This group of bacteria can collectively be referred to as ‘rhizobia’, referring to bacteria of several genera that induce and infect nodules on the roots and/or stems of plants of the familyLeguminosae. According to this definition, bacteria may be considered as rhizobia irrespective of whether they actually fix N2. However, this definition excludes bacteria that may be very closely related to nodule-forming bacteria either in other phenotypic properties or according to data derived from nucleic acid sequences, but which do not form nodules on any legume plant. As we shall see, this grouping of bacteria based on occupation of a common ecological niche does in fact encompass an enormous amount of genetic and phenotypic diversity.
Common elements of the symbiosis
Before discussing the variations in bacterial identity, host range, pathway of infection, nodule structure, efficiency of N2-fixation and so on, we shall begin by describing the common core elements of the legume symbiosis. Here a note of
semantic caution must be sounded: the word ‘symbiosis’ is being used loosely on occasion, as our definition of rhizobia includes bacteria able to induce and infect nodules without actually fixing N2, and therefore in effect behaving as parasites.
Perhaps the most important general principle is that the symbioses established between rhizobia and their host plants are specific. That is, only certain strains of rhizobia can form a symbiosis with a given legume – and these may be termed
‘compatible’ rhizobia. Incompatible rhizobia, which by definition (as rhizobia) do form a symbiosis with some other legume species, cannot form a symbiosis with the host plant in question. This observation indicates that there must be recognition between the plant and bacteria.
Thus the first step in the establishment of a rhizobial–legume symbiosis is an interaction between a legume species that is susceptible to nodulation and compati- ble rhizobia. For the time being the discussion will be restricted to root nodules, and so it can further be specified that this is an interaction between the roots of the plant and rhizobia present in the soil. These rhizobia may have been introduced deliberately into the soil by inoculation, or they may have been already present in the soil as free-living bacteria, in which case they are termed ‘indigenous’ rhizobia. As will be discussed in Chapter 14, most soils contain some indigenous rhizobia capable of nodulating most legume species that are planted in them. While this is a testament either to the diversity of the bacterial population in any given soil, or to the promiscuity of certain rhizobia, it is also a major handicap to the improvement of legume yield through inoculation technology.
A great deal is now understood about the molecular events underlying the recognition between bacterial and plant partners (for reviews see van Rhijn and Vanderleyden, 1995; Denariéet al., 1996; Heidstra and Bisseling, 1996; Long, 1996;
Cohnet al., 1998). A major component of this initial interaction consists of stimula- tion of biochemical activity in the rhizobial strains by flavonoid and isoflavonoid molecules in the plant root exudate. These compounds stimulate the activity ofnod (nodulation) genes – that is, genes whose products are required to enable nodulation of the cognate legume host. There is some specificity in this interaction as different flavonoid and isoflavonoid compounds from different legumes have been shown to activate thenodgenes of their compatible rhizobia preferentially, and thenodgenes of the broad-host-rangeRhizobiumstrain NGR234 are activated by a correspondingly broad range of phenolic compounds. However, this stimulation is by no means completely specific, as exudates from incompatible legume species can often activate thenodgenes of a givenRhizobiumstrain to some degree, and in some cases exudates even from non-legumes may cause this stimulation.
These flavonoids or isoflavonoids enter the bacterial cell, where they bind to a protein termed NodD. The effect of this binding is to convert NodD into a transcriptional activator – that is, a factor that stimulates expression of specific genes.
NodD then activates the remaining nodulation genes, protein products of which cooperate to synthesize a ‘Nod factor’, a signal molecule that is secreted into the plant rhizosphere. These Nod factors share a common ground plan, but it is the chemical details that confer specificity of recognition. The basic structure is ab-1,4-linked N-acetyl-D-glucosamine backbone with three to six sugar units which is acylated on
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the non-reducing terminal sugar residue. The degree of saturation of this acyl group and the presence of other groups varies between rhizobial species and appears to determine host specificity (Fig. 2.2). An example would be the first Nod factor characterized, NodRm-1 fromSinorhizobium meliloti. The recognition by the host plant is so exquisite that absence of a critical sulphate group switches the specificity of this molecule from recognition byMedicagoto recognition byVicia(Denariéet al., 1996; Schultze and Kondorosi, 1998).
