• Arbuscular mycorrhizal fungi colonize the roots of most monocotyledons and dicotyledons despite their different root architecture and cell patterning. Among the cereal hosts of arbuscular mycorrhizal fungi, Oryza sativa (rice) possesses a peculiar root system composed of three different types of roots: crown roots; large lateral roots; and fine lateral roots. Characteristic is the constitutive formation of aerenchyma in crown roots and large lateral roots and the absence of cortex from fine lateral roots. Here, we assessed the distribution of colonization by Glomus intraradices within this root system and determined its effect on root system architecture.
• Large lateral roots are preferentially colonized, and fine lateral roots are immune to arbuscular mycorrhizal colonization. Fungal preference for large lateral roots also occurred in sym mutants that block colonization of the root beyond rhizodermal penetration.
• Initiation of large lateral roots is significantly induced by G. intraradices colonization and does not require a functional common symbiosis signaling pathway from which some components are known to be needed for symbiosis-mediated lateral root induction in Medicago truncatula.
• Our results suggest variation of symbiotic properties among the different rice root-types and induction of the preferred tissue by arbuscular mycorrhizal fungi. Furthermore, signaling for arbuscular mycorrhizal-elicited alterations of the root system differs between rice and M. truncatula.
Plant root systems serve two major functions: water and mineral uptake; and anchoring of the plant to the ground. In nature, mineral nutrient acquisition by plant roots is often assisted by symbioses with beneficial arbuscular mycorrhizal (AM) fungi of the phylum Glomeromycota (Schüßler et al., 2001). During this intimate association, the extraradical hyphal mycelium acquires minerals from the soil beyond the zone accessible by the root itself. Inside the root, a considerable proportion of the minerals are delivered to the host in exchange for carbohydrates. The AM symbiosis is one of the most prevalent interactions of terrestrial plants, including important crops such as rice.
Arbuscular mycorrhizal colonization follows a series of distinct steps (for review, see Smith & Read, 1997; Harrison, 2005; Paszkowski, 2006) starting with a presymbiotic molecular dialogue that involves root-released strigolactones (Akiyama et al., 2005; Besserer et al., 2008) and as yet unidentified AM fungal signaling molecules (Kosuta et al., 2003, 2008; Olah et al., 2005). The first contact at the root surface is marked by the differentiation of the fungal hypha into a swollen and/or branched structure, called a hyphopodium, from which a penetration hypha invades the rhizodermal cell layer (Genre et al., 2005). After the fungus has traversed the outer cell layers it spreads longitudinally in the inner cortex and forms highly ramified structures, called arbuscules, inside cortical cells. Arbuscules are hypothesized to be the main site of nutrient transfer from the fungus to the plant (Harrison et al., 2002).
Most dicotyledonous plants possess an allorhizic root system where the embryonic radicle develops into a primary root that dominates the root system. The numerous lateral roots that depart from it remain shorter in length. By contrast, the root system of monocotyledons is secondarily homorhizic with numerous shoot-borne main roots, which might bear lateral roots of several orders being equally important in the root stock (Hochholdinger et al., 2004). However, monocotyledons and dicotyledons not only differ in the architecture of their root systems but also in the cellular patterning of certain root types (Hochholdinger & Zimmermann, 2008). The consequences of these alterations on, for example, the distribution of AM colonization within a dicotyledon or monocotyledon root system are not known but might impact the nutritional benefit of AM colonization.
