A. M. Innocenti

Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 μM 6-benzyladenine, 1.0 μM α-naphthaleneacetic acid and 250 mg l−1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

Abstract. The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h−1) compared with the control (8.4 m h−1). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants.

Ascorbic Acid Effect on Pericycle Cell Line in Allium Cepa Root

SUMMARYThe pericycle cells in Allium cepa root tip undergo differentiation at 2C level. The present study indicates two clear effects of ascorbic acid treatment on pericycle cell line: i) the extension of the upper limit of mitosis from the tip; ii) the increase of the labelling index. In a previous paper we have just found that, in Allium cepa, treatment with ascorbic acid stimulates the G1 nuclei of the quiescent centre to undergo DNA synthesis. Our present results suggest the hypothesis that ascorbic acid could have a regulatory role of cell proliferation during the G1 phase.

Peach [Prunus persica (L.) Batsch] KNOPE1, a class 1 KNOX orthologue to Arabidopsis BREVIPEDICELLUS/KNAT1, is misexpressed during hyperplasia of leaf curl disease

Class 1 KNOTTED-like (KNOX) transcription factors control cell meristematic identity. An investigation was carried out to determine whether they maintain this function in peach plants and might act in leaf curliness caused by the ascomycete Taphrina deformans. KNOPE1 function was assessed by overexpression in Arabidopsis and by yeast two-hybrid assays with Arabidopsis BELL proteins. Subsequently, KNOPE1 mRNA and zeatin localization was monitored during leaf curl disease. KNOPE1 and Arabidopsis BREVIPEDICELLUS (BP) proteins fell into the same phyletic group and recognized the same BELL factors. 35S:KNOPE1 Arabidopsis lines exhibited altered traits resembling those of BP-overexpressing lines. In peach shoot apical meristem, KNOPE1 was expressed in the peripheral and central zones but not in leaf primordia, identically to the BP expression pattern. These results strongly suggest that KNOPE1 must be down-regulated for leaf initiation and that it can control cell meristem identity equally as well as all class 1 KNOX genes. Leaves attacked by T. deformans share histological alterations with class 1 KNOX-overexpressing leaves, including cell proliferation and loss of cell differentiation. Both KNOPE1 and a cytokinin synthesis ISOPENTENYLTRANSFERASE gene were found to be up-regulated in infected curled leaves. At early disease stages, KNOPE1 was uniquely triggered in the palisade cells interacting with subepidermal mycelium, while zeatin vascular localization was unaltered compared with healthy leaves. Subsequently, when mycelium colonization and asci development occurred, both KNOPE1 and zeatin signals were scattered in sectors of cell disorders. These results suggest that KNOPE1 misexpression and de novo zeatin synthesis of host origin might participate in hyperplasia of leaf curl disease.

Molecular cloning, characterisation and expression of two catalase genes from peach

Two cDNA clones encoding catalase (Cat1 and Cat2) from peach [Prunus persica (L.) Batsch] were identified, that show homologies to other plant catalases. The nucleotide sequences of the two coding regions showed 88% identity to each other. The amino acid sequences predicted from the two full-length clones showed the highest homology to a catalase from cotton and Nicotiana plumbaginifolia L. and included C-terminal tri-peptides typical of those used to target proteins to peroxisomes. Southern hybridisation analysis suggested the existence of two catalase genes in peach. The expression of Cat1 and Cat2 was determined in seeds, vegetative tissue, leaves during the seasonal cycle and in leaves in response to light / dark treatments. Cat1 had high levels of expression only in leaf tissue and was responsive to light and seasonal changes. Cat2 had high levels of expression in in vitro shoots and was also responsive to seasonal changes, but not to light. In situ hybridisations to leaf tissue indicated that the expression of Cat1 was localised mainly in palisade cells, while Cat2 mRNA was present in the vascular tissue. The results of the expression analysis and in situ hybridisation suggest a role for Cat1 in photorespiration and for Cat2 in stress responses.