The role of the Nod factors appears to be to trigger responses in the plant that lead to rhizobial infection and development of a root nodule. Nod factors trigger a number of responses in the plant epidermis: membrane depolarization of cells in the zone of emerging root hairs; deformation of root hairs; and induction of expression of nodule specific genes (‘nodulins’). In addition, Nod factors contribute to induc- tion of infection thread formation. Nod factors are also able to mitotically reactivate quiescent cells in the root cortex that become the nodule meristem and in some cases purified Nod factors are sufficient to induce development of a complete nodule structure. Finally, Nod factors also induce expression of nodulins in the pericycle (Heidstra and Bisseling, 1996). A major focus of current research is the search for the plant proteins that recognize and initiate the response to the Nod factors – the Nod factor receptors. Factors that bind or otherwise interact with chemically synthesized Nod factors have been identified from lucerne andDolichus biflorus, but further data are required to demonstrate whether these candidates are the true receptors (Etzler et al., 1999; Gressentet al., 1999; Robertset al., 1999; Stacey, 2000). Moreover,
Fig. 2.2. A generic structure for rhizobial nodulation factors (Pacios Braset al., 2000). These molecules can elicit formation of nodules on legume hosts when present at very small concentrations and with a high degree of host specificity.
genetic studies suggest that legumes may actually have two Nod factor receptors – one that is responsible for allowing entry of the bacteria into the plant and one that transmits the signal to the nodule cortex to initiate cell division (Ardourelet al., 1994; Minamiet al., 1996; Geurtset al., 1997; Bonoet al., 2000).
The net effect of the chemical signalling between plant and bacterium is that compatible rhizobia that are closely attached to the root surface gain entry to the root, and the two processes of plant cell division and concurrent rhizobial invasion continue until the mature, N2-fixing nodule is formed. There are many variations on the pathway of development and in the final structure of the nodule. All that can be said to be in common is that the bacteria eventually, at some location within the mature nodule, undergo biochemical and morphological differentiation and begin to fix N2. It is at this point that they are referred to as bacteroids (Oke and Long, 1999b).
Variations on the theme
Infection mechanism
Three different pathways of legume root infection have been described: root hair infection; crack entry; and direct penetration between epidermal cells. The first is clearly different from the second two, but the evidence to distinguish the latter two mechanisms is more circumstantial.
Root hair infection
Root hairs are outgrowths of root epidermal cells, the primary function of which is to increase the volume of soil that may be exploited by the root for nutrients and for water. In many legumes, these outgrowths serve as the conduit by which rhizobia first gain entry to the plant root. The Nod factors produced by the rhizobia cause changes in the growth patterns of the root hairs, producing many deformations, including the characteristic ‘shepherd’s crooks’. At the centre of the crook, disruption of the plant cell wall occurs, enabling the rhizobia to enter the root hair.
As they do so, a new structure, the infection thread, forms within the plant cell and encloses the rhizobia. Thus the rhizobia remain topologically outside the plant cell cytoplasm. The wall of the infection thread is composed of plant cell wall mate- rial, and the bacteria inside the thread are embedded in a glycoprotein matrix that is probably of both plant and bacterial origin (VandenBoschet al., 1989). At the same time, cells within the root cortex undergo cytological rearrangements to create a series of radially aligned cytoplasmic bridges termed ‘pre-infection threads’, which are tra- versed by the actual infection thread as it passes through the root (van Brusselet al., 1992). The infection thread grows by continual deposition of plant cell wall material at its tip, and ramifies within the root tissue, so delivering the rhizobia to numerous plant cells within the emerging root nodule. The rhizobia meanwhile divide within the infection thread, and eventually are ‘released’ from unwalled segments of the infection thread, thus gaining entry to the plant cell cytoplasm. This release process is essentially endocytosis. The rhizobia become surrounded by fragments of the
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host-cell plasma membrane that formerly separated the host cytoplasm from the infection thread wall and this forms the peribacteroid membrane (Newcomb, 1981).
The bacteria generally undergo significant morphological change, which appears to be host determined (see Fig. 2.4), and are now termed bacteroids. These visible changes are accompanied by biochemical changes: nitrogenase is synthesized, the bacterial enzymes of ammonia assimilation are repressed, and N2-fixation begins.
Crack entry and epidermal penetration
In these two mechanisms of infection, penetration does not occur via root hairs, but directly at the root surface. Key evidence for this pathway is provided by nodulation in legume species that have no or very few root hairs. This is particularly common among woody legume species and epidermal infection has been directly demon- strated for the Brazilian tree speciesMimosa scabrella(de Fariaet al., 1988). In this example rhizobia were seen to penetrate the primary cell wall at the junction of two epidermal cells, and no infection threads were ever observed in the rare root hairs that did occur.
