Björn J. Sieberer

A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca2+ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca2+ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca2+ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca2+ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca2+ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca2+ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca2+ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca2+ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Mechanism of Infection Thread Elongation in Root Hairs of Medicago truncatula and Dynamic Interplay with Associated Rhizobial Colonization

In temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). To study IT development in living root hairs, a protocol has been established for Medicago truncatula that allows confocal microscopic observations of the intracellular dynamics associated with IT growth. Fluorescent labeling of both the IT envelope (AtPIP2;1-green fluorescent protein) and the host endoplasmic reticulum (green fluorescent protein-HDEL) has revealed that IT growth is a fundamentally discontinuous process and that the variable rate of root hair invagination is reflected in changes in the host cell cytoarchitecture. The concomitant use of fluorescently labeled Sinorhizobium meliloti has further revealed that a bacteria-free zone is frequently present at the growing tip of the IT, thus indicating that bacterial contact is not essential for thread progression. Finally, these in vivo studies have shown that gaps within the bacterial file are a common feature during the early stages of IT development, and that segments of the file are able to slide collectively down the thread. Taken together, these observations lead us to propose that (1) IT growth involves a host-driven cellular mechanism analogous to that described for intracellular infection by arbuscular mycorrhizal fungi; (2) the non-regular growth of the thread is a consequence of the rate-limiting colonization by the infecting rhizobia; and (3) bacterial colonization involves a combination of bacterial cell division and sliding movement within the extracellular matrix of the apoplastic compartment.

A switch in Ca2+ spiking signature is concomitant with endosymbiotic microbe entry into cortical root cells of Medicago truncatula.

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

Endoplasmic Microtubules Configure the Subapical Cytoplasm and Are Required for Fast Growth of Medicago truncatula Root Hairs

To investigate the configuration and function of microtubules (MTs) in tip-growing Medicago truncatularoot hairs, we used immunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similar results and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages. During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hair and remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional array throughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmic dense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, the only remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growing hairs with 1 μm oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes very short, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at a much larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense region remains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with 1 μm oryzalin. The role of EMTs in growing root hairs is discussed.

Cytoarchitecture and pattern of cytoplasmic streaming in root hairs of Medicago truncatula during development and deformation by nodulation factors.

SummaryThe cytoarchitecture and the pattern of cytoplasmic streaming change during the development of root hairs ofMedicago truncatula and after a challenge with nodulation (Nod) factors. We measured the speed and orientation of movement of 1–2 μm long organelles. The speed of organelle movement in cytoplasmic strands in the basal part of growing root hairs is 8–14 μm/s and is of the circulation type like in trichoblasts, bulges before tip-growth initiation, and full-grown hairs. In the subapical area of growing hairs, reverse-fountain streaming occurs discontinuously at a slower net speed. The reason for the slower speed is the fact that organelles often stop and jump. Reverse-fountain streaming is a pattern in which the main direction of organelle transport reverses 180 degrees before the cell tip is reached. Within minutes after their application to roots,Rhizobium leguminosarum-derived Nod factors, cause an increase and divergence in the subapical cytoplasmic strands. This phenomenon can best be observed in the growth-terminating hairs, since in hairs of this developmental stage, subapical cytoplasmic strands are transvacuolar. First, the tips of these hairs swell. The organelle movement in the swelling tip increases up to the level normal for circulation streaming, and the number of strands with moving organelles increases. When a new polar outgrowth emerges, reverse-fountain streaming is set up again, with all its characteristics like those seen in growing hairs. This outgrowth may obtain a new full root hair length, by which these hairs may become twice as long as nonchallenged hairs.

Nod Factors Alter the Microtubule Cytoskeleton in Medicago truncatula Root Hairs to Allow Root Hair Reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

Microtubule dynamics during preprophase band formation and the role of endoplasmic microtubules during root hair elongation.

Plant cells possess a number of distinct microtubule cytoskeleton configurations that are successively being formed, remodeled and broken down during the life of a cell: the interphase cortical microtubules and those encaging the nucleus, the cortical preprophase band, the spindle and the phragmoplast. In order to understand whether and how dynamic instability of microtubules contributes to the transition from a cortical array to the preprophase band, we studied the dynamic instability of individual microtubules in BY2 cells expressing a GFP-microtubule binding domain (GFP-MBD) fusion protein. The step from cortical array to preprophase band was also studied at the level of the microtubule population dynamics. The results showed that microtubule dynamic instability is altered during preprophase band formation and that the process is biphasic. Interphase plant cells have not been reported to possess endoplasmic microtubules, except around the nucleus. However, using both a GFP-MBD fusion protein and immuno-cytochemistry after freeze fixation/ freeze substitution, we found endoplasmic microtubules in the sub-apical area of root hairs of the legume Medicago truncatula. Drug experiments showed that these microtubules are less stable than the cortical microtubules. Real-time 4D confocal microscopy revealed that the endoplasmic microtubules in the subapical region are very dynamic, constantly changing between phases of growth and shrinkage. Moreover, single microtubules were observed to grow from the sub-apical region into the very tip of the root hair. The endoplasmic sub-apical microtubules contribute to the organization of the cell, i.e. the cell architecture, which includes keeping the nucleus at a certain distance from the growing root hair tip. After depolymerization of the endoplasmic microtubules only, the distance between the growing root hair tip and the nucleus became larger, but the nucleus still followed the expanding tip, albeit at a larger distance than before depolymerization of the endoplasmic microtubules. Depolymerization of sub-apical endoplasmic microtubules, alone or along with the cortical microtubules, retarded, but did not stop, hair elongation. This novel microtubule array has not been reported for Arabidopsis root hairs. Further study should show whether it is specific for legumes and related to their interaction with Rhizobium bacteria. * Corresponding author. Tel.: +31-317-484329; fax: +31-317-485005. E-mail address: annemie.emons@pcb.dpw.wag-ur.nl (A.M.C. Emons). Cell Biology International 27 (2003) 295 Cell Biology International

Culturing immobilized plant cells for the TUBUL space experiments on the DELTA and 12S Missions

For the TUBUL experiments during the DELTA mission in April 2004 and 12S mission in March/April 2006 on board the Soyuz capsule and the International Space Station we developed a method to culture and chemically fix plant suspension culture cells. The aim of the ten day experiment was to investigate the effect of microgravity on single plant cells. Fully automated experiment cassettes (Plunger Box Units) were developed by Centre for Concepts in Mechatronics (Nuenen, the Netherlands). Tobacco BY- 2 cells were immobilized in a semi- solid agarose matrix that was reinforced by a nylon mesh. This assembly allowed liquid medium refreshment, oxygen supply and chemical fixation, including a post- fixative wash. The method was optimized for post- flight analysis of cell structure,shape and size, cell division, and the microtubule cytoskeleton. The viability of cells in the agarose matrix was similar to cells grown in liquid medium under laboratory conditions, only the stationary growth phase was reached six days later.