases are high before the start of drying maturation, after which they decrease. Moreover, analysis of the redox state of ASC and GSH pairs and the sulphydryl to disulfide transition into proteins suggests that these three parameters are tightly related during kernel maturation, thus confirming the involvement of the two redox pairs in protein maturation as well as in protection against ROS.
50
AMMENDAMENTO CON RESIDUI DELL'INDUSTRIA AGRUMARIA: RISPOSTA FISIOLOGICA E PRODUTTIVA DI PIANTE DI GIRASOLE
M. G. Di Leo, G. Avola, S. Giannetto, V. Abbate, A. Belligno
Il comparto agrumicolo rappresenta per le regioni dell'area mediterranea uno dei settori più importanti dal punto di vista socio-economico. La relativa problematica della gestione dei rifiuti derivanti dai processi della loro trasformazione sta assumendo, così, sempre maggior rilievo. Questi processi di trasformazione danno origine a tre prodotti principali: succhi, oli essenziali e "pastazzo". Quest'ultimo, considerato un sottoprodotto a basso valore o scarto di lavorazione, ricade nella voce spesa nei bilanci aziendali con, in più, un impatto ambientale da non sottovalutare. E' stato valutato in un impianto lisimetrico a cielo aperto l'effetto dell'ammendamento con due differenti dosi di pastazzo (3 e 9 kg m-2) in confronto ad un testimone (non ammendato) su due varietà di girasole. A completamento dei risultati sul comportamento bioagronomico del girasole, oggetto di una precedente nota, si riferisce sulle variazioni a livello biochimico occorse durante l'intero ciclo biologico e su alcune caratteristiche della produzione. La distribuzione di differenti dosi di pastazzo ha determinato già alla dose minima un considerevole aumento del contenuto in nitrato, zuccheri, acidi tricarbossidici, acidi grassi e degli elementi nutritivi quali potassio, calcio, sodio e magnesio con un corrispondente aumento della resa in olio degli acheni. The cultivation of citrus grove represents, in the Mediterranean areas, one of the more important agricultural sectors under both social and economic point of view. The relative problematic of the management of the citrus waste produced from the processes of their transformation (known as "pastazzo") is raising, today, more and more importance. The citrus waste, considered as a trash of processing, represent a cost in the companies budgets and, moreover, it has an environmental impact that is not more possible undervalue. The aim of this research was to investigate the changes caused by the application of 2 level of the citrus waste (3 and 9 Kg m-2 dry matter in comparison to a control in which no treatments was applied) and two sunflower cultivars. Sunflower was grown on an open-air lysimeter. In order to complete the results on the bioagronomic behaviour, object in a previous communication, this paper reports the biochemistry changes recorded during the whole biologic cycle and on some characteristics of the relative production. The distribution of citrus waste resulted in a high increase, already in the lower dose, in the content of nitrate, sugars, tricarbossilic acids, fat acids and the nutritive elements (potassium, calcium, sodium and magnesium) with a correspondent increase of the achene oil percentage and oil yield.
51
VASCULAR TISSUE REGENERATION IN PEAR/QUINCE GRAFTS WITH DIFFERENT COMPATIBILITY.
Luca Espen, Marizio Cocucci e Gian Attilio Sacchi
Dip. di Produzione Vegetale, UNI Milano
Structural events occurring at the interface of a compatible graft union comprise the formation of a callus bridge, the differentiation of new vascular cambium from parenchyma cells and thereafter of secondary xylem and phloem cells connecting the two partners. Aim of this work was to verify whether in in vitro grafting of shoot internodes from pear and quince in vitro cultured plants mimics in short time the (in)compatibility responses that they show in wooden grafts. In particular, the time-course of functional phloem connection between the partners was analysed. Immuno-histological approach has been applied to verify whether programmed cell death (PCD), a process related to differentiation of vascular system, occurs after internode grafting, with the aim to relate it to (in)compatible characteristics. In vitro grafting of shoot internodes excised from pear cv Kaiser (K) and cv Butirra Hardy (BH), and quince EM clone C (C) well mimics the histological and functional responses of (in)compatibility exhibited by the respective wooden plants. In the incompatible K/C combination an evident delay in the cohesion of the graft partners and, over all, a poor differentiation of functional wound-phloem connections were observed with respect to the compatible BH/ C ones. In the compatible combination, but not in the incompatible ones, the onset of the PCD was detectable in the graft region. The results suggest that K/C incompatibility might be related to some effect of the graft union on the active program of PCD and in turn on the processes driving to differentiation of vascular bridges in the graft region.
52
DAG1 and DAG2: two Arabidopsis Dof transcription factors involved in germination
Giuliana Gualberti, Maura Papi, Iolanda Ricci, Paolo Costantino and Paola Vittorioso
Dipartimento di Genetica e Biologia Molecolare, UNI Roma 1
The Dof proteins are plant transcription factors characterized by a conserved domain (a C2-C2 zinc finger and a basic region). The unusual conservation of this domain, coupled with the presence of the Dof transcription factors in all plants, suggests a crucial role of these proteins in regulating different functions typical of plants. We demonstrated that the Arabidopsis Dof protein DAG1 is involved in the control of red light-dependent seed germination. The T-DNA insertion line dag1 shows maternal control of seed germination. We identified a T-DNA insertion line in another gene of the Arabidopsis Dof family, denominated DAG2 for its sequence similarity with DAG1 (76,2% aminoacidic identity). dag2 is a knock-about mutant, as the N-terminus of DAG2 is expressed in translational fusion with the GUS gene. The DAG1 and DAG2 genes show also a very conserved organization, although the two genes map on different chromosomes. Furthermore, both genes show a tissue-specific expression localized in the whole vascular tissue. The seed germination characteristics (sensitivity to vernalization, light and GAs) of the dag2 mutant are opposite to those of dag1, but similar to those of plants overexpressing DAG1. DAG1 and DAG2 might be involved in interacting pathways or regulate with opposite roles the same genes, based on their overlapping expression patterns and on the opposite phenotype of the respective mutants. We have performed a microarray screen to identify genes that are differentially expressed in the dag1 and dag2 mutant plants as compared to WT, with the aim of identifying their targets to understand if they control the same regulatory circuits.
53
DEVELOPMENT OF MAIZE ISOGENIC LINES DIFFERING FOR A QTL WHICH AFFECTS LEAF ABA CONCENTRATION
Pierangelo Landi, Silvio Salvi, Maria Corinna Sanguineti, Roberto Tuberosa
Dipartimento di Scienze e Tecnologie Agroambientali, Via F. Re 6, 40126, Bologna.
Previous studies on the genetic control of L-ABA concentration (Tuberosa et al., Theor. Appl. Genet., 97, 744-755; Sanguineti et al., J. Exp. Bot., 50, 337, 1289-1297) and a divergent selection for this trait (Salvi et al., 1997, Maize Genetics Newsletter, 71, 15-16) in a maize population derived from the cross Os420 × IABO78 allowed us to identify an important QTL for L-ABA on chromosome 2 between the RFLP loci umc34 and csu4. Based on these results, we undertook a backcross program aimed at developing isogenic lines at this QTL with the objective to further investigate its effects on L-ABA concentration and on related traits. Two parallel backross procedures were followed; in the first one, Os420 (contributing the + allele increasing L-ABA concentration at the target QTL) was used as recurrent parent and IABO78 (contributing the - allele decreasing L-ABA concentration at the target QTL) as the donor, while in the second one the role of the parents was reversed. The mean values of the two BC5F2 homozygotes derived from Os420 were 336 (+ +) and 252 (- -) ng ABA g-1 dw, while the mean values of the two BC5F2 homozygotes derived from IABO78 were 222 (+ +) and 184 (- -) ng ABA g-1 dw., these results indicate that the target QTL was successfully transferred by the marker-assisted backcross and confirm the relative importance of its additive effect. In the current year we are testing the isogenic BC5F3 lines (+ +) and (- -) in field trials conducted at different levels of water stress.
54
THE GLUTENIN POLYMER STRUCTURE: ANALYSIS OF TRANSGENIC PLANTS AND IN VITRO RE-OXIDATION OF GLUTENIN SUBUNITS
Masci S., , Patacchini C., Scossa F., D'Ovidio R., Lafiandra D.
DIPARTIMENTO DI AGROBIOLOGIA E AGROCHIMICA, UNIVERSITÀ DELLA TUSCIA, VIA S.C. DE LELLIS, VITERBO
THE GLUTENIN POLYMERS PRESENT IN WHEAT ENDOSPERM ARE AMONG THE LARGEST PROTEIN MOLECULES KNOWN. THEIR MOLECULAR WEIGHT MAY RANGE FROM 40.000 UP TO MILLIONS. THEIR MAIN COMPONENTS ARE THE GLUTENIN SUBUNITS, BOTH HIGH (HMW-GS) AND LOW (LMW-GS) MOLECULAR WEIGHT, STABILIZED BY DISULFIDE BONDS. THEIR NATURAL ROLE IS TO PROVIDE THE GROWING EMBRYO WITH NITROGEN AND CARBON, BUT THEY ARE MAINLY STUDIED BECAUSE OF THEIR PIVOTAL ROLE IN DETERMINING GLUTEN VISCO-ELASTIC PROPERTIES. Their huge size has hampered the possibility to study their structure in detail. In this work we present the characterization of several LMW-GS genes, and their use to manipulate gluten composition. Homozygous T3 and T4 seeds from one transformed bread wheat line overexpressing a LMW-GS gene have been characterized in order to determine its influence on polymer structure. Analysis of endosperm proteins of transgenic seeds show that the transgene product is overexpressed about 10-15 times in comparison to endogenous LMW-GS. Biochemical analyses demonstrated that the transgenic LMW-GS is part of the glutenin polymers and its presence contribute to the increasing of its molecular size. We are pursuing the definition of the glutenin polymer structure also through in vitro re-oxidation of native or heterologously expressed HMW-GS and LMW-GS, differing in structural characteristics, such as cysteine distribution and size of the repetitive domain.
