Wild-type nodule morphogenesis
Various authors (e.g. Bond, 1948; Yang et al., 1994; Timmers et al., 1999) have demonstrated that, in indeterminate legume nodules, the development of the wild-type nodule starts with the resumption of cell proliferation in the pericycle and then in the inner root cortex. This results in the formation of the nodule primordium (NP). At the distal end of the NP, an apical NM develops that is ‘uninfected’, that is, free of ITs and cells containing intracellular bacteria. By contrast, in cells with determinate NMs, for example in Phaseolus and Lotus spp., the meristematic cells within the developing nodule are infected, in the sense that they harbour either ITs or released bacteria, or both (Brewin, 1991).
It has been demonstrated by genetic dissection that the developmental processes governing bacterial ‘entry’ and nodule organogenesis are closely co-ordinated but distinct (Tsyganov et al., 2002). There are instances in which bacterial infection can occur in the absence of nodule organogenesis (Murray et al., 2007), and, conversely, there are instances in which nodule organogenesis can occur in the absence of bacterial infection (Truchet et al., 1989; Gleason et al., 2006; Tirichine et al., 2006). Furthermore, it has been postulated that the epidermal and cortical processes leading to nodule development must be closely coupled so that a nodule primordium occurs close to the site of bacterial infection in order to generate a nodule containing intracellular bacteria (Oldroyd & Downie, 2008).
In general, this model of nodule initiation has been confirmed in the present study, but with certain previously unobserved additions. By analysis of symbiotically defective pea mutants, it was discovered that IT development and plant cell proliferation are tightly coupled during the development of cylindrical nodules with apical uninfected meristems (indeterminate type). In order to develop a mature nodule with an active apical meristem, a group of dividing cells on the proximal face of the nodule primordium apparently have to be penetrated by an IT and undergo the consequent process of topological reorganisation. Therefore, a key condition for formation of the apical NM, discovered in this study and apparently absent from mutant lines SGEFix−-2 (sym33) and RisFixA (sym41), is the ability of plant cells to form transcellular branching ITs without leaving the mitotic cycle (Figs 1g,h, 4d). These mutant lines also form abnormal lateral meristems instead of a normal apical NM.
At the transition from nodule primordium to a mature nodule, five histological zones were revealed in the present study (Fig. 1c):
- • a zone of NM with uninfected distal cell layers providing nodule growth;
- • a proximal layer of NM with infected proliferating cells providing both nodule growth and propagation of ITs;
- • the pathway of ‘entrance’ of the ‘main’ IT crossing plant cells which, based on their small size and presence of mitotic figures (Fig. 1f–h), are still in the mitotic cycle;
- • the central zone of endoreduplicated plant cells containing intracellular bacteria taken up from ITs;
- • peripheral tissues which were not analysed in detail in this study.
Based on the results of this study, it can be postulated that plant cells from the nodule primordium that are still in the mitotic cycle can start forming ITs when adjacent cortical cells have been invaded by the original IT. In the maturing nodule, cell-to-cell spreading of ITs through the nodule primordium culminates in the development of proximal layers of NM with similar properties (Fig. 1g,h), that is, they are proliferating cells harbouring ITs. It is clear from Fig. 1h that cells in the mitotic cycle can harbour ITs. This raises the topological issue of how a cell plate can be laid down at the same time as an IT, as the formation of both structures seems to involve components of the same cytoskeletal machinery. However, our data indicate that these two processes could probably occur simultaneously, or at least successively in a single host cell.
Furthermore, we postulate that proper functioning of the apical NM requires the continual presence of mitotic cells harbouring ITs. The existence of this interzone is thought to be a key condition for proper histological differentiation, development and functioning of the indeterminate nodule. It is interesting to note that, in spherical nodules with determinate meristems, the presence of infected proliferating cells has always been regarded as a key component of nodule development (Brewin, 1991). The molecular basis of the developmental processes governing cell proliferation and nodule development may be discovered when the genes corresponding to Sym33 and Sym41 have been cloned and sequenced. Furthermore, it is still unclear how the hormonal state of the tissues involved in nodule formation controls such a process, although it is obvious that hormone-mediated mechanisms of regulation could be involved (e.g. Ferguson et al., 2005; Gonzalez-Rizzo et al., 2006).
