JXB Advance Access originally published online on February 27, 2008
Journal of Experimental Botany 2008 59(4):917-925; doi:10.1093/jxb/ern015
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© 2008 The Author(s).
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RESEARCH PAPER |
Does legume nitrogen fixation underpin host quality for the hemiparasitic plant Rhinanthus minor?
1College of Life Sciences, Beijing Normal University, Xin Jie Kou Wai Street 19, 100875 Beijing, China
2Julius von Sachs Institut für Biowissenschaften der Universität, Lehrstuhl Botanik I, Julius von Sachs Platz 2, D-97082 Würzburg, Germany
3Department of Animal and Plant Sciences, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK
* To whom correspondence should be addresses. E-mail: d.cameron{at}sheffield.ac.uk
Received 14 November 2007; Revised 7 January 2008 Accepted 8 January 2008
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Abstract |
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The high quality of leguminous hosts for the parasitic plant Rhinanthus minor (in terms of growth and fecundity), compared with forbs (non-leguminous dicots) has long been assumed to be a function of the legume's ability to fix atmospheric nitrogen (N) from the air and the potential for direct transfer of compatible amino compounds to the parasite. Using associations between Rhinanthus minor and Vicia faba (Fabaceae) that receive N either exclusively via symbiotic associations with rhizobia supplying organic N fixed from N2 or exclusively through the supply of inorganic nitrate to the substrate, the underlying reasons for the quality of legumes as hosts for this parasite are unravelled. It is shown that sole dependence of the host, V. faba, on N fixation results in lower growth of the attached parasite than when the host is grown in a substrate supplied exclusively with inorganic N. In contrast, the host plants themselves achieved a similar biomass irrespective of their N source. The physiological basis for this is investigated in terms of N and abscisic acid (ABA) partitioning, haustorial penetration, and xylem sap amino acid profiles. It is concluded that legume N fixation does not underpin the quality of legumes as hosts for Rhinanthus but rather the well-developed haustorium formed by the parasite, coupled with the lack of defensive response of the host tissues to the invading haustorium and the presence of sufficient nitrogenous compounds in the xylem sap accessible to the parasite haustoria, would appear to be the primary factors influencing host quality of the legumes.
Key words: ABA, haustorium, legume, nitrogen fixation, nodules, parasitic plant
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Introduction |
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Rhinanthus minor is a root hemiparasitic plant with a wide host range estimated to be in excess of 20 species (Gibson and Watkinson, 1989). Rhinanthus minor abstracts carbon (C), nitrogen (N), and other minerals from its hosts by penetrating the xylem through the organ of attachment called the haustorium, which provides a physical as well a physiological bridge between parasite and host (Riopel and Timko, 1995). Rhinanthus minor is a catholic parasite forming haustoria on a taxonomically diverse range of plants (Cameron et al., 2006), but it is most successful, in terms of growth and reproduction, when growing in association with N-fixing legumes (Hwangbo, 2000; Westbury, 2004; Cameron et al., 2006). This differential success of R. minor across functional groups: grasses, legumes, and forbs (non-leguminous perennial dicots), is known to be a function of the host's ability to defend itself; forbs generally represent the worst hosts as they are able to employ a range of successful defence responses, including encapsulation of the parasite's endophytic tissues and localized cell death (Cameron et al., 2006), which prevent the abstraction of host solutes (Cameron and Seel, 2007). In contrast, few, if any, defence responses are shown by grasses and legumes (Cameron et al., 2006; Rümer et al., 2007).
Previous studies have mapped solute fluxes from the host to R. minor (Jiang et al., 2003, 2004a, 2005; Cameron and Seel, 2007) but, to date, no study exists which investigates the physiological basis for why legumes are so much better hosts than the grasses. It has, however, been postulated that the readily available and compatible organic N (in the form of amino acids) resulting from N2 fixation by the bacterial symbionts of legumes underpins their quality as hosts (Hwangbo, 2004). The hypothesis that the availability of organic N, a product of N2 fixation, indeed underpins the quality of legumes (grown in an N-free medium) as hosts for R. minor was therefore investigated.
