Francesco Carimi

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H2O2 is necessary.

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line.

Past and present role of the Indian-fig prickly-pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the agriculture of sicily

Of prickly-pear cacti occurring in Sicily, the most widespread and economically important isOpuntia ficus-indica (L.) Miller. In Sicily it has, since its introduction, played an important role in the exploitation of marginal areas. The Sicilian experience is described with reference to the historical outlines and the present intensive production of late fruit. Information on historical and actual uses of the plant and its products (flowers, cladodes, fruits) is given.AbstractEntre los nopales que se pueden encontrar en Sicilia la mas difundida es laOpuntia ficus-indica (L.) Miller. Esta especie desde hace su introduccìon ha sido la mas disfrutada en las explotaciones de las zonas marginales. En este trabajo los Autores refiren sobre la historia y sobre la actual situacion productiva, finalisada a la producion de higos de retallo. Se refire tambien sobre su uso y sus productos (flores, cladodios, frutos).

Molecular Characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD Markers on Eggplant

Fusarium oxysporum f. melongenae is a major soil-borne pathogen of eggplant (Solanum melongena). ISSR and RAPD markers were used to characterize Fusarium oxysporum f. melongenae isolates collected from eggplant fields in southern Turkey. Those isolates were not pathogenic to tomato. Pathogens were identified by their morphology, and their identity was confirmed by PCR amplification using the specific primer PF02-3. The isolates were classified into groups on the basis of ISSR and RAPD fingerprints, which showed a level of genetic specificity and diversity not previously identified in Fusarium oxysporum f. melongenae, suggesting that genetic differences are related to the pathogen in the Mediterranean region. The primers selected to characterize Fusarium oxysporum f. melongenae may be used to determine genetic differences and pathogen virulence. This study is the first to characterize eggplant F.oxysporum species using ISSR and RAPD.

Transcriptome analysis of Medicago truncatula leaf senescence: similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling

Here, for the first time, a comprehensive transcriptomics study is presented of leaf senescence in the legume model Medicago truncatula, providing a broad overview of differentially expressed transcripts involved in this process. The cDNA-amplification fragment length polymorphism (AFLP) technique was used to identify > 500 genes, which were cloned and sorted into functional categories according to their gene ontology annotation. Comparison between the datasets of Arabidopsis and M. truncatula leaf senescence reveals common physiological events but differences in the nitrogen metabolism and in transcriptional regulation. In addition, it was observed that a minority of the genes regulated during leaf senescence were equally involved in other processes leading to programmed cell death, such as nodule senescence and nitric oxide signalling. This study provides a wide transcriptional profile for the comprehension of key events of leaf senescence in M. truncatula and highlights a possible regulative role for MADS box transcription factors in the terminal phases of the process.

Physical, morphological and chemical changes during fruit development and ripening in three cultivars of prickly pear, Opuntia ficus-indica (L.) Miller

Late ripening fruits of prickly pear cvs Gialla, Rossa and Bianca took 80-90 days from flowering to harvestable maturity. A decrease in fruit growth rate occurred from 30 to about 60 d after flowering, during the period of seeds development and hardening. The fruit continued to grow both on a fresh and dry weight basis until maturity. The greatest development of the core occurred from 50 d after flowering. During the same period there were consistent changes in biochemical activity: the total sugar content and the total soluble sugars (TSS) sharply increased whilst the fruit firmness and the total tritratable acids (TTA) decreased. Fruits reached the optimum maturity stage when TSS reached a “steady state” over 13% Brix; this stage corresponds to the period of peel colour breakage. From 90 d after flowering onwards the fruits became tender and unsuitable for processing and storage. At that stage the peel was fully coloured.

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil.

Somatic embryogenesis in Citrus from styles culture

Abstract Styles (including the stigma) of Citrus aurantium L. (cvs. ‘AA 12’, ‘AA 30’ and ‘AA31’), C. deliciosa Tenore (cvs. ‘Avana’ and ‘Tardivo di Ciaculli’), C. paradisi Macf. (cvs. ‘Marsh seedless’ and ‘Star Ruby’) and C. sinensis (L.) Osb. (cvs. ‘Bonanza’, ‘Brasiliano 92’, ‘Sanguinello’ and ‘Valencia’) were cultured for induction of somatic embryogenesis. Explants were excised from flower buds which were collected during full bloom, and cultured on Murashige and Skoog (MS) basal medium supplemented with 146 mM sucrose, 500 mg/l malt extract and 13.3 μM 6-benzylaminopurine (BAP) as well as the same medium without BAP. Callus development was observed from the style base 4 weeks after treatment initiation, and embryogenesis occurred 2–3 months later. Embryogenesis has been induced from the style-derived callus of all the cultivars tested except for the cultivars ‘Avana’ and ‘Star Ruby’. The best results for callus growth and embryo regeneration was obtained from explants of ‘Brasiliano 92’ cultured on medium containing BAP. Somatic embryos were isolated from callus and placed on MS medium supplemented with 146 mM sucrose, 500 mg/l malt extract and 0.27 μM α-naphthaleneacetic acid (NAA) where they formed entire plants. Two months later plants were successfully established in soil.

Microsatellite analyses for evaluation of genetic diversity among Sicilian grapevine cultivars

A total of 82 grapevine genotypes were sampled from several areas of the Italian region of Sicily where vineyards are widely spread. The grapevines were characterized using six microsatellite markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62 and VrZAG79) to evaluate genetic diversity. Thirty-seven of the 82 cultivars sampled had their names quoted in historical and literary sources, while 45 cultivars from old vineyards did not have their names reported in ancient literature. According to their genetic profiles at SSR loci, 70 different cultivars were found, while interesting cases of synonymies (Regina and Moscato bianco, Alicante and Dolcetta or among different clones of Zibibbo and Catarratto) and cases of homonymy (Frappato and Nerello Mascalese) were discovered. Several genetic parameters were calculated to assess the efficacy of the loci chosen in this work. Pairwise genetic distances between all cultivars were calculated. A dendrogram representing the genetic similarities among cultivars was depicted using the UPGMA method to investigate their relationships, explaining them from a historical point of view. The cluster distribution of cultivars clearly does not reflect their current geographical distribution, suggesting successive introductions of cultivars in Sicily from different areas of origin.

A proteomic approach provides new insights into the control of soil-borne plant pathogens by Bacillus species.

Beneficial microorganisms (also known as biopesticides) are considered to be one of the most promising methods for more rational and safe crop management practices. We used Bacillus strains EU07, QST713 and FZB24, and investigated their inhibitory effect on Fusarium. Bacterial cell cultures, cell-free supernatants and volatiles displayed varying degrees of suppressive effect. Proteomic analysis of secreted proteins from EU07 and FZB24 revealed the presence of lytic enzymes, cellulases, proteases, 1,4-β-glucanase and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum. Our findings provide new knowledge on the mechanism of action of Bacillus species and insight into biocontrol mechanisms.