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First published online December 29, 2005; 10.1104/pp.105.072876 Plant Physiology 140:661-670 (2006) © 2006 American Society of Plant Biologists
Architecture of Infection Thread Networks in Developing Root Nodules Induced by the Symbiotic Bacterium Sinorhizobium meliloti on Medicago truncatula1,[W]Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269–3125
During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10- and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.
The development of biological branching structures has been studied from theoretical, biomechanical, cellular, and molecular points of view, using model systems that include plant branches, plant roots, blood vessel networks, neuron dendrites, and tracheal development in invertebrates (Farnsworth and Niklas, 1995  ; Uylings and van Pelt, 2002; Uv et al., 2003; Masters, 2004; McColloh and Sperry, 2005; Wu et al., 2005). What these networks share are common solutions to the problems of optimizing resource uptake and waste dispersal. Many biological branching structures develop their final size and form in response to gradients in light, water, nutrients, or signaling molecules emanating from target and nontarget tissues. Work in this article describes the system of branched tubules, infection threads (ITs), that develop during symbiotic nodulation of certain plant roots. These branched networks share features in common with many other well-studied branched networks, including growth by tip extension and directed growth to deliver resources (the bacterial symbiont).
The symbiosis between Medicago truncatula, alfalfa (Medicago sativa), and their nodulating symbiont, Sinorhizobium meliloti, begins when the bacterium detects flavonoids and other compounds released by host plants. These activate S. meliloti transcriptional regulators, which then induce the expression of about 25 bacterial nod genes required for the biosynthesis of the lipooligosaccharide signaling molecule, Nod factor, which is obligatory for nodulation (Mulligan and Long, 1989
The extension of root hairs is one of the few plant developmental processes that takes place by tip growth. Vesicles containing plant cell wall and cell membrane material travel in an actin- and microtubule-dependent fashion to the root hair tip where they fuse with the cell membrane, thus adding cell membrane to the tip and depositing cell wall material outside the membrane (Peterson and Farquhar, 1996
The IT itself, both in root hairs and in host cells that are infected at later stages, is a bacteria-filled invagination of the plant cell wall and its underlying plasma membrane. Thus, the lumen of the IT contains bacteria, secreted bacterial products such as exopolysaccharide and lipopolysaccharide, and material similar to that which makes up the plant extracellular matrix (Fraysse et al., 2003
Early during infection, the growth of the IT network is highly polarized. Threads initiate in root hairs and then branch and ramify, to some degree, in both epidermal and cortical cells as they grow inward toward the developing nodule (Gage, 2004
Most of the volume of outer cortical cells is occupied by a large, centrally placed vacuole. Upon activation, cytoplasm moves from the cell periphery to a central position in those outer cortical cells located in columns above activated cells in the pericycle, inner, and middle cortex (van Brussel et al., 1992
Once mature, indeterminate nodules contain at least five recognized zones (Vasse et al., 1990 Little is known about how ITs grow and develop in nodules. A fundamental question about the organization of IT networks inside nodules is whether, as a whole, they show growth toward the nodule meristem or whether their growth is random within the infection zone with only some branches developing toward the meristem. It is also unknown whether characteristics of IT networks remain fairly constant over time or change as nodules develop. Examination of the architecture of IT networks in nodules of different ages was thought likely to provide insight into how IT network architecture changes as nodules develop from immature to mature forms.
