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Ethanol fermentation is considered as one of the main metabolic adaptations to ensure energy production in higher plants under anaerobic conditions. Following this pathway, pyruvate is decarboxylated and reduced to ethanol with the concomitant oxidation of NADH to NAD+. Despite its acknowledgement as an essential metabolic strategy, the conservation of this pathway and its regulation throughout plant evolution have not been assessed so far. To address this question, we compared ethanol fermentation in species representing subsequent steps in plant evolution and related it to the structural features and transcriptional regulation of the two enzymes involved: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). We observed that, despite the conserved ability to produce ethanol upon hypoxia in distant phyla, transcriptional regulation of the enzymes involved is not conserved in ancient plant lineages, whose ADH homologues do not share structural features distinctive for acetaldehyde/ethanol-processing enzymes. Moreover, Arabidopsis mutants devoid of ADH expression exhibited enhanced PDC activity and retained substantial ethanol production under hypoxic conditions. Therefore, we concluded that, whereas ethanol production is a highly conserved adaptation to low oxygen, its catalysis and regulation in land plants probably involve components that will be identified in the future., Transcriptional and biochemical comparisons in different phyla suggest the existence of alternative strategies of ethanol fermentation and its regulation in land plants.

As non-photosynthesizing organs, roots are dependent on diffusion of oxygen from the external environment and, in some instances, from the shoot for their aerobic metabolism. Establishment of hypoxic niches in the developing tissues of plants has been postulated as a consequence of insufficient diffusion of oxygen to satisfy the demands throughout development. Here, we report that such niches are established at specific stages of lateral root primordia development in Arabidopsis thaliana grown under aerobic conditions. Using gain- and loss-of-function mutants, we show that ERF-VII transcription factors, which mediate hypoxic responses, control root architecture by acting in cells with a high level of auxin signaling. ERF-VIIs repress the expression of the auxin-induced genes LBD16, LBD18, and PUCHI, which are essential for lateral root development, by binding to their promoters. Our results support a model in which the establishment of hypoxic niches in the developing lateral root primordia contributes to the shutting down of key auxin-induced genes and regulates the production of lateral roots.

The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions.

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

The suspensor of Phaseolus coccineus L. is a short-living tissue, which begins to degenerate at the early cotyledon stage of embryogenesis when it has become no more necessary for further embryo development. The degeneration of terminally differentiated suspensor is considered one of the most typical examples of developmental programmed cell death in plants. We report the occurrence of some hallmarks of animal apoptosis such a leakage of cytochrome C from the mitochondria to the cytosol and an induction of caspase-like proteases, which are sensitive to pH and to specific inhibitors but not affected by generic protease inhibitors.

