ENDOGENOUS CHANGES OF PHOTOCHEMICAL ACTIVITIES OF SPINACH LEAVES

The activities of green cell-free extracts of spinach leavesin performing photochemical transphosphorylation, photosyntheticphosphorylation, the HILL reaction and the light-induced formationof the endogenous reducing substance (ascorbic acid) were followedin parallel during the growth process of the plant. There wasa certain parallelism between the development of the activitiesof photochemical transphosphorylation, of photo-synthetic phosphorylationand of the HILL reaction, activities being low in the earlierstage of growth, reaching a maximum just before efflorescence,and showing thereafter a more or less sharp decline. The activityin the light-induced formation of endogenous reducing substancewas undetectable for the first 35 day-period of growth, reacheda maximum about one week earlier than the other three activities,and again disappeared after 60 days of growth. (Received September 9, 1960; )     

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes

 Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H⁺-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H⁺ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H⁺-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins. Received: 10 February 2000 / Accepted: 31 March 2000     

Further studies on the role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles

A fungal endo-ß-l,3-glucanase was compared with afungal exo-ß-1,3-glucanase with respect to their effectson elongation and cell-wall extensibility in oat coleoptilesegments. The exo-enzyme enhanced elongation and extensibilityof the cell wall. Its effect was not additive to the effectof indole-3-acetic acid when given together with the latter,at least during 3 hr of incubation. Endo-glucanase showed nosignificant effect on elongation and no interaction with theexo-enzyme. Auxin and exo-glucanase increased extensibilityof the cell wall. The exo-glucanase was separated by isoelectricfocusing. The two fractions which were separated and showedglucanase activity induced elongation and cell wall loosening. (Received March 16, 1970; )     

Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the <Emphasis Type="Italic">Lolium</Emphasis>-<Emphasis Type="Italic">Festuca</Emphasis>species complex

Background  

The agriculturally important pasture grass tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] and centroradialis [CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-located matK gene. Other taxa within the closely related Lolium-Festuca species complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events.     

Comparison of homoeolocus organisation in paired BAC clones from white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and microcolinearity with model legume species

Background  

White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.     

Proton Pumping in Growing Part of Maize Root: Its Correlation with 14-3-3 Protein Content and Changes in Response to Osmotic Stress

The spatial pattern of mitotic activity, cell elongation, rate of H+ fluxes, and 14-3-3 protein content were determined in Zea mays roots. We found that the regions along the apical part of the growing root conversely differ in their proton pumping activity. Higher rate of H+ efflux coincides with higher growth rate and correlates with increased 14-3-3 protein content in membrane preparations. The segment consisting of the root cap and the apical part of the meristem exerts net inward proton pumping, which can be inverted under fusicoccin treatment or osmotic stress. In the latter case, this inversion is accompanied by accumulation of 14-3-3 protein in plasma membranes. The results obtained highlight 14-3-3 protein as an obvious candidate for the fine regulation of plasma membrane H+-ATPase in root apex.     

iTRAQ-based Proteomics Profiling Reveals Increased Metabolic Activity and Cellular Cross-talk in Angiogenic Compared with Invasive Glioblastoma Phenotype

Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.Glioblastoma multiforme (GBM)¹ is the prevalent and most fatal brain tumor in adults with an average patient survival time between 12 and 14 months under current treatment regimens. Invasion and angiogenesis are two defining hallmarks of GBM that are largely responsible for the aggressive nature of the disease (1). Invasion is likely triggered by signals that prompt tumor cells to egress from the tumor mass, including those that are activated by an acidic and hypoxic environment (e.g. hypoxia-inducible factor) (2). These highly infiltrative glioma cells escape neurosurgical resection and are the seeds for tumor recurrence. Oxygen limitation in the tumor microenvironment is also responsible for the active recruitment of new blood vessels from preexisting vessels, a process termed angiogenesis. Absence of angiogenesis is considered a rate-limiting factor in solid tumors. Although high grade gliomas show extensive infiltration of the normal brain they are also among the neoplasms with the highest degree of vascularization (3–5). Antiangiogenic treatment is considered a promising therapeutic strategy against malignant brain tumors and is currently being evaluated in clinical trials (6).In solid tumors the angiogenic switch is thought to occur when the balance between proangiogenic and antiangiogenic molecules is shifted in favor of angiogenesis, permitting rapid tumor growth and subsequent development of invasive and metastatic properties (7). Thus, aggressive tumor growth depends on a successful adaptation of the tumor cells to the host microenvironment. In brain tumors no biomarkers are currently available that define different cell populations within human GBMs (for instance tumor cells that show infiltrative growth and those that trigger angiogenesis) or that predict the propensity of low grade (non-angiogenic) gliomas to develop into malignant angiogenic gliomas. We have recently generated a xenograft model for human GBM that displays a highly invasive phenotype and stem cell characteristics (8). By serial transplantation in nude rats new cell clones eventually develop that generate a more rapidly growing aggressive, angiogenesis-dependent phenotype. The transition to an angiogenic phenotype is accompanied by a reduced infiltrative growth (8). Thus, we are able to initiate two distinct phenotypes from human GBMs that classify their growth and progression. Our model is extremely useful for identifying mechanisms causing the switch from angiogenesis-independent to angiogenesis-dependent tumor growth.This work was aimed at identifying cell membrane markers and molecular pathways that characterize the two phenotypes and may underlie the angiogenic switch. Such markers may represent potential therapeutic targets toward specific cellular subsets within GBMs. Here we applied iTRAQ peptide labeling on membrane-enriched tumor fractions followed by MALDI-TOF/TOF protein identification and bioinformatics analysis to quantify large scale species-specific protein expression over four consecutive generations of the glioma xenograft model.In a search for disease biomarkers, there has been a rapid development of quantitative protein expression technologies including isobaric peptide tagging (iTRAQ) combined with multidimensional LC and MS/MS analysis (9). This approach allows for sample multiplexing (currently 4- or 8-plex at the time). iTRAQ is particularly powerful when applied on a subfraction of the proteome, thereby increasing the possibility of identifying less abundant proteins (10). Because more than a third of all known biomarkers as well as more than two-thirds of known and potential antitumor protein targets are membrane-related proteins (11–14), we focused on membrane-enriched fractions of the tumor xenografts. In four different iTRAQ experiments we were able to identify over 7000 (redundant) proteins of which 1460 proteins were extracted based on quantifiable and species-specific expression. Correspondence analysis and unsupervised cluster analysis confirmed consistent protein expression profiles in the different xenograft phenotypes generated from different patient samples. The expression of a selection of identified candidates was confirmed by immunohistochemical methods on tissue microarrays (TMAs) from a large number of xenograft tumors and patient gliomas. The differentially expressed proteins identified in the two phenotypes represent unique candidate biomarkers that may represent novel therapeutic targets in GBMs. The information generated also provides novel insight into the molecular networks governing the infiltrative and the angiogenic tumor properties and reveals new mechanisms involved in the angiogenic switch in GBMs.     

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.     

The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins

The vacuolar H(+)-ATPase (V-ATPase) is a key enzyme that controls the electrochemical proton potential across endomembranes. Although evidence suggests that V-ATPase is important for photo-morphogenesis, little is known about short-term regulation of V-ATPase upon initiation of the photo-morphogenetic programme by exposure of dark-grown plants to light. In this study, etiolated coleoptiles were given a short blue light treatment and V-ATPase characteristics were determined. The effectiveness of the light treatment was assessed by means of fusicoccin binding to the plasma membrane; this increased 5-fold. The short light treatment also induced a 2-fold to 3-fold increase in the hydrolytic activity of V-ATPase. Members of the 14-3-3 protein family are involved in both blue light perception and the subsequent activation of the P-type ATPase. We provide evidence that 14-3-3 proteins specifically interact with the catalytic A-subunit of the V-ATPase. First, the isolated V1-part of the V-ATPase co-purifies with 14-3-3 on a gel filtration column. Secondly, in an overlay experiment, 14-3-3 interacts with a 68 kDa band that was identified as the V1 A-subunit by mass spectrometry. Thirdly, in 14-3-3 affinity chromatography, both A- and B-subunits of the catalytic moiety of the V-ATPase were identified by matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI TOF/TOF MS) as 14-3-3-interacting proteins. It was shown that the A-subunit can be phosphorylated in vitro by a tonoplast-bound kinase, whose properties are affected by blue light. Taken together, the data show that besides the P- and F-type H(+)-ATPases, the V-type H(+)-ATPase also interacts with 14-3-3 proteins.