Super-Resolution Fluorescence Imaging of Arabidopsis thaliana Transfer Cell Wall Ingrowths using Pseudo-Schiff Labelling Adapted for the Use of Different Dyes

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.

Ultrastructure of fertilization in Plumbago zeylanica

The synergidless female gametophyte of <em>Plumbago zeylanica</em> receives the pollen tube through specialized cell wall ingrowths at the base of the egg; tube growth continues between egg and central cells. Pollen tube discharge occurs between egg and central cell and results in release of two male gametes, vegetative nucleus, and some pollen cytoplasm. Except for the location of gamete discharge, details of transmission and fusion of gametic nuclei appear to conform to reports of these processes in taxa possessing conventional embryo sacs.

Structure of syncytia induced by Heterodera schachtii Schmidt in roots of susceptible and resistant radish (Raphanus sativus L., var. oleiformis)

The structure of syncytia induced by <i>Heterodera schachtii</i> Schmidt in roots of susceptible <i>Raphanus sativus</i> L. cv. "Siletina" and resistant radish cv. "Pegletta" was investigated. In the radish cultivar "Siletina" the syncytia most often appeared in the elongation zone of lateral roots. They were initiated in the procambium and pericycle but also included the parenchyma cells of vascular cylinder. In the susceptible cultivar "Siletina" the cells forming the female's syncytia were subject to hypertrophy. Their cytoplasmic density increased. The cytoplasm contained numerous organella. The proliferation of the smooth endoplasmic reticulum took place. Branched cell wall ingrowths were formed next to the vessels. In the male's syncytia the cells were only slightly increased. Their protoplasts contained few organelles. The cell wall ingrowths were poorly developed. In the syncytia of the resistant cultivar "Pegletta" there was only a slight increase of the cell volume. A well developed system of rough endoplasmic reticulum was observed in the protoplast. Distended ER cisterns contained fine fibrillar material. Material of similar structure also appeared in numerous small vacuoles. In resistant plants only some, not numerous, syncytia spreading in procambium fully developed and functioned long enough for the parasite females to mature. At an advanced stage of infection a well developed system of a rough ER was observed also in those syncytia and numerous vacuoles appeared.

Nectary ultrastructure and secretory modes in three species of Tillandsia (Bromeliaceae) that have different pollinators

The floral nectaries of three Tillandsia L. spp. having different pollinators were investigated with transmission electron microscopy (TEM) to describe the previously unstudied ultrastructure of the nectar-producing tissues (primarily the epidermis) and also to determine if any differences in the ultrastructural features could be correlated to pollination mode. We determined that there were variations in nectaries among the three species, and that these may be linked to pollinator choice. Tillandsia seleriana Mez, which has a strict relationship with ants, had a nectary epithelium characterized by abundant dictyosomes and endoplasmic reticulum (ER), and a final degeneration stage possibly leading to holocrine secretion. The presence of protein crystals in epithelial plastids was correlated to a nectar enriched with amino acids and proteins, likely functioning to provide a protein-enriched diet and possibly defence against pathogens. Epithelial cells of the hummingbird-pollinated Tillandsia juncea (Ruiz et Pav.) Poir. nectary displayed cell wall ingrowths and dictyosomes and also contained cytoplasmic lipid droplets and protein crystals within plastids, both of which would enrich the nectar for hummingbirds. The nectary epithelium and the parenchyma of bat-pollinated Tillandsia grandis Schltdl. possessed a few cubic protein crystals in the plastids and its secretion product appeared electron transparent.

The root–fungus interface as an indicator of symbiont interaction in ectomycorrhizae

Seedlings of Alnuscrispa (Ait.) Pursh, Alnusrubra Bong., Eucalyptuspilularis Sm., and Betulaalleghaniensis Britt. were grown in plastic pouches and subsequently inoculated with Alpovadiplophloeus (Zeller & Dodge) Trappe & Smith (two different strains), Pisolithustinctorius (Pers.) Coker & Couch, and Laccariabicolor (R. Mre) Orton, respectively, to form ectomycorrhizae insitu. Alnus seedlings were inoculated with Frankia prior to inoculation with the mycosymbiont. The interface established between A. crispa and A. diplophloeus was complex, involving wall ingrowth formation in root epidermal cells and infoldings in Hartig net hyphae. Alnusrubra – A. diplophloeus ectomycorrhizae had an interface lacking epidermal cell wall ingrowths but with infoldings in Hartig net hyphae. The interface between E. pilularis –. tinctorius consisted of branching Hartig net hyphae between radially enlarged epidermal cells lacking wall ingrowths. Ectomycorrhizae between B. alleghaniensis and L. bicolor developed unique interfaces with radially enlarged epidermal cells near the apical meristem, which synthesized dense vacuolar deposits. Very fine branchings occurred in Hartig net hyphae.

Megagametophyte development in Hordeum vulgare. 2. Later stages of wall development and morphological aspects of megagametophyte cell differentiation

The micropylar quartet of nuclei in the barley megagametophyte is first partitioned by a vertical wall between the synergid nuclei and by an initially horizontal wall between the micropylar polar and egg nuclei. The latter wall continues to grow in an expanding horizontal plane forming much of the upper wall of all three egg apparatus cells and eventually fusing with the megagametophyte wall peripherally. A branch of the egg – polar nucleus wall grows in a micropylar direction and becomes attached to the megagametophyte wall. After partitioning, the egg apparatus is composed of three flat cells having a ceiling wall and two upright supporting walls, which are fused centrally. The micropylar polar nucleus lies just chalazal to the ceiling wall. Expansion of the egg apparatus results in rounding of all three cells followed by lengthening and thinning of their walls in contact with the central cell. Probable membrane contacts may facilitate sperm transmission after pollination. Partitioning of the chalazal quartet of nuclei exhibits many similarities to that of the egg apparatus but with a different cellular arrangement. Transfer cell wall ingrowths appear in cells at both poles of the megagametophyte. Such ingrowths appear in the two synergid cells, representing the filiform apparatus. They also develop in two of the original three antipodal cells where these cells are in contact with the megagametophyte wall. Either the micropylar or chalazal polar nucleus migrates to a position close to the other polar nucleus. Partial fusion of polar nuclei occurs later.

Dissipative structures and morphogenetic pattern in unicellular algae

Patterns of cell wall growth and ornamentation in unicellular algae, mainly in desmids, are compared with patterns generated by Tyson’s Brusselator, a two-morphogen reaction-diffusion model. The model generates hexagonal arrays of points in two dimensions, according well with the observed patterns of surface ornamentation on desmid zygospores. Computed patterns in one dimension and of branching on a circular disc account both qualitatively and quantitatively for morphogenetic patterns that develop following cell division in several desmid genera. Cell wall ingrowths appear to be under similar pattern control to wall outgrowths during morphogenesis, which suggests the involvement of a reaction-diffusion mechanism in establishing and correctly positioning the cell division septum. The application of the model to morphogenesis in Acetabularia and diatoms is also discussed.