A meta-clorofenilpiperazina (1-(3-clorofenil)piperazina; mCPP) é uma piperazina sintética que vem sendo apreendida de forma crescente no mercado de drogas ilícitas, primeiro na Europa e, a partir de 2006, também no Brasil. Recentemente há relatos de aumento significativo da apreensão de comprimidos vendidos como ecstasy e que na realidade contêm mCPP, porém estudos sobre potenciais riscos dessa utilização à saúde ainda são escassos. O papel da mCPP como agonista de receptores serotoninérgicos já está bem descrito na literatura, razão pela qual esta substância é amplamente empregada em trabalhos científicos, principalmente psiquiátricos. No entanto, poucos são os trabalhos realizados com o intuito de investigar mais profundamente as ações desencadeadas no organismo pela substância em si. É sabido que a serotonina é um neurotransmissor liberado por plaquetas no local de inflamação e que exerce papel imunomodulatório importante sobre as células imune Assim, considerando (1) a relevância atual da mCPP no contexto de apreensão de drogas de abuso, (2) a necessidade de estudos sobre efeitos das drogas de abuso no organismo, (3) escassez de avaliações dos efeitos da mCPP sobre o sistema imunológico e (4) sua provável correlação com este sistema a partir de suas ações em receptores serotoninérgicos, o presente trabalho pretendeu iniciar investigações acerca da atividade da mCPP sobre as respostas imune inatas e sobre os mecanismos da mobilização leucocitária. Para tanto, ratos Wistar machos foram tratados com mCPP (1mg/kg, v.o.) e foram realizadas quantificações de leucócitos na medula óssea, no compartimento circulante e no foco inflamatório (peritônio), em presença ou ausência de estímulo inflamatório (LPS, 1 mg/mL, i.p.), como também da mieloperoxidase presente em tecido hepático, pulmonar e do baço. Complementando os estudos in vivo, ainda foi quantificada a cortisona plasmática em animais que receberam tal tratamento tendo seus receptores de glicocorticoides previamente antagonizados (RU38486). Neutrófilos coletados do exsudato peritoneal foram incubados nas concentrações de 10 µM, 100 µM e 1000 µM in vitro e investigados a migração neutrofílica, a expressão das moléculas de adesão na superfície neutrofílica, a quantificação de mediadores inflamatórios no sobrenadante de cultura de neutrófilos e o processo de adesão neutrófilo-endotélio, com células endoteliais coletados a partir do cremáster. Os resultados demonstram que a mCPP diminuiu a quantificação de leucócitos no exsudato peritoneal e, concomitantemente, aumentou o influxo de PMN para o tecido pulmonar, em vigência de estímulo inflamatório. Ainda, in vivo, não foi possível observar diferenças na concentração de cortisona sérica entre animais cujos receptores de glicocorticóides foram antagonizados e aqueles cujos receptores em questão encontravam-se normais. In vitro, a mCPP, nas três concentrações empregadas, e tanto na vigência quanto na ausência de estímulo (LPS ou fMLP), causou alterações na migração dos neutrófilos, na adesão deste tipo celular ao endotélio, na expressão de moléculas de adesão (Lselectina, β2-integrina e PECAM-1) na superfície neutrofílica e na quantificação das concentrações de mediadores inflamatórios (NO, IL-1β, IL-10, TNF-α) no sobrenadante de cultura de neutrófilos. Os resultados obtidos sugerem, em conjunto, que a mCPP exerce atividade pró-inflamatória no que se refere à atividade neutrofílica, bem como sugerem que o fármaco seja capaz de amplificar a resposta inflamatória em modelo animal de rato, tanto in vivo quanto in vitro. The meta-chlorophenylpiperazine is a synthetic piperazine which has been seized increasingly in the illicit drug market, primarily in Europe and after 2006, in Brazil. Recently, there has been a significant increase on the apprehension of tablets sold as ecstasy but that in reality contain mCPP. Despite its importance, studies over its potential health risks are few. The mCPP role as serotonergic receptor agonist is well known in the literature, reason why this substance is widely employed in scientific research, particularly in psychiatric studies. But there are few reports that aim to investigate thoroughly the actions that this substance triggers in the organisms. It is also known that serotonin is a neurotransmitter released by platelets at the site of inflammation and plays important immunomodulatory role on immune cells. Therefore, considering (1) the relevance of mCPP in the drug abuse context nowadays, (2) the necessity of researching the effects of the drug abuse in the organisms, (3) the lack of studies containing analysis of the mCPP effects over the immune system and (4) the probable correlation between mCPP and this system, through its activity in serotoninergic receptors, this report intended to lead investigations about the mCPP activity over the innate immune response and over the leukocyte mobilization mechanisms. For this purpose, Wistar male rats were treated with mCPP (1mg/kg, v.o.), their leukocytes were measured in the bone marrow, in the circulating compartment and in the inflammatory foci (peritoneum) in the presence or absence of inflammatory stimuli (LPS, 1 mg/mL, i.p.) and the myeloperoxidase present in liver tissue, lung and spleen was also quantified. In addition to in vivo studies, the cortisone plasma was quantified in animals receiving such treatment with glucocorticoid receptors antagonized previously (RU38486). Neutrophils collected from the peritoneal exudate were incubated at concentrations of 10 mM, 100 mM and 1000 mM in vitro. It was then investigated the neutrophil migration and the expression of adhesion molecules on the surface of neutrophils as well as the quantification of inflammatory mediators in the culture supernatant. The process of neutrophil-neutrophil adhesion to endothelium was also analyzed and endothelial cells were collected from the cremaster. Results reflect that mCPP decreased the amount of leukocytes at the peritoneal exudate and, concomitantly, increased the influx of PMN into the lung tissue during an inflammatory stimulus. Also, in vivo, it was possible to observe differences in the concentration of serum from animals which cortisone glucocorticoid receptors were previously antagonized and those in which the receptors in question were normal. In vitro, mCPP in three concentrations employed, and both in the presence or absence of stimulus (LPS or fMLP) caused changes in the migration of neutrophils, in the cell adhesion to the endothelium, in the expression of adhesion molecules (L-selectin, integrin-β2 and PECAM-1), on the surface of neutrophils and in the concentrations of inflammatory mediators (NO, IL-1β, IL-10, TNF-α) in the culture supernatant of neutrophils. The results suggest that, together, mCPP exerts pro-inflammatory activity with respect to neutrophil activity. They also suggest that the drug is able to amplify the inflammatory response in an animal model rat both in vivo and in vitro.