At the stage of the young nodule, the NM identity is being determined by means of at least two genetic factors (Voroshilova et al., 2004; Combier et al., 2006). Also, at this stage the ITs change the direction of growth: when the IT enters a nodule primordium the plant creates ITs in a different direction, with subsequent IT growth following behind the NM, colonizing proximal layers of it. Reversed growth of ITs was previously reported for indeterminate nodules of P. sativum (Libbenga & Harkes, 1973) and Medicago truncatula (Timmers et al., 1999; Monahan-Giovanelli et al., 2006). How these developmental processes, which are also observed in the mutants studied (data not shown), influence the formation of typical NM with proliferating cells harbouring ITs will be a subject of further studies.
Incidentally, in this paper we have tended not to use the term ‘meristematic cells’ because it is based on a topological definition rather than a functional definition. The term ‘meristematic cells’ simply implies a location within the region of the meristem. It could include cells in the mitotic cycle, cells undergoing endoreduplication, and even terminally differentiated cells. The term ‘proliferating cells’ is preferred because it provides a precise description for cells in the mitotic cycle. For example, nodule primordia contain ‘proliferating cells’ but these cannot be described as meristematic cells for the reasons described above.
Another new feature described in the present study is the position of the primary IT penetration site, relative to the apex of the mature nodule. It was shown in this study that, as a rule, the ‘entrance’ of an IT in a mature pea nodule is situated laterally (Fig. 1e). A similar position was found previously for pea nodules invaded by lipopolysaccharide-defective strains of R. leguminosarum (Perotto et al., 1994). Accordingly, it is suggested that the growth of the nodule is usually supported by only one sector of the hemispheric NM of a young nodule.
Development of nodules in symbiotically defective mutants
In previous work, the term Nmd (for nodule meristem development) was used to describe mutants affected in NM formation (Tsyganov et al., 2002), but the mutants analysed in the present study allow us to dissect this process still further. It is proposed that two new terms should be used to describe specific stages of NM development: (1) Anm, for development of the apical nodule meristem at the distal part of the nodule primordium, and (2) Nmp, for the subsequent stage of mature nodule meristem functioning (i.e. nodule meristem persistence).
It has been shown that, during interactions of the RisFixA (sym41) mutant with rhizobia, the nodule primordium consisting of the plant cells in the mitotic cycle is formed. However, proliferating nodule primordium cells are not penetrated by ITs. The only cells that are penetrated by ITs are cells in the five to six cell layers of the outer cortex of the root (Fig. 2c,d). As a result, the NM at the distal end of the nodule primordium does not arise (Anm− phenotype). This can be explained by the observation that the mutation in the gene Sym41 prevents IT formation in nodule primordium cells that are still in the mitotic cycle. Instead of forming an apical NM, an abnormal lateral NM grows around cells containing ITs (Fig. 2c), and nodule development is usually blocked at the stage of nodule emergence from the root as a result of the low ‘mitotic potential’ of this lateral NM.
A block at a similar stage of nodule development –‘emergence from the root’– was reported for Afghanistan peas carrying the Sym2A allele (Degenhardt et al., 1976; Geurts et al., 1997) and for pea symbiotic mutants in the genes Sym36 (Sagan et al., 1994), Sym37 and Sym38 (Tsyganov et al., 2002). It is important to note that, in the case of mutations in these genes, the growth of ITs was aborted in the root hair cells (‘infection thread differentiation inside root hair cells’ (Ith−) phenotype) which led to a block in NM initiation. By contrast, ITs of the RisFixA (sym41) mutant develop fairly normally in root hairs and in the cells of the outer cortex but do not penetrate the nodule primordium. Moreover, occasionally, if the lateral NM persists, the mutant RisFixA (sym41) can form few nodules, but they are ineffective as a result of abnormal IT growth inside the young nodule and lack of bacteroid differentiation (Morzhina et al., 2000).