An adult rice plant has a complex root system (Fig. 1). Postembryonic crown roots (CRs) emerge from the nodes on the stem and tillers and give rise to two types of lateral roots: large lateral roots (LLRs), which display indeterminate growth and elongate downwards; and fine lateral roots (FLRs), whose growth is determinate and agravitropic (for review, see Hochholdinger et al., 2004; Rebouillat et al., 2008). Large lateral roots support FLRs and can form further LLRs of several orders (Kawata & Soejima, 1974). The radial anatomy of rice roots is typical of that found in semi-aquatic plants, with a sclerechymatic cell layer underneath the rhizodermis and exodermis serving as a diffusion barrier, and extensive aerenchyma formation in CRs and LLRs (Clark & Harris, 1981; Ranathunge et al., 2003). Aerenchyma development is constitutive in rice roots and most extensive in CRs. However, in CRs, several layers of cortical cells are present in the proximity of emerging LLRs (Fig. 1, Clark & Harris, 1981). Interestingly, in contrast to CRs and LLRs, FLRs lack cortex tissue (Fig. 1, Rebouillat et al., 2008), the tissue type where AM fungi form arbuscules in higher plants.
The development and functioning of AM symbioses have so far mainly been studied in dicotyledonous leguminous and solanaceous plants (for review, see Paszkowski, 2006; Javot et al., 2007). It has been reported that AM colonization itself influences the architecture of the host root system (Price et al., 1989; Yano et al., 1996; Berta et al., 2002; Paszkowski & Boller, 2002; Olah et al., 2005). In Medicago truncatula, perception of a diffusible signal released by AM fungi before contact was sufficient to induce lateral root formation; however, the cellular target for the stimulation is unknown (Olah et al., 2005). Interestingly, root induction depended on the signaling components DMI1 and DMI2, positioned upstream of a calcium-spiking signal, which is central to the common symbiosis (SYM) signaling pathway required for establishment of both root symbioses with rhizobia and AM fungi (Parniske, 2008). The signaling component, DMI3, positioned downstream of calcium spiking (DMI3), was not necessary for lateral root induction (Olah et al., 2005).
Although it is known that rice associates readily with AM fungi, the participating root-types have not been determined. Here we show that Glomus intraradices preferentially colonizes LLRs. Furthermore, we show that AM colonization induces the formation of LLRs, the preferred tissue for colonization. Induction of LLRs was independent of the SYM signaling protein, POLLUX, indicating a fundamental difference from dicotyledonous M. truncatula where the AM-induced increase in lateral roots required POLLUX (DMI1) (Olah et al., 2005).
Materials and Methods
Plant material, plant growth and inoculation conditions
Oryza sativa L. ssp. japonica cv. Nipponbare wild-type and the homozygous mutant lines containing retrotransposon Tos17 insertions (Miyao et al., 2003) pollux-2, ccamk-2 and cyclops-1 (Gutjahr et al., 2008) were used for inoculation with G. intraradices. Plant growth and inoculation conditions have previously been described (Gutjahr et al., 2008).
Root staining and quantification of mycorrhizal distribution within the root system
Roots were stained with Trypan blue. Images were taken using a Leica DM 5000 B microscope equipped with a Leica DFC420 camera (Leica, Wetzlar, Germany). Mycorrhizal colonization of different root types was quantified as described previously (Gutjahr et al., 2008).
Root types of 10 entire root systems were quantified for each genotype and treatment. Means and standard errors (SEs) were calculated using Microsoft Excel 2003. Significance of differences between mock-inoculated plants and G. intraradices-inoculated plants were assessed by the nonparametric Mann-Whitney statistical test using the statistical software systat 10, SPSS Inc. 2000, Chicago, USA .
Large lateral roots are preferentially colonized by G. intraradices
To define the distribution of colonization by G. intraradices in O. sativa ssp. japonica cv. Nipponbare root systems, the roots of five plants were microscopically inspected at 6 wk post-inoculation (wpi). The overall colonization ranged from 30 to 50% of total root length, and the presence of arbuscules and vesicles was observed frequently at all stages of colonization. However, closer examination of the distribution of colonization revealed that while LLRs were often colonized over their full length (Fig. 2a,b), CRs were colonized in small patches (Fig. 2c,d). The majority of CR colonization patches, namely 75.4% out of 60 patches, occurred close to both types of lateral roots (Fig. 2c) or to lateral root primordia (Fig. 2d). There was no colonization of FLRs including the absence of external structures such as hyphopodia (Fig. 2a–c). After extended co-cultivation to 12 wpi, FLRs remained void of extraradical and intraradical fungal structures, and inoculation with a different fungus (Gigaspora rosea) did not lead to colonization of FLRs (data not shown). Fine lateral roots therefore appear to be resistant to colonization by taxonomically different AM fungi.
