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Nodules taken from ten-week old inoculated Listia, Leobordea and Lotononis

s. str. host species (Chapter 2) were sectioned and examined by light microscopy to

compare their internal structure. In all cases, effective nodules contained uniformly infected central tissue with no uninfected interstitial cells, whether from lupinoid nodules of L. angolensis and L. bainesii (Figure 3.5 a – f) or from the indeterminate nodules of Lotononis s. l. species outside the genus Listia (Figure 3.6 a – f). The degree of vacuolation of bacteroid-containing cells appeared to vary between species (Figure 3.5 a and f; Figure 3.6 a and d). In some nodules (Figures 3.5 b and 3.6 f), amyloplasts could be seen in the cortical uninfected cells next to the nodule central tissue. L. angolensis inoculated with WSM2598 produced ineffective nodules in which the bacteria were not differentiated into nitrogen-fixing bacteroids (Figure 3.5 c – e). Interestingly, bacteroids of WSM2598 in nitrogen-fixing nodules of L.

bainesii (Figure 3.7 a) appeared to be far more swollen than the WSM3557

Figure 3.5. Light microscopy of sections of ten-week-old Listia angolensis and Listia bainesii nodules. In both species, the central tissue (*) of effective nodules contains infected cells only.

a, b) Nitrogen-fixing nodule of L. angolensis inoculated with WSM3557. Amyloplast structures (arrow) can be seen in the peripheral cortical uninfected cells.

c, d, e) Non-fixing nodule of L. angolensis inoculated with WSM2598. f) Nitrogen-fixing nodule of L. bainesii inoculated with WSM2598. S, stele; VB, vascular bundle

Figure 3.6. Light microscopy of sections of ten-week-old Leobordea spp.and Lotononis s. str. spp.nodules. In all nodules, the central tissue (*) of effective nodules contains infected cells only.

a) Leobordea bolusii inoculated with WSM2632. b) Leobordea mollis inoculated with WSM2667. c) Leobordea platycarpa inoculated with WSM2653. d) Lotononis crumanina inoculated with WSM2653

e, f) Lotononis pungens inoculated with WSM2653. Structures resembling amyloplasts (arrow) can be seen in the peripheral cortical uninfected cells.

Figure 3.7. Light microscopy of sections of ten-week-old nodules, showing bacteroids. a) Listia bainesii inoculated with WSM2598.

b) Listia angolensis inoculated with WSM3557.

3.4 Discussion

Nodulation in L. angolensis and L. bainesii occurs mainly on the hypocotyl and taproot. Remarkably, the infection site appears to be independent of the age of the plant at the time of inoculation, as the same pattern of nodulation was observed for plants inoculated at 85 days old as those inoculated at seven days old. This is in contrast to other studies of both root-hair-curl- and epidermally-infected legumes.

Rhizobial invasion in legumes infected via root hair curling is confined to a small zone of root hairs that have nearly finished growing (root hair zone II) and are susceptible to deformation and infection (Bhuvaneswari et al., 1980, 1981; Gage, 2004; Heidstra et al., 1994; Maunoury et al., 2008). In vetch, about 80% of zone II root hairs were deformed three hours after exposure to NodRlv factor (Heidstra et al., 1994). Similarly, rhizobial invasion in the epidermally infected genus Lupinus appears to be localised within a transient zone. In Lupinus angustifolius, nodules appeared most frequently in the region between the smallest emergent root hairs and the root tip at the time of inoculation. Epidermal root cells aged 13 h or over appeared not to be infected (Tang et al., 1992). Rhizobia preferentially accumulate in the root hair portion of the main root of Lupinus albus seedlings, c. 10–15 mm from the root tip (González-Sama et al., 2004).

Although infection and nodule formation in dalbergioid and genistoid legumes does not proceed via root hair curling and infection thread development, root hair deformation and transient infection threads have been observed in some species belonging to these clades. Bradyrhizobium strain BTA-1 induced root hair deformation and curling, and the development of infection threads (that subsequently aborted), in the genistoid legume tagasaste (Cytisus proliferus; formerly

Chamaecytisus proliferus) (Vega-Hernández et al., 2001). Deformation and curling

of root hairs has been observed in Arachis, Stylosanthes and Lupinus species (Boogerd & van Rossum, 1997; Chandler et al., 1982; González-Sama et al., 2004; Łotocka et al., 2000). Structures similar to short, wide infection threads have occasionally been found in L. angustifolius (Tang et al., 1992) and L. albus nodules (James et al., 1997). Infection threads were not found during the nodulation process

in the Listia species (this work) and similarly were not seen in Lupinus albus nodulation (González-Sama et al., 2004) or in the invasion zone of Crotalaria

podocarpa nodules (Renier et al., 2011).

