Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.     

Soluble and cell-wall peroxidases and auxin destruction in normal and habituated tobacco callus

Summary A comparison of the soluble and cell-wall-bound isoperoxidases of normal auxin-requiring and auxin-independent (habituated) tobacco callus revealed that normal tissues contained a higher level of isoperoxidases. There were also qualitative differences in these isoperoxidases. Partially purified soluble and ionically bound isoperoxidases of normal callus likewise exhibited higher auxin-oxidase activities. Normal tissues also were found to contain higher levels of auxin-oxidase inhibitors (auxin protectors). Overall, however, the data indicate that there is a higher rate of auxin destruction in normal tobacco callus than in habituated tissue. This presumably leads to insufficient endogenous auxin for growth. This study was supported in part by grants to T.G. from the Center IRSIA d'Etude de la Reproduction végétale and the FRFC Contract No. 2.9009. it Was carried out while T.A.T. was the holder of a senior Fellowship under the NATO senior Scientists Program.     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

Direct Measurements of the Pressure and Flow in the Xylem Vessels of Nicotiana tabacum and their Dependence on Flow Resistance and Transpiration Rate

The relationships between xylem tension, velocity of water ascending and transpiration in tobacco plants were measured by means of the “xylem pressure probe technique” (Balling, A. and Zimmermann, U., Planta 182, 325–338, 1990). The flow velocity was determined by suction or injection of fluorescein (or FITC-labelled dextrans of various molecular weights) from the microcapillary of the pressure probe into the punctured xylem vessel, followed by serial-sectioning of the stem after a given propagation time. The distance travelled was defined as the distance from the injection point to the uppermost xylem section in which the dye could be detected. For a transpiration rate of 0.52 ± 0.12 ml . h^?1, a linear dependence between the flow velocity and the tension gradients was found as expected from the Hagen-Poiseuille law. The slope of the straight lines decreased with increasing molecular weight of the fluorescent labelled compound, presumably because of (partial) plugging of the pit membranes. The average value of the flow velocity (2.5 . 10^?4 ± 0.9 . 10^?4 m . s^?1) was one magnitude smaller than the value estimated from the geometric dimensions of the xylem vessels, but agreed well with the literature value of 2.8 . 10^?4 m . s^?1 for herbs (determined by the heat pulse technique; Huber, B. Ber. deutsch. bot. Ges. 50, 89–109, 1932). The average pressure gradient was determined to be 0.39 ± 0.23 MPa . m^?1, in agreement with the literature (Begg, J. E. and Turner, N. C. Plant Physiol. 46, 343–346, 1970). The first response of xylem pressure (or tension) and of flow velocity to a reduction of the transpiration rate (0.14 ± 0.06 ml . h^?1) occurred after about 24 h, when an increase of the xylem pressure towards higher values associated with a decrease in flow velocity was observed. In contrast, re-establishment of the normal transpiration rate brought the pressure (or tension) and the flow velocity back to normal values within half an hour. Similary, introduction of a transverse cross-sectional cut into the stem did not lead during the first 10 h to a change in xylem tension (or velocity). However, during the following day the pressure fell to relatively low values (about ?0.13 MPa). The velocity increased 10-fold. In the next two days the xylem pressure increased again to normal values (average +0.03 MPa), whereas the flow velocity assumed higher values than normal. The data are discussed in terms of the water status and storage of the adjacent tissue cells.     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Induction of Expression Instability of the nptII Gene in Transgenic Tobacco Plants

A comparative analysis of the neomycin phosphotransferase (nptII) gene expression was performed in two groups of transformed tobacco plants, one of which included plants with direct and inverted tandem uidA gene repeats in the T-DNA insertion. This insertion of inverted repeats was shown to reduce the level of stable nptII gene expression to 20%, as compared with 65% in the control transformants. The level of unstable expression of this gene substantially increased (up to 71.4% vs. 5.5% in the control group) when homologous sequences were brought together with direct tandem repeats in the genome of hybrid plants.     

A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.     

Chloride as a macronutrient increases water‐use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO2

Chloride (Cl^?) has been recently described as a beneficial macronutrient, playing specific roles in promoting plant growth and water‐use efficiency (WUE). However, it is still unclear how Cl^? could be beneficial, especially in comparison with nitrate (NO₃^?), an essential source of nitrogen that shares with Cl^? similar physical and osmotic properties, as well as common transport mechanisms. In tobacco plants, macronutrient levels of Cl^? specifically reduce stomatal conductance (g_s) without a concomitant reduction in the net photosynthesis rate (A_N). As stomata‐mediated water loss through transpiration is inherent in the need of C₃ plants to capture CO₂, simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C₃ crop productivity. Our results showed that Cl^?‐mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces g_s and water consumption. Conversely, Cl^? improves mesophyll diffusion conductance to CO₂ (g_m) and photosynthetic performance due to a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, possibly as a consequence of the stimulation of chloroplast biogenesis. A key finding of this study is the simultaneous improvement of A_N and WUE due to macronutrient Cl^? nutrition. This work identifies relevant and specific functions in which Cl^? participates as a beneficial macronutrient for higher plants, uncovering a sustainable approach to improve crop yield.