Rootstocks of fruit trees, commonly used to regulate growth, precocity, fertility, and yield, can also be used to solve problems associated with soils pollution such as copper excess. Despite their potential use for phytoremediation, information on the physiological, biochemical, and molecular aspects of heavy metal toxicity stress on fruit tree rootstocks remains limited. In this study of Prunus cerasifera, a peach rootstock, growth responses, CAT and SOD activity levels, and the modulation of transcription of catalase and superoxide dismutase genes were analyzed after exposure to various copper concentrations. P. cerasifera plantlets tolerated copper concentrations up to 50 mM and unexpectedly showed improved iron uptake under low to moderate concentrations (from 0.1 to 50 mM). At 100 mM of copper, plantlets reduced relative growth rate for both fresh and dry weight and developed severe browning which progressed to necrosis. Stress due to copper toxicity resulted in an increase in total catalase and superoxide dismutase activity and a simultaneous induction of Sod and Cat gene expression. This study demonstrated that P. cerasifera is quite tolerant to copper and mobilizes catalase and superoxide dismutase in order to mitigate copper-stress damages. # 2004 Elsevier Ireland Ltd. All rights reserved.

On calcareous soils, carbonate induced iron deficiency can have heavy effects on growth and development of several fruit crops. Leaf chlorosis, biochemical, and ultrastructural alterations are the first symptoms causing severe damages to yield and productivity. In this work, some physiological, biochemical, and molecular aspects of iron deficiency stress induced on the peach rootstock Mr.S 2/5 in vitro were studied. The aim was to demonstrate if in vitro culture can be used as a valid and fast method to evoke iron deficiency symptoms and to study plant responses to this kind of abiotic stress. Two different treatments were carried out and compared to a control (MS medium): plantlets grown on MS medium completely free of iron and on MS medium containing 1 mM potassium bicarbonate. After 10 and 20 days from the beginning of the treatments fresh and dry weight, chlorophyll, and carotenoids content were measured. Superoxide dismutase and catalase total activity was measured, and gene expression was a...

Summary Strong evidence is presented for auxin regulating lily tepal abscission timing in relation to senescence. Transcriptome data was used to correlate auxin levels with expression of auxin-related genes., Petal wilting and/or abscission terminates the life of the flower. However, how wilting and abscission are coordinated is not fully understood. There is wide variation in the extent to which petals wilt before abscission, even between cultivars of the same species. For example, tepals of Lilium longiflorum wilt substantially, while those of the closely related Lilium longiflorum×Asiatic hybrid (L.A.) abscise turgid. Furthermore, close comparison of petal death in these two Lilium genotypes shows that there is a dramatic fall in fresh weight/dry weight accompanied by a sharp increase in ion leakage in late senescent L. longiflorum tepals, neither of which occur in Lilium L.A. Despite these differences, a putative abscission zone was identified in both lilies, but while the detachment force was reduced to zero in Lilium L.A., wilting of the fused tepals in L. longiflorum occurred before abscission was complete. Abscission is often negatively regulated by auxin, and the possible role of auxin in regulating tepal abscission relative to wilting was tested in the two lilies. There was a dramatic increase in auxin levels with senescence in L. longiflorum but not in Lilium L.A. Fifty auxin-related genes were expressed in early senescent L. longiflorum tepals including 12 ARF-related genes. In Arabidopsis, several ARF genes are involved in the regulation of abscission. Expression of a homologous transcript to Arabidopsis ARF7/19 was 8-fold higher during senescence in L. longiflorum compared with abscising Lilium L.A., suggesting a conserved role for auxin-regulated abscission in monocotyledonous ethylene-insensitive flowers.