55
DO 14-3-3 PROTEINS REGULATE APOPTOSIS IN PLANT CELLS?
Massimo Malerba and Renato Bianchetti
Dipartimento di Biologia, sez. Botanica Generale, Università degli Studi di Milano, via Celoria 26, 20133 Milano
Recent works showed that a primary function of the mammalian 14-3-3 proteins is to inhibit apoptosis. In a previous work we showed that Brefeldin A and Tunicamicyn are strongly inducers of apoptosis in cultured plant cells. Here we report data of immunological studies on the level and distribution of the 14-3-3 proteins in cultured Acer pseudoplatanus cells. Preliminary data show that both Brefeldin A and Tunicamicyn treatment bring to an evident change in cell 14-3-3 pattern.
56
ENDOBACTERIA OF A MYCORRHIZAL FUNGUS POSSESS NIF GENES
D. Minerdi, A.M. Cantisani, V. Bianciotto e P. Bonfante
Dip. Biologia Vegetale e CSMT-CNR, Viale Mattioli 25 10125 Torino Italy.
Arbuscular mycorrhizal (AM) fungi are ancient Zygomycetes, which have colonised the roots of the first land plants and are nowadays associated with about 80% of plant species. The symbiosis they form is a complex system where interactions between fungi, plants and bacteria occur. Bacteria belonging to the genus Burkholderia have been described in the cytoplasm of one isolate of the AM fungus Gigaspora margarita (BEG 34) and identified on the basis of their 16S rDNA sequences. To understand the role of the intracellular Burkholderia which cannot be grown on a cell-free medium, we took advantage of a genomic library constructed with DNA extracted from Gi. margarita spores, and also representative of the bacterial genome. Bacterial DNA regions containing putative nif H,D,K genes organised in a single operon were identified and characterised. RT-PCR experiments with primers designed on the nifD and nifK demonstrate the genes expression during the germination step (Minerdi et al., 2001 AEM 67:725-732) . SDS-Page analysis of proteins extracted from spores showed some bands differently expressed during the fungal life cycle. Microsequencing of some of the most expressed proteins suggests homology with bacterial proteins. Antibodies raised against nitrogenase from Rodospirillum rubrum and Azotobacter vinelandii were used in immunofluorescence and immunogold experiments, showing labelling on the bacterial cells. Taken in their whole, molecular, biochemical and immunocytochemical experiments suggest that the endobacteria have some of the molecular tools required to fix nitrogen.
57
OLIGOSACCHARIDE ELICITORS AND INDUCTION OF CYTOSOLIC CALCIUM TRANSIENTS IN AEQUORIN-TRANSFORMED SOYBEAN CELLS
Lorella Navazio*, Roberto Moscatiello*, Piera Linguanotto*, Marisa Brini+, Barbara Baldan*, Paola Mariani*
*Dip. di Biologia, UNI Padova; +Dip. di Chimica Biologica, UNI Padova
Oligosaccharides released from plant or fungal cell walls during plant-pathogen interactions rapidly generate intracellular signals that trigger defense responses. Calcium ions are important intracellular messengers involved in the transduction pathway of several oligosaccharide signals. The Ca2+ message is encoded in transient changes of cytosolic Ca2+ concentration. Soybean cells stably expressing the Ca2+-sensitive photoprotein aequorin were used to test the effect of both endogenous and exogenous elicitors. Oligogalacturonides (with different degree of polymerization or fully methylated), chitosan and b-glucan oligomers were found to induce cytosolic Ca2+ transients characterized by unique kinetic parameters. When combinations of the above oligosaccharide elicitors were simultaneously administered to cells, changes in the individual Ca2+ signatures were observed.
58
IAA biosynthesis in immature seeds of Phaseolus coccineus
F.Paolicchi+, C.Solfanelli+, P.Picciarelli+, R.Lorenzi+, N.Ceccarelli+.
+ Dip. Biologia delle Piante Agrarie, UNIPisa
In a previous work we described the distribution of free and conjugated indole-3 acetic acid (IAA) in the seed of Phaseolus coccineus at various developmental stages (Picciarelli et al., A. J. P. P. 28, 2001). Results show that the IAA content in the single seed parts is quite different, and changes with different patterns during seed growth We hypothesized that the different auxin concentration gradient in the embryo-suspensor could play a significant role in the establishment of embryo polarity. With the aim to investigate the role of auxin in this process we have now studied the biosynthesis of IAA in the endosperm, suspensor and embryo proper of coccineus seeds. The biosynthesis experiments were performed both with cell free preparations and by incubating in vitro the labelled precursors. In order to test both tryptophan and non-tryptophan biosynthetic ways, different labelled precursors have been used: tryptophan, indole and deuterated water at different concentration and incubation time. The precursors were marked with heavy isotopes (13C or D) or with 14C. The different metabolites were identified using GC-MS techniques and quantified by isotopic enrichment. A higher biosynthetic activity was found in suspensors than in the other seed parts. The metabolic approach has confirmed that in the embryo-suspensor system of P. coccineus the free IAA represents the main auxin form. These data are discussed in relation to the hypothized role of auxins in the establishment of early pattern formation.
59
OPTIMISATION OF TRANSGENE ACTION AT THE POST-TRANSCRIPTIONAL LEVEL: HIGH QUALITY PARTHENOCARPIC FRUITS IN INDUSTRIAL TOMATOES.
Pandolfini, T., Rotino, G.L.1 and Spena, A..
Faculty of Science, University of Verona, 1Research Institute for Vegetable Crops, via Paullese 28, Montanaso Lombardo (LO)
Genetic engineering of parthenocarpy confers to horticultural plant species the ability to produce fruits under environmental conditions which curtail fruit productivity and quality. The DefH9-iaaM parthenocarpic gene confers auxin synthesis specifically in the placenta, ovules and derived tissues. UC82 tomato plants, a typical cultivar used by the processing industry, transgenic for the DefH9-iaaM gene produce malformed parthenocarpic fruits, whilst UC82 plants transgenic for the DefH9-RI-iaaM, a DefH9-iaaM derivative modified in its 5'ULR by replacing 53 nucleotides immediately upstream the AUG initiation codon with an 87 nucleotides-long sequence derived by the rol A intron sequence, produce parthenocarpic fruits of high quality. In an in vitro translation system, the iaaM mRNA, modified in its 5'ULR is translated 3-4 times less efficiently than the original transcript. An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds. Thus, by using a parthenocarpic transgene downregulated at the post-trascriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene.
60
STUDY ON GENES EXPRESSION IN COMPATIBLE AND INCOMPATIBLE PEAR/PEAR AND PEAR/ QUINCE GRAFT BY mRNA DIFFERENTIAL DISPLAY
Livia Pirovano1, Gian Attilio Sacchi1, Alessandro Abruzzese1, Stefano Musacchi2, G. Bernardi2, Silviero Sansavini2, Maurizio Cocucci1
1Dipartimanto Produzione Vegetale,Universita di Milano, 2Dipartimento di Colture Arboree, Università di Bologna
Graft union between a scion and stock is a normal and economically important practical in fruit plant cultivation. Graft between genetically unrelated plants are often incompatible, this is a wide spread phenomenon not yet fully elucidated, especially at molecular level. The aim of this work was to obtain information about molecular basis of compatible/incompatible graft, using the in vitro model and applying as molecular approach the mRNA Differential Display RT-PCR technique. The in vitro model include a co-culture of pear calli with suspension cells of pear or quince, the two culture were physically separated by a solute permeable membrane. This "in vitro" model simulate the compatible/incompatible graft. The molecular approach applied allowed direct isolation and cloning of cDNA fragments corresponding to mRNAs differently expressed in calli of pear co-cultured with compatible and incompatible genotype. Results: forty differentially expressed cDNA fragments were obtained in the first amplification. To eliminated the false-positive, the 40 cloned fragments were examined by Reverse Northern blot analysis. To further verify the expression pattern of the 10 positive differentially display fragments, Northern blot analysis were performed. The same fragments were sequenced and the sequences were compared with those in the GeneBank database using the BLAST program.
61
TRANSGENIC TOBACCO PLANTS EXPRESSING HUMAN GAD65 ARE AFFECTED IN PLANT GROWTH AND DEVELOPMENT
Laura Pistelli*, Lorenzo Guglielminetti*, Linda Avesani+, Alberto Falorni#, Mario Pezzotti+, Amedeo Alpi*
*Dip. Biologia Piante agrarie, UNI Pisa, +Dip. Scientifico e tecnologico, UNI Verona, # Dip. Medicina Interna, Scienze Endocrine e Metaboliche, UNI Perugia
GAD (glutamic acid decarboxylase) catalyzes the decarboxylation of L-glutamate into gamma-aminobutiric acid (GABA); The enzyme is ubiquitously expressed in prokaryotes and eukariotes, including plants. Two isoforms of GAD are known in humans, GAD65 and GAD67; the smaller isoform (GAD65) is expressed in human pancreatic islets and represents a major autoantigen in human IDDM (insulin-dependent diabetes mellitus). In plants the accumulation of GABA is a typical response to many diverse stimuli and stress. Plant GAD is however peculiar in its ability to bind calmodulin (CaM), and the presence of a functional CaM binding domain is of importance for the expression of GAD in transgenic plants, since transgenic tobacco plants expressing a full length petunia GAD are indistinguishable from the wild-type plants, while the lack of the CaM binding motif produce transgenic tobaccos with altered phenotype (Baum et al. 1996, EMBO J, 15, 2988-2996) Transgenic tobacco plants expressing hGAD65, which lack the CaM-binding domain, exhibit normal phenotype, as already described (Porceddu et al. 1999, Mol. Breeding,1-8), and dwarf phenotype. The aim of this study was to evaluate the discrepancy between the two lines. Physiological studies show some differences between the two lines in organ accumulation of GABA and glutamate contents well as of GAD activity. Expression studies and organellar distribution of hGAD65 and plant GAD are under investigation in order to evaluate a physiological meaning of the altered phenotype.