‘Crack entry’ refers to a similar pathway of infection where the rhizobia also gain entry between two epidermal cells rather than via a root hair (Boogerd and van Rossum, 1997). The key difference is the purported necessity for a ‘wound’ at the root surface before penetration of rhizobia can occur. This wounding is believed to be caused by the emergence of lateral roots, as nodules are found only at the junctions between lateral and main roots in the three species for which crack entry has been described:Arachis hypogaea(Chandler, 1978), andStylosanthes capitataandS. hamata (Chandleret al., 1982). The presence of axillary root hairs appears to be involved in the infection process, as these are absent in non-nodulating genotypes of Arachis (Nambiar et al., 1983b). Given the difficulty in obtaining direct evidence for wounding, the fact that it is clearly not considered essential for all forms of direct epidermal penetration, and the fact that non-nodulating mutants ofA. hypogaea still develop lateral roots and presumably the associated ‘wounds’ (Nigamet al., 1980), this hypothesis may be treated with caution. It is of interest to note that both host plant genera known to exhibit crack entry are members of the tribe Aeschynomeneae.
In either case, as with root hair infection, the rhizobia within the developing nodule are initially extracellular. InA. hypogaeafiles of bacteria can be seen filling the spaces between the cells in the young nodule. Eventually, presumably by some form of endocytotic process at areas where the host cell wall is weakened, these rhizobia gain entry into the host cell cytoplasm, again surrounded by peribacteroid mem- branes derived from the plant cytoplasmic membrane. N2-fixation is not believed to begin until this stage. InStylosanthes, infection appears to occur by a system of progressive infection and then collapse of cortical cells, enabling inward spread of the rhizobia. InMimosa, bacteria were seen to progress in the cortical region actually through the cell walls, rather than by separating cells at the middle lamella. Where intracellular infection occurred, the bacteria remained surrounded by cell wall material (de Fariaet al., 1988).
Persistent infection threads
The idea that N2-fixation by rhizobia is always carried out by bacteroids present in the host cell cytoplasm was first challenged when the structure of nodules induced on the non-legume treeParasponia andersonii was examined (Becking, 1992). It was found that infection threads persisted, and N2-fixation by the bacteria took place within these structures without bacterial release (Trinick, 1979). The structure of the threads was seen (in the transmission electron microscope, following staining with osmium tetraoxide) to change from a tightly packed thread with a darkly staining wall to a more loosely packed thread with a lightly stained wall (Trinick, 1979; Price et al., 1984). The latter threads were assumed to be the sites of active N2-fixation and were therefore termed fixation threads (Priceet al., 1984).
Persistent infection threads have subsequently been found in legume species, including many nodulated legumes from the subfamilyCaesalpinioideae (de Faria et al., 1987). Infection threads therefore represent an alternative pathway of nodule development rather than an isolated exception.
Nodule structure
The variation in rhizobial infection and nodule development already described begs the question: what is a legume root nodule? Sprent (1989) suggested that the only property held in common by all legume nodules is the stem-like character of the peripheral vascular system, which contrasts with the central vascular system observed in roots, and in actinorhizal andParasponianodules. Nevertheless, a basic nodule structure can be described. In all cases there is an outer, uninfected cortical region containing the peripheral vasculature. This is separated from the inner, infected zone by a nodule endodermis. The central zone contains the infected cells where active N2-fixation takes place. These are often interspersed with uninfected cells. In plants that assimilate fixed N in the form of ureides (Chapter 4) these are the sites of ureide synthesis, but the specific role, if any, in plants that assimilate fixed N as amides is unknown. In nodules of some legumes, such asA. hypogaea, all cells in the central zone are infected.
Two primary types of nodule structure can be discerned: the determinate type and indeterminate type (Fig. 2.3). These terms refer to differences in the nodule meristem, which is persistent in the latter but not in the former case. Determinate nodules grow for a fixed period, all parts of the nodule differentiating at the same time; thus senescence also occurs at one time and they have a finite life span. In contrast, indeterminate nodules have an apical meristem which continues to be active throughout the lifetime of the nodule, producing new zones of infection, and so giving rise to a gradient of differentiation progressing back towards the root – the apical meristem being most distal, followed by the invasion zone where infection thread growth continues (now reversed in direction to follow the apical meristem) with concomitant bacterial release. Progressing towards the root are then found the early, mature and late symbiotic zones, respectively. Perennial nodules are always of the indeterminate type, the persistent meristem being able to resume activity in each new growing season.