In mutant SGEFix−-2 (sym33), the development of nodule tissue is arrested at the same stage of meristem development as in the RisFixA (sym41) mutant (Anm− phenotype), but the development of mutant nodules is much more advanced because their lateral NMs have a higher potential for mitotic activity than those of RisFixA (sym41). Consequently, these nodules develop abnormally with the large plant cells in the central part filled with ITs and representing the area of penetration from an original IT (Fig. 3c). The same morphology was also observed for nodules that developed occasionally on the roots of the mutant RisFixA (sym41) (Fig. 3c). Therefore, in pea, apical NM formation is apparently (directly or indirectly) controlled by at least two genes: Sym33 and Sym41. It is important to note that mutations in both these genes also cause impaired IT growth in comparatively mature nodules (‘infection thread formation in nodule primordium’ (Itn−) phenotype) (Tsyganov et al., 1998; Morzhina et al., 2000; this study), demonstrating an inability to form ITs in cells in the mitotic cycle.
In the case of the pea mutant SGEFix−-1 (sym40), the development of nodule tissue does not differ from that in the wild-type plants up to the stage of apical NM formation (Fig. 4a,b,d,e). However, the NM apparently stops functioning prematurely and the resultant nodules are much smaller than those of wild-type plants (Fig. 4c). Previously, it was shown that IT development of this mutant is blocked at the stage of infection droplet differentiation (Idd− phenotype) (Tsyganov et al., 1998). Thus, the expression of the pea gene Sym40 is not only important for IT functioning but also for supporting the persistence of the NM (Nmp− phenotype).
Sequential functioning of pea symbiotic genes with respect to nodule tissue development
In Table 3, the observations from the present study are set alongside other proposed schemes of development for nodules with indeterminate meristems, as suggested for early (Guinel & Sloetjes, 2000; Tsyganov et al., 2002) and late developmental stages (Borisov et al., 1997a,b; Tsyganov et al., 1998; Morzhina et al., 2000; Voroshilova et al., 2001). Altogether, eight pea genes have been found to date to be co-operatively involved in NM formation. The genes Sym2 (Degenhardt et al., 1976; Geurts et al., 1997), Sym36 (Sagan et al., 1994), Sym37 and Sym38 (Tsyganov et al., 2002) control the colonization process, that is, the growth of ITs before they enter the nodule primordium (Tsyganov et al., 2002), and mutations in them lead to the arrest of nodule primordium formation and NM formation. The genes Sym21 (Markwei & LaRue, 1997) and Sym39 (Sagan et al., 1994) probably function in the programme of nodule tissue development.
Table 3. Sequential expression of phenotypes for regulatory Pisum sativum symbiotic genes during nodule formation
|Sym9|| ||Sym35||Sym36||Sym16||Sym39||Sym41|| ||Sym32||Sym25|
|Sym10|| || ||Sym37||Sym34|| || || || ||Sym26|
|Sym19|| || ||Sym38|| || || || || ||Sym27|
|Sym30|| || || || || || || || ||Sym42|
In this research, it has been found that mutations in the pea genes Sym33 and Sym41 result in the inability of plant cells to form ITs while still in the mitotic cycle. In addition to abnormal growth of ITs in root cortical cells and young nodule tissue (Tsyganov et al., 1998; Morzhina et al., 2000; this study), the phenotype of such mutants is described as Anm− (Table 3). The gene Sym40 functions at a later stage with respect to NM formation, at the stage of NM persistence (Nmp−). The phenotype of the double mutant RBT3 (sym33, sym40) is basically similar to that of SGEFix−-2 (sym33), indicating that Sym33 is epistatic to Sym40 with respect to its effects on nodule primordium development. However, these inferences are based on a single mutant allele at each locus and therefore the conclusions should be regarded as preliminary. Further characterization of these genes at the level of primary structure will reveal molecular mechanisms involved in the cellular function of these genes and how they control the interaction between cell proliferation and the formation of ITs.