62
EXPRESSION OF GA 3*-HYDROXYLASE IN DEVELOPING SEEDS OF PHASEOLUS COCCINEUS BY IN SITU HYBRIDISATION
C. Solfanelli1, F. Ceron1, L. Giorgetti2, M. Ruffini Castiglione2, C.E. Geri2, N. Ceccarelli1, P. Picciarelli1.
1 Dip. Biologia delle Piante Agrarie, UNI Pisa 2 Istituto di Mutagenesi e Differenziamento, CNR, Area della Ricerca di Pisa
Several experimental evidence demonstrates that the suspensor plays an active role in embryo development. In previous work we investigated all plant hormones in embryo-suspensor system of P. coccineus during embryogenesis. Related to gibberellins we demonstrate that bioactive GAs (especially the GA1) are predominant in the young suspensors and embryos, while biologically inactive GA8 is relevant in senescing suspensor. The level of GA1 is regulated by GA 3*-hydroxylase that catalyse the conversion from GA20 to GA1 and by GA 2*-hydroxylase that converts GA1 to GA8. Last year our group successfully isolated the gene coding for GA 3*-hydroxylase from P.coccineus seeds. With the aim to understand how GA1 biosynthesis is regulated in the embryo-suspensor system of runner bean, we are now trying to localise, at tissue level, the expression of genes involved in GAs metabolism performing in situ hybridisation experiments. Such experiments were carried out at different developmental stages of the seed, from globular to cotyledonary embryo. A non-radioactive labelling method was used to obtain the GA 3*-hydroxylase RNA probe. The results obtained indicate a higher GA 3*-hydroxylase expression in young suspensor than in senescing ones. These data will be discuss in relation to the role of the suspensor in the embryo pattern formation.
63
ROLE OF INDOLEACETIC ACID IN PEACH (PRUNUS PERSICA (L.) BATSCH) EMBRYOGENESIS
Carlo Sorce, Fabio Paolicchi and Roberto Lorenzi
Dip. Biologia Piante Agrarie, UNI Pisa
The time course of indoleacetic acid (IAA) concentration was investigated in peach developing seeds, in order to obtain informations on the possible role of this hormone in seed growth. Teguments, endosperm and cotyledons (with the attached embryos) from seeds of PsA5 clone were analysed starting 45 days after 50 % full blooming until embryo complete maturity. The highest levels of free IAA were detected in teguments, followed by endosperm and cotyledons. Changes of IAA concentration followed the same course in teguments and endosperm. Less samples were collected for cotyledons, owing to their delayed development, therefore the time course of IAA concentration did not coincide with that of the other tissues. Cotyledons showed a peak of IAA just prior to a period of fast growth. Such a peak coincided with the peaks of maximum IAA concentration detected both in teguments and in endosperms. Therefore, as suggested also by previously published results from other species, it can be hypothesised that the auxin is translocated from endosperm (and teguments) toward the embryo during seed growth, and this could represent a fundamental mechanism of control of embryo development. We are also performing biosynthesis studies which should help to clarify the role of auxin in this process.
64
ß-galattosidasi con un dominio simile a lectine sono espresse in frutti di fragola.
Livio Trainotti, Riccardo Spinello, Anna Piovan, Silvia Spolaore, Giorgio Casadoro
Dipartimento di Biologia, Università di Padova, Viale G. Colombo 3, I-35121 Padova, Italy
E' noto dalla letteratura che il rammollimento dei frutti di fragola si accompagna ad un rilascio di zuccheri liberi fra cui è presente anche galattosio. Partendo da estratti proteici di parete, attività ß-galattosidasica è stata misurata in frutti a diverso stadio di sviluppo. Tre cDNA completi (Fabgal1, Fabgal2 and Fabgal3), codificanti differenti ß-galattosidasi, sono stati isolati da una libreria di cDNA di frutto rosso. L'analisi di espressione dei trascritti relativi ai tre cDNA ha dimostrato che essi hanno un pattern di espressione diverso, anche se tutti e tre vengono espressi sia in frutti che in tessuti vegetativi. In particolare, mentre Fabgal1 mostra un'espressione crescente nel corso della maturazione, gli altri due (Fabgal2 e Fabgal3) hanno un massimo di espressione nei frutti verdi e diventano scarsamente rilevabili nei frutti maturi. L'auxina ha un effetto negativo sull'espressione dei tre geni per b-galattosidasi, così come già visto per altri geni di maturazione del frutto non climaterico di fragola. I tre cDNA sono stati usati per esprimere le relative proteine nel lievito Pichia pastoris ed è stato così possibile dimostrare che ciascuno di essi effettivamente codifica una b-galattosidasi. Una interessante caratteristica è stata evidenziata all'estremità C-terminale di due delle b-galattosidasi di fragola. Sia Faßgal1 che Faßgal2 presentano infatti un dominio che è strutturalmente correlato con peptidi noti di origine animale i quali hanno la capacità di legare zuccheri.
65
Meccanismi di controllo dell'apoptosi in colture cellulari di carota.
Michela Zottini, Elide Formentin, Michela Scattolin, Francesco Carimi, Fiorella Lo Schiavo and Mario Terzi
Dipartimento di Biologia - Università degli Studi di Padova - Via U. Bassi, 58/B - 35131 Padova. zottini@civ.bio.unipd.it
Programmed cell death is a biological process intimately correlated to the cell cycle. Several experimental data demonstrate that alterations at the cell cycle level can either prevent or induce apoptosis. The cell cycle arrest is a form of cellular response to stress, as it has been shown in synchronized tobacco cells. The production of reactive oxygen species is an early and characteristic feature of hypersensitive response (HR) triggered by the plant upon a pathogen attack. We are characterizing the metabolic pathways activated during programmed cell death and our data show that NO, that increases cell death due to H2O2, induces a block and a slowing down of cell proliferation. The data presented here suggest possible targets and cell death mechanisms of NO. In this work we show that mitochondria are one of the early and important target of NO during HR.
PHYSIOLOGICAL AND PRODUCTIVE RESPONSE IN SUNFLOWER (cv. KATHARINA), IRRIGATED WITH DIFFERENT SEA-WATER CONCENTRATION
G. Avola, M.G. Di Leo, F. Sambuco, A. Belligno
In the coastal zones characterizated by hot-dry climate, the utilization of sea-water for irrigation could be a solution in relation to reduced water availability. The response of the plants to the sea-water supply changes in relationship to the species, to the cultivars, to the physiological stadium in which the plant stays, to the concentration and type of salts present in the water. Sunflower could constitute a possible alternative crop to the fall-winter cereals in the Mediterranean areas in relationship to his ability of valorise modest waters resources. The aim of this study was to value the utilization of sea-water irrigation on the growth of sunflower, on some its metabolic aspects and on some its productive characteristics. The experiment, carried out in a open-air lysimeter, has valued 2 controls (fresh water equal to the ETM and ETM+20%) and 2 different sea-water supply (16 and 33%) with a leaching factor (FL) equal to the ETM and to ETM+20%. In addition to bioagronomic parameters, pointed out in a previous communications, the content of sugars, of tricarbossilic acids and proline were studied. The enzymatic activity was determined in order to study the trend of glucidic and nitrogen metabolism as the nitrate reductase and the saccharose phosphate sintetase. The application of sea-water resulted in a increase in sugars content during the vegetative cycle, more pronounced on the 16% treatment. With regard to acids content, no differences was pointed out neither in relationship to the salt concentration, nether in relationship to the leaching. As concern the proline content, it was showed a higher content in the 33% and 16% treatments as regards the controls, where values significantly lower were recorded. In conclusion and in consideration that the 33% treatment has not carried to the end of whole vegetative cycle, the 16% treatment, with both the leaching factor, is a dose solution that could be used without to compromise the productivity and the physiological functions of the plant.
67
Differential expression of manganese superoxide dismutase during vegetative development in peach
F. Bagnoli, D. Giannino*, S. Caparrini, M.L. Racchi
Dipartimento di Biotecnologie Agrarie, Laboratorio di genetica, Università degli Studi di Firenze, Piazzale delle Cascine 24 , 50144 Firenze, Italia; *Istituto di Biochimica ed Ecofisiologia Vegetali del CNR, via Salaria km 29,300, 00016 Monterotondo Scalo, Roma, Italia.
All organisms exposed to an aerobic environment are subjected to the toxic effects of active oxygen species (AOS) such as superoxide, H2O2, and the hydroxyl radical. In plants the cellular concentration of oxygen is the highest among organisms, due to their oxygen-evolving capabilities. Consequently, the antioxidant enzymes in plants have a crucial role in oxidative stress protection. In fact superoxide dismutases (SOD), togheter with catalases and peroxidases, represent the major defence against cell oxidation. Moreover, SODs have been reported to be very sensitive to metabolic changes during cell life and respond to developmental stimuli. In the present study we report the expression analysis of MnSod, whose cDNA was previously isolated and characterised. The results showed that MnSod transcript was developmentally regulated in vegetative structures of the aerial part during the seasonal growth and in relation to tree age. Furthermore to establish MnSod copy number in peach, Southern and PCR analysis was performed on genomic DNA restricted with several endonucleases which do not cut in the probe, and hybridised to a 592-bp cDNA fragment which is in the MnSod ORF. Two distinct bands were signalled in all the cases, thus suggesting the presence of two MnSod copies.
68
ADATTAMENTO DEL CLOROPLASTO DI KOLIELLA ANTARCTICA ANDREOLI ET AL. SOTTOPOSTA A SIMULATA NOTTE AUSTRALE: STUDIO MICROSPETTROFLUORIMETRICO ED ULTRASTRUTTURALE.
Costanza Baldisserotto*, Lorenzo Ferroni*, Nicoletta La Rocca°, Carlo Andreoli° e Simonetta Pancaldi*.