Overview of the Analyses
Three-dimensional (3-D) representations of IT networks were reconstructed from nodules harvested 5, 10, and 30 d after spot inoculation. These time points were chosen because they represent distinct stages of nodule development (Vasse et al., 1990
IT networks in 5-, 10-, and 30-d-old nodules shared many common structural features. They were generally made up of one or more fairly simple tree-like branching structures that occupied 1.5% to 3.0% of the volume of nodule tissue in zone II and interzone II-III (Table I). In some cases, network subtrees were likely connected by branches that were outside the nodule volume that had been reconstructed (Fig. 2). This was especially true for the larger 10- and 30-d-old nodules. One 5-d-old nodule had network subtrees that may have arisen from separate infection events (Fig. 2). Main ITs were generally oriented along the long axis of the nodule and exhibited lumps and protrusions, some of which were infection droplets, the sites of bacterial release. These could be discerned because they had a rather characteristic, wide, rectangular shape in the 3-D reconstructions, were contained within one nodule cell, and appeared to not be bounded by plant cell wall material in the stained sections (Supplemental Fig. 1). The average length of terminal (youngest) branches and the branches from which they arose (first and second order, respectively), and the average of all branches (including branches with orders >2) were similar among nodules of different ages (Fig. 3). The only major exceptions to this were the shorter branches found in one 5-d-old nodule that was relatively small (Figs. 2 and 3). Second-order branches were consistently longer than the terminal branches.
Structure of IT Networks in 5-d-Old M. truncatula Nodules Five-day-old nodules were developmentally immature. Meristems had not yet developed to the point where they occupied a majority of the nodule apex. They contained developing zones I and II and relatively few nodule cells with released bacteria. Filled nodule cells with elongated bacteroids characteristic of interzone II-III had not yet developed. Network structure in the 5-d-old nodules was more complex than that seen in the older nodules in that the direction of branch growth was oriented in two main directions. Most of the branches of the IT networks were directed inward toward the root (Figs. 1 and 4), even though meristematic regions were developing near the nodule apexes. These inward-directed branches were not merely remnants of threads that developed in the root cortical layers, but likely developed in nodule primordium during the development of the outward-growing meristem. Both 5-d-old nodules showed well-defined regions where ITs were not growing inward, but rather were growing toward the nodule apex (Fig. 1, E, H, and I). In one case, the outward-growing branches were directed toward the only part of the nodule with obvious meristematic activity (Fig. 1H). While it is impossible to state for certain when, and by what mechanisms, the branches growing toward the nodule apex developed, their orientations and positions with respect to the branches from which they arose suggest that they originated by branching off of older ITs, rather than by changing their direction of growth from inward to outward during development.
Bacterial release into nodule cells was seen over a region in the 5-d-old nodules that was broader than that seen in the older nodules (Fig. 1E). This zone was five to six cell layers wide in the 5-d-old nodules, two to four cell layers wide in the 10-d-old nodules, and two to three cell layers wide in the 30-d-old nodules. The zone seen in the 5-d-old nodules may have been relatively broad because bacteria were being released independently from putative infection droplets seen on both inward- and outward-directed branches. Surprisingly, the density, cross-sectional area, and surface-to-volume ratios of ITs in the 5-d-old nodules were not qualitatively different from those seen in the older, 10- and 30-d-old nodules (Table I; Fig. 5).
Structure of IT Networks in 10- and 30-d-Old M. truncatula Nodules
Ten- and thirty-day-old nodules exhibited classic nodule structure with well-defined meristems, infection, and nitrogen-fixation zones. In these nodules, the IT network appeared to be growing toward the nodule apex and meristematic regions. This was confirmed by measuring the growth direction of the network branches with respect to the center of the meristematic region (Fig. 4). The density of ITs was greatest near the distal part of the infection zone, about 50 to 100 µm from the advancing tips of the IT network (Figs. 1, 2, and 5). This same IT-dense region held the youngest nodule cells containing intracellular S. meliloti (Fig. 1, E–G) and likely corresponded to the distal part of zone II described by Vasse et al. (1990) The surface-to-volume ratio of the threads in the network was close to 1.0 (Table I) and showed surprisingly little variation along the length of the nodules in spite of differences in cross-sectional area and network density (Fig. 5). An exception to this was near the advancing edge of the networks, where threads tended to have a higher surface-to-volume ratio than the rest of the threads in the network (Fig. 5).
