Piero Picciarelli

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons.

Crude particulate homogenates from leaves of barley (Hordeum vulgare L.), rice (Oryza sativa L.), leaf-beet (Beta vulgaris var. cicla L.) and pumpkin (Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm−3 and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and β-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta 176, 548–550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence).

Hormonal relationships in xylem sap of grafted and ungrafted Prunus rootstocks

Three Prunus species, characterised by different levels of growth vigour, were used as rootstocks for peach [Prunus persica (L.) Batsch] grafting and compared for their hormone transport rates in the sap extracted from both the grafted scions and the ungrafted rootstocks. Two of these species were hybrid rootstocks widely used in peach orchards, while the third was a commercial cultivar, which was used both as rootstock and as scion. The xylem exudates were analysed for the concentrations of indoleacetic acid and for the main cytokinins, namely zeatin, zeatin riboside, isopentenyl-adenine and isopentenyl-adenosine. In ungrafted rootstocks the growth potential was positively correlated with both indoleacetic acid and zeatin riboside transport rates. In the xylem sap of peach scions grafted on the same rootstocks, the growth potential and the zeatin riboside levels were also positively correlated, while a negative correlation was found with indoleacetic acid levels. The results are discussed in view of the most widely accepted hypothesis concerning the control exerted by rootstocks on scion growth.

Fruit-set and Early Fruit Growth in Tomato are Associated with Increases in Indoleacetic Acid, Cytokinin, and Bioactive Gibberellin Contents.

Fruit-set, defined as the activation of a developmental program which converts the ovary into a developing fruit, depends on the crosstalk among plant hormones. Here we show that in pollinated ovaries fruit-set is associated with an increase in indole-3-acetic acid (IAA) content and high transcript levels of ToFZY, a gene involved in the tryptophan-dependent auxin biosynthesis pathway. In unpollinated ovaries developed parthenocarpically in response to the synthetic auxin chlorophenoxyacetic acid (4-CPA), ToFZY mRNA levels and IAA content slightly increase. The most likely sequence of events after fertilization involves auxin-mediated activation of gibberellin (GA) synthesis. Fertilization events not only strongly increase SlGA20ox1 and SlGA20ox3 message levels but also increase SlGA2ox2, SlGA2ox4, and SlGA2ox5 mRNA levels, suggesting a concerted regulation to modulate the level of bioactive GAs, GA1 and GA3. 4-chlorophenoxyacetic acid was found to mimic the fertilization events in the stimulation of SlGA20ox1 and SlGA20ox3 mRNA levels, which were also enhanced and increased earlier, but in contrast with pollinated ovaries, SlGA2ox2, SlGA2ox4, and SlGA2ox5 mRNA levels were repressed leading to higher levels of bioactive GAs. We have also analyzed the content of abscisic acid (ABA) and its metabolites dihydrophaseic acid, phaseic acid, and ABA-glucosyl ester and the level of cytokinins (CKs) (free bases and their corresponding ribosides and ribotides) in pollinated and auxin-treated tomato fruits. We show that ABA levels decrease whereas the levels of free CKs increase immediately after pollination or auxin treatment.

Changes in ultrastructure, protease and caspase-like activities during flower senescence in Lilium longiflorum

The last phase of flower development is senescence during which nutrients are recycled to developing tissues. The ultimate fate of petal cells is cell death. In this study we used the ethylene-insensitive Lilium longiflorum as a model system to characterize Lily flower senescence from the physiological, biochemical and ultrastructural point of view. Lily flower senescence is highly predictable: it starts three days after flower opening, before visible signs of wilting, and ends with the complete wilting of the corolla within 10 days. The earliest events in L. longiflorum senescence include a fall in fresh and dry weight, fragmentation of nuclear DNA and cellular disruption. Mesophyll cell degradation is associated with vacuole permeabilization and rupture. Protein degradation starts later, coincident with the first visible signs of tepal senescence. A fall in total protein is accompanied by a rise in total proteases, and also by a rise of three classes of caspase-like activity with activities against YVAD, DEVD and VEID. The timing of the appearance of these caspase-like activities argues against their involvement in the regulation of the early stages of senescence, but their possible role in the regulation of the final stages of senescence and cell death is discussed.

Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H(2)O(2)). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H(2)O(2) cause an induction of caspase-like proteases previously characterized in physiological nucellar PCD.

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (Ka=1.5 × 109 L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.

Cerato-Platanin Induces Resistance in Arabidopsis Leaves through Stomatal Perception, Overexpression of Salicylic Acid- and Ethylene-Signalling Genes and Camalexin Biosynthesis

Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1–5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψβ-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.

Gibberellin-like activity in suspensors of Tropaeolum majus L. and Cytisus laburnum L.

Gibberellins in the embryo-suspensor system have been considered so far only in Phaseolus coccineus. We present in this report the localization of gibberellin-like substances in the suspensors of Tropaeolum majus L. and Cytisus laburnum L. The total gibberellin activity (expressed as gibberellic-acid equivalent in the α-amylase bioassay) in 2000 suspensors (106 mg fresh weight; FW) of C. laburnum and in 600 suspensors (236 mg FW) of T. majus were 50.9 μg g-1 FW and 8.9 μg g-1 FW respectively.

Proteolytic Activities in Alfalfa (Medicago sativa L.) Leaves

Autodigestion in alfalfa (Medicago sativa L. cv Messe) leaves takes place at a higher rate in the acidic range of pH suggesting that acid proteases are probably involved in protein breakdown. Several lines of evidence show that alfalfa leaves contain various proteolytic activities. A carboxypeptidase active on CBZ-Phe-Met and on other CBZ-dipeptides with a pH optimum of 5.5 has been partially purified by CM-cellulose chromatography. Another acid protease, probably endopeptidase, is active on hemoglobin with an optimum at pH 4.5; perhaps it is not a single protease since it shows another peak of activity at pH 7-7.5. In the neutral-alkaline range of pH there are at least three peptidases active on naphthylamides. One of these, active on Lys-NA, is most active in very young leaves and probably is aSH dependent protease.