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An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.

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By 2025, more than 500 M people worldwide will suffer from diabetes; 125 M will develop foot ulcer(s) and 20 M will undergo an amputation, creating a major health problem. Understanding how these wounds become chronic will provide insights to reverse chronicity. We hypothesized that oxidative stress (OS) in wounds is a critical component for generation of chronicity. We used the db/db mouse model of impaired healing and inhibited, at time of injury, two major antioxidant enzymes, catalase and glutathione peroxidase, creating high OS in the wounds. This was necessary and sufficient to trigger wounds to become chronic. The wounds initially contained a polymicrobial community that with time selected for specific biofilm-forming bacteria. To reverse chronicity we treated the wounds with the antioxidants α-tocopherol and N-acetylcysteine and found that OS was highly reduced, biofilms had increased sensitivity to antibiotics, and granulation tissue was formed with proper collagen deposition and remodeling. We show for the first time generation of chronic wounds in which biofilm develops spontaneously, illustrating importance of early and continued redox imbalance coupled with the presence of biofilm in development of wound chronicity. This model will help decipher additional mechanisms and potentially better diagnosis of chronicity and treatment of human chronic wounds.

SUMMARYOsmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.

Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis.

Arabinogalactan-proteins (AGPs) are cell wall proteoglycans and are widely distributed in the plant kingdom. Classical AGPs and some nonclassical AGPs are predicted to have a glycosylphosphatidylinositol lipid anchor and have been suggested to be involved in cell-cell signaling. Yariv phenylglycoside is a synthetic probe that specifically binds to plant AGPs and has been used to study AGP functions. We treated Arabidopsis suspension cell cultures with Yariv phenylglycoside and observed decreased cell viability, increased cell wall apposition and cytoplasmic vesiculation, and induction of callose deposition. The induction of cell wall apposition and callose synthesis led us to hypothesize that Yariv binding of plant surface AGPs triggers wound-like responses. To study the effect of Yariv binding to plant surface AGPs and to further understand AGP functions, an Arabidopsis whole genome array was used to monitor the transcriptional modifications after Yariv treatment. By comparing the genes that are induced by Yariv treatment with genes whose expressions have been previously shown to be induced by other conditions, we conclude that the gene expression profile induced by Yariv phenylglycoside treatment is most similar to that of wound induction. It remains uncertain whether the Yariv phenylglycoside cross-linking of cell surface AGPs induces these genes through a specific AGP-based signaling mechanism or through a general mechanical perturbation of the cell surface.Arabinogalactan-proteins (AGPs) are widely distributed in plant species and are located at the plasma membrane and cell wall and in the media of cell cultures. These proteoglycans are typically composed of at least 90% carbohydrate by weight. The AGP core polypeptide is usually rich in Hyp, Ser, Thr, and Ala. Extended motifs comparable to those of extensins are not generally found in AGPs, although short stretches of Hyp alternating with Ala or Ser occur in many AGPs. The sugar moieties are composed of (1/3)-b-D-galactan backbones and (1/6)-b-D-galactan side chains with terminal sugars of Ara or GlcUA (Nothnagel, 1997). In the classical AGPs, the nascent polypeptide chain is synthesized with a C-terminal hydrophobic sequence that is later replaced with a glycosylphosphatidylinositol lipid anchor in the mature protein (Gaspar et al., 2001). The Arabidopsis genome contains approximately 47 genes encoding AGP core polypeptides (Schultz et al., 2002).The abundance of AGP genes and the high degree of posttranslational modifications of AGPs suggest a high genome investment in the synthesis of AGPs, which indicates that these macromolecules have conserved and important roles in plants. Although several possible roles of AGPs have been suggested (MajewskaSawka and Nothnagel, 2000), the detailed biological functions of AGPs currently remain unknown. Many experiments have demonstrated that the expression of AGPs is developmentally regulated in tissueand organ-specific manners (Majewska-Sawka and Nothnagel, 2000). Other experiments showed that AGPs are involved in s...