AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue,Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK AFM. Specifically, in this protocol we measure the apparent Young’s modulus of cell walls at subcellular resolutions across regions of up to 100 µm x 100 µm in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.However, several limitations remain: the method can only be used on fairly small samples (around 100 µm in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue’s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.     

Fitness of resprouters versus seeders in relation to nutrient availability in two Plantago species

Two contrasting strategies of plants from disturbed areas are reported to depend on nutrient availability. Resprouters, investing into storage and capable of vegetative regeneration after disturbance, are predicted to be enhanced in nutrient poor environments. This contrasts to seeders, which invest preferentially into seed production and regenerating only from seeds, and are thought to prevail in nutrient rich environments. To test such predicted dichotomy, we set up an experiment with two facultative resprouters with contrasting nutrient demands and assessed the fitness of individuals regenerated from seeds and root fragments in differently productive environments. We hypothesized that 1) plants with higher nutrient demands have a higher fitness as seeders irrespectable of nutrient availability and/or 2) both species will have a higher fitness as resprouters under lower nutrient availability and as seeders when nutrient availability is higher. Nutrient availability was also manipulated prior to and after disturbance to evaluate the impact of changing nutrient availability on the strategy of resprouting. The results of our pot experiment with Plantago lanceolata and Plantago media supported the first but not the second hypothesis. Moreover, high nutrient availability prior to disturbance negatively affected resprouting success, but the growth and fitness of successfully regenerated individuals were enhanced under higher nutrient availability. We concluded that resprouting from roots after disturbance is affected by nutrient availability, but this effect considerably differs between individual life-history stages.     

Mutational analysis of Cak1p, an essential protein kinase that regulates cell cycle progression

In Saccharomyces cerevisiae, entry into S phase requires the activation of the protein kinase Cdc28p through binding with cyclin Clb5p or Clb6p, as well as the destruction of the cyclin-dependent kinase inhibitor Sic1p. Mutants that are defective in this activation event arrest after START, with unreplicated DNA and multiple, elongated buds. These mutants include cells defective in CDC4, CDC34 or CDC53, as well as cells that have lost all CLB function. Here we describe mutations in another gene, CAK1, that lead to a similar arrest. Cells that are defective in CAK1 are inviable and arrest with a single nucleus and multiple, elongated buds. CAK1 encodes a protein kinase most closely related to the Cdc2p family of protein kinases. Mutations that lead to the production of an inactive kinase that can neither autophosphorylate, nor phosphorylate Cdc28p in vitro are also incapable of rescuing a cell with a deletion of CAK1. These results underscore the importance of the Cak1p protein kinase activity in cell cycle progression. Received: 2 January 1997 / Accepted: 20 June 1997     

Localized accumulation of cell wall mannoproteins and endomembranes induced by brefeldin A in hyphal cells of the dimorphic yeastCandida albicans

Summary Candida albicans, a dimorphic yeast, has the abililty to switch its growth form between budding growth and hyphal growth. Since fungal growth involves secretory processes, spatial control of secretion should play a crucial role in such a morphogenetic transition. Brefeldin A (BFA), an inhibitor of the membrane trafficking system of eukaryotes, increases the occurrence of Golgi-like cisternae in the yeast. In the present study, BFA was used to obtain further insights into the spatial organization of secretory processes in hyphal growth ofC. albicans. BFA completely inhibited the formation and growth of germ tubes at a concentration of 35 M or higher. Electron microscopy of BFA-untreated germinated cells revealed many vesicles in the apical region and Golgi-like cisternae in the cytoplasm. In cells treated with 35 M BFA, the vesicles disappeared from the apical region, and, instead, stacked membrane cisternae and membrane-enclosed spherical dense bodies accumulated in the subapical region. These accumulated structures were positive for both polysaccharide staining and immunocytochemical staining with antibodies raised against cell surface antigens ofC. albicans, as were Golgi cisternae in BFA-untreated cells. In cells treated with a higher concentration of BFA (140 M), the structures that appeared in cells treated with 35 M BFA were no longer observed and the endoplasmic reticulum was extended and positive for polysaccharide staining. These results suggested that BFA affects different steps of membrane trafficking in a concentration-dependent manner. The accumulated structures induced by 35 M BFA seemed to be the altered forms of Golgi cisternae. Their accumulation in the subapical region of the germ tube might indicate that the step(s) in membrane trafficking that are associated with the Golgi pathway are vectorially organized in hyphal growth ofC. albicans.Abbrevations BFA brefeldin A - BSA bovine serum albumin - CBB Coomassie brilliant blue - Con A concanavalin A - HRP horseradish peroxidase     

