The incorporation of radioactive proline into cultured cells. Interpretations based on radioautography and electron microscopy.

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-(3)H; 24 hr for proline-(14)C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.

The labeling of cultured cells of Acer with (14C)proline and its significance.

The distribution of the radioactivity from [(14)C]proline that is bound in cultured cells of Acer has been determined by electron microscope autoradiography. In this way proline may be related to the cell wall as a morphological entity rather than as a fraction in a biochemical separation of a heterogeneous crop of cells. The cells in culture may vary greatly. Some are active growing, turgid cells, with thin protoplasts tightly pressed against their walls; in others the protoplasts may spontaneously withdraw from the wall; in still others the protoplasts disorganize, and walls thicken and become sculptured as the cells differentiate and even senesce. Different culturing practices may affect the status of the cells, and this, in turn, affects the distribution of radioactivity from proline in the cells. Cells which are actively growing, turgid, and nucleated have the highest grain density in their protoplasts and nuclei; as the protoplasts of such cells withdraw from their walls, they retain the bulk of the radioactivity. On the other hand, in cells which have thickened walls and sparse protoplast contents, the radioactivity is accumulated in their walls. A high content of proline and hydroxyproline-rich protein is, therefore, not a necessary or invariable feature of the cell walls of cultured Acer cells but depends on the state of development of these cells.

Intracellular localization of arginase in chick kidney.

Although chickens are uricotelic and do not have significant urea-ornithine cycle in any tissue, the kidneys contain a high concentration of arginase which apparently functions to regulate degradation of dietary arginine. A series of investigations has been made to determine the intracellular localization of this arginase in chicken kidney. Tissue fractionation using sucrose density gradients and differential centrifugation showed as association of arginase activity with certain marker enzymes and with fractions identified as mitochondria by electron microscopy. This is consistent with the localization of the arginase in the mitochondrial matrix of chicken kidney cells. Such a finding has significance in understanding the regulation of arginine degradation in chickens.

Pinwheel inclusions in morphogenesis: a possible alternative to induction by viruses.

Pinwheel inclusions (PWs) were found in cells of callus tissue derived from explants of secondary phloem parenchyma of carrot (Daucus carota) storage root and grown on a basal medium containing zeatin and indoleacetic acid or coconut milk, naphthalene acetic acid, or combinations of these. Preliminary attempts to demonstrate the presence of viruses in the callus tissue failed. The possibility that the tissles were infected by a low titer or unstable conventional virus or by a defective mutant has not been ruled out. However, two lines of evidence suggest that the PWs in these tissues may be a result of culture conditions and not of virus infection. First, no PWs or other cytoplasmic inclusions were found in cells of otherwise similar tissue cultured on basal medium alone, and multifibrillar bundles (MFBs) but not PWs were found when the tissues were cultured on a medium that stimulates differentiation and morphogenesis. Second, culture stimulated to differentiate and containing MFBs only were returned to the supplemented basal medium and subsequently found to contain both PWs and MFBs.

Cell wall appositions and plant disease resistance: Acoustic microscopy of papillae that block fungal ingress.

Plant cells react to localized stress by forming wall appositions outside their protoplasts on the inner surface of their cellulose walls. For many years it has been inferred that appositions elicited by encroaching fungi, termed "papillae," may subsequently also deter them and thus represent a disease-resistance mechanism. Recently, it has been shown that preformed, oversized papillae, experimentally produced in coleoptile cells of compatible barley, Hordeum vulgare, can completely prevent direct entry of Erysiphe graminis f. sp. hordei that ordinarily penetrates and causes disease. To discover how these papillae may function, acoustic microscopy was used to contrast their in vivo elastic properties with those of ineffective normal papillae and contiguous cell wall. Raster and line scans showed intense acoustic activity at sites of preformed papillae; scans in selected focal planes identified this activity with the papillae, not with subtending cell wall. Minimal acoustic activity was found in normal papillae. It is suggested that some wall appositions could serve in disease resistance as viscoelastic barriers to mechanical forces exerted by the special penetration structures of advancing pathogenic fungi.