Robert W. Ridge

Microtubule Dynamics in Living Root Hairs: Transient Slowing by Lipochitin Oligosaccharide Nodulation Signals

The incorporation of a fusion of green fluorescent protein and tubulin-α 6 from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs. Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting dynamic instability as the main type of microtubule behavior in Lotus root hairs. Comparison of the four standard parameters of in vivo dynamic instability—the growth rate, the disassembly rate, and the frequency of transitions from disassembly to growth (rescue) and from growth to disassembly (catastrophe)—revealed that microtubules in young root hairs were more dynamic than those in mature root hairs. Either inoculation with Mesorhizobium loti or purified M. loti lipochitin oligosaccharide signal molecules (Nod factors) significantly affected the growth rate and transition frequencies in emerging and growing root hairs, making microtubules less dynamic at a specific window after symbiotic inoculation. This response of root hair cells to rhizobial Nod factors is discussed in terms of the possible biological significance of microtubule dynamics in the early signaling events leading to the establishment and progression of the globally important Rhizobium/legume symbiosis.

Recent developments in the cell and molecular biology of root hairs

Recent results in root hair research show that these tip-growing cells are useful models in plant cell biology research. The review covers a range of topics, but there is particular emphasis on the use of mutants in molecular (genetic) analysis.

Micro-vesicles, pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hairs ofVicia hirsuta after freeze-substitution

After freeze-substitution, micro-vesicles were found only in close proximity to the plasma membrane. Macro and pyriform vesicles were found throughout the cytosol, but also ‘packaged’ close to the plasma membrane, the package delineated by electron transparent outlines similar to the endoplasmic reticulum. These outlines appeared to be continuous with nearby endoplasmic reticulum and were always associated with Golgi bodies and microtubules. Micro-vesicles were found only in grazing sections of the plasma membrane made between the apical dome and the region of the nucleus, where the cell is the most cytoplasmic, and only in close proximity to the plasma membrane. Micro-vesicles were also found in close proximity to microtubules as well as other vesicle types. From the results it is suggested that pyriform and micro-vesicles may have specialised roles in root hair tip growth.

Reactive oxygen species from type‐I photosensitized reactions contribute to the light‐induced wilting of dark‐grown pea (Pisum sativum) epicotyls

Type-II, singlet oxygen-mediated photosensitized damage has already been shown to occur in epicotyls of dark-germinated pea (Pisum sativum L.) seedlings upon illumination, resulting in fast turgor loss and wilting. In this study we show evidence that the palette of reactive oxygen species (ROS) is more complex. Hydrogen peroxide, superoxide and hydroxyl radicals are also formed, suggesting the occurrence of type-I reactions as well. Moreover, hydrogen peroxide injection into the epicotyls in the dark was able to provoke wilting directly. Formation of hydroxyl radicals could also be triggered by the addition of hydrogen peroxide in the dark, preferentially in the mid-sections where wilting occurs, showing that potential mediators of a Fenton reaction are present in the epicotyls, but unevenly distributed. Localization of light-inducible ROS formation fully (hydrogen peroxide) or partially (superoxide radicals) overlaps with the distribution of monomer protochlorophyllide complexes, showing that these pigment forms are capable of provoking both type-I and type-II reactions.

The diversity of lectin-detectable sugar residues on root hair tips of selected legumes correlates with the diversity of their host ranges for rhizobia

SummaryFour legumes and two nonlegumes were investigated for the presence of sugars at the tips of their root hairs, using commercially available lectins which have specific affinities for certain sugars. It was found that while only one lectin (RCA-I, which binds to β-D-galactose) bound to narrow-host-range legumes and one nonlegume, five out of ten lectins tested bound to the root hair tips of the broad-host-range legume siratro (Macroptilium atropurpureum). None of the lectins tested bound to any part ofArabidopsis roots. Binding of lectins (and therefore the presence of sugars) only at the tips of growing root hairs has led us to deduce that the sugars are the glyco moiety of membrane-bound glycoproteins that are recycled at the base of the tip apical dome, along with excess plasma membrane that is known to be recycled there. As many kinds of signal transduction molecules are membrane-bound glycoproteins, we suggest that these sugars may be involved in early interactions with rhizobia, and that the broad-host-range legume siratro has more kinds of sugars to cope with the wide range of rhizobia it is able to accept for symbiotic interactions. As far as we know, this is the first report of multiple sugars at the same surface area of a tip-growing plant cell.

Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.

Hormonal Control of Root Hair Growth and Development

Physiological and morphological studies on root hairs trace back to the 18th Century (see Farr 1927a,b,c). The pattern of root hair growth and development is under the influence of various factors including genetic, physiological and environmental factors (see Ridge 1995b; Peterson and Farquhar 1996). Involvement of plant hormones in the control of root hair development and elongation has been the topic of much study. Whether or not a plant hormone is required for normal progress of the developmental process of root hairs, there are at least three criteria to be considered: (1) Production of the hormone by root hairs or supply of the hormone from other sources to root hairs. For this, though it is not easy to demonstrate root hairs per se produce plant hormones, root tissues can supply them to root hairs. In addition, under natural conditions, root hairs are exposed to microorganisms which produce plant hormones (Wang et al. 1982; Pegg 1985). (2) Stimulation of the normal process by exogenous supply of the hormone and/or suppression of the process by application of inhibitors of the hormone and its recovery by the hormone. For this, supportive evidence has been reported. (3) Suppression of the process in mutants related to the hormone such as those deficient in the hormone or hormone response.

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation.

AbstractThe endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.

Analysis of Flagellar Movement in Ginkgo biloba Sperm by High Speed Video Microscopy

The discovery of a freely swimming sperm in Ginkgo biloba at the end of the nineteenth century by Sakugoro Hirase [1–3] was one of the most significant moments in botany, because it led to the establishment of Ginkgo as the true and sole link between the non-flowering primitive plants and the advanced seed plants. Hirase, as both amateur botanist and artist, was able to beautifully depict the sperm [2] and fertilization process in Ginkgo [3], and although he was able to describe the ornamentation of the spiral, from which emanate thousands of flagella, the instruments of the day prevented him from more fully understanding the complexity of the apparatus.

Molecules at the Tips of Root Hairs

Most of the work on this subject is associated with the Rhizobium/legume symbiosis, because the earliest interactions between rhizobia and legume host, at least for many important legume crops, are at the root hair-rhizosphere interface. Despite this, it is surprising that so few laboratories have published in this area of research: the presumed receptor(s) for communication molecules emanating from rhizobia would almost certainly be at the surface of the root hair.