Wound Healing in Plant Cells

Wound Healing in Plant Cells

The Current study will attempt to further clarify and utilize Arabidopsis thaliana in studying wound healing in plants as well as the most effective means in studying the process. Root hairs are not essential for plant growth and development and are convenient to study since they are on the exterior of the root. The simplicity of the patterning and the range of mutants with defects in hair pattern and morphology make the Arabidopsis root hair a useful model for the study of plant cell growth and for tip growth in particular. There are likely to be many parallels between the growths of the different types of tip-growing cells, e.g. plant pollen tube growth, fungal hyphal growth, and algal rhizoids. Previous studies indicate that, mutations have been identified that affect Arabidopsis root epidermis development. These mutations define genes that influence early stages of epidermal cell type specification, genes that affect the initiation of root hairs, and genes that affect root hair enlargement.In root hairs, calcium must flow into the tip from outside the hair in order for the hair to grow. If the external calcium is removed or if the calcium ion channels are blocked, a young root hair stops growing. Old hairs stop growing because of modifications to the number or activity of their calcium ion channels - calcium simply stops flowing into the hairs, and growth stops. However, calcium ions are not needed for the outgrowth of a new root hair, just for tip growth. Since rhd7 root hairs usually rupture during outgrowth, we can manipulate calcium ions to determine if calcium is needed for wound plugging. This will be tested in the current study.

INTRODUCTION:

Arabidopsis thaliana has been utilized in several previous studies due to its small size and the ability to conduct molecular genetic analysis easily. (Galway, Heckman, & Schiefelbein, 1996, p.209) Arabidopsis thaliana is interesting to use due to the previous use as a model plant in the study of cell differentiation. Researchers have reported that Arabidopsis root consists of root hair-producing cells they are derived from atrichoblasts. (Ryan, Steer, & Dolan, 2000, p.140)

Root hairs are not essential for plant growth and development and are convenient to study since they are on the exterior of the root. The simplicity of the patterning and the range of mutants with defects in hair pattern and morphology make the Arabidopsis root hair a useful model for the study of plant cell growth and for tip growth in particular. There are likely to be many parallels between the growths of the different types of tip-growing cells, e.g. plant pollen tube growth, fungal hyphal growth, and algal rhizoids. (Carol, 2002, p.815)

In most cells of the root, hypocotyls and leaf epidermal trichomes, growth is spread over a large proportion of the cell surface, producing diffusely growing cells. Development of some cells, such as leaf epidermis pavement cells, might involve a combination of localized tip growth and diffuse growth to produce strangely shaped interlocking cells. In higher plants, amongst all other cellular components, the two major cytoskeleton elements actin microfilaments and microtubules appear to play a decisive role in the shape-determining process. Treatment of plant cells with cytoskeleton-interacting drugs has been particularly helpful in developing this view and has allowed particular changes in cell morphology to be linked to altered activity of specific cytoskeletal proteins; microtubules are implicated in the establishment and maintenance of polar growth directionality. (Mathur, 2004, p.584)

Many of the important unanswered questions in root development involve events that occur at the root apex. Little about the nature of the stem cells, how cell files are established, how cell numbers and vascular patterning are determined, what controls the organization and size of the meristem, how root hair initials are formed, how cell expansion is regulated, and, perhaps most important, what controls the cell cycle and the planes of cell division. Many of these are general questions that apply equally well to morphogenesis in other parts of the plant. However, several aspects of root morphogenesis serve to simplify the study of these basic questions. (Schiefelbein & Benfey, 1991, p.1147)

Insights into cell growth and patterning obtained from the root hair are derived from a multifaceted approach to understanding the biology of this cell type. The characterization of proteins required for various cellular activities that localize to the growing tip is providing instructive information regarding tip growth. Researchers have shown that Rop GTPases localize to the tip of the growing hair, and disruption of their function results in the development of abnormal hairs. Likewise, the genetic dissection of the process of tip growth is providing a genetic framework on which to build a molecular analysis of tip growth. (Dolan, 2001, p.553)