*Dipartimento di Biologia - Sezione Botanica, Università di Ferrara, C.so Porta Mare, 2 - 44100 Ferrara °Dipartimento di Biologia, Università di Padova, Via Ugo Bassi, 58/b - 35131 Padova
Koliella antarctica Andreoli et al. (Trebouxiophyceae, Chlorophyta) è stata isolata nell'estate australe 1989/90, alla profondità di 3 m sotto il pack-ice, presso la Baia di Terra Nova (Antartide). Trasferita presso il Dip. di Biologia dell'Università di PD, è stata mantenuta in condizioni riconducibili a quelle del sito di origine (T = 4°C, salinità = 34, irradianza = 20 µmol m-2 s-1). Essa possiede un unico cloroplasto parietale/cellula con lunghi tilacoidi agranali e privo di pirenoide. Al fine di valutare i cambiamenti adattativi del plastidio a simulate condizioni di notte australe, colture liquide cresciute alla luce sono state poste al buio per 2 mesi, senza variare gli altri parametri di coltura. L'esame ultrastrutturale, l'analisi microspettrofluorimetrica dell'assetto del PSII, l'analisi spettrofotometrica dei pigmenti fotosintetici hanno mostrato che l'alga nel periodo di buio attua un rimodellamento del suo plastidio. Questo comprende: 1. una specifica sequenza di degradazione di costituenti non necessari per la vita al buio ed il loro immagazzinamento in strutture intraplastidiali specializzate; 2. il mantenimento di buona parte delle membrane tilacoidali, strettamente appressate in fascetti alla periferia dell'organulo; 3. la comparsa di emissioni di fluorescenza proprie di protoclorofille associabili a strutture simili a corpi prolamellari poco evoluti. Tali cambiamenti appaiono come risultato di due momenti di adattamento, uno che si realizza nei primi 6 giorni di buio, e l'altro, più lento, che si instuara nelle settimana successive.
69
SPECIFICITA' TISSUTALE DI RISPOSTA AL MANGANESE NELLA LAMINA FOGLIARE DI TRAPA NATANS L.
*Baldisserotto C., *Ferroni L., °Pagnoni A., *Medici V. e *Pancaldi S.
*Dipartimento di Biologia - sez. Botanica, Università di Ferrara, C.so Porta Mare, 2 - 44100 Ferrara °Dipartimento di Chimica, Università di Ferrara, via L. Borsari, 46 - 44100 Ferrara
La tolleranza delle piante ad alte concentrazioni di metalli pesanti implica meccanismi morfo-fisiologici, non ancora perfettamente definiti, che sono specie-specifici. Nel presente studio si riporta che Trapa natans L. (Trapaceae), idrofita annuale a foglie natanti e sommerse, possiede caratteristiche di specie bioaccumulatrice, essendo in grado di sequestrare nel mesofillo della lamina delle foglie natanti l'elemento Mn, come rilevato dalle analisi di spettroscopia ICP, ottenute da esemplari cresciuti in condizioni controllate presso l'Orto Botanico dell'Università di Ferrara. Esami ultrastrutturali di cloroplasti di cellule appartenenti a ciascuno strato mesofillare, analisi microspettrofluorimetriche effettuate in vivo a temperatura ambiente su ogni singolo strato per la valutazione dell'assetto del PSII ed analisi spettrofotometriche degli estratti acetonici per la determinazione del pattern dei pigmenti fotosintetici hanno evidenziato che T. natans presenta una specificità tissutale di risposta all'eccesso di Mn. In particolare, le cellule del primo strato del palizzata e gli idioblasti del lacunoso contengono precipitati osmiofili vacuolari ascrivibili a composti fenolici tipici di cellule geneticamente predisposte alla chelazione di metalli pesanti. Tali cellule non mostrano alterazioni morfologiche dei cloroplasti, ma variazioni del rapporto di emissione di fluorescenza LHCII/PSII rispetto ai controlli. Al contrario, le cellule del secondo strato del palizzata, il cui vacuolo non presenta inclusioni, reagiscono alla presenza del metallo modificando la morfologia plastidiale, senza variare i rapporti di emissione di fluorescenza.
70
EFFECT OF THE PRESENCE OF CADMIUM ON ASCORBATE AND GLUTATHIONE METABOLISMS IN WHEAT SEEDLINGS.
R. Berardino1, A. Paradiso1, A. Stefani2, L. De Gara1
1Dip. Biologia e Patologia Vegetale. UNI Bari; 2Scuola Superiore S. Anna Pisa
Plants can absorb a variety of heavy metals produced by antropic activities and not essential for their growth. Even though plants can be damaged by the presence of these metals, they are able to accumulate them and they can be utilised in the so-called phytoremediation for the scavenger of heavy metals from polluted soils. Since these pollutants induce an oxidative stress in the tissues in which they are accumulated, the phytoremediation efficiency also depends on the plant's capability to cope with oxidative stress. In a previous paper, we reported that sunflower plants grown in thepresence of cadmium, one of the more common pollutants, increase their ascorbate metabolism (Di Cagno, Guidi, De Gara Soldatini New Phytol. In press 2001). Here we report a wider study aimed at analysing the relationship between ascorbate and glutathione systems in the defence responses against the oxidative stress induced by the presence of Cd (II). The lipid peroxidation, protein oxidation, pigment degradation, the redox state of ascorbate/dehydroascorbate and glutathione/glutathione disulphide pairs and the enzymes of their oxido-reduction have been analysed in wheat seedlings grown in the presence of different Cd(II) concentration. This analysis was also carried out on plants in which the biosynthesis of ascorbate and glutathione has been experimentally altered. Our results confirm the involvement of these two redox systems in the protection against Cd(II) and underline that plants with enriched antioxidant capability better tolerate the presence of heavy metals.
71
THE FATE OF ULTRAVIOLET-B-DAMAGED D1 PROTEIN DURING RECOVERY IN VISIBLE LIGHT: EVIDENCE FORE A MIXED PHOTOLYTIC AND PROTEOLYTIC DEGRADATION MECHANISM
Elena Bergo+, Giorgio M. Giacometti+, Roberto Barbato*
+Dip. di Biologia, UNI Padova and *DISTA, UPO Alessandria
Irradiation of plants with ultraviolet-B light brings about inactivation of Photosystem II activity, paralleled by the degradation of the reaction center D1 protein to a 20-kDa C-terminal breakdown product. The mechanism by which D1 proteins is degraded to this immunodetectable protein fragment is here seggested to be photolytic in nature. When ultraviolet B-tretaed plants are transferred back either to darkness or low light, recovery is observed only in the latter. These two conditions differently and affect the fate of the ultraviolet-B induced 20-kDa breakdown product: while stable in darkeness, it is rapidly degraded in low light. Evidence is provided for the involvement in this process of an as yet unidentified proteolytic activity, apparently activated by light and peripherically bound to the thylakoid membrane. Therefore, a mixed mechanism based on both photolytic and proteolytic degradation of D1 is observed during recovery from ultraviolet-B induced stress.
72
UV-B INDUCES GENE EXPRESSION OF SOME OPPP ENZYMES, PAL, CHS, AND REPRESSES GENE EXPRESSION OF TRP SYNTHASE IN CUCUMBER (Cucumis sativus)
Cinzia M. Bertea§, Marco Mucciarelli+, Marisa Gallino+ e Massimo Maffei§
§Dip. Biologia Vegetale, +Dip. Morfofisiologia Veterinaria, Università di Torino.
In a previous work we observed that UV-B irradiation induced in cucumber changes in enzyme activity of some oxydative pentose phosphate pathway (OPPP) enzymes, PAL as well as Tryptofan and Tyrosine levels, resulting in the production of UV-B absorbing flavonoids. Here we report new results confirming that biochemical responses are paralleled by gene expression and suppression. RNA was extracted by using a lysis buffer, followed by phenol:chloroform:isoamylalcohol extractions to remove proteins, LiCl precipitation of RNA to remove DNA, and finally ethanol precipitation. First-strand cDNA was synthesized from RNA using a reverse primer, designed on the highly conserved domains in the sequences of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, tryptofan synthase, chalcone synthase and phenylalanine ammonia-lyase cDNA from different plants. The first-strand cDNA was amplified by adding Taq DNA polymerase (Promega) and a further forward primer designed on highly conserved domains as described above. PCR products were analyzed on polyacrylamide gels and detected by silver nitrate staining. No PCR products were amplified without reverse-transcription reactions. In response to UV-B treatment, cucumber plants induces high expression of several genes involved in the OPPP-pathway and in the phenolic compound biosynthesis, while suppressing expression of Trp synthase. No PCR products derived from RT-PCR on control plants were detected. These results are in accordance with previous biochemical analyses.
73
CYSTEINE SYNTHESIS IN S-STARVED CHLORELLA SOROKINIANA CELLS: EFFECTS OF O-ACETYLSERINE SUPPLY.
Simona Carfagna 1, Graziella Massaro 1, Sergio Esposito1, Vincenza Vona1, Vittoria Di Martino Rigano 2, Carmelo Rigano 1
1 - Dipart. di Biologia Vegetale - Università di Napoli "Federico II" Via Foria, 223 - 80139 Naples - ITALY. 2 - Dipart. Farmacologia Sperimentale - Università di Napoli "Federico II"- Via Montesano - 80131 Naples - ITALY Email: simcarfa@cds.unina.it
Cysteine is the endproduct of the assimilatory sulfate reduction pathway in bacteria, fungi and plants. L-cysteine biosynthesis proceeds via two enzymic reactions. Serine acetyl transferase (SAT) first catalyzes the acetylation of L-serine by acetyl-CoA. The product, O-acetylserine, then reacts with sulfide to yield L-cysteine in a reaction catalyzed by O-acetylserine(thiol)lyase (OASTL). Chlorella sorokiniana cells were grown under different sulfur nutritional conditions. Sulfur deprivation (24 hours) caused a strong decrease in levels of cysteine, methionine and glutathione, and a more than 3-fold increase in the content of serine and O-acetylserine (OAS). The OASTL activity was increased in S-starved cells when compared to a sulfur repleted cell culture. The addition of 1.2 mM sulfate to S-starved cells caused an immediate increase in the concentration of cysteine and a concomitant decrease of O-acetylserine, after which cysteine slowly decreased while O-acetylserine content remained constant. S-starved cells were able to absorb and accumulate OAS added exogenously to the nutrient solution showing an increase in intracellular content of cysteine at the same extent as in cells added with the sulfate alone. In contrast, S-sufficient cells were not able to absorb OAS supplied; however a significant but transitory increase in the intracellular content of cysteine was observed. These data will be discussed in the light of the key role of OAS in cysteine synthesis.
