Chlorella saccharophila cytochrome f and its involvement in the heat shock response

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

<p>Abstract</p> <p>Background</p> <p>Calcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. <it>Trichoderma </it>spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.</p> <p>Results</p> <p>Here, we investigated the effects on plant cells of metabolite complexes secreted by <it>Trichoderma atroviride </it>wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca²⁺and activation of defense responses. <it>Trichoderma </it>culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen <it>Botrytis cinerea</it>, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (<it>Glycine max </it>L.) cell suspension cultures, <it>Trichoderma </it>and <it>Botrytis </it>metabolite mixtures were distinctively perceived and activated transient intracellular Ca²⁺elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca²⁺signature and cellular effects were modified by the culture medium from the knock-out mutant of <it>Trichoderma</it>, defective for the production of the secreted 42 kDa endochitinase.</p> <p>Conclusion</p> <p>New insights are provided into the mechanism of interaction between <it>Trichoderma </it>and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca²⁺changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.</p

Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in <i>Arabidopsis</i>

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation

Apreçamento de debêntures ilíquidas combinando técnicas de aprendizado não supervisionado e supervisionado

Marking illiquid assets to market is challenging due to the scarcity of information that could indicate their fair price. In the case of illiquid debentures, one of the hindrances to figuring out the proper spread for these kinds of assets is the lack of values from prior trading. This paper proposes to assess the spread of illiquid debentures based on their characteristics, information on the issuers' financial health, and market conditions. The presented method uses unsupervised and supervised learning techniques, combining their respective characteristics to solve the illiquid debentures pricing problem, with applications extending to other types of illiquid assets. In experiments, the proposed methodology proved effective for pricing illiquid debentures with no previous spread value.A marcação a mercado de ativos ilíquidos é um desafio, dada a escassez de informações e negociações no mercado que possam indicar qual deve ser o seu preço justo. No caso de debêntures ilíquidas, uma das dificuldades na determinação do spread apropriado para esses ativos é a falta de valores de negociações anteriores. Este trabalho busca determinar o spread de debêntures ilíquidas com base nas suas características, nas informações sobre a saúde financeira dos emissores e na situação do mercado. O método apresentado utiliza técnicas de aprendizado não supervisionado e supervisionado, aliando suas respectivas características para a solução do problema de apreçamento de debêntures ilíquidas, podendo ser aplicada para outros tipos de ativos ilíquidos. Nos experimentos, a metodologia proposta se mostrou efetiva para apreçar debêntures ilíquidas sem valor anterior de spread

Functional specialization of calreticulin domains

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought