Potential role of presenilin 1 in regulation of synaptic function

One of the earliest neuropathological symptoms of Alzheimer's disease is the loss of synapses, which preceed the formation of amyloidosis and neurodegeneration. Although most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic disfunction are not yet completely understood. In this paper we analysed of the intracellular and extracellular distribution of PS1 in the cultures of mouse cortical embryonic neurons. We found that PS1 is concentrated on the surface of the growth cone and at neurite contact sites. PS1 was also found in synapses where it is co-localized with synaptophysin. Independent evidense of involvement of PS1 in synaptic function we obtained by transfection of neurons with GFP-PS1 cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained processed N-cadherin. This result presents an additional proof of involvment PS1 in synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare synaptic density in neuronal cell cultures from PS1 knockout mice PS1 (-/-) and wild type mice PS1 (+/+). Our results clearly show that PS1 (-/-) displayed a low number of morphological synapses in comparing with wild type culture PS1 (+/+). In summary, our results indicate a role of PS1 in synaptic function.     

Interactions of Npc1 and amyloid accumulation/deposition in the APP/PS1 mouse model of Alzheimer’s

Although Niemann-Pick C1 disease has frequently been called “juvenile Alzheimer’s”, the effects of introducing Npc1 mutations into a mouse model of Alzheimer’s have not previously been performed. We have crossed Npc1 ^+/− mice with APP/PS1 “Alzheimer’s” mice and studied Aβ42 accumulation and amyloid plaque formation. Mice heterozygous for Npc1 and positive for the APP and PS1 transgenes accumulated Aβ42 more rapidly than the APP/PS1 controls and this correlated, as expected, with the area of amyloid plaques. We conclude that the alterations of intracellular cholesterol present in Npc1 ^+/− mice can influence the progress of Alzheimer’s disease in the APP/PS1 mouse model.     

Genetic influences on Alzheimer's disease: evidence of interactions between the genes APOE, APOC1 and ACE in a sample population from the South of Brazil

Alzheimer’s disease is a complex neurodegenerative disorder. Several genes have been suggested as Alzheimer’s susceptibility factors, the apolipoprotein E (APOE) gene being an established susceptibility gene and the genes coding angiotensin-converting enzyme (ACE) and apolipoprotein C1 (APOC1) being considered possible candidate genes for the disease. The objective of this study was to investigate the association of ACE and APOC1 gene polymorphisms with susceptibility to Alzheimer’s disease and dementia in general, both alone and combined with the APOE gene. Forty-seven patients with dementia in general (35 of them with Alzheimer’s disease) and 85 controls were investigated. The haplotypes E*3/−317*ins and E*4/−317*ins of APOE/APOC1 genes were significantly more frequent in the groups with Alzheimer′s disease and dementia in general (P < 0.001). The frequency of the ACE*ins allele was also greater in the groups with Alzheimer’s disease and dementia in general (P = 0.022; P = 0.045), but genotype frequencies were only different in groups without the E*4/−317*ins haplotype (P = 0.012 for Alzheimer’s disease; P = 0.04 for dementia). Our data point to important genetic interactions involved in these diseases.     

Morphological and functional abnormalities in neuromuscular junctions of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> induced by human <Emphasis Type="Italic">APP</Emphasis> gene expression

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in increased production of amyloid-β-protein (Aβ). To gain insight into the synaptic effects of APP protein in AD patients, wild-type APP, its mutant form APP-Swedish responsible for familial AD, and human beta-secretase gene were expressed in motor neurons of Drosophila melanogaster larvae. It was found that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons caused significant morphological and functional changes in neuromuscular junctions (NMJs)-a dramatic increase in the number of synaptic boutons and altered exocytosis revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed with APP (APP-Swedish) expression, as well as for double transgenes bearing APP (APP-Swedish) and human beta-secretase (BACE), which caused secretion of amyloid beta protein (Aβ). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from Aβ neurotoxic effects.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Applicability of Peak-Scaled Nonstationary Fluctuation Analysis to the Study of Inhibitory Synaptic Transmission in Hippocampal Cultures

At present, there are no direct methods to determine the number of synaptic receptor-related channels activated in the course of synaptic transmission (N) or a value of the single-channel conductance (γ). Peak-scaled nonstationary fluctuation analysis (PS NSFA) should be considered the most well-developed indirect approach used for estimating these parameters. Despite the relatively wide using of this approach for the analysis of various synaptic currents, some aspects of possible errors that can occur in the course of data acquisition or their subsequent processing have not been studied. We examined in detail the problem of applicability of PS NSFA in the study of spontaneous and evoked GABA-ergic inhibitory postsynaptic currents (IPSCs). IPSCs were recorded using a dual patch-clamp technique from hippocampal neurons growing in low-density cultures. Parameters of the recorded IPSCs and values for different components of GABA-ergic synaptic transmission reported earlier were used for simulations and PS-NSFA analysis. In Monte Carlo computer simulations of evoked IPSCs, the influence of series resistance, background noise, asynchronicity of transmitter release, GABA_A channel properties, dendritic attenuation, and instrumental filtering on γ estimates obtained by PS NSFA was examined. We concluded that the γ and, consequently, N values may be satisfactorily estimated by the suggested approach using spontaneous and evoked IPSCs recorded in inhibitory synaptic connections in hippocampal cultures within a wide range of experimental conditions. We also estimated the mean of the single-channel conductance of synaptic GABA_A receptors in neurons from primary hippocampal cultures and found that this value (29 ± 5 pS) agrees well with the high conductance of single synaptic GABA_A receptors observed in acute hippocampal slices. This indicates that dissociated cultures are an adequate model for studying the properties of synaptic GABA_A receptors. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 379–388, July–August, 2004.     

Colocalization between caveolin isoforms in the intestinal smooth muscle and interstitial cells of Cajal of the Cav1+/+ and Cav1−/− mouse

Confocal microscopic images were obtained from the immunohistochemical sections of jejeunum to determine the localization/colocalization between caveolin-1, caveolin-2 and caveolin-3 in intestinal smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC) of Cav1^+/+ and Cav1^−/− mouse. Intestinal regions were segmented [inner circular muscle (icm), outer circular muscle (ocm), myenteric plexus region (mp), and longitudinal muscle (lm)] by LSM 5 and analyzed by ImageJ to show Pearson’s correlation (r _p) and overlap coefficient (r) of colocalization. In the intestine of Cav1^+/+, caveolin-1 (cav1) was colocalized with caveolin-2 (cav2) and caveolin-3 (cav3). Cav2 also was well colocalized with cav3. In the intestine of Cav1^−/−, cav1 and cav2 were absent in all images, but reduced cav3 was expressed in ocm. Caveolae were present in cell types with cav1 in Cav1^+/+, and present with cav3 in ocm of Cav1^−/−. C-kit occurred in deep muscular plexus (ICC-DMP) and myenteric plexus (ICC-MP), in both Cav1^+/+ and Cav1^−/−, and colocalized with cav1 and cav2 in the intestine of Cav1^+/+. Cav3 was absent/present at low immunoreactivity in ICC-DMP and ICC-MP of the intestines of Cav1^+/+ and Cav1^−/−. To conclude, cav1 is necessary for the expression of cav2 in SMC and ICC of intestine and facilitates, but is not necessary for the expression of cav3.     

Cofilin-mediated neurodegeneration in alzheimer’s disease and other amyloidopathies

Transport defects may arise in various neurodegenerative diseases from failures in molecular motors, microtubule abnormalities, and the chaperone/proteasomal degradation pathway leading to aggresomal-lysosomal accumulations. These defects represent important steps in the neurodegenerative cascade, although in many cases, a clear consensus has yet to be reached regarding their causal relationship to the disease. A growing body of evidence lends support to a link between neurite transport defects in the very early stages of many neurodegenerative diseases and alterations in the organization and dynamics of the actin cytoskeleton initiated by filament dynamizing proteins in the ADF/cofilin family. This article focuses on cofilin, which in neurons under stress, including stress induced by the amyloid-β (Aβ) 1–42 peptide, undergoes dephosphorylation (activation) and forms rod-shaped actin boundles (rods). Rods inhibit transport, are sites of amyloid precursor protein accumulation, and contribute to the pathology of Alzheimer’s disease. Because rods form rapidly in response to anoxia, they could also contribute to synaptic deficits associated with ischemic brain injury (e.g., stroke). Surprisingly, cofilin undergoes phosphorylation (inactivation) in hippocampal neurons treated with Aβ_1–40 at high concentrations, and these neurons undergo dystrophic morphological changes, including accumulation of pretangle phosphorylated-τ. Therefore, extremes in phosphoregulation of cofilin by different forms of Aβ may explain much of the Alzheimer’s disease pathology and provide mechanisms for synaptic loss and plaque expansion. An erratum to this article is available at .     

Subcellular localization of PS1 based on PS1/GFP fusing protein

Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer’s disease (EOFAD). The fusion genes, GFPPS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT II (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution. __________ Translated from China Biotechnology, 2006, 26(6): 17-22 [译自: 中 国生物工程杂志]     

<Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as an experimental tool for the study of complex neurological diseases: Parkinson’s disease,Alzheimer’s disease and autism spectrum disorder

The nematode Caenorhabditis elegans has a very well-defined and genetically tractable nervous system which offers an effective model to explore basic mechanistic pathways that might be underpin complex human neurological diseases. Here, the role C. elegans is playing in understanding two neurodegenerative conditions, Parkinson’s and Alzheimer’s disease (AD), and a complex neurological condition, autism, is used as an exemplar of the utility of this model system. C. elegans is an imperfect model of Parkinson’s disease because it lacks orthologues of the human disease-related genes PARK1 and LRRK2 which are linked to the autosomal dominant form of this disease. Despite this fact, the nematode is a good model because it allows transgenic expression of these human genes and the study of the impact on dopaminergic neurons in several genetic backgrounds and environmental conditions. For AD, C. elegans has orthologues of the amyloid precursor protein and both human presenilins, PS1 and PS2. In addition, many of the neurotoxic properties linked with Aβ amyloid and tau peptides can be studied in the nematode. Autism spectrum disorder is a complex neurodevelopmental disorder characterised by impairments in human social interaction, difficulties in communication, and restrictive and repetitive behaviours. Establishing C. elegans as a model for this complex behavioural disorder is difficult; however, abnormalities in neuronal synaptic communication are implicated in the aetiology of the disorder. Numerous studies have associated autism with mutations in several genes involved in excitatory and inhibitory synapses in the mammalian brain, including neuroligin, neurexin and shank, for which there are C. elegans orthologues. Thus, several molecular pathways and behavioural phenotypes in C. elegans have been related to autism. In general, the nematode offers a series of advantages that combined with knowledge from other animal models and human research, provides a powerful complementary experimental approach for understanding the molecular mechanisms and underlying aetiology of complex neurological diseases.     

Familial Alzheimer’s disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer’s disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial HEp2 cells. It is assumed that mutations in familial Alzheimer’s disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

A Comparative Evaluation of the Response to Peroxynitrite by a Brain Endothelial Cell Line and Control of the Effects by Drug Targeting

The potent oxidant peroxynitrite (ONOO⁻) is formed after the combination of nitric oxide with superoxide and has been closely associated with the pathology of inflammatory disease. In particular, the generation of ONOO⁻ has been linked to central nervous system disorders including Alzheimer’s and Parkinson’s disease, multiple sclerosis and bacterial and viral meningitis. Specifically, ONOO⁻ has been implicated in the loss of blood–brain barrier (BBB) integrity during neuroinflammation, but the precise mechanisms through which the molecule acts to mediate neurovascular breakdown have not been established. The disruptive effects of ONOO⁻ could be mediated by either direct or indirect actions on the endothelial cells that comprise the major component of the BBB. The current study has comparatively assessed the direct toxic effects of ONOO⁻ on the brain endothelial cell line, b.End3 and C6 astrocytoma and NA neuroblastoma preparations. b.End3 cells were relatively resistant to ONOO⁻-induced cell death compared with C6 and NA cultures. The indirect involvement of ONOO⁻ in neuroendothelial disruption was pharmacologically determined via adhesion molecule expression and immunocompetent cell attachment to b.End3 cells. ONOO⁻-targeted drugs, including the selective free radical scavenger, uric acid, the decomposition catalyst 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinatoiron (III) (FeTPPS) and the poly(ADP-ribose) polymerase inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino) acetamide hydrochloride (PJ34) revealed that ONOO⁻ was only partly involved in E-selectin, ICAM-1 and VCAM-1 expression on b.End3 cells and also cytokine-induced T-lymphocyte attachment to the cell line. The results indicate that ONOO⁻ contributes to b.End3 cell disruption but is not exclusively responsible for the breakdown of neuroendothelial function.     

