Francesco Maria Restivo

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus.

Aims:  To develop a multiplex reverse transciption‐polymerase chain reaction (RT‐PCR) protocol to discriminate aflatoxin‐producing from aflatoxin‐nonproducing strains of Aspergillus flavus.

Resolving the role of plant glutamate dehydrogenase. I. In vivo real time nuclear magnetic resonance spectroscopy experiments.

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time 15N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [15N]Glu or 15NH4+ respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

Sex determination and differentiation in Asparagus officinalis L.

Abstract The paper summarizes the coordinated researches conducted by three Italian groups in the area of sex determination and differentiation in the dioecious species Asparagus officinalis . Morphological evidence indicates that sex differentiation in Asparagus consists essentially of selective abortion of gynoecium or androecium of initially hermaphroditic floral primordia occurring in genotypically determined male and female individuals. Abortion occurs in pollen-mother cells and anthers in females and in megaspore-mother cells but not in the vegetative tissues of the ovary in males. The differential developmental pathway is accompanied by changes in relative abundance of auxin and cytokinins. The genetic ssytem controlling abortion of male or female organs is apparently monogenic (possibly a bipartite gene) with factor(s) associated with the homomorphic chromosome pair L5. Other genes influence the development of reproductive structures as indicated by the presence of genetic factors controlling stylar growth in male plants. The presence of extensive polymorphism in isoenzyme and DNA restriction fragment length patterns (RFLP) allows the search for markers associated with ‘sex genes’: a locus encoding a malic dehydrogenase (MDH) isoenzyme has been found about 20 cM from sex genes implying that chromosomes in which sex factors are located could pair and recombine. Searches for messages specifically expressed in reproductive structures were conducted by 2D-electrophoresis of existing and newly synthesized polypeptides or of in vitro translation products of poly(A) + RNA from male and female flowers and by isolating specific monoclonal antibodies against sex specific floral antigens.

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

Abstract An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM.

Isozyme gene markers in the dioecious species Asparagus officinalis L.

SummaryExtracts from phylloclads of Asparagus officinails were electrophoretically analyzed for isozyme polymorphism. Fourteen enzyme systems were examined using four buffer systems: seven enzymes (acid phosphatase, catalase, glutamate-oxaloacetate transaminase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase) exhibited clear and consistent banding patterns. Isozyme polymorphism was studied in seven pairs of male and female doubled haploids and in their male F1s. Segregation of polymorphic loci was examined in the backcross progenies and was found to be consistent with a simple Mendelian inheritance in all cases, except for three anodical peroxidases, where two factors have been hypothesized. No linkage could be found between isozyme markers that were segregating in the same cross, but association was demonstrated between one malate dehydrogenase locus and the sex determining genes. The availability of isozyme markers may be useful in breeding and, in particular, the localization of one malate dehydrogenase locus on the sex chromosomes may be helpful in mapping the sex genes.

The effects of carbon starvation on cellular metabolism and protein and RNA synthesis in Gerbera callus cultures

Abstract Gerbera jamesonii H. Bolus var. hybrida callus cultures subjected to carbon starvation show alteration in some physiological parameters and protein synthesis. Five days of continuous starvation induce a decrease of protein and carbohydrate contents to respectively, 27% and 12% of the values of the control, while specific activities of glutamate dehydrogenase and proteases reached levels about 5-fold and 2-fold higher than the control ones. Electrophoresis of proteins labelled by in vivo and in vitro translation showed the appearance of several starvation-related proteins (STP). At day 5 from the onset of starvation the induction of STPs was related with levels of in vitro translatable mRNAs, whereas at day 1 the synthesis of STPs depended upon translational regulation. The induction of STPs was also analyzed under conditions of specific protein synthesis inhibition by heat-shock. Protein synthesis in heat-shocked starved cells showed (i) the induction of heat-shock proteins (HSPs), and (ii) the disappearance of all but three STPs of molecular mass 70, 69 and 36 kDa. A similarity between one HSP and one STP is hypothesized on the basis of comigration on two-dimensional gels (70 kDa). During recovery from heat-shock starved cells showed the reappearance of all the STPs and the simultaneous maintenance of most of the HSPs. The data presented support a role of STPs in starvation and the existence of interactions between heat-shock and nutrient deprivation.

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones.

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 1∶1 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.

Identification of chloroplast associated heat-shock proteins in Nicotiana plumbaginifolia protoplasts

SummaryNicotiana plumbaginifolia protoplasts grown at 25 °C and transferred at 40 °C synthesized new temperature-dependent polypeptides called heat-shock proteins (hsp) distributed in a wide range of molecular weight. The cellular localization of hsp showed a concentration of the low molecular weight hsp in a fraction enriched in membranes and chloroplasts. Purified chloroplasts obtained by a Percoll cushion and by a discontinuous sucrose density gradient centrifugation, were therefore analyzed for hsp distribution. Several hsp were found to be associated with this subcellular compartment: in particular three hsp were associated with the soluble fraction of the chloroplasts, whereas six hsp were associated with organelle membranes. The involvment of chloroplast information in the synthesis of some of these organelle associated hsp and therefore in the response to heat-shock is discussed.

Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications.

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.