Fil: Mignolli, Francesco. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. Fil: Mignolli, Francesco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina. Fil: Vidoz, María Laura. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. Fil: Vidoz, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina. Fil: Mariotti, Lorenzo. Universidad de Pisa. Departamento de Biología; Italia. Fil: Lombardi, Lara. Universidad de Pisa. Departamento de Biología; Italia. Fil: Picciarelli, Piero. Universidad de Pisa. Departamento de Agricultura; Italia. In tomato (Solanum lycopersicum L.), auxin and gibberellins (GAs) cross-talk plays an important role during fruit-set. The entire tomato mutant has been previ- ously reported to carry a deletion in the coding region of the SlIAA9 gene, a member of the auxin signal repressor family Aux/IAA. In this paper, we examined the role of ENTIRE gene in controlling GAs metabolism and directing spontaneous fruit initiation and early ovary growth. It was shown that, similarly to pollinated fruits, facultative par- thenocarpy in entire depends on active GA metabolism, since fruit growth is suppressed when GA biosynthesis is blocked. Analysis of endogenous GAs during the first 10 days after flower emasculation revealed that entire fruits accumulated higher amounts of active GAs (GA1 and GA3) in comparison to wild type pollinated fruits, sug- gesting that a different GA homeostasis regulation occurs. Transcript analysis of the main GA biosynthesis genes showed that differently from unpollinated and non par- thenocarpic wild type ovaries, in entire active GA flux modulation is regulated by the activation of SlGA20ox1 and SlGA20ox2 and also by a marked reduction of GA catabolism (reduced transcription of GA 2b-oxidase genes) during the early fruit expansion phase.

DNA methylation is associated with transcriptional silencing in vertebrates and plants. In mammals, the effects of methylation are mediated by a family of methyl-CpG-binding proteins. In plants the mechanisms by which methylation represses transcription are still not clear. In this paper we describe protein factors in carrot nuclear extracts exhibiting specific affinities for conventional or non-conventional methylation acceptor sites. We characterized two classes of proteins: the first, dcMBP1 (Daucus carota methylated DNA-binding protein 1), shows high affinity for sequences containing 5-methylcytosine; the second, dcMBP2 (Daucus carota methylated DNA-binding protein 2), efficiently complexes sequences containing 5-methylcytosine in both CpXpX and CpXpG trinucleotides and shows much lower affinity for 5-methyl CpG dinucleotides. Both dcMBP1 and dcMBP2 are abundant proteins differing in molecular weight and binding features. Their activities are modulated during carrot vegetative cell growth and somatic embryo development. This is the first time that, in either plants or mammals, proteins exhibiting specific binding affinities for conventional or non-conventional DNA methylation have been shown. Based on these results, the possibility that both the extent and the context of the methylation might contribute to modulate gene expression is discussed.

The complete assignment of the 1H and 13C NMR spectra of benzoylecgonine, a cocaine metabolite, was performed, with the aid of some 2D experiments such as gCOSY and gHSQC.

Senescence is a tightly regulated process and both compartmentalisation and regulated activation of degradative enzymes is critical to avoid premature cellular destruction. Proteolysis is a key process in senescent tissues, linked to disassembly of cellular contents and nutrient remobilisation. Cysteine proteases are responsible for most proteolytic activity in senescent petals, encoded by a gene family comprising both senescence-specific and senescence up-regulated genes. KDEL cysteine proteases are present in senescent petals of several species. Isoforms from endosperm tissue localise to ricinosomes: cytosol acidification following vacuole rupture results in ricinosome rupture and activation of the KDEL proteases from an inactive proform. Here data show that a Lilium longiflorum KDEL protease gene (LlCYP) is transcriptionally up-regulated, and a KDEL cysteine protease antibody reveals post-translational processing in senescent petals. Plants over-expressing LlCYP lacking the KDEL sequence show reduced growth and early senescence. Immunogold staining and confocal analyses indicate that in young tissues the protein is retained in the ER, while during floral senescence it is localised to the vacuole. Our data therefore suggest that the vacuole may be the site of action for at least this KDEL cysteine protease during tepal senescence.