74
EFFECT OF NITRATE ON NITROGEN METABOLISM OF DURUM WHEAT SEEDLINGS IRRIGATED WITH SALT WATER
Petronia Carillo and Amodio Fuggi
Dipartimento di Scienze della Vita- Seconda Università di Napoli. Via Vivaldi 43 - 81100 Caserta, ITALY
Salinity limits crop productivity by inducing osmotic and ionic stress that affects many physiological plant processes among which nitrogen metabolism. To reduce the effect of salinity on plant productivity, due to competition between Cl- and NO3-, it has been suggested to supplement the irrigation water with nitrate. In this view we studied the conbined effect of different concentrations of nitrate and NaCl on seedlings of durum wheat (Triticum durum Desf. cv ofanto). In a two week treatment salt (100 mM in Hoagland solution) reduced growth of plants more at shoot than at root level. The effect was independent of nitrate concentration. There was no significant change of chlorophyll and carotenoids depending on the stress. In cortical root cells nitrate concentration determined by microelectrodes, was quite constant in the cytoplasm and was affected neither by the salt treatment nor by nitrate concentration (1- 10 mM in the culture solution). In the vacuols, instead, nitrate concentration was correlated positively at nitrate and negatively at salt concentration of the culture solution. Nitrate reductase activity, higher in the root than in the shoot, was induced by salt when the culture solution was at 1 mM NO3- , but not at 10 mM NO3-, suggesting that chloride could be involved in the induction of the enzyme. Nitrite reductase activity, instead, was reduced by the treatment. Salt reduced the activity of glutamine synthetase in the leaves, but increased that of glutamate dehydrogenase. Phosphoenolpyruvate carvboxylase activity increased in salt treated plants, supporting its role in replenishing the TCA cycle of intermediate compounds for the synthesis of amino acids and derivatives that increased in the stressed plants.
75
CADMIUM EFFECTS ON RICE (ORYZA SATIVA L.) CV. VIALONE NANO PLANTLETS.
Francesca Dalla Vecchia, Andrea Bortolozzo e Nicoletta Rascio
Dip. Di Biologia, UNI Padova
Cadmium is one of the most phytotoxic among the heavy metals introduced into the environment. It is taken up by plants and enters the food chain, thus becoming a dangerous source of contamination also for man. We have studied some effects of cadmium 50 and 250 mM on the shoots of a rice cultivar (Vialone nano) of considerable agronomical interest. The results obtained showed that, already at 50 mM, cadmium altered the gaseous exchanges between leaf and environment, leading to a water imbalance. This accounted for the increase in dry weight/fresh weight ratio of the tissues and for the limited growth of the plantlets. The curtailed CO2 supply to the photosynthetic cells due to a rise in stomatal resistance caused a great fall in carbon assimilation. A reduced photosynthetic functionality was evidenced by both the fluorescence emission values of chloroplasts and the O2 evolution from leaf tissues. The western analyses carried out on main components of PSII did not reveal photooxidative damage to the photosystem. In chloroplasts a decrease in chlorophyll and carotenoid amounts and the number of rather damaged thylakoidal membrane occurred. Cadmium 50 and 250 mM did not affect the activity of RUBISCO, nitrate reductase and glutamine synthetase, while the activity of ATP sulphurylase was already reduced in the plants treated with cadmium 50 mM.
76
Caratterizzazione elettroforetica, assistita da analisi di immagine, di proteine sintetizzate in embrioni di frumento durante la germinazione
Antonio Dell'Aquila+ e Marisa Di Turi*
+Ist. del Germoplasma, CNR Bari;* Dip. Di Produzione Animale, UNI Bari
Trattamenti di stress, ottenuti con temperature di 5-40°C o con imbibizione in soluzioni di glicole polietilenico o NaCl (-1,5 MPa di PO ) e applicati prima dell'emergenza della radichetta, sono efficaci nel controllare il processo della germinazione in semi di frumento. Anche l'invecchiamento del seme, ottenuto in condizioni di conservazione non ottimali, influisce sulla germinazione. Le proteine totali embrionali sintetizzate durante l'emergenza della radichetta sono modificate dai vari trattamenti, come evidenziato da analisi elettroforetiche bidimensionali in gel di poliacrilamide dopo incorporazione in vivo di [35S]-metionina. Le variazioni qualitative e quantitative di un gruppo di polipeptidi, selezionati in un'area del gel con PI di pH 5,9 - 5,4 e con MM di 75 - 54 kDa, sono state valutate mediante analisi di immagine assistita da computer. Quando i semi sono trattati con stress imbibizionali, è osservata una diffusa variazione dei polipeptidi in esame, non legata quantitativamente alla inibizione della germinazione per se ma al tipo di stress. Al contrario, se la germinazione viene ridotta per effetto dell'invecchiamento, un progressivo aumento o riduzione dei polipeptidi selezionati può essere correlato con il ritardo dell'inizio dell'emergenza. L'analisi elettroforetica bidimensionale, assistita da quella di immagine, sembra indicare che alcune di queste proteine possono essere considerate marcatori biochimici della sopravvivenza del seme a condizioni di stress imbibizionali o di invecchiamento.
77
Glycine betaine synthesis in spinach irrigated with salt water.
Catello Di Martino1, Amodio Fuggi 2
1. Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università del Molise, 86100 Campobasso, Italy. 2. Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy.
Plants respond to salt stress by inducing the synthesis of compatible low molecular compounds for osmotic adjustment as well as for protecting the cell components. Glycine betaine is one of the compatible nitrogen containing compounds accumulated in the tissues of some plant species under stress conditions. In this work the increase of glycine betaine in spinach leaves during the onset of salt stress has been determined. Spinach (Spinacia Oleracea L. cv. Matador) were stressed by restoring the daily evapotranspiration loss of the soil with saline water (1%). The first effects of the stress was the reduction of the photosynthetic activity essentially due to stomata closure. When photosynthesis was reduced at about 60% of the control and still the leaves and the photosynthetic apparatus did not differ significantly from those of the unstressed plants, their free amino acid pool was strongly increased due mainly to the increase of glycine and serine. A strong increase of glycine betaine was observed subsequently when the rubisco level began to decrease, and the overall amino acid pool was reduced. The increase of glycine betaine balanced the decrease of glycine and serine content, suggesting that this metabolite replaced those amino acids in the osmotic adjustment of the cell. .Serine and ultimately glycine are precursors of glycine betaine synthesis, supporting a role for photorespiration in stress response, not only because it provided a sink for photosynthetic electron transport, dissipating the excess of photochemical energy, but also because it mobilized carbon reserves through the glycolate oxidation pathway to produce compatible solute to control the stress.
78
EFFECT OF PHYTOTOXINS PRODUCED BY ASCOCHYTA CAULINA ON THE ASCORBATE SYSTEM OF CHENOPODIUM ALBUM L.
Nunzio Dipierro*, Silvana De Leonardis*, Laura Bianco*, Costantino Paciolla*, Maria Chiara Zonno°, Maurizio Vurro°, Silvio Dipierro*
*Dip. di Biologia e Patologia Vegetale, UNI Bari; °Ist. Tossine e Micotossine da Parassiti Vegetali, CNR, Bari
Chenopodium album L. (fat hen or lambsquarter) is one of the most widespread and important weeds. Ascochyta caulina (P. Karst.) v.d. Aa and v. Kest., a deuteromycetes fungus, has been proposed to be involved in the biological control of Chenopodium plants. In fact, the treatment of young Chenopodium plants with suspensions of the fungus's spores results in necrosis of leaves and stems. The same symptoms are induced by the treatment of the plants with culture filtrates of the fungus. With the aim to understand the molecular mechanism(s) by which the phytotoxins produced by Ascochyta caulina affect Chenopodium plants, we have start studying the effect of such phytotoxins on the components of the ascorbate system. This system is normally involved in the scavenging of reactive oxygen species (ROS). Since the production of ROS is often one of the first events set up by toxic substances in plants, it is possible that toxic substances induce modifications of the inherent detoxifying systems of attached plants. The data reported here, though preliminary, show that the activities of the enzymes of the ascorbate system, particularly of dehydroascorbate reducing proteins, of Chenopodium plants treated with Ascochyta phytotoxins are changed.
79
THE BEAN PGIP GENE FAMILY IS COMPOSED BY FOUR MEMBERS SPANNING A 50 KB REGION
D'Ovidio R.$, Capodicasa C.$, Roberti S.$, O'Sullivan D.*, Raiola A.+, Favaron F.^, Melaragni M.$, De Lorenzo G.+, Cervone F.+
$Dip. ABAC, UNI Tuscia; +Dip. Biologia vegetale, UNI Roma1;*IARC-LARS, UK; ^Dip. TeSAF, UNI Padova.