To determine quantitative differences in root-type colonization, we recorded the percentage of root-length colonization in different root types. Total root-length colonization was about four-fold higher in LLRs than in CRs, and no colonization was detected in FLRs (Fig. 2d). In LLRs, arbuscules were by far the most abundant fungal structure, while in CRs the percentage root-length colonization by intraradical hyphae, arbuscules and vesicles was similar (Fig. 2d). In summary, G. intraradices prefers a specific rice root type for colonization, reflected by the total colonization and by the high proportion of arbuscules.
Arbuscular mycorrhizal colonization induces changes in rice root architecture
To examine the effect of colonization by G. intraradices on root architecture, we quantified the different root types in mock-inoculated and G. intraradices-inoculated roots at 6 wpi. While the absolute number of CRs did not change upon G. intraradices colonization (Fig. 3a), the absolute quantity of LLRs and FLRs originating from CRs was one-third higher in colonized root systems than in the absence of the fungus (Fig. 3a). Also, the absolute number of FLRs originating from LLRs was 20% elevated by colonization with G. intraradices. The increase in absolute quantities of lateral roots was normalized against the root of origin (Fig. 3b). Arbuscular mycorrhizal colonization led to a 50% augmentation of LLRs and FLRs per CR. However, the amount of FLRs per LLR did not change significantly upon AM colonization. The observed increase in absolute numbers was therefore caused by the increase in LLRs. The higher quantity of lateral roots originating from CRs was accompanied by an increase in root system dry weight of approx. 20% (Fig. 3c).
The AM-induced augmentation of LLRs and FLRs emerging from CRs could be caused by an increase in lateral root density or in CR length with unchanged lateral root density, or a combination of both. To differentiate between these possibilities, CR length and lateral root density were monitored. Colonization by G.intraradices led to a significant increase in CR length of 30% (Fig. 3d). Additionally, the density of LLRs and FLRs on CRs was approx. 15% higher in colonized root systems compared with noncolonized root systems (Fig. 3e). The ratio between the number of CR-derived FLRs and the number of CR-derived LLRs was equal for both treatments (Fig. 3f). In summary, we demonstrated that AM colonization changes rice root architecture by stimulating the elongation of CRs and additionally increasing their initiation frequency for both types of lateral roots. The formation of LLRs and FLRs originating from CRs appears to be coupled independently of the AM stimulus.
Arbuscular mycorrhiza-induced changes in rice root architecture are independent of common SYM signaling
Roots of legume and rice mutants affected in components of the common SYM pathway block AM colonization at the outer cell layers and do not allow cortex colonization (Gutjahr et al., 2008; Parniske, 2008 and references therein). We investigated whether common SYM components are required for preferential LLR colonization. Mutant alleles of SYM genes were selected that arose in the Nipponbare background. The three mutants, pollux-2, ccamk-2 and cyclops-1, all lack wild-type gene function and display an equivalent mycorrhizal penetration phenotype (Gutjahr et al., 2008). Colonization attempts, corresponding to extraradical fungal hyphae and hyphopodia, appeared with similar distribution across the three mutants and the wild-type root system (Fig. 4a). The number of colonization attempts was five- to eight-fold higher in LLRs than in CRs.
We also addressed the relevance of the common SYM signaling pathway for AM-mediated lateral root induction. Large lateral root numbers and root system dry weight served as markers for changes in root system architecture of mock-inoculated and G. intraradices-inoculated root systems of rice sym mutants. The number of CRs of the pollux-2, ccamk-2 and cyclops-1 mutants was not altered by AM colonization, but the total number of CRs was lower in the mutants than in the wild-type plants (Fig. 4b), a result that demands further investigation. However, the absolute and relative numbers of LLRs significantly increased upon inoculation with G. intraradices in all three mutants (Fig. 4c,d). Both numbers increased by approx. 45% for the wild-type plant, by approx. 35% for the pollux-2 mutant and by approx. 15% for ccamk-2 and cyclops-1 mutants. Future studies will address variation in responsiveness across the genotypes. Furthermore, all genotypes responded to AM inoculation with an increase in root dry weight of approx. 15% (Fig. 4e).