Three-dimensional representations of IT networks were constructed from 2-µm-thick, stained, serial sections of M. truncatula nodules harvested 5, 10, and 30 d after inoculation with wild-type S. meliloti strain Rm1021. These revealed the structure of IT networks in nitrogen-fixing root nodules as well as information about their growth characteristics. While fragments of ITs can be seen in standard semithin sections of nodule tissue, these give a very limited view of the architecture of the IT networks because ITs occupy only 1.5% to 3.0% of a nodule's total volume (Table I). Thus, any single nodule cross section reveals little about the rest of the network.
In all nodules documented here, and in other nodules not presented, the IT networks exhibited simple, open tree-like structures in which fusion between separate branches was rare or nonexistent (Fig. 2; Supplemental Fig. 4). ITs in nodules displayed a preponderance of lumps and protrusions, some of which were infection droplets where bacteria were released from the threads into nodule cell cytoplasm (Rae et al., 1992
Bacterial release from ITs takes place in the infection zone (Vasse et al., 1990 Analysis of the reconstructed IT networks showed that thread branch lengths, average cross-sectional areas, and average surface-to-volume ratios were remarkably similar in nodules of different ages. Such constancy may make future analysis of network development easier than it might have otherwise been if IT size, density, or surface area had changed radically with nodule age. Based on the data presented here, if a bacterial or plant mutant is shown to induce nodules that differ greatly from the wild type in one of the age-constant characteristics listed above, it is unlikely that the difference will be due to mutation-dependent delays or advances in nodule development, but rather because the mutation directly affects thread growth or development.
The work described in this article was initiated to characterize the structure of IT networks in nodules and determine whether IT networks displayed polarized development as they propagated through nodule tissue. It is known from the work of others that early during development ITs branch and display polarized growth as they develop in cortical tissue (Dudley et al., 1987
It is not known what is required for reorientation of IT networks in response to meristem development or for their progression through the infection zone. Outward-directed, polarized network growth could be supported if cells between the meristem and the infection zone were polarized, much like the cortical cells that support polarized IT growth early during infection (van Brussel et al., 1992
Alternatively, IT development may respond to a diffusible targeting signal. The hypothesis of response to a diffusible signal and the hypothesis of polarized cells regulating the direction of IT development in the nodule are not mutually exclusive because cells between the meristem and infection zone could be polarized in response to diffusible signals arising from the meristem or from signals arising from bacteria in ITs. It was recently shown that developing M. truncatula nodules form symplastic fields connected to phloem that are capable of trafficking large molecules. The location of these fields changes as nodules develop, and they correspond to regions of nodules that, in this study, supported IT growth. In young nodules, all internal regions formed a phloem-connected symplastic field, whereas only the meristem and infection zones did so in mature nodules (Complainville et al., 2003
ITs are composite structures, cooperatively built by both the symbiotic, nitrogen-fixing bacteria and their plant host. Their role is to deliver the bacteria to the interior of the developing nodule. ITs, and the bacterial populations inside them, have been studied in root hairs and in cortical cell layers near the surface of the root early during infection. To our knowledge, the networks that result from IT branching and development inside nodules have never been described, analyzed, or even visualized as a whole. We present 3-D reconstructions of IT networks in nodules of different developmental stages. This work shows how IT networks are structured inside nodules, the average size and surface-to-volume ratios of ITs in different parts of the nodule, where bacterial release occurs with respect to the whole IT network, and how the directional development of IT networks change as nodules mature. The analysis of network structure and development we present in this article is interesting in its own right, but it may also form a basis for the quantitative analysis of the development of the internal structure of nodules. In addition, the work presents techniques that can be used for future analyses of IT networks. The quantitative data and the introduced techniques will be important because large-scale genomics projects utilizing the model legume M. truncatula are starting to identify genes needed for proper IT growth, nodule formation, and nodule development.