Polygalacturonase-inhibiting protein (PGIP) is a leucine rich repeat protein present in the cell wall of many plants. The in vitro ability to interact with fungal endopolygalacturonases (PGs) and its capacity to favour the formation of oligogalacturonides, able to induce some defence responses, suggests a role for PGIP in plant-pathogen interactions. PGIPs are encoded by gene families and functional analysis of their encoded products demonstrated that they can have different recognition capabilities against fungal PGs. In bean, such specificity can be controlled by a single amino acid substitution within the predicted b-sheet/b-turn region. To understand the role of different PGIP members in plant defence, we have characterized the bean pgip gene family by isolating and shot-gun sequencing two overlapping BAC clones covering about 150 Kbp. Sequence analysis of this region showed that the bean pgip gene family is composed by four different members spanning a 50 Kbp region. Sequence comparison of coding regions show an 85% identity between members of a gene family and a narrow variability between corresponding members of a gene family from a different bean cultivars. Non synonymous substitutions or small deletions are responsible for sequence polymorphism, that in some cases resides within the predicted b-sheet/b-turn structure, in position potentially involved in PG recognition. The extent of PGIP specificities from a single genotype will be tested against several fungal PGs
80
EXPRESSION GENE ANALYSIS IN Fe-DEFICIENT CUCUMBER ROOTS
Valentina Ditadi, Livia Pirovano, Patrizia De Nisi, Graziano Zocchi
DiPROVE, UNI Milano
Strategy I plants, respond to Fe-deficiency by inducing physiological and biochemical modifications in order to increase Fe uptake. In fact, under Fe deficient conditions, we can observe in plants roots an increased in RNA and proteins synthesis in plants roots. Moreover, the spectrophotometric analysis of polysomes extracted from Fe deficient roots, shows a greater resolution of the polysomal peaks compared to the control and which was interpreted as a better functioning of the translational machinery. In this study, differential display method was used to identify and analyse altered gene expression at the mRNA level in cucumber Fe deficient roots. This method is based on the assumption that every individual mRNA molecule can be reversely transcribed and amplified by the PCR. After that, we used six anchored primers in combination whit six arbitrary primers for PCR amplification of cDNA generated by reverse transcription from mRNA. The PCR products were resolved on a sequencing gel. Those products will then be cloned and, successively, sequenced.
81
BIOCHEMICAL AND GENETIC CHARACTERIZATION OF TWO PAPAVER RHOEAS BIOTYPES RESISTANT TO SULFONYLUREA HERBICIDES
G. Forlani*, A. Campani*, M.A. Gasparetto§, L. Scarabel#, S. Varotto# and M. Sattin §
*Dept of Biology, UNI-FE; §CeBI-CNR, PD; #Dept of Agronomy, UNI-PD
Acetolactate synthase (ALS, EC 4.1.3.18) is the enzyme that catalyses the first committed step of branched-chain amino acid synthesis, and the target of different classes of herbicides, such as sulfonylureas (SUs), imidazolinones (IMs) and triazolpyrimidines (TPs). An increasing utilization of these effective compounds led to the spontaneous emergence of resistant biotypes among weeds. Italy, unlike most countries with advanced agriculture, had been little affected by the development of herbicide resistant populations. However, during the last years there has been an unexpected and constant increase of both reported cases and the land area involved. Two Papaver rhoeas L. biotypes were identified upon the ability to survive field treatments with the SUs Tribenuron (TB) and Chlorsulfuron (CS). Agronomic characterization showed high level of resistance to all the SUs tested, and a slight tolerance to the IM Imazethapyr and the TP Florasulam. The resistance was not dose-dependent, suggesting a target site mechanism. The results of genetic and biochemical analyses were consistent with such hypothesis. A single nucleotide substitution at the variable Pro codon at position 257 of the ALS gene was identified in both cases. When ALS activity in extracts from cell cultures established from w.t. and resistant seedlings was assayed in the presence of increasing doses of CS and TB, a remarkable variation in enzyme sensitivity was found, with a 600-fold difference in concentrations causing 50%-inhibition of the catalytic rate.
82
RESPONSE TO COPPER SHORT TIME EXPOSURE IN LYCOPERSICUM LICOPERSICON
Galardi F., Gonnelli C., Gabbrielli R.
Dipartimento di Biologia Vegetale Via Micheli 50121 Firenze
Plant respond to biotic and abiotic stresses, such as those caused by wound, foraging insect, ozone, heavy metals etc, by synthesising proteins that play a role in general defence and tissue repair. Ethylene, jasmonic acid, salicylic acid and H2O2 are important secondary signal molecules thus serving to amplify and spread the response after the recognition of injury. Tomato plants treated with 1mM, 5mM, 10mM CuSO4 showed an increase in ethylene production after 15m of treatment, independently from the copper concentration used for the treatment. Moreover, after 15 minutes of treatment the H2O2 concentration was three times higher than the control. Salicylic acid, determined by HPLC analysis, decreased after 15 minutes with 1mM CuSO4. On the other hand Atomic spectroscopy analysis of the metal showed that the amount of copper taken up by the treated plant did not significantly differ from the untreated one, indicating that metal did not reach the internal tissues, so these results could indicate the presence of an intracellular signalling pathway which is activated before the metal reaches a toxic concentration inside the plant and that involves ethylene, hydrogen peroxide and salicylic acid
83
CARBOHYDRATE-ETHANOL TRANSITION IN CEREAL GRAINS UNDER ANOXIA
Lorenzo Guglielminetti*, Héctor Abel Busilacchi*, Pierdomenico Perata+, and Amedeo Alpi*
*Dip. Biologia Piante Agrarie UNI Pisa; +Dip. Scienze Agrarie, UNI Modena & Reggio Emilia
The conversion of carbohydrates to ethanol under anaerobic conditions was investigated in rice, barley, and wheat grains. We also analyzed the level of alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) activities in these cereal grains under aerobic and anaerobic incubation. Our data suggest that rice grains are able to produce ethanol under anoxia for the whole length of anoxic treatment, while barley and wheat grains are able to produce this terminal product of fermentation only during the first days of anaerobiosis. The level of enzymes involved in the fermentation pathway increases strongly under anoxic conditions in all the three cereals under investigation. We also demonstrate that, concerning the three cereals under investigation, hexose-CO2 conversion was nearly unaffected by anoxia, while only rice grains are able to degrade and utilize sucrose efficiently under anoxia. Wheat and barley, on the contrary, do not utilize sucrose efficiently under anaerobic conditions.
84
GENES INVOLVED IN OXIDATIVE STRESS RESPONSES FROM THE ENDOMYCORRHIZAL FUNGUS GIGASPORA MARGARITA
Luisa Lanfranco, A. Benedetto, S. Gabella, P. Bonfante
Dipartimento di Biologia Vegetale, Università di Torino
Arbuscular mycorrhizal (AM) fungi are one of the most widespread soil microbial community since they associate with the roots of about 80% of land plants. The knowledge on the presymbiotic phase of their life cycle, which includes spore germination and vegetative hyphal growth, is still poor due to their limited saprotrophic capability. The isolate Gigaspora margarita BEG34 is able to germinate in vitro and to produce a limited amount of free living mycelium. In the frame of an EST (Expressed Sequence Tags) project from germinating spores of this AM fungus we identified clones showing high similarity with proteins involved in oxidative stress responses, like superoxide dismutases Cu-Zn, glutathione S-transferases and metallothioneins. Complementation assays in a yeast strain sensitive to heavy metals demonstrated that the cDNA showing similarity to metallothioneins is able to provide a high level of tolerance to CdSO4. Expression analysis, obtained with semi-quantitative RT-PCR, indicated that these genes are constitutively expressed during the different phases of the fungal life cycle (spore, germinated spores and mycorrhizal roots). However, transcripts coding for the superoxide dismutase and for the glutathione S-transferase are more abundant in germinated spores, suggesting that the in vitro growth conditions are not favourable and thus could mime a stress situation.
85
KINETICS OF REFILLING OF CAVITATED MINOR VEINS IN LEAF BLADES OF CERATONIA SILIQUA L.
M. A. Lo Gullo+, S. Salleo*, P. Trifilò+ e A. Nardini*
+Dip. di Sc. Botaniche, UNI Messina; *Dip. di Biologia, UNI Trieste
Previous studies had shown that leaf minor veins of Carob undergo substantial cavitation during air-dehydration of 3-year-old leafy shoots maintained in the light. Extensive leaf vein cavitation in field-growing plants, can be expected either to be recovered or to lead to stomatal closure, thus preventing leaf dehydration. In the present study, potted Carob plants were deprived of irrigation until their pre-dawn leaf water potential (Yl) reached levels of -1.5, -2.0,-2.5 and -3.0 MPa corresponding to values recorded in the field. Measurements of maximum.diurnal leaf conductance to water vapour (gL) showed that gL decreased from 270 to 20 mmolm-2s-1at increasing stress levels. At the pre-established Yl's, leaflets were infiltrated under vacuum with a fluorescein solution (0.7mM) and were observed under a binocular equipped with UV excitation filter (360/40nm). The images were then processed using an image software to estimate changes in the fluorescent leaf area as the expression of the loss of the functional integrity of leaf minor veins. Analogous measurements were performed after plants were supplied with water and maintained in the dark for 30 and 90 min and 12h, thus simulating rainy night conditions. Simultaneous measurements, of the hydraulic conductance (K) of leaves, leaflets and rachides were also performed. Our data strongly suggest that: a) the major loss of K measured in leaves depended on rachis cavitation; b) within given water stress levels, leaf vein embolism was repaired in a matter of 30 to 90 min. Some evidence exists that leaf cavitation can represent a signal for stomatal closure.
86
The role of a-amylases in anaerobiosis tolerance: hormonal induction and metabolic repression under anoxia
Elena Loreti1, Amedeo Alpi1 , Pierdomenico Perata2
1 Department of Crop Plant Biology, University of Pisa, Via Mariscoglio 34, Pisa, Italy. 2 Department of Agricultural Sciences, University of Modena and Reggio Emilia, Via Kennedy 17, Reggio Emilia, Italy.
Anoxia exerts a strong effect on a-amylase induction in cereal grains. While gibberellic acid (GA3) induces a-amylase in rice (Oryza sativa L.) half-grains under either aerobic or anaerobic conditions, barley (Hordeum vulgare L.) half-grains are insensitive to this hormone when applied under anoxia. The possible repressive role of ethanol and abscisic acid (ABA) were investigated, and the results indicate that these two compounds cannot be held responsible for the failure of barley grains to respond to gibberellic acid. Furthermore, anoxia represses the induction of a-amylase downstream of the slender mutation, indicating that the repression is independent of effects related to gibberellin perception. Overall, the results suggest that the ability of rice to respond to gibberellins under anoxia is an adaptative trait independent of known negative regulators of a-amylase induction. Cereal a-amylases are modulated by both hormones and sugars. Interestingly, carbohydrates are able to influence a-amylase induction not only under aerobic conditions, but also under anoxia, indicating that sugar-sensing is active also under oxygen deprivation. The physiological consequences of sugar sensing under anoxia will be discussed.