Photosystem 2 photochemistry and pigment composition of a yellow mutant of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) under different irradiances

A yellow leaf colouration mutant (named ycm) generated from rice T-DNA insertion lines was identified with less grana lamellae and low thylakoid membrane protein contents. At weak irradiance [50 μmol(photon) m⁻² s⁻¹], chlorophyll (Chl) contents of ycm were ≈20 % of those of WT and Chl a/b ratios were 3-fold that of wild type (WT). The leaf of ycm showed lower values in the actual photosystem 2 (PS2) efficiency (Φ_PS2), photochemical quenching (q_P), and the efficiency of excitation capture by open PS2 centres 1 (F_v′/F_m′) than those of WT, except no difference in the maximal efficiency of PS2 photochemistry (F_v/F_m). With progress in irradiance [100 and 200 μmol(photon) m⁻² s⁻¹], there was a change in the photosynthetic pigment stoichiometry. In ycm, the increase of total Chl contents and the decrease in Chl a/b ratio were observed. Φ_PS2, q_P, and F_v′/F_m′ of ycm increased gradually along with the increase of irradiance but still much less than in WT. The increase of xanthophyll ratio [(Z+A)/(V+A+Z)] associated with non-photochemical quenching (q_N) was found in ycm which suggested that ycm dissipated excess energy through the turnover of xanthophylls. No significant differences in pigment composition were observed in WT under various irradiances, except Chl a/b ratio that gradually decreased. Hence the ycm mutant developed much more tardily than WT, which was caused by low photon energy utilization independent of irradiance.     

Glycine input induces the synaptic facilitation in salamander rod photoreceptors

Glycinergic synapses in photoreceptors are made by centrifugal feedback neurons in the network, but the function of the synapses is largely unknown. Here we report that glycinergic input enhances photoreceptor synapses in amphibian retinas. Using specific antibodies against a glycine transporter (GlyT2) and glycine receptor β subunit, we identified the morphology of glycinergic input in photoreceptor terminals. Electrophysiological recordings indicated that 10 μM glycine depolarized rods and activated voltage-gated Ca²⁺ channels in the neurons. The effects facilitated glutamate vesicle release in photoreceptors, meanwhile increased the spontaneous excitatory postsynaptic currents in Off-bipolar cells. Endogenous glycine feedback also enhanced glutamate transmission in photoreceptors. Additionally, inhibition of a Cl⁻ uptake transporter NKCC1 with bumetanid effectively eliminated glycine-evoked a weak depolarization in rods, suggesting that NKCC1 maintains a high Cl⁻ level in rods, which causes to depolarize in responding to glycine input. This study reveals a new function of glycine in retinal synaptic transmission.     

Photosynthesis of lichen symbiotic alga Trebouxia erici as affected by irradiance and osmotic stress

The relation between oxygen evolution rate (OER) and quantum yield of photochemical reactions in photosystem 2 (Φ_PS2) was examined in lichen symbiotic alga Trebouxia erici Ahmadjian (strain UTEX 911) exposed to different irradiances and osmotic stress (2 M sucrose for 60 h). Linear relationship was found between OER and Φ_PS2 in control cell suspension within irradiance range of 0 – 500 μmol m⁻² s⁻¹. Under osmotic stress, OER and Φ_PS2 were significantly reduced. Relation between OER and Φ_PS2 was curvilinear due to strong osmotically-induced inhibition of OER at high irradiance. The highest used irradiance (500 μmol m⁻² s⁻¹) was photoinhibitory for osmotically-stressed T. erici because non-photochemical quenching (NPQ) increased substantially. Energy-dependent quenching represented major part of NPQ increase. Osmotic stress led also to the reduction of capacity of photochemical processes in PS 2 (F_V/F_M) and increase in F₀/F_M. These changes indicated negative effects of osmoticum on structure and function of photosynthetic apparatus.     