The study evaluated the partial substitution of soybean meal by faba beans (18%) or peas (20%) as additional protein sources in diets destined for typical Italian heavy pig production. It compared animal performances, meat quality, the presence of residual anti-nutritional factors (ANF) and phytoestrogens in plasma and meat and the possible effects on pig health, by evaluating oxidative, inflammatory and pro-atherogenic markers. The results showed that the productive performances, expressed as body weight and feed conversion ratio, of pigs fed with faba bean and pea diets were similar to those of pigs fed only the soybean meal. Meat quality of pigs fed with the three diets was similar in colour, water-holding capacity, tenderness and chemical composition. Despite the higher levels of phytoestrogen in the plasma of pigs fed only the soybean meal, phytoestrogen concentration in the muscle was equivalent to that of animals fed diets with faba beans, whereas pigs fed a diet with peas showed a lower concentration. Inflammation and pro-atherogenic parameters did not show significant differences among the three diets. Overall, the partial substitution of soybean meal by faba beans appears more interesting than with peas, particularly in relation to the higher amount of polyphenols in the diet and the highest concentration of phytoestrogens found in the plasma and muscle of animals, while the pyrimidine anti-nutritional compounds present in the diet did not appear to accumulate and had no effect on the growth performance of animals.

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.

In tomato (Solanum lycopersicum Mill.), auxin is believed to play a pivotal role in controlling fruit-set and early ovary growth. In this paper we investigated the effect of the reduced auxin sensitivity exhibited by the diageotropica (dgt) tomato mutant on ovary growth during early stage of fruit development. Here we show that in hand-pollinated ovaries fruit-set was not affected by the dgt lesion while fruit growth was reduced. This reduction was associated with a smaller cell size of mesocarp cells, with a lower mean C values and with a lower gene expression of the expansin gene LeExp2. When a synthetic auxin (4-CPA, chlorophenoxyacetic acid) was applied to the flowers of wild type plants, parthenocarpic ovary growth was induced. Onthe contrary, auxinapplicationto theflowers of dgtplants failed to induceparthenocarpy. Hand-pollinated ovaries of dgt contained higher levels of IAA compared to wild type and this was not associated with high transcript levels of genes encoding a key regulatory enzyme of IAA biosynthesis (ToFZYs) but with lower expression levels of GH3, a gene involved in the conjugation of IAA to amino acids. The expression of diverse Aux/IAA genes and SAUR (small auxin up-regulated RNA) was also altered in the dgt ovaries. The dgt lesion does not seem to affect specific Aux/IAA genes in terms of transcript occurrence but rather in terms of relative levels of expression. Transcript levels of Aux/IAA genes were up regulated in auxin-treated ovaries of wild-type but not in dgt. Together, our results suggest that dgt ovary cells are not able to sense and/or transduce the external auxin signal, whereas pollinated dgt ovary cells are able to detect the IAA present in fertilized ovules promoting fruit development. Fil: Mignolli, Francesco. Universita Degli Studi Di Pisa; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina Fil: Mariotti, Lorenzo. Universita Degli Studi Di Pisa; Italia Fil: Lombardi, Lara. Universita Degli Studi Di Pisa; Italia Fil: Vidoz, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina Fil: Ceccarelli, Nello . Universita Degli Studi Di Pisa; Italia Fil: Picciarelli, Piero. Universita Degli Studi Di Pisa; Italia

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 analysed the role of the endosperm as main regulator of nucellus PCD. We demonstrated that endosperm produces high amount of ethylene, nitric oxide and indoleacetic acid. We examined the role of these small and diffusible signalling molecules in the regulation of nucellus PCD and we tried to elucidate how they can cooperate and regulate each other into the endosperm. We showed that ethylene acts a positive regulator of nucellus PCD and its synthesis can be in part induced by nitric oxide. High levels of IAA were detected both in the endosperm and in dying nucellus but this hormone is not directly involved in the execution of PCD.

Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ethylene accumulating within the submerged plant tissues, although other signalling mechanisms may also co-exist. In this study, aerenchyma development was analysed in two rice (Oryza sativa) varieties, 'FR13A' and 'Arborio Precoce', which show opposite traits in flooding response in terms of internode elongation and survival.The growth and survival of rice varieties under submergence was investigated in the leaf sheath of 'FR13A' and 'Arborio Precoce'. The possible involvement of ethylene and reactive oxygen species (ROS) was evaluated in relation to aerenchyma formation. Cell viability and DNA fragmentation were determined by FDA/FM4-64 staining and TUNEL assay, respectively. Ethylene production was monitored by gas chromatography and by analysing ACO gene expression. ROS production was measured by using Amplex Red assay kit and the fluorescent dye DCFH(2)-DA. The expression of APX1 was also evaluated. AVG and DPI solutions were used to test the effect of inhibiting ethylene biosynthesis and ROS production, respectively.Both the varieties displayed constitutive lysigenous aerenchyma formation, which was further enhanced when submerged. 'Arborio Precoce', which is characterized by fast elongation when submerged, showed active ethylene biosynthetic machinery associated with increased aerenchymatous areas. 'FR13A', which harbours the Sub1A gene that limits growth during oxygen deprivation, did not show any increase in ethylene production after submersion but still displayed increased aerenchyma. Hydrogen peroxide levels increased in 'FR13A' but not in 'Arborio Precoce'.While ethylene controls aerenchyma formation in the fast-elongating 'Arborio Precoce' variety, in 'FR13A' ROS accumulation plays an important role.

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.

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.

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.

Plant growth regulators are involved in the control of potato (Solanum tuberosum) tuber dormancy. Evidence concerning the role of IAA is controversial; we therefore investigated its role by analyzing two cultivars with varying lengths of dormancy. We examined the time course of free and conjugated IAA in tuber tissue isolates from the final stages of tuber growth to the end of dormancy, the distribution of free IAA in tuber tissues by in situ analysis, and the biosynthesis of the hormone by feeding experiments. The time course of free IAA showed marked differences between the examined cultivars, although the concentration of the auxin generally was the highest at the early stages of tuber dormancy. Immunodetection showed a similar pattern of IAA distribution in both genotypes: in dormant buds from freshly harvested tubers, the free hormone accumulated mostly in apical meristem, leaf and lateral bud primordia, and differentiating vascular tissues underlying the apical meristem, while at the end of the storage period only axillary bud primordia from growing buds displayed appreciable auxin levels. Feeding experiments indicated that changes in IAA biosynthesis rate were a major cause of auxin variation in buds. In both cultivars, dormancy apparently ceased when free IAA fell below a threshold value. Despite this, our data led us to conclude that IAA would not be directly responsible for inhibiting sprouting. Instead, auxin might shorten dormancy, in a cultivar-dependent manner, by enhancing early developmental processes in buds, ultimately leading to dormancy termination.

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.

The suspensor of Phaseolus coccineus L. degenerates at the cotyledonary stage of embryogenesis when it is no longer necessary for continued embryonic development; this degeneration is considered to be a typical example of the so-called developmental programmed cell death (PCD) in plants. The presence of specific hallmarks of PCD as it occurs during the degeneration of P. coccineus suspensor was investigated in the current study. By using the TUNEL assay and electrophoretic analysis, we found evidence of nuclear DNA degradation, a known feature of PCD, in the endosperm and degenerating suspensors. Degeneration of the suspensor begins after degeneration in the endosperm and it starts in the neck region, spreading basipetally towards the knob. We conclude from this study that suspensor degeneration in P. coccineus occurs by means of PCD and displays typical hallmarks of PCD, such as DNA fragmentation. PCD in the suspensor is a highly asynchronous process, originating first in the neck cells and subsequently spreading to the basal cells.

Stomata respond to blue light and it is generally believed that the photoreceptor for this response is located inside the guard cells. Only a small number of blue light photoreceptors have been identified so far, namely cryptochromes and phototropins, and they show overlapping functions in regulating many different responses to light. The possibility that plants may possess other receptors regulating blue light responses under different light conditions cannot be excluded. In this paper we show the presence of two retinal-binding proteins in Commelina communis and we report the identification of retinal, a chromophore usually bound to the photoreceptor rhodopsin and previously identified in algae and other higher plants. We show that, under our experimental conditions, stomata open promptly when exposed to blue light and we demonstrated that this response is dependent on retinal. We hypothesise that rhodopsin-like retinal-binding proteins might be involved in stomatal response to blue light.