Several loci involved in Arabidopsis root hair tip growth have been isolated, including RHD2, RHD3, RHD4, and TIP1. These genes may encode products that affect known tip growth factors, such as the cytoskeleton associated proteins, or calcium ion transport. The rhd2 mutants possess "stubby" hairs that are apparently caused by an inability to expand past the initial swelling stage. The phenotypes rhd3 and rhd4 are "wavy hairs" and "bulging hairs" respectively, and have been interpreted as having defects in the control of cell expansion polarity at the root hair tip. By crossbreeding and producing double mutants, Researchers were able to show that the RHD2 gene product is required before the RHD3 or RHD4 products and that RHD3 and RHD4 products probably act in separate pathways. Although the RHD3 and RHD4 genes apparently affect root hairs specifically, the RHD3 gene is required for normal cell expansion in many plant tissues, and has a similar action to the COBRA gene. (Ridge, 1995, p.402)

Mutants with defects in potassium transport are providing insight into the role of potassium during root hair elongation. Tiny root hair mutants form short root hairs and frequently initiate more than one root hair per trichoblast. (Foreman & Dolan, 2001, p.3) Iron deficiency causes a number of visible symptoms in plants, such as severe chlorosis and changes in the morphology of the roots, enhanced formation of root hairs and the development of transfer cells in the root apical zones. In the same root zones, iron deficiency enhanced proton extrusion and induced the "Turbo reductase" system, a description that comes from the enhancement of the reduction of external ferric compounds by intact roots. This reducing capacity (in vivo activity) has been identified as the enhanced activity of a plasma membrane bound ferric chelate reductase. (Moog, van der Kooij, Bruggemann, Schiefelbein, & Kuiper, 1994, p.505)

Researchers state in journal article (#10), that rhd7 mutations are new alleles of CslD3 Two root hair mutants independently identified by J.S. Schiefelbein (rhd7-1) and G.O. Wasteneys (rhd7-4) both exhibited root hair rupture. rhd7-1 was one of three rhd7 alleles, and was previously designated RM57 (Moog et al., 1995). Complementation tests revealed that rhd7-1 and rhd7-4 were alleles. Subsequent crosses between rhd7 mutants and the kjk-2 mutant also failed to complement the mutant phenotype. Hence, Rhd7 is identical to Kojak, also designated CslD3. Here we will use rhd7, kjk and csld3 plus the allele number in order to refer to specific alleles of this gene, and CslD3 to refer to the non-mutated gene. Single point mutations were identified in the coding region of CslD3 in both rhd7-1 and rhd7-4. The mutation in rhd7-4 is identical to that reported for kjk-1, although the latter was isolated from a mutagenized Landsberg erecta population. C is replaced by T, converting the arginine codon in the highly conserved QXXRW motif to a stop codon. The novel point mutation in rhd7-1 similarly replaces an initial C. with a T, converting a glutamine codon Q951 to a stop codon. This mutation is located in the short sequence between the predicted third and fourth membrane spanning domains. (#10)

Previous studies indicate that, mutations have been identified that affect Arabidopsis root epidermis development. These mutations define genes that influence early stages of epidermal cell type specification, genes that affect the initiation of root hairs, and genes that affect root hair enlargement. Mutations affecting the RHD3 gene are unique because they alter the enlargement of root hairs as well as the root proper, the root hairs exhibit a short and wavy morphology, and the roots possess a reduced length. Detailed ultra structural analyses of growing rhd3 root hairs have shown that vacuole formation is reduced and secretory vesicle distribution is altered. Because the rhd3 mutations alter the size of roots and root hairs, the RHD3 product may be involved in a fundamental plant cell expansion mechanism. (Wang, Lockwood, Hoeltzel, & Schiefelbein, 1997, p.800)

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHDP, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. 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 complexes during cell wall deposition. 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 than three per file) There are quite a few dyes that do not enter live cells, but do enter dead cells (example: propidium iodide, FB28, trypan blue, possibly Congo red). Alternatively, they enter live cells, and undergo a reaction in live cells, but not in dead cells. For example, stain the seedling roots in a non-fluorescent compound fluorescein diacetate. This diffuses into living cells and is converted to fluorescein, a fluorescent compound. The live cells are green fluorescent and the dead cells look dark. What is the best dye to use? I successfully determine the live and dead cells using propidium iodide, because it is not controlled by the plant like the fluorescein produced from fluorescein diacetate, nor is it unstable on exposure to light, like FB28 and possibly Congo red).Do we only look at files of hair-bearing cells? (No) Could there be a bystander effect on non-hair forming cells? (the data will tell us, but cell death and crushing by neighbors may be restricted to hair-bearing cells)