2-DE polypeptide mapping of Posidonia oceanica leaves, a molecular tool for marine environment studies

Abstract The aim of this research is to provide a molecular tool based on polypeptide mapping to investigate the flowering marine plant Posidonia oceanica. This plant is very vulnerable to contaminants; thus it is considered a valuable bio-indicator of water quality in bio-monitoring of coastal environments. Posidonia oceanica was found to be recalcitrant to the common protein extraction methods. In the present work, three different extraction procedures were compared to obtain high yield and quality protein extracts suitable for mono-dimensional and bi-dimensional electrophoresis (1-DE and 2-DE). Proteins were extracted from juvenile, intermediate and adult leaves in order to assess the influence of tissue differentiation on protein yield. The highest protein yield was obtained with 20% trichloroacetic acid (TCA) precipitation of proteins. The best extraction efficiency was found in juvenile leaves as compared with intermediate and adult ones. However, as a large amount of juvenile leaves is required for obtaining sufficiently large protein samples, these were considered not suitable for the electrophoretic analysis. Extensive sampling could introduce further damage in the meadows under study. High quality 2-DE polypeptide mappings were obtained only from intermediate and adult leaves; the good reproducibility of protein patterns indicates that this approach could be used to explore changes in protein expression of P. oceanica in response to altered environmental conditions.

Magnetic field affects meristem activity and cell differentiation in Zea mays roots

Abstract Exposure of Zea mays seedlings to a continuous electromagnetic field (EMF) for 30 h induced a 30% stimulation in the rate of root elongation compared with the controls. It also resulted in a significant increase of cell expansion, in both the acropetal (metaxylem cell lineage) and basipetal (root cap cells) direction. In addition, in EMF-exposed roots a precocious structural disorder was observed both in differentiating metaxylem cells and root cap cells. All these features may be consistent with an advanced differentiation of root cells that are programmed to die. EMF treatment also resulted in a significant reduction in the size of the quiescent centre in the root apical meristem. The extent to which these responses are causally linked is discussed.

Differente invecchiamento nelle cariossidi « chiare » e « scure » di Haynaldia villosa Schur. Uno studio citofotometrico nei meristemi radicali quiescenti

Abstract Different ageing pattern in « normal » and « red » seeds of Haynaldia villosa Schur. A cytophotometric analysis in resting root meristems. – Cytophotometric determinations of DNA and histones content per nucleus were carried out in the 2C root meristematic nuclei from « yellow » and « red » seeds of Haynaldia villosa of different ages; the histones/DNA ratio increases during the storage period in the « yellow » seeds while it is almost constant in the « red » ones. The possible significance of these observations in relation to seed ageing is discussed.

Different Duration of the Mitotic Cycle in Seedlings from Brown and Black Caryopses of Haynaldia Villosa Schur.

SUMMARYThe duration of the mitotic cycle and its phases has been determined in the root tips of seedlings from brown and black caryopses of Haynaldia villosa, by autoradiographic technique after labelling with m3H-thymidine. The total duration of the mitotic cycle was found to be 12h in brown caryopses and 9.5h in black caryopses. As to the time length of single phases, the greatest difference between the two kinds of caryopses was foun dat the level of G1 phase. The possible significance of these differences is discussed.

Altered cytokinin distribution in the shootless maize mutant ed*41

The distribution of cytokinins is strongly altered in seedlings of the shootless ed*41 mutant of maize (Zea mays L.), compared with wild-type. Immunolocalisation of zeatin and analysis of cytokinin levels clearly indicate that these hormones are not present in the shoot apex zone of the mutant. Since an anomalous differentiation of vascular tissues has also been observed in the mutant, a major role of vascular connection in hormone translocation affecting development of the shoot apical meristem is proposed. Immunolocalisation of zeatin was confined to the root cap, cortex and vascular tissues of both mutant and wild-type seedlings suggesting a tissue-specific synthesis of this hormone in the root. A time-course of cytokinin distribution in the wild-type developing shoot provided evidence that the tunica and corpus zones become competent to respond to cytokinins in subsequent periods, very probably in conjunction with photomorphogenesis. On the contrary, this pathway is totally disrupted in the mutant. Taken together, the data point to a relationship between the ed*41 mutation, inadequate vascular connection and disrupted hormone translocation.