The influence of branches and leaf area on rooting and development of Cotinus coggygria cv. Royal Purple cuttings

The influence of branches and mature leaves on the rooting and subsequent development of cuttings was examined, using Cotinus coggygria cv. Royal Purple. A model system was developed, whereby branched cuttings could be harvested from stock hedges and manipulated to alter leaf area, the number of actively‐growing, lateral branches and thus the source: sink ratio for photoassimilates. Highest percentage rooting ((80%) was promoted by retention of branches and a full leaf area. Reducing leaf area resulted in a lower rooting percentage (44%); however, greatest reductions in rooting were associated with the removal of lateral branches ((22%). Applying exogenous auxin (indole‐3‐butyric acid) at the excision point where branches had been removed significantly improved rooting potential, but did not fully substitute for the presence of branches with active shoot tips. Negative effects associated with removing a proportion of mature leaves appeared to relate to alterations in carbon balance rather than an influence on the supply of endogenous auxin to the potential rooting zone. The use of branched cuttings accelerated root and shoot development and resulted in a finished plant being produced more rapidly than is achieved from conventional, non‐branched cuttings. The results presented indicate a means for improving the efficiency of production of Cotinus coggygria, which may be applicable to a wider range of ornamental plants.     

茎尖和芽的超低温保存

介绍了植物茎尖和芽超低温保存的意义和现状。影响超低温保存的一些主要因素及其所采取的措施,主要包括预培养、低温锻炼、使用冰冻保护剂以及适当采用不同的降温方法和化冻洗涤方法,并就今后的研究提出了一些看法。     

Changes of Endogenous Hormone Contents During Floral Bud and Vegetative Bud Differentiation in Thin Cell Layer Culture of Cichorium intybus L. Explant

The sectioned thin cell layers (TCL) of flower stalk of Cichorium intybus L. were cultured in MS medium supplemented with NAA and BA or IAA and BA where floral and vegetative buds were developed from the explant. Endogenous IAA, DHZ+DHZR, iPA increased significantly during the floral bud formation, while Z+ZR remained changed. The levels of cytokinins, DHZ +DHZR, iPA, and Z-f-ZR all increased significantly during the vegetative bud formation, however IAA level was reduced during the first 7 days of culture and increased to two-thirds of initial values on the day when the bud primordia were formed. The results suggested that the initiation of floral buds was associated with a high IAA/CTK ratio, whereas the induction of vegetative bud differentiation was related to a low IAA/CTK ratio.     

Preliminary Study on Transformation of Wheat Apical Point

Apical points of young seedlings of wheat (Triticum aestivum) cultivar "Jing 411” and somatic calli of cultivar "FK8” were transformed with plasmids pBI121 and (or) pBIAH-A+ by using microprojectile bombardment. Histochemical assay of GUS activity showed positive reaction on some of the transformation processed apical points and calli. This demonstrated that foreign genes were introduced into the apical meristematic cells as well as the callus cells. The plantlets of cv. "Jing 411” survived after apical point transformation with pBIAH-A+ were transplanted into the field and the progenies were screened with kanamycin. 4 % of the screened seeds germinated into green seedlings with kanamycin resistance. Dot hybridization of total DNA from kanamycin resistant plants showed the existence of foreign DNA in some of the detected plants.     

杜仲雌雄株细胞学,顶芽及叶含胶量的比较

杜仲（ＥｕｃｏｍｍｉａｕｌｍｏｉｄｅｓＯｌｉｖ．）为严格的雌雄异株植物,其雌雄株的比例近似于１：１,说明其性别可能由性染色体决定。但在形态上看不到特异的性染色体,雄株花粉母细胞整个减数分裂过程中,同源染色体的配对和分离是正常的。偶尔可发现个别细胞有染色体桥和环状或链状四价体。从１９９３年和１９９４年的１２月到翌年４月芽完全展开前对顶芽的测量说明,雄株顶芽的长度和最大直径都明显大于雌株的（Ｐ＜０．０１）,而整个生长季节中雌株叶子的杜仲胶含量却明显高于雄株的（Ｐ＜０．０１）。不管雄株还是雌株,其叶的含胶量都随季节变化和叶子的长大而降低。实践证明可用芽的大小鉴别杜仲幼株的性别。