NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco

The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.     

Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro

Targeted expression of Vitreoscilla haemoglobin (VHb) has been analysed in Nicotiana tabacum plants and suspension cultures under various growth and stress conditions. VHb localization to different cell compartments (cytoplasm, chloroplast and mitochondria) was successful, as judged by signal peptide cleavage. The presence of VHb in subcellular compartments did not result in phenotypical differences between these plant lines. In contrast with previous reports, we were unable to discern any significant changes in growth and other phenotypical characteristics between VHb-expressing and transformed control plants under standard growth conditions. When exposed to nitrosative stress, growth of VHb-expressing cultures was less affected relative to transformed controls. Furthermore, a diminished inactivation of the NO-sensitive enzyme aconitase was observed in the presence of VHb. In contrast, no protective effect of VHb expression against oxidative stress could be detected.     

Restoration of stamen development and production of functional pollen in an alloplasmic CMS tobacco line by ectopic expression of the Arabidopsis thaliana SUPERMAN gene

The alloplasmic male-sterile tobacco line Nta(rep)S, combining the nucleus of Nicotiana tabacum with the cytoplasm of Nicotiana repanda, exhibits cadastral-type anomalies due to a fusion of several stamens with the pistil. These anomalies share similarities with Arabidopsis superman mutants. SUPERMAN (SUP) is a cadastral gene controlling the boundary between whorls 3 (androecium) and 4 (gynoecium). Thus we hypothesized that the expression of the tobacco SUP orthologue might be impaired in the alloplasmic Nta(rep)S line, and that the deficiency could be complemented by the Arabidopsis SUP gene. Here we show that the ectopic expression of SUP in the alloplasmic male-sterile tobacco line Nta(rep)S significantly increases the frequency of flowers possessing free stamens, inducing the recovery of a proper structure for whorls 3 and 4. Furthermore, flowers of transgenic plants show a significant improvement of the morphology of stamens, and more particularly of the anthers, which are able to produce few but functional pollen. The data show that ectopic expression of Arabidopsis SUP reactivates the regulatory cascade of anther development. The plausible causes of the developmental defects of anthers in the alloplasmic male-sterile tobacco line are discussed in relation to the model of regulation of the Arabidopsis SUP gene.     

Impact of the C-N status on the amino acid profile in tobacco source leaves

This paper investigates the influence of the carbon (C) and nitrogen (N) status on the amino acid profile in tobacco source leaves. Treatments used included growing plants at different light intensities, using an antisense RBCS (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) construct to inhibit Rubisco activity, growing plants on 12 or 0.5 mM nitrate, comparing wild-types with genotypes that have small and large decreases in nitrate reductase (NIA) activity, and sampling plants at different times during the diurnal cycle. This combination of experiments provides information on how amino acid levels respond to several inputs including the C and N status, nitrate, excess light and light-dark transitions. The data set was analysed using principal component analysis, regression analysis and by normalizing the level of each individual amino acid on the total amino acid pool. Most amino acids show a downward trend when the C or the N status is decreased, and rise during day and fall at night during the diurnal cycle. However, individual amino acids often showed deviating responses. Furthermore, no evidence was found for feedback inhibition of minor amino acid synthesis, either within or between pathways, when 18 individual amino acids were supplied to detached leaves. Results indicate that regulation of amino acid metabolism, for example by the C and N status, leads to qualitatively similar responses of many amino acids, but homeostatic mechanisms involving feedback inhibition within or between individual amino acid biosynthesis pathways are not stringent. All of the above inputs affect the level of phenylalanine, an amino acid that is also the substrate for an important sector of secondary metabolism. The levels of glutamate were remarkably constant, indicating that unknown mechanisms stabilize the concentration of this key central amino acid. Analyses of metabolite levels and feeding experiments indicated that 2-oxoglutarate plays an important role in regulating glutamate levels. Glutamate was the most effective inhibitor of NIA activity when 18 individual amino acids were supplied to detached leaves. Feeding glutamate, and other downstream amino acids, led to an increase of glutamine, indicating glutamate exerts feedback regulation on ammonium metabolism.     

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.     

Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells

Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants.     

IMAC/TiO(2) enrich for peptide modifications other than phosphorylation: implications for chromatographic choice and database searching in phosphoproteomics

Protein regulation by reversible phosphorylation is fundamental in nature, and large-scale phosphoproteomic analyses are becoming routine in proteomics laboratories. These analyses utilise phosphopeptide separation and enrichment techniques linked to LC-MS/MS. Herein, we report that IMAC and TiO(2) also enrich for non-phosphorylated modified peptides such as acetylated, deamidated and carbamylated peptides. Urea and digestion conditions commonly used in phosphoproteomic workflows are the likely sources of the induced modifications (deamidation and carbamylation) and can easily modify phosphopeptides. Including these variable modifications in database searches increased the total number of identified phosphopeptides by 15%. We also show that strong cation exchange fractionation provides poor resolution of phosphopeptides and actually enriches these alternatively modified peptides. By switching to reverse-phase chromatography, we show a significant improvement in the number of identified phosphopeptides. We recommend that the users of phosphopeptide enrichment strategies avoid using urea as a denaturant and that careful consideration is given to chromatographic conditions and the types of variable modifications used in database searches. Thus, the capacity of IMAC and TiO(2) to enrich phosphopeptides bearing modifications other than phosphorylation is a previously unappreciated property of these chromatographies with practical implications for the field of phosphoproteomics.     

Expression and localization of FAD2 desaturase from spinach in tobacco cells

The sites of desaturation in plant cells were studied by expressing the fusion gene composed of the genes encoding FAD2 (Δ12 desaturase) from spinach (Spinacia oleracea) and enhanced green fluorescent protein (EGFP) under the control of the 35S cauliflower mosaic virus promoter. The chimeric protein was functional in tobacco (Nicotiana tabacum) BY-2 cells. The temporal changes in distribution and localization of fusion FAD2-EGFP protein were studied. According to the results obtained, we can conclude that the sites of desaturation in plant cells are associated with both the endoplasmic reticulum and plasma membrane.