87
NITRIC OXIDE MEDIATES IRON INDUCTION OF FERRITIN ACCUMULATION IN ARABIDOPSIS THALIANA
Irene Murgia1, Massimo Delledonne2 and Carlo Soave1.
1Sezione di Fisiologia e Biochimica delle Piante, Dip. di Biologia, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italia. 2 Istituto di Genetica, Università Cattolica Sacro Cuore, via Emilia Parmense 84, Piacenza 29100 Italia.
Nitric oxide (NO) is a signaling molecule involved in many different functions in mammal cells, among which the regulation of iron homeostasis. The recent discovery of NO-mediated regulation of aconitase activity also in plant cells prompted us to investigate the possible role of NO as regulator of iron homeostasis in plant cells, in particular its involvement in the regulation of A.thaliana ferritin, an iron-storage protein which accumulates in response to iron increase. The infiltration of the NO-donor SNP in Arabidopsis leaves caused a sustained accumulation of the transcript encoding ferritin; protein accumulation was also observed, although at much lower extent. The NO-dependent accumulation of ferritin transcript was observed also in iron-depleted Arabidopsis cells, whereas CPTIO, a NO scavenger, was able to completely abolish ferritin transcript accumulation caused by iron loading. To establish a possible involvement of cGMP as signal in in NO-mediated ferritin accumulation, we treated cells with 8 Br-cGMP, a cGMP analogous able to permeate cells. 8Br-cGMP was not sufficient to either induce ferritin accumulation nor to act synergistically with iron or SNP cGMP was not even found to participate to this signalling pathway since ODQ, an inhibitor of NO-dependent guanilate cyclase was not able to repress iron- nor SNP-induced ferritin accumulation. Furthermore calyculin, a Ser/Thr phosphatase inhibitor, completely abolished iron- or SNP-induced transcript accumulation. We propose that NO locates downstream of iron in the signalling pathway leading to ferritin accumulation which proceeds, beyond NO, through cGMP independent, Ser/Thr phosphatase dependent steps.
88
ISOLATION OF AN ARABIDOPSIS THALIANA MUTANT KNOCKED-OUT IN THE FERRITIN (ATFER1) GENE
Irene Murgia, Maddalena Fustinoni and Carlo Soave.
Sezione di Fisiologia e Biochimica delle Piante, Dip. di Biologia, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italia.
Iron is a critical element in photosynthetic tissues: it is an essential constituent of the photosynthetic complexes involved in electron transport but it is toxic when it is in a free, not complexed form, because it acts as a catalyst in the production of hydroxyl radicals during the Haber-Weiss reaction. The ability of the leaves to store iron in the chloroplasts in a soluble, readily available and non toxic form is provided by ferritins, a class of proteins present in plants as well as in bacteria, fungi and animals. Goal of our research is to better understand ferritin involvement in the plant protection against various oxidative stresses, like photoinhibition or iron excess. For that purpose we isolated, from the KO Wisconsin mutants collection, an Arabidopsis mutant bearing a T-DNA insertion in the ferritin Atfer1 promoter, 40 bp upstream the starting ATG. A description of the molecular steps through which the isolation of the mutant was performed and a preliminary physiological characterization will be given.
89
Monitoring large-scale changes in transcript abundance in barley under drought and high-salinity
Z. Neslihan Ozturk1,2, Valentina Talamé1,3, Christine B. Michalowski1,4, Nermin Gozukirmizi2, Roberto Tuberosa3 and Hans J. Bohnert1,4
1Dept. of Biochemistry and Molecular Biophysics, U. of Arizona, USA, 2TUBITAK, Marmara Research Center, Turkey, 3Dept. of Agroenvironmental Science and Technology, U. of Bologna, Italy; 4Dept. of Plant Biology and of Crop Sciences, U. of Illinois, USA
Responses to drought and salinity in barley (Hordeum vulgare L., var. Tokak) were monitored by microarray hybridization of 1463 unique transcripts derived from cDNA libraries of 6h and 10h drought-stressed and 24h salt-stressed plants. Approximately 38% of the transcripts were novel and functionally unknown. Microarray hybridization experiments were analyzed for drought/salinity regulated sequences, with significant changes defined as a deviation from the control exceeding 2.5-fold (log2 = 1.5). Nearly 15% of all transcripts were either up- or down-regulated under drought stress, while 150 mM NaCl (24h) led to a change in 5% of the transcripts. Transcripts that showed significant upregulation under drought stress are exemplified by jasmonate-responsive, metallothionein-like, late embryogenesis abundant (LEA) and ABA-responsive proteins. Most drastic down-regulation in a category was observed for photosynthesis-related functions. Upregulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress. Many functionally unknown transcripts from cDNA libraries of drought-stressed plants showed upregulation by drought but down-regulation by salt stress, documenting how closely transcript profiles report different growth conditions.
90
POSSIBILE RUOLO DELL'H2O2 NELL'INDUZIONE DELLA PCD IN PIANTE DI TABACCO SOTTOPOSTE A TRATTAMENTO CON OZONO
Claudia Piccioni, Stefania Pasqualini, Luisa Ederli, Lara Reale, Francesco Ferranti e Marisa Antonielli
Dip. Di Biologia Vegetale e Biotecnologie Agroambientali, UNI Perugia
La PCD (Programmed Cell Death) è un processo fisiologico che interviene durante il normale sviluppo e in conseguenza di condizioni patologiche in sistemi vegetali ed animali. Numerose osservazioni suggeriscono che le specie reattive dell'ossigeno (ROS) prodotte da fumigazione con ozono, radiazioni UV, infezione da patogeni, possono mediare la PCD. Allo stesso tempo la PCD può essere stimolata dalla incapacità delle cellule di detossificare le ROS. Da nostri esperimenti preliminari è stato possibile osservare che la fumigazione di piante di tabacco con O3 (300 ppb per 3h) induce la formazione di lesioni sulle foglie della cultivar Bel W3 (O3-sensibile) e, in misura molto minore, nella cultivar Bel B (O3-resistente). In questo studio viene esaminata la relazione tra ROS prodotte in seguito a trattamento con O3 e morte cellulare. In un primo momento, si cercherà di stabilire se la formazione di lesioni indotta dall'ozono è conseguenza di un processo di PCD o di necrosi. A tale scopo verranno esaminati alcuni parametri che ci consentano di distinguere tra questi due processi (analisi della frammentazione del DNA mediante TUNEL, elettroforesi su gel d'agarosio ed osservazione dei nuclei al microscopio elettronico; analisi dell'attività enzimatica delle proteasi). Contestualmente sarà determinato il contenuto di H2O2 e l'espressione di enzimi strettamente correlati al metabolismo dell'H2O2 per comprendere il ruolo delle ROS nello sviluppo delle lesioni ed eventualmente nella PCD.
91
La via di trasduzione del segnale per l'accumulo anaerobico di Gaba in radici di Riso (Oryza sativa L.)
R. Reggiani e P. Laoreti
Istituto Biosintesi Vegetali, CNR, Via Bassini 15, 20133 Milano
L'acido g-aminobutirrico (Gaba) è sintetizzato mediante l'enzima calmodulina (CaM)-dipendente glutammato decarbossilasi e si accumula durante lo stress anaerobico. In precedenza (Aurisano et al., 1995, 1996), abbiamo stabilito che l'accumulo di Gaba è inibito da rosso rutenio (RR, inibitore di canali Ca2+), da antagonisti della CaM (trifluoperazine, W-7) e da inibitori di G proteins (GDP, GDPbS). In questo lavoro abbiamo cercato di identificare altri segnali implicati con il rilascio di Ca2+. Il pre-trattamento di radici di riso per 2 ore in aria con antagonisti della fosfolipasi C (PLC, neomicina, composto 48/80) inibiva l'accumulo di Gaba durante 3 ore di trattamento anossico. La presenza di Ca2+ e A23187 (ionoforo del Ca2+) aboliva l'effetto degli inibitori della PLC. L'aumento anaerobico nella concentrazione di inositolo 1,4,5-trifosfato (IP3, prodotto della PLC) era annullato dal trattamento con inibitori della PLC. La neomicina e il composto 48/80 riducevano l'effetto di stimolazione della via di trasduzione del segnale anaerobico ottenuta con alluminio fluoruro (attivatore delle G proteins). Questi risultati indicano che la PLC agisce a valle delle G proteins e che l'IP3 è verosimilmente implicato nel rilascio di Ca2+ nel citoplasma attraverso canali RR-sensibili. Aurisano N., Bertani A. & Reggiani R. (1995) Plant Cell Physiol. 36: 1525-1529. Aurisano N., Bertani A. & Reggiani R. (1996) J. Plant Physiol. 149: 517-519.
92
HEAVY METALS ACCUMULATION AND PHOTOSYNTHETIC CHANGES IN TWO POPLAR CLONES EXPOSED TO INDUSTRIAL WASTE.
Luca Sebastiani1 and Roberto Tognetti2
1SSSUP "S.Anna", Pisa; 2Dip.to SAVA, UNI Molise.
Phytoremediation is an emerging cleanup technology which uses green or higher terrestrial plants for treating environmental contaminants as heavy metals, trace elements, organic compounds or radioactive compounds in soils, groundwater, and wastewater. Early studies on phytoremediation closely associated this technology with the use of hyperaccumulator species, which are able to accumulate in their tissues unusual levels of metals. Most of the hyperaccumulators are small herbaceous plants producing very low-biomass levels, which strongly limit the phytoremediation potential. For this reason high-biomass plants such as tree and grass species are now being actively evaluated. In this study one-year-old actively growing cuttings of two poplar clones (Populus deltoides x maximowiczii - clone Eridano and P. x euramericana - clone I-214) were grown in soil: a) amended with heavy metal-enriched industrial waste; b) not-amended. Heavy metals accumulation and photosynthetic changes during the growing season were recorded. Results showed significant changes in heavy metals accumulation and photosynthesis in plants grown on amended soil when compared with control plants. Genotypc variability in heavy metals accumulation and photosynthetic parameters were also observed.