PS1 Expression is Downregulated by Gonadal Steroids in Adult Mouse Brain

Mutations in presenilin (PS) 1 and PS2 genes are associated with early onset (≤65 years) of Alzheimer’s disease (AD). PS1 is involved in γ-secretase mediated cleavage of β-amyloid precursor protein (APP), but its regulation is poorly understood. Sex steroids influence APP cleavage pathways resulting in reduced burden of both intra- and extra-cellular nonamyloidogenic products. As gonadal hormones are implicated in AD and their levels change with age, we have analyzed the effect of 17β-estradiol and testosterone on PS1 expression in the cerebral cortex of adult and old AKR mice of both sexes. Northern and Western-blot analysis revealed that PS1 mRNA and protein expression followed similar pattern of regulation. PS1 expression was downregulated by 17β-estradiol and testosterone in the cerebral cortex of females and adult male, but upregulated in old male mice. Such sex-dependent regulation of PS1 expression during aging by gonadal steroids might account for the PS-related brain functions.     

<Emphasis Type="Italic">In vivo</Emphasis> injected mitochondria-targeted plastoquinone antioxidant SkQR1 prevents β-amyloid-induced decay of long-term potentiation in rat hippocampal slices

Addition of 200 nM β-amyloid 1–42 (Abeta) to a rat hippocampal slice impairs the induction of a long-term post-tetanic potentiation (LTP) of population spike (PS) in pyramidal neurons of the CA1 field of hippocampus. Intraperitoneal injection into the rat of the mitochondria-targeted plastoquinone derivative SkQR1 (1 μmol/kg of weight given 24 h before the slices were made) abolishes the deleterious effect of Abeta on LTP. These data demonstrate that SkQR1 therapy is able to compensate the Abeta-induced impairments of long-term synaptic plasticity in the hippocampus, which are the main cause of loss of memory and other cognitive functions associated with Alzheimer’s disease.     

Gradual Disassembly of Photosystem 2 In Vivo Induced by Excess Irradiance. A Hypothesis Based on Changes in 77 K Fluorescence Spectra of Chlorophyll a in Barley Leaves

Effects of short-term exposure to different irradiances on the function of photosystem 2 (PS2) were studied for barley grown at low (LI; 50 μmol m⁻² s⁻¹) and high (HI; 1 100 μmol m⁻² s⁻¹) irradiances. HI barley revealed higher ability to down-regulate the light-harvesting within PS2 after exposure to high irradiance as compared to LI plants. This ability was estimated from the light-induced decreases of F685/F742 and E476/E436 in emission and excitation spectra of 77 K chlorophyll (Chl) a fluorescence in vivo which was 65 and 10 % for HI plants as compared to 30 and 2 % for LI plants, respectively. For LI plants this protective down-regulation of the light-harvesting of PS2 was saturated at 430 μmol m⁻² s⁻¹, and progressive PS2 photodamage was induced at higher irradiances. After exposure of LI segments to 2 200 μmol m⁻² s⁻¹ a pronounced maximum at 700 nm appeared in emission spectrum of 77 K Chl a fluorescence. Based on complementary analysis of 77 K excitation spectra measured at the emission wavelength 685 nm we suggest that this emission maximum may be attributed to the formation of aggregates of light-harvesting complexes of PS2 (LHC2) with part of PS2 core during progressive PS2 photodamage. Our results can be explained assuming different contributions of LHC2 and PS2 core to the total nonradiative dissipation of absorbed excitation energy for the LI and HI barley. This revised version was published online in August 2006 with corrections to the Cover Date.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.