We conclude that AM-induced changes in rice root system architecture are independent of the signaling components POLLUX, and also of CCAMK and CYCLOPS. Furthermore, the equivalent changes of root architecture in wild-type plants and all three mutants suggest that the presence of extraradical fungal hyphae and hyphopodia or an earlier event is sufficient for the induction of changes in root system architecture, while cortex colonization and/or arbuscule formation are not required. The existence of possible additional root system-modulating effects at later stages of colonization is difficult to determine owing to the nature of the AM invasion process that simultaneously includes the presence of early stages and the associated signals.
We investigated the distribution of AM colonization within the rice root system and found LLRs to be preferentially colonized. Large lateral roots might have a specific competence for colonization through tissue composition and plasticity. Also, in dicotyledoneous plants, AM fungi generally favour lateral roots for colonization (Harrison, 2005). Tissue penetration by AM fungi requires localized cell-wall flexibility and expansion to accommodate the fungus. In many plant species, AM fungi invade the growing and elongating root zone, which corresponds to an already expanding tissue (for review, see Mathesius, 2003), rather than mature and less plastic cells. In rice, the combination of elongating tissue and the presence of cortex, necessary to support arbuscule formation (Harrison, 2005), exists in LLRs. Crown roots instead contain large amounts of aerenchymatic lacunae (Clark & Harris, 1981), which are void of intact cortex cells and were not observed to harbour AM fungal structures (data not shown). Close to lateral roots and lateral root primordia, a limited number of cortex cells are usually present (Clark & Harris, 1981) and here most AM colonization of CRs is observed. Interestingly, also rhizobia do not induce nodules in mature legume roots except at sites of lateral root initiation and emergence, even in a legume species that does not form aerenchyma in primary roots (Mathesius et al., 2000 and references therein).
As there is no cortex layer in rice FLRs (Rebouillat et al., 2008), the absence of arbuscule formation in this root type was expected. However, it is surprising that hyphopodia were not observed. The complete absence of mycorrhizal structures from FLRs suggests that fungal recognition of the appropriate root-type occurs before contact. Strigolactones, which are constitutively released from plant roots, induce AM fungal activity before root contact and are required for efficient AM colonization (Akiyama et al., 2005; Besserer et al., 2008; Gomez-Roldan et al., 2008). As they are highly unstable in soil and function as a signal over a short distance it is tempting to speculate that the spatial specificity of AM colonization might be regulated by root-type specific variation in strigolactone exudation. However, this is difficult to test experimentally owing to the minute amounts of strigolactones released. Alternatively, FLRs might lack a specific surface signal required for fungal attachment and hyphopodium induction.
We demonstrate here that the architecture of rice root systems changes profoundly in response to AM colonization. It has been previously reported that AM colonization leads to increased root system development, especially by enhancing the formation of lateral roots (Price et al., 1989; Yano et al., 1996; Berta et al., 2002; Paszkowski & Boller, 2002; Pritchard et al., 2004; Olah et al., 2005). However, while many dicotyledonous and monocotyledonous root systems possess lateral roots of different order with identical cellular patterning, rice forms lateral roots that are differently composed. In rice, at the stage of a fully established symbiosis, the density of both types of lateral roots originating from CRs was significantly enhanced and was accompanied by the simultaneous increase in CR length. In M. truncatula, AM-induced lateral root formation occurs before physical contact in response to a diffusible fungal signal (Olah et al., 2005). It has been suggested that this induction serves to raise the availability of the tissue preferred for colonization (Harrison, 2005; Olah et al., 2005), which is also consistent with fungal preference for actively growing tissue. The increase in density of FLRs on CRs of rice seems inconsistent with this interpretation as these roots are immune to AM fungi. However, an inherent root developmental program might determine a fixed relationship between FLRs and LLRs. Indeed, the ratio of FLRs to LRRs in mock-inoculated and G. intraradices-inoculated root systems remained unchanged, while the ratio of LLRs to CRs was increased.
