Continuous isolation of plasmid DNA by annular chromatography.

Continuous chromatographic separations, especially of multicomponent mixtures, constitute interesting options for biotechnological downstream processing. Taking the separation of plasmid DNA from clearified lysates on hydroxyapatite as a pertinent example, we discuss the potential of continuous annular chromatography (CAC) in comparison with conventional (preparative) batch chromatography. In CAC the column is realized in the form of a thin (5 mm, height 210 mm) slowly rotating annulus. The performance of such a CAC column is compared to that of an ("analytical") batch column of similar thickness (diameter) and length (4 x 250 mm) and that of a ("preparative") batch column of similar cross-sectional surface area and height (50 x 210 mm). The quality of the obtained plasmid as defined by the appearance of the corresponding agarose gels (native and linearized plasmid), the 260/280 ratio and the biological activity (transient transfection of HEK 293 cells) was found to be identical in all three cases. The yields are also shown to be equivalent. The loading factor is found to be the most decisive parameter for the transfer of a given separation method between the continuous and the batch columns. Under nonoptimized conditions, plate numbers tended to be lower in the continuous compared to the batch columns. This is shown to be largely due to an artifact created by the CAC design (collection of averaged fractions at the outlets) and can be overcome by optimizing the rotation speed. Surprisingly the large batch column consistently gave better plate numbers than either the small batch or the CAC column. Compared to the preparative batch column, wall effects are more pronounced in the CAC (respectively the small diameter batch column), which may translate into better bed stability but conceivably also contributes to an increase in plate height, due to the reduction in bed density usually observed in the proximity of the wall. The CAC is shown to be a powerful approach to continuous chromatography, which allows a direct and straightforward upscale of chromatographic bioseparation methods.     

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Microbial pathogens with impaired ability to acquire host iron

Successful microbial pathogens must be adept in obtaining growth-essential iron from healthy hosts. Some potential pathogens, however, are sufficiently impaired in iron acquisition ability so as to be dangerous mainly in hosts with such iron loading conditions as alcoholism, asplenia, hemochromatosis, -thalassemia major, or tobacco smoking. The association of six impaired pathogens (Capnocytophaga canimorsis, Yersinia enterocolitica and Y. pseudotuberculosis, Vibrio vulnificus, Tropheryma whippelii, and Legionella pneumophila) with iron loaded humans is described.     

Minor vein structure and sugar transport in Arabidopsis thaliana

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex. Received: 5 October 1999 / Accepted: 20 November 1999     

Herbivory could unlock mutations sequestered in stratified shoot apices of genetic mosaics

Many higher plants have shoot apical meristems that possess discrete cell layers, only one of which normally gives rise to gametes following the transition from vegetative meristem to floral meristem. Consequently, when mutations occur in the meristems of sexually reproducing plants, they may or may not have an evolutionary impact, depending on the apical layer in which they reside. In order to determine whether developmentally sequestered mutations could be released by herbivory (i.e., meristem destruction), a characterized genetic mosaic was subjected to simulated herbivory. Many plants develop two shoot meristems in the leaf axils of some nodes, here referred to as the primary and secondary axillary meristems. Destruction of the terminal and primary axillary meristems led to the outgrowth of secondary axillary meristems. Seed derived from secondary axillary meristems was not always descended from the second apical cell layer of the terminal shoot meristem as is expected for terminal and primary shoot meristems. Vegetative and reproductive analysis indicated that secondary meristems did not maintain the same order of cell layers present in the terminal shoot meristem. In secondary meristems reproductively sequestered cell layers possessing mutant cells can be repositioned into gamete-forming cell layers, thereby adding mutant genes into the gene pool. Herbivores feeding on shoot tips may influence plant evolution by causing the outgrowth of secondary axillary meristems.     

A symplasmic flow of sucrose contributes to phloem loading in Ricinus cotyledons

External sucrose, supplied by the endosperm in vivo, is the physiological source of sucrose for Ricinus communis L. seedlings. It is taken up by the cotyledons and exported via the sieve tubes to the growing hypocotyl and root. Two parallel pathways of external sucrose to the sieve tubes, directly via the apoplasm and indirectly after transit through the mesophyll, have already been established (G. Orlich and E. Komor, 1992). In this study, we analysed whether a symplasmic flow of sucrose contributes to phloem loading. Uptake of external sucrose into the mesophyll and into the sieve tubes, and export of total sucrose were measured with intact and exuding seedlings in the presence of p-chloromercuribenzenesulfonic acid (PCMBS). Sucrose uptake into the mesophyll and into the sieve tubes was inhibited by 80–90%. Consequently, export of total sucrose slowed down. However, after the addition of PCMBS, sucrose was transiently exported in such a high amount that could not be accounted for by the residual uptake activity nor by the amount of sucrose confined to the sieve element-companion cell complex (seccc). From the results, we conclude that most of the sucrose exported transiently had moved to the sieve tubes from a symplasmic domain larger than the seccc, comprising at least all the cells of the bundle including the bundle sheath. We suggest that the symplasmic flow of sucrose observed is a mass flow driven by a turgor pressure. As a structural prerequisite for a symplasmic flow, plasmodesmata interconnect all the cells from the bundle sheath to the sieve tubes and also occur between the bundle sheath and the mesophyll. The phloem loading pathway of Ricinus cotyledons can thus be classified as a combination of three different routes. Received: 17 October 1997 / Accepted: 9 March 1998