93
CHEMICAL INDUCERS OF SYSTEMIC ACQUIRED RESISTANCE REDUCE THE SUSCEPTIBILITY OF PEAR TREES TO Erwinia amylovora
Francesca Sparla, Lorenza Rotino, Maria Chiara Valgimigli, Paolo Pupillo e Paolo Trost
Lab. of Plant physiology, Dept. Biology, UNI Bologna
As a consequence of the interaction with a necrogenic pathogen, certain plants exhibit a systemic acquired resistance (SAR) effective versus different pathogens. The establishment of SAR correlates with the stimulated expression of a particular set of genes, including those encoding pathogen related (PR) proteins. For the induction of SAR, salicylic acid (SA) plays a crucial role as a signalling molecule. A synthetic analog of SA, benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester (BTH), mimics the effects of SA in activating SAR. Although BTH as no direct effect on pathogen viability, Arabidopsis plants treated with BTH are more resistant to the same set of pathogens and overexpress the same set of genes as biological inducers of SAR such as SA. Erwinia amylovora is a necrogenic bacterium causing fire blight of meloideae subfamily of Rosaceae such as pear and apple. We have found that the pre-treatment of 2-years old pear trees with BTH induces partial resistance to Erwinia amylovora inoculated on the apical bud. Best results were obtained when BTH was sprayed 10 days before inoculation. Following BTH pre-treatment and inoculation, the percentage of asymptomatic individuals was doubled in respect to controls. Also, BTH-treated symptomatic individuals showed relatively shorter shoot cankers. The effect of BTH was found to be systemic. Gene expression studies are in progress with the aim of understanding whether the BTH-induced resistance in pear may be classified as SAR.
94
HOW THE ANTIOXIDANT SYSTEMS WORK DURING STARVATION OF RECALCITRANT SEEDS OF GINKGO BILOBA L.?
Franca Tommasi, Costantino Paciolla, Maria C. de Pinto and Laura DeGara.
Dipartimento di Biologia e Patologia Vegetale - UNI Bari tommasi@botanica.uniba.it
The long term conservation of recalcitrant seeds is a great problem because they are damaged by dehydration and sometime by chilling and cannot survive for long periods (Pammenter and Berjak, Seed Sci. Res, 9,13, 1999). One question remains to be answered: why and how do recalcitrant seeds die? Some data connect the loss of the germination capability with a drop in antioxidant system efficiency (Hendry et al., New Phytol., 122, 273, 1992), but the molecular mechanisms involved in recalcitrance are not completely understood. Ginkgo biloba, an ancient species used as an ornamental plant and for its derivatives, produces recalcitrant seeds containing a developed embryo plugged in a haploid endosperm The behaviour of the G. biloba seeds was studied during starvation at 4 °C. Data reported in this work point out that their germinability decreases progressively after six months of starvation although the water content was only moderately affected. The two major antioxidant systems, Ascorbate (ASC) and glutathione (GSH), show different behaviour and also differences are evident between the two main structures of the seed, the embryo and the endosperm. The GSH, present prevalently in the reduced form, decreases progressively during starvation both in the embryos and in the endosperm although the GR reductase activity raises in the embryos. The ASC content is not affected by cold starvation in the embryo while it decreases in the endosperm. The ASC peroxidase activity, after an initial decrease remains constant. The activities of the enzymes involved in ascorbate recycling (Ascorbate free radical reductase and Dehydroascorbate reductase) markedly decrease in the embryos during cold starvation. The role of these two enzymes as well as the inability of the antioxidant systems to counteracts the deterioration of the seed are discussed.
95
CLONING AND CHARACTERIZATION OF TWO GENES ENCODING POLYGALACTURONASE-INHIBITING PROTEIN (PGIP) IN Arabidopsis thaliana
D.Vairo1, S.Ferrari2, F.Cervone1, F.M.Ausubel2, G. De Lorenzo1
1Dipartimento di Vegetale, Università di Roma "La Sapienza", Roma (Italy), 2Department of Molecular Biology Massachussetts General Hospital, Boston, MA (USA)
The cell wall is the first line of defence of plants against pathogens. Polygalacturonases (PG) are the first enzymes to be secreted by phytopathogenic fungi during infection processes. Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins capable of inhibiting fungal PGs. Plants have evolved PGIPs with specific recognition abilities against the many PGs produced by fungi. We have isolated and characterised two PGIPs genes from Arabidopsis plants. AtPGIP1 and AtPGIP2 are closely related genes (78% and 76% of nucleotide and amino acidic identity, respectively) encoding for two 330 amino acids (aa) predicted proteins with a 22 aa putative signal peptide. In Arabidopsis genome the two genes are located in tandem, about 500bp apart, mapping on chromosome 5. Expression of both AtPGIPs genes is developmentally regulated. Interestingly despite sharing elevated degree of homology AtPGIP1 and AtPGIP2 are both induced by pathogen infection and wounding but differently regulated by chemical signals and environmental conditions. For instance AtPGIP1 is induced by oligogalacturonide elicitors and cold stress and AtPGIP2 is sensitive to methyl jasmonate.
96
Expression of a maize 14-3-3 protein in tomato plants: effect on the response to biotic and abiotic stress.
Fullone M.R.1, Visconti S.2, Bettini P.3, Buiatti M.3, Capogna F.4, Garufi A.2, Guerriero I.3, Manes F.4, Michelotti S.3, Pellegrini M.G.3, Ragusa S.5 e Aducci P.2
1 Dip.to di Biochimica "Rossi Fanelli" Università "La Sapienza" Roma 2 Dip.to di Biologia Università "Tor Vergata" Roma 3 Dip.to di Biologia Animale e Genetica "Leo Pardi" Università di Firenze 4 Dip.to di Biologia Vegetale Università "La Sapienza" Roma 5 Dip.to Farmacobiologico Università di Messina
14-3-3 are a family of proteins, ubiquitous in all eukaryotes, involved in the control of several physiological and cellular processes; through the interaction with a number of proteins, they play an important role in cell cycle regulation, differentiation, in targeting to cellular location and in the coordination of multiple signal transduction pathways. In plants, 14-3-3 proteins are mainly known to regulate the activity of enzymes of carbon and nitrogen metabolism and the plasma membrane H+-ATPase. Recently it has been demonstrated a variation in the expression of 14-3-3 genes during the pathogen attack and in different abiotic stress conditions, thus suggesting a possible role of 14-3-3 proteins in the response of plant to environmental stress. In order to investigate this possibility, we have inserted the cDNA encoding the maize 14-3-3 GF14-6 isoform in tomato plant through the Agrobacterium-mediated transformation and we have analysed the effect of 14-3-3 overexpression during the plant response to different stress condition. In particular, the response of transgenic and control plants to drought, by gas exchange and chlorophyll a fluorescence measurements, and the production of the ethylene, which is known to have an important role in the response of plant to pathogen and to abiotic stress, have been valued.
97
Overespression of lhcb4 in Arabidopsis thaliana induces an altered NPQ response.
Francesco Vantini, Luigi Cattivelli and Roberto Bassi
Dipartimento scientifico e tecnologico- Università di Verona
Nonradiative dissipation of excitation energy is the major photoprotection mechanism in higher plants. De-epoxidation of violaxanthin to zeaxanthin in the antenna of photosystem II (PSII) has been shown to be responsible for a component of nonphotochemical quenching (NPQ) in vivo. The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4) is one of the major violaxanthin/zeaxanthin binding proteins in PSII. To study the photoprotective activity of CP29 in vivo, we have transformed Arabidopsis thaliana with Zea mays Lhcb4 gene under the control of a constitutive promoter. The new plants (ZM-cp29) overexpress CP29 and the mature protein is correctly localised into thylakoid membranes of the chloroplast. Transgenic lines and WT have been analysed during exposure to actinic light and subsequent dark recovery. NPQ measurements were performed in vivo through a fluorescence kinetic video-imaging apparatus. ZM-cp29 plants overexpessing CP29 clearly showed an alteration of dark recovery from NPQ. This phenotype was accompanied by an alteration of zeaxanthin re-epoxidarion. These results suggest that incorporation of zeaxanthin into Lhc proteins constitute a mechanism of memory for "excess light experience" aimed to prevent repeated high light shocks to PSII RC.
98
EVIDENCE FOR THE PRESENCE OF FERRITIN IN PLANT MITOCHONDRIA
Marco Zancani1, Carlo Peresson1, Irene Murgia2, Carlo Soave2, Angelo Vianello1, and Francesco Macrì1.
1Dip. Biologia ed Econ. Agro-ind., Università di Udine; 2Dip. di Biologia, Università di Milano.
Iron is an essential element for all living organisms, although it can be very toxic to cell because of its high reactivity with H2O2 which leads to the generation of hydroxyl radical. In plants, chloroplasts and mitochondria are two of the major sites of H2O2 generation. To prevent this risk, two strategies can be employed, namely scavenging of H2O2 or sequestration of iron. Ferritins are a class of ubiquitous proteins able to store iron, thus providing a mechanism to control its homeostasis. In plant cells, ferritins have been found only in plastids. In this work we tried to verify whether plant mitochondria also contain these iron-storage proteins. It was found that the proteins of etiolated pea stem mitochondria cross-reacted with a polyclonal antibody raised against pea seed ferritin. The band identified corresponded to a protein with a molecular mass of 29 kDa, a value which is identical to that of ferritin. These mitochondria were devoid of contaminations from other types of membranes. In addition, by means of a specific net program (www.inra.fr/servlets/WebPredotar), it was shown that a "putative ferritin" of Arabidopsis thaliana, could be present in mitochondria with a score of 0.974. Therefore, to confirm the above result on pea mitochondria and the latter observation, the proteins of mitochondria from A. thaliana cell cultures were cross-reacted with pea ferritin antibody. Again, the reaction involved a protein of about 29 kDa, thus confirming the presence of ferritins also in this type of plant material.
Last Updated - 23/07/01