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Other obvious key differences are in nodule shape, which is always spherical for determinate nodules but can vary from cylindrical to coralloid for indeterminate nodules, and the location of the root cells that initially form the nodule meristem: in determinate nodules these are cells of the outer root cortex, whereas in indeterminate nodules the initial nodule is formed of cells of the inner root cortex. There are a number of detailed reviews of the biology and molecular biology of the infection process and subsequent nodule development (Nap and Bisseling, 1990; Hirsch, 1992; Brewin, 1998; Cohnet al., 1998).
Many features of the symbiosis are host controlled
Most, if not all, of these differences in nodule development and structure are host controlled. This is clear because a single rhizobial strain is often capable of infecting different host plants by different means, and giving rise to nodules of different structure. It may be released into the host cell in one species and remain in fixation threads in another (e.g. Priceet al., 1984). In fact, the symbiosis appears to be so closely controlled by the plant that even bacteroid shape is determined by the plant host. Figure 2.4 illustrates such differences, showing light and scanning electron micrographs of nodule structure and bacteroids in a groundnut plant and a siratro plant, both induced by the same strain,Bradyrhizobiumsp. (Arachis) strain NC92.
Stem nodulation
Stem nodulation occurs in three genera of legumes that have the capacity to grow in waterlogged conditions:Aeschynomene(several species),Discolobium(at least two species) andSesbania(S. rostrataonly). Nodulation can be induced along the whole length of the stem (Fig. 2.5), which inS. rostratacan mean that nodules sometimes form up to 3 m above the ground. Each of these plants also has the ability to form root nodules.
Infection and development of stem nodules are quite different from those of root nodules, even though both may be induced by the same rhizobial strain. In all Fig. 2.3. Structure of (a) determinate and (b) indeterminate legume nodules.
N2-fixing Organisms25
Fig. 2.4. Structure of nodules induced on (a, c) siratro and (b, d) groundnut byBradyrhizobiumsp. (Arachis) strain NC92. Light (a, b;
bar 100mm) and transmission electron (c, d; bar 1mm) micrographs. Note the absence of infected cells in the infected zone of groundnut nodules. B, bacteroid; CW, plant cell wall; I, infected cell; N, nucleus; S, starch grains; U, uninfected cell; V, vacuole; VB, vascular bundle.
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species examined, stem nodules form at regular intervals along the stem at preformed incipient but dormant primordia. In different species these primordia may be com- pletely hidden within the stem or they may be slightly protruding, the latter showing much greater susceptibility to infection. These dormant primordia have a typical root structure, and in waterlogged conditions, if nodules do not form, adventitious roots may develop from them (Dreyfuset al., 1984).
S. rostratashows the most profuse stem nodulation. The root primordia pro- trude up to 3 mm in three or four vertical rows up the stem. These nodulation sites, in contrast to sites for root nodule formation, remain susceptible to infection throughout the life of the plant. Thus infection can be induced simply by spraying the plants with an aerial suspension of compatible rhizobia. In the field, infection is probably achieved by epiphytic rhizobia – in one study as many as 5´105 stem- nodulating rhizobia were found on each square centimetre of leaf (Adebayoet al., 1989) – but is often sporadic. Thus, there is a good likelihood of increasing stem nodulation ofS. rostrataby spray inoculation, particularly as nodule development is favoured by increasing humidity (Parsonset al., 1993a). Epiphytic rhizobia occur in greater numbers on leaves ofS. rostratathan other plants found in the same habitats, suggesting that their association with Sesbania is also beneficial to the rhizobia (Robertsonet al., 1995).
Infection actually occurs in three steps. In the first, rhizobia colonize the intercellular spaces of the root primordia and form infection pockets. At the same time meristematic activity is induced in some of the primordial cells (Ndoyeet al., 1994). An infection thread then forms within the root primordium and this penetrates host cells and enables eventual intracellular release of the rhizobia. Once Fig. 2.5. Stem nodulation on (a)Sesbania rostrataand (b)Aeschynomene
afraspera. (Photographs: M. Becker.)