Root hair cells seem to be important in the initial infection process in some dalbergioid and genistoid legumes. Rhizobia invade the middle lamella between enlarged basal hair cells and adjacent root hair or epidermal cells in Arachis and

Lupinus species (Boogerd & van Rossum, 1997; González-Sama et al., 2004; Tang et

al., 1992). In Arachis, rhizobial infection is associated with tufts of root hairs that arise in the axils of young lateral roots and non-nodulation is strongly correlated with an absence of these hairs. Successful infection is restricted to penetration sites where enlarged root hair basal cells are found (Boogerd & van Rossum, 1997; Uheda et al., 2001). Conversely, in Aeschynomene afraspera stem-nodulation sites, root hairs are not observed in the epidermis of the lateral root primordium (Alazard & Duhoux, 1990). Further study is required to determine whether or not root hair cells are involved in rhizobial infection of the hypocotyl in Listia angolensis and Listia

bainesii. It could be hypothesised that in these Listia species, the ability of the

hypocotyl to remain potent for infection may be correlated with the putative lack of a role for root hair cells.

Nodule organogenesis in Listia angolensis and Listia bainesii appears to follow a process similar to that observed in Lupinus albus and Lupinus angustifolius (González-Sama et al., 2004; Tang et al., 1992). In the Lupinus studies, rhizobia penetrate intercellularly at the junction between epidermal cells and subsequently invade a cortical cell immediately beneath the epidermis. The infected cell divides

rapidly, as do the rhizobia within the cell, to produce a nodule with characteristic uniformly infected cells in the central infected zone (Fernández-Pascual et al., 2007). The incidence of cell division spreads progressively from the infection focus towards the inner cortex, with cellular division in the deeper cortical layers occurring several days after the initial infection (González-Sama et al., 2004; Tang et al., 1992). The same pattern was seen for nodule morphogenesis in the Listia species. The proliferation of outer cortical cells formed the nodule primordium and cellular division subsequently spread to the inner cortex. In this, the lupinoid nodule primordium is more similar to that of the desmodioid nodule, in that desmodioid nodule initiation (seen for example in Lotus japonicus)is typically found in the first outer cortical layer, immediately beneath the primary infection site (Guinel, 2009; Szczyglowski et al., 1998). Conversely, both aeschynomenoid and indeterminate hologalegoid nodules arise from divisions in the root inner cortex (Alazard & Duhoux, 1990; Guinel, 2009; Voroshilova et al., 2009). As well, in the aquatic robinioid legume Sesbania rostrata (where infection can occur intercellularly at lateral root bases) and in the aeschynomenoid genera Aeschynomene and

Stylosanthes, the infection process is associated with the collapse and death of

cortical plant cells (Alazard & Duhoux, 1990; Chandler et al., 1982; D'Haeze et al., 2003). In contrast, cell death does not appear to occur during the nodulation process

in Listia species.

In keeping with the morphology found in typically genistoid nodules (Sprent, 2009) nodule sections of all Lotononis s. l. host species, whether of lupinoid or indeterminate nodules, had a mass of central, uniformly infected tissue, with no uninfected interstitial cells. It is interesting to note that nitrogen-fixing bacteroids of

WSM2598 within L. bainesii nodules should appear to be more swollen than those of WSM3557 within L. angolensis nodules. It has been suggested that swollen bacteroids may confer advantages to the host, such as enhanced nitrogen fixation capability (Mergaert et al., 2006; Oono & Denison, 2010). The symbiosis between WSM2598 and L. bainesii is highly effective (Yates et al., 2007), but further study would be required to determine if a relationship between bacteroid size and effectiveness exists in this system.

In summary, the mechanism of morphogenesis in lupinoid nodules, in which cell division is initiated in the outer cortex beneath the infection site, appears to be

the same for both genistoid Lupinus and crotalarioid Listia species. The salient

characteristics of infection and nodule organogenesis in Listia species – epidermal

infection, the lack of infection threads and a central mass of uniformly infected tissue - are consistent with the nodulation process observed in the dalbergioid and genistoid clades. The ability of the root and hypocotyl epidermal cells to remain potent for

infection is an interesting feature of the infection process in Listia, and one that