LHPP, the light-harvesting NADPH:protochlorophyllide (Pchlide) oxido¬reductase:Pchlide complex of etiolated plants, is developmentally expressed across the barley leaf gradient

NADPH:protochlorophyllide oxidoreductase is a key enzyme for the light-induced greening of etiolated angiosperm plants. In barley, two POR proteins exist termed PORA and PORB that have previously been proposed to structurally and functionally cooperate in terms of a higher molecular mass light-harvesting complex named LHPP, in the prolamellar body of etioplasts [Nature 397 (1999) 80]. In this study we examined the expression pattern of LHPP during seedling etiolation and de-etiolation under different experimental conditions. Our results show that LHPP is developmentally expressed across the barley leaf gradient. We further provide evidence that LHPP operates both in plants that etiolate completely before being exposed to white light and in plants that etiolate only partially and begin light-harvesting as soon as traces of light become available in the uppermost parts of the soil. As a result of light absorption, in either case LHPP converts Pchlide a to chlorophyllide (Chlide) a and in turn disintegrates. The released Chlide a, as well as Chlide b produced upon LHPP’s light-dependent dissociation, which leads to the activation of the PORA as a Pchlide b-reducing enzyme, then bind to homologs of water-soluble chlorophyll proteins of Brassicaceae. We propose that these proteins transfer Chlide a and Chlide b to the thylakoids, where their esterification with phytol and assembly into the photosynthetic membrane complexes ultimately takes place. Presumably due to the tight coupling of LHPP synthesis and degradation, as well as WSCP formation and photosynthetic membrane assembly, efficient photo-protection is conferred onto the plant

The effect of mitochondrial uncoupling (CCCP, DNP e FCCP) on bovine sperm function

A variável touro tem apresentado um efeito significativo nas taxas de blastocisto podendo variar entre 6,9 e 51,2%. A elucidação dos fatores responsáveis por essas diferenças de desempenho entre touros tem impulsionado diversas linhas de pesquisas dentro da produção in vitro de embriões (PIVE). O estresse oxidativo é descrito como um dos fatores que podem levar a estas baixas taxas no sistema in vitro, causado por uma elevada produção de espécies reativas de oxigênio (EROs). Neste estudo a hipótese testada foi de que o tratamento com desacopladores de membrana mitocondrial reduz a produção mitocondrial de EROs, porém mantém a viabilidade espermática. Os objetivos foram comparar diferentes desacopladores de membrana mitocondrial, em relação à dose e o efeito do tempo de exposição sobre os atributos espermáticos. Para tanto foram realizados tratamentos com diferentes desacopladores [Carbonil Cianeto M-Clorafenilhidrazona (CCCP), 2,4-Dinitrofenol (DNP) e Carbonil Cianeto 4-(Trifluorometoxi) Fenilhidrazona (FCCP)] no sêmen descongelado de bovinos. Os resultados demonstraram que o DNP, dentre os testados, é um desacoplador fraco. A citometria de fluxo evidenciou que o FCCP e CCCP apresentaram redução significativa (p=0,0019, p<0,001, respectivamente) de células espermáticas manifestando marcação negativa para o estresse oxidativo celular e membrana plasmática íntegra. Já a análise computadorizada (CASA) revelou acentuada queda de motilidade com a utilização dos desacopladores CCCP e FCCP, provavelmente pela supressão da produção de ATP através da ATP sintase. O estudo em questão, demonstrou que a motilidade é o atributo mais sensível para o espermatozoide quando submetido ao tratamento com os referidos desacopladores e sugere a suplementação com glicose para promover a estimulação da via glicolítica e assim manter a viabilidade e motilidade destas células. O sêmen quando tratado antes do gradiente de Percoll®, mantém a motilidade constante devido a alguma influência não diagnosticada, mesmo quando suplementada com glicose (5 µM).The bull variable has had a significant effect on blastocyst rates, ranging from 6.9 to 51.2%. Elucidate the factors responsible for these differences in different bull′s performance has been driven several lines of research in in vitro embryo production (IVP). Oxidative stress is described as one of the factors that can lead to these low rates in the in vitro system, caused by a high production of reactive oxygen species (ROS). In this study, the hypothesis tested was that treatment with mitochondrial membrane uncouplers reduces mitochondrial production of ROS, but maintains sperm viability. The aims were compare different mitochondrial membrane uncouplers, in relation to the dose and the effect of time exposure on sperm attributes. Treatments with different uncouplers [Carbonyl Cyanide M- Chloraphenylhydrazone (CCCP), 2,4-Dinitrophenol (DNP) and Carbonyl Cyanide 4- (Trifluoromethoxy) Phenylhydrazone (FCCP)] were carried out in thawed bovine semen. The results showed that the DNP, among those tested, is a weak uncoupler. Flow cytometry demonstrated that FCCP and CCCP showed a significant reduction (p=0.0019, p<0.001, respectively) of the sperm cells negative labeled for cellular oxidative stress and intact plasma membrane. Computerized assisted analysis (CASA) revealed a decrease in motility with the use of the uncouplers CCCP and FCCP, probably due to the suppression of ATP production through ATP synthase. The study in question demonstrated that motility is the most sensitive attribute for sperm when subjected to a treatment with the aforementioned uncouplers and suggests that supplementation with glucose can promote the stimulation of the glycolytic pathway and thus maintain the viability and motility of these cells. Semen, when treated before Percoll® gradient, keeps a constant motility due to some undiagnosed influence, even when supplemented with glucose (5 µM)