Nodule Fixation, Sectioning, and Staining
Wild-type Sinorhizobium meliloti strain Rm1021, grown in TY (Tryptone-Yeast) medium, was rinsed and suspended in Nod3 medium at 109 cells/mL, and spot inoculated onto Medicago truncatula roots of plants growing on slides covered with a layer of Nod3 medium (Bhuvaneswari et al., 1981
The main processes used to generate data and perform growth analyses are outlined in Figure 1, A to D. Images of serial sections were captured with a Qimaging Retiga 12-bit cooled CCD camera, using a 40x objective, which resulted in a resolution in the x and y planes of 0.162 µm/pixel. Images were aligned using ALIGN software (Fiala et al., 2002 Branch growth vectors were defined as beginning at a branch point and going to the next, more terminal branch point. Growth vectors of terminal branches went from their branch points to the ends of the branches (Fig. 1, C and D). Incomplete branches that left the sample volume were included in growth analysis because they retained directional information. ITs were 5.5 µm in width and 29 µm in length, on average. The error in assigning branch points and end points is expected to be no more than one-half of the diameter of the threads. For example, a maximum error in assigning an end point would be to place it on the surface of the thread rather than on the centerline of the thread. The assigned angle of an average branch, with a true branch angle of 90°, could be off by a value of 90° – arctan [(1/2 average length)/(1/2 average width)] = 11°. Average branches at other angles would also have a maximum possible error of 11°. The average error of the measured angles presented in the "Results" section is certainly much lower than 11° because care was taken to place branch and end points near the IT centerlines.
The growth vectors were transformed to reflect the direction each branch grew with respect to the nodule meristem (Supplemental Fig. 3). This was done by attaching the base of a new orientation vector to the base of the growth vector under consideration. The tip of the orientation vector was placed at the meristem center. The common base of the two vectors, and the attached vectors, were rotated around the tip of the orientation vector so that the y and z coordinates of the common base were the same as those of the meristem center. This resulted in an orientation vector that pointed horizontally (180°) at the meristem center. The altered growth vector, resulting from the rotations, was termed the transformed growth vector. If a growth vector had been pointing directly toward the meristem center from anywhere in the network, its transformed direction would point at 180° due west. Conversely, if it had been pointing directly away from the meristem center, its transformed direction would point at 0° due east. Growth vectors pointing neither at, nor away from, the meristem center had transformed vectors pointing in directions other than 180° or 0°. Transformed vectors were plotted with a common start point resulting in a sunburst plot (Fig. 1D) or their angles in the x-y plane were plotted in radial histograms. In cases where a well-defined meristem had not yet formed, vectors were transformed to show their growth with respect to a point five to six cell layers below the growing apex of the nodule (the approximate location of the meristem center in older nodules). In cases where a well-defined arc of meristematic cells had developed, transformation was done with respect to the meristem center. In these cases, transformed vectors from approximately 135° to 225° were growing toward meristematic tissue. The translations and rigid body rotations of datasets and their coordinate systems needed to generate transformed growth vectors, and generation of the radial histograms were done using MATLAB (Fisher, 1993 To determine how IT number, area, and surface-to-volume ratio varied along the length of analyzed nodules, 3-D representations of IT networks were sampled by extracting 100 evenly spaced cross-sectional slices from the apexes of the networks to their bases. The cross sections were oriented perpendicular to the long axis of the nodules. Total IT area per slice, average number of IT cross sections per slice, average area of each IT cross section in each slice, and average surface-to-volume ratio of each IT cross section in the slice, assuming a slice thickness of 0.1 µm, were calculated. The error associated with these measurements is dependent on the accuracy of the segmentation. Segmentation was done by hand and care was taken to accurately demark objects of interest. We estimate that the maximum error associated with segmenting areas of interest was less than 25%.
The authors would like to thank Dr. Cynthia Jones and Dr. Marie Cantino for advice on tissue sectioning. Received October 13, 2005; returned for revision November 22, 2005; accepted November 23, 2005.
1 This work was supported by the National Science Foundation (grant no. IBN9974483) and by the University of Connecticut Research Foundation.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Daniel J. Gage (daniel.gage{at}uconn.edu).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.072876. * Corresponding author; e-mail daniel.gage{at}uconn.edu; fax 860–486–4331.
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