The RHDl gene product appears to be necessary for proper initiation of root hairs, whereas the RHDS, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.(Schiefelbein & Somerville, 1990, p.235) The genetic analysis of root hair development has identified several genes that are required for the initiation and growth of the root hair. RHL1, RHL2, and RHL3 genes are active during the formation of a bulge early in root hair growth. RHL1 encodes a nuclear protein of unknown function that is required for the formation of the polarized outgrowth. RHD6 activity is necessary to localize the site of hair initiation in the trichoblast. RHD6 acts through an auxin/ethylene pathway, as the rhd6 mutant phenotype can be rescued by the application of either auxin or ethylene. RHD1 strengthens the cell wall near the bulge. RHD2 is necessary for hair outgrowth, as plants homozygous for recessive loss of function alleles stop growing soon after the formation of a bulge. (Favery et al., 2005, p.80) member of the cellulose syntheses-like gene family of Arabidopsis, AtCSLD3, has been identified by T-DNA tagging. The analysis of the corresponding mutant, csld3-1 showed that the AtCSLD3 gene plays a role in root hair growth in plants. Root hairs grow in phases: First, a bulge is formed and then the root hair elongates by polarized growth, the so-called "tip growth." In the mutant, root hairs were initiated at the correct position and grew into a bulge, but their elongation was severely reduced. The tips of the csld3-1 root hairs easily leaked cytoplasm, indicating that the tensile strength of the cell wall had changed at the site of the tip. Based on the mutant phenotype and the functional conservation between CSLD3 and the genuine cellulose syntheses proteins, we hypothesized that the CSLD3 protein is essential for the synthesis of polymers for the fast-growing primary cell wall at the root hair tip. The distinct mutant phenotype and the ubiquitous expression pattern indicate that the CSLD3 gene product is only limiting at the zone of the root hair tip, suggesting particular physical properties of the cell wall at this specific site of the root hair cell. (Wang et al., 2001, p.575)

Despite previous research, cell rupture is more common in metazoans than previously noted, so rupture and repair processes are now receiving more attention. Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2C triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. (McNeil & Steinhardt, 2003, p.697)

McNeil & Terasaki, conduct research regarding the significance of cell repair. The same criteria used to identify a successfully microinjected or otherwise transiently permeablized cell in vitro can be used to detect and quantify sub-lethal plasma membrane disruption, or 'cell wounding', event in vivo. (2001, p.E124) Research indicates that resealing in plants indicates that many different compartments might be involved depending on the cell type and the size of the lesion. (McNeil & Kirchhausen, 2005, p.500)

Hall et al., discuss in their research that the wound response involves the production of reactive oxygen species, including soperoxide and its production hydrogen peroxide. Hydrogen peroxide is produced both locally and systemically in the plant within 1 hour of wounding. The wound response also involves the proteolytic cleavage of prosystemin into systemin. Linoleic acid is converted into jasmonic acid, which accumulates locally in the plant within 2 hours of wounding. This signaling cascade leads to the activation of wound-induced defense genes within hours. Yet angiosperm epidermal cells may be subject to rupture from mechanical stress, pathogens, and predators. (Hall, MacGregor, Nijsse, & Bown, 2004, p.441)

Actin filaments and microtubules are the two main components of the cytoskeleton in plant and algal cells and generally control distinct processes. Along with their associated proteins, actin filaments generate cytoplasmic streaming and organelle transport whereas microtubules co-ordinate mitosis, cytokinesis, and the guidance of cellulose syntheses...

Orientation of regenerated cortical microtubules was previously studied in small- and medium-sized windows, up to 50 µm in diameter, using microinjection of fluorescently labeled brain tubulin. In contrast, microtubules did not persist but disappeared from the window shortly after chloroplast bleaching. Following re-growth of actin bundles parallel to the streaming direction, microtubules regenerated, and their orientation was random, irrespective of microtubule orientation outside the window. (Foissner & Wasteneys, 1998, p.480)
OBJECTIVES & METHODS:

SPSS software will be utilized in the data collection and compilation process regarding methods used with rhd7 as a model of cell injury and repair in angiosperms. The following questions will be considered regarding testing: First, What percentage of ruptured rhd7-4 root hairs can recover and resume growth when embedded in agarose in standard microscope slide chambers. The researcher expects 100% of newly initiated hairs to rupture based on previous time course studies. To determine if mutant hairs rupture at or before the transition to tip growth, 5 d old seedlings were placed on microscope slides in cooled 0.3% Type VII low gelling temperature agarose (Sigma) in APW. They were then covered with CoverWell perfusion chambers (20 mm diameter X 0.5 mm deep; Grace Bio-Labs Inc., Bend, or, USA) modified by cutting off one side to allow the seedling cotyledons to project out of the chamber. Addition of the chambers produced a thin layer of agarose of uniform thickness, which held the seedlings in place on the slide. Because physical manipulation of seedlings usually inhibits root hair growth, the slides were placed in humid; Para film sealed square Petri plates at an angle of 45 degrees or more from the vertical for at least 3 hours to ensure formation of a new set of root hairs. The perfusion chambers were removed and a drop of fresh APW solution was added, followed by a glass cover slip. A series of digitized images of 10 newly initiated wild type and rhd7-1 root hair outgrowths were recorded at 3 min intervals for up to 63 minutes as previously described. Recording was terminated when rhd7-1 hairs ruptured. Root hair growth over time was determined from the recorded images using image analysis software (#10).

Determinations need to be made regarding what percentage can resume growth under the given circumstances. If using standard methods, incubate for a long period (all day or all night) before determining the percent of recovered roots. Secondly, can the method of growing the roots for microscopy (especially confocal microscopy) be improved? Current method causes roots and root hairs to stop growing when they are transferred to agarose on microscope slides. Therefore, roots must resume normal growth by growing for a minimum of 3 hours on microscope slides in agarose before they can be observed or used in experiments. Researchers indicated the following option, germinate, and grow seedlings in sterile microscope slide chambers (silicon slide chambers), currently in progress, testing, with and without sucrose, without sucrose. Equipment includes: glass slides - standard, Long cover slips e.g. 50 x 22 mm, Silicone sealant - e.g. bathroom mastic, 0.1 M. EGTA solution, pH 8.0, Half strength M&S medium or liquid Arabidopsis growth medium, Petri dishes, Plastic 1ml pipette tips, Glass Pasteur pipettes (#19)

In correlation to research conducted by Ketelaar the wild-type Arabidopsis Col-0 will be utilized in the controls. Seeds will be potted in general-purpose compost and sand and grown in a glass house. After 3 weeks, the plants will be fed with 25 ml of 0.5% ethanol every 3 days, and the phenotype will be recorded. To observe the effects on the root development, seeds will be sterilized by soaking in 10% bleach plus.o5% Triton x-100 for approximately 15 minutes, followed by three washes in sterile distilled water. Germinate and grow seedlings on cover slips (biofoil method) (Ketelaar et al., 2004, p.148) Then germinate and grow seedlings on cover slips in Petri dishes, which can then be removed from the dish and combined with slide for microscopy. (Wymer, Bibikova, & Gilroy, 1997)

What percentages of mature (fully developed) epidermal cells are normally alive or dead on 5 or 6-day-old seedlings? This information is needed to determine requirement for both callose and calcium. We now have a fully developed method to use, Initial questions included: How best statistically to do this? (the current plan is to look epidermis 3 mm from hypocotyls, 10 cells per root, 5 hair-forming cells and 5 non- hair forming cells, no more…