Phosphate starvation induces lateral root development while inhibiting primary root growth (Williamson et al., 2001; Lopez-Bucio et al., 2002). Thus, at first sight it seems counterintuitive that AM fungi increase plant phosphate uptake and at the same time promote an increase in lateral root density. However, the AM-induced lateral root response might be developmentally uncoupled from phosphate-related responses (Harrison, 2005) or steered through signaling pathways that lead to lateral root proliferation in phosphate-rich soil pockets (Drew, 1975). Indeed, the simultaneous elongation of CRss argues against a phosphate starvation response and an involvement of auxin signaling, at least at the whole root system level (Olah et al., 2005). Observations of AM-induced responses in roots of the maize mutant, lrt1, which fails to initiate lateral roots during early postembryonic root formation (Hochholdinger & Feix, 1998), support this view. Although this mutant is defective in a component of the default pathway of lateral root initiation, AM colonization induced bushy lateral roots, even at higher levels of external phosphate (Paszkowski & Boller, 2002).
The induction of lateral root formation in M. truncatula by AM fungal signaling factors was shown to be dependent on the SYM pathway components DMI1 (POLLUX) and DMI2 (SYMRK), which act upstream of Ca2+-spiking, but not on DMI3 (CCAMK), which acts downstream of Ca2+-spiking (Olah et al., 2005). Interestingly, in our study, enhanced LLR formation occurred in all mutants independently of their positioning relative to Ca2+-spiking. Despite having a similar colonization phenotype (Catoira et al., 2000; Gutjahr et al., 2008), the developmental root responses of Medicago and rice POLLUX mutant alleles might suggest partial variation of POLLUX function between the two species. Indeed, the attempt to restore AM colonization or nodulation in a Lotus japonicus pollux mutant by cross-species complementation with the OsPOLLUX gene failed (Banba et al., 2008). Furthermore, OsPOLLUX did not restore rhizobial infection in an M. truncatula dmi1 mutant (Chen et al., 2009), suggesting differential function of OsPOLLUX and the legume version of POLLUX. Another possibility is that, in rice, AM-induced lateral root formation is independent of Ca2+-spiking, while in M. truncatula this response is dependent on the second messenger. However, it remains to be shown whether Ca2+-spiking is part of the signaling pathway that leads to lateral root induction.
Importantly, AM-induced changes in root system architecture of rice sym mutants were similar to those observed in wild-type and indicate that cortex colonization is not necessary for these changes to occur. Although we cannot exclude that root system architecture modulation continues beyond the hyphopodial stage, signal exchange during the presymbiotic stage, or preceding hyphopodia formation, is sufficient for the architectural changes to be induced. It is thus likely that, as for M. truncatula (Olah et al., 2005), presymbiotic AM-fungal signaling factors induce the formation of lateral roots in rice. In contrast to M. truncatula these changes are independent of common SYM signaling. These data thus suggest differences in signal transduction between M. truncatula and rice in AM-mediated induction of lateral root formation.
We thank Christophe Périn (CIRAD, Montpellier, France) for stimulating discussions on rice root architecture, and Ruairidh Sawers (University of Lausanne) for critically reading the manuscript. This work was supported by the Swiss National Science Foundation (SNF) grant no. 3100AD-104132, the NCCR grant ‘Plant Survival’, PhD scholarships of the ‘Studienstiftung des deutschen Volkes’ (German National Academic Foundation) and the Roche Research Foundation to C.G. and the SNF ‘professeur boursier’ grant PP00A-110874 to U.P.