Speed of back-swimming of Lymnaea

The pond snail, Lymnaea stagnalis , can locomote on its back utilizing the surface tension of the water. We have called this form of movement ‘back-swimming’. In order to perform this behavior, the snail must flip itself over on its back so that its foot is visible from above. Little is known about the mechanism of this back-swimming. As a first step for the elucidation of this mechanism, we measured the speed of back-swimming of Lymnaea at the different times of the day. They back-swam significantly faster in the morning than just before dark. These data are consistent with our earlier findings on circadian-timed activity pattern in Lymnaea. Lymnaea appear to secrete a thin membrane-like substance from their foot that may allow them to back-swim. To confirm the existence of this substance and to examine whether this substance is hydrophobic or hydrophilic, we applied a detergent onto the foot during back-swimming. A single drop of 1% Tween 20 drifted Lymnaea away that were still kept at the water surface. These results suggest that Lymnaea secrete a hydrophobic substance from their foot that floats to the water surface allowing Lymnaea to back-swim

Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris

The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation of floral transition.Peer reviewedFinal Published versio

The molecular basis of heme oxygenase deficiency in the pcd1 mutant of pea

The pcd1 mutant of pea lacks heme oxygenase (HO) activity required for the synthesis of the phytochrome chromophore and is consequently severely deficient in all responses mediated by the phytochrome family of plant photoreceptors. Here we describe the isolation of the gene encoding pea heme oxygenase 1 (PsHO1) and confirm the presence of a mutation in this gene in the pcd1 mutant. PsHO1 shows a high degree of sequence homology to other higher plant HOs, in particular with those from other legume species. Expression of PsHO1 increased in response to white light, but did not respond strongly to narrow band light treatments. Analysis of the biochemical activity of PsHO1 expressed in Escherichia coli demonstrated requirements for reduced ferredoxin, a secondary reductant such as ascorbate and an iron chelator for maximum enzyme activity. Using the crystal structure data from homologous animal and bacterial HOs we have modelled the structure of PsHO1 and demonstrated a high degree of structural conservation despite limited primary sequence homology. However, the catalytic site of PsHO1 is larger than that of animal HOs indicating that it may accommodate an ascorbate molecule in close proximity to the heme. This could provide an explanation for why plant HOs show a strong and saturable dependence on this reductant.<br/