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Materials and methods |
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Host plant material
Vicia faba (Faba bean) seeds were germinated in moistened washed sand in a glasshouse and allowed to grow for a further 2 d post-germination when eight seedlings were individually transplanted into pots containing washed quartz sand. Prior to transplanting, half of the plants were inoculated with a commercially available rhizobium culture specifically compatible with V. faba (Rhizobium leguminosarum strain DSM 2108 in Medium 98: Rhizobium Medium; DSMZ, Braunschweig, Germany) to induce nodulation. These plants were then re-inoculated with rhizobia at 7, 9 12, and 14 d after transplanting (DAT). All pots were supplied with 40% Long Ashton (LA) solution (Hewitt, 1966); the LA solution was N free for plants inoculated with rhizobia (25% strength LA solution was applied until 14 DAT). The cotyledons and the remainder of the seed were removed from all individuals of V. faba 5 d after germination to prevent remobilization of N from the seed reserves. Four pre-germinated seedlings of the parasitic plant R. minor were then transferred to each pot 14 DAT; this was subsequently reduced to one parasite once they had showed signs of attachment (see Klaren and Janssen, 1978). The host–parasite associations were harvested after a further 12 weeks. Xylem sap was extracted from each host and parasite (see method below), and host plant tissues were separated into stems, leaves, and roots. The tissues were freeze-dried and the biomass was then recorded, after which the tissues were finely ground.
Germination of R. minor
Rhinanthus minor seeds were germinated according to Keith et al. (2004). Briefly, seeds (collected from a grassland on the banks of the River Danube near Linz, Austria) were surface-sterilized with 5% (v/v) sodium hypochlorite solution for 5 min and washed three times with sterile distilled water. Seeds were transferred to sterile Petri dishes containing moistened capillary matting overlaid with a sheet of filter paper (Whatman No. 1). Light was excluded from Petri dishes which were incubated at 4 °C until germination (
12 weeks).
Xylem sap collection
Xylem sap was collected using the methods developed by Seel and Jeschke (1999) for the simultaneous collection of xylem sap from R. minor and the co-associated host plant. Briefly, the pot containing moistened sand substrate was sealed into a pressure chamber with the shoots of the host and parasite protruding from two holes in the surface plate; gas-tight seals were made around the bases of each plant using a silicone-based, two-component dental impression gum (Blend-a-med Forschung, Schwalbach, Germany). The shoots of both host and parasite were then excised near to the base (the point at which they were sealed into the pressure chamber). The pneumatic pressure in the chamber containing the pot was increased until xylem sap was exuded from the cut stems. The first few drops of sap were discarded as this may have been contaminated with damaged cell contents; the remaining sap was collected and analysed for abscisic acid (ABA) content and amino acid composition.
Amino acid profiles
Xylem sap samples were diluted with biochrom dilution buffer (Biochrom, Cambridge, UK) and the amino acid composition quantified using an amino acid analyser (Biochrom 20 Plus, Biochrom, Cambridge, UK).
Carbon and nitrogen composition
A 5 mg aliquot of freeze-dried, finely ground plant material was weighed into aluminium cups and the C and N content determined using a CHN-Analyser (Firma Elementar, Hanau, Germany).
ABA analysis
Freeze-dried tissue samples were homogenized and extracted in 80% (v/v) methanol–water solution. Extracts were passed through a Sep Pak C18-cartridge. Methanol was removed under reduced pressure and the aqueous residue was partitioned three times against ethyl acetate at pH 3.0. The ethyl acetate of the combined organic fractions was removed under reduced pressure. This fresh residue was dissolved in TBS buffer (TRIS-buffered saline; 150 mmol l–1 NaCl, 1 mmol l–1 MgCl2, and 50 mmol l–1 TRIS at pH 7.8) and subjected to an immunological ABA assay [enzyme-linked immunosorbent assay (ELISA)] as described earlier by Mertens et al. (1985) and Peuke et al. (1994). The accuracy of the ELISA has been verified in earlier investigations (Hartung et al., 1994). Recoveries of ABA during the purification procedures were checked routinely using radioactive ABA and found to be >95%.
Haustorial anatomy
Haustoria were harvested from host roots and fixed in a formaldehyde, ethanol, glacial acetic acid mixture. The samples were prepared for microtomy as described in detail by Wacker (2006). In summary, haustoria were dehydrated in an isopropanol series, transferred to methylbenzoate, and embedded in wax (Paraplast plus). Sections of 8 µm thickness were cut with a sliding mictrotome (HN40, Jung, Heidelberg, Germany). Samples were stained with acridine red, acriflavin, and astrablue as described in detail by Wacker (2006). Nuclei and lignified and suberized cell walls appear red, and cellulose blue-green. De-waxed cross-sections were stained in oil blue N, dissolved in 60% isopropanol for 15 min. Stained sections were washed with 60% isobutanol and warm water. Images of the sections were then taken using a Visitron microscope and digital camera (Visitron systems, Puchheim, Germany).
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Results |
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Biomass
Rhinanthus minor achieved the lowest shoot biomass when attached to host plants inoculated with rhizobia (and hence able to fix atmospheric N2 as their only N source) compared with those parasites attached to host plants fed exclusively (i.e. no rhizobia) with exogenous inorganic N (t-test: df=7; T=4.56; P=0.003, Fig. 1). Similarly, a significantly lower biomass of haustoria was formed by the parasite on the host plants inoculated with rhizobia compared with the host plants fed with inorganic N (df=5; T=4.66; P=0.003, Fig. 1). There was no significant difference in the biomass of parasite roots from either treatment (df=7; T=0.27; P=0.79, Fig. 1).
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ABA content
There was no significant effect of the parasite on the amount (pmol g–1 dry weight) of ABA in the tissues of host plants inoculated with rhizobia fixing atmospheric N2 as the only N source (Fig. 2). In contrast, there was significantly more ABA in uninfected host plants supplied with inorganic N compared with the parasitized hosts with the same nutrient regime (Fig. 2). Furthermore, uninfected host plants fed exclusively with inorganic N contained the greatest amount of ABA in all tissues, with the exception of internodes 4–6 (Fig. 2).
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In the parasite, there was significantly more ABA in the shoots and roots attached to host plants supplied with inorganic N as the only N source compared with the host plants inoculated with rhizobia fixing atmospheric N2 as the only N source (Fig. 3).
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The ABA content of xylem sap was only measured in host plants supplied with inorganic N as the only N source as host plants inoculated with rhizobia fixing atmospheric N2 as the only N source did not achieve enough biomass to permit sap collection. There was no significant difference in the ABA contents of the xylem sap collected from infected compared with uninfected host plants (Fig. 4). In contrast, there was significantly (
10 times) more ABA in the parasite than in either the infected or uninfected host (Fig. 4).
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Nitrogen content
There was no significant effect of the parasite on the concentration (% element) of N in any host tissue (Fig. 5). In contrast, the host plants inoculated with rhizobia (and hence able to fix atmospheric N2 as the only N source) were significantly less concentrated in N in leaves 1–3, buds, and roots than those plants fed exclusively (i.e. no rhizobia) with exogenous inorganic N (Fig. 5). There was no significant effect of the N source on the N concentration in internodes 1–3 or leaves 4–6 (Fig. 5), whereas internodes 4–6 had a significantly lower concentration of N in plants supplied with exogenous inorganic N compared with those inoculated with rhizobia (Fig. 5).
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The concentration of N (% N) in the root and shoot tissues was significantly affected by the N source. Roots and shoots of the parasites attached to host plants receiving organic N exclusively through their association with rhizobia had a significantly lower concentration of N, containing 1.89 (±0.28) and 2.87 (±0.85) % N, respectively, compared with the roots and shoots of parasites attached to hosts receiving inorganic N which contained 3.83 (±0.46) and 7.04 (±1.29) % N, respectively.
Amino acid profiles of xylem sap
A full spectrum of amino acids was detected in the host and parasite xylem sap (see Supplementary Table S1 at JXB online). Cystathionine and asparagine acid were the most common amino compounds in the xylem sap of both uninfected and infected hosts fixing atmospheric N2 as their only N source (Fig. 6; see Supplementary Table S1 at JXB online). In contrast, there was a much lower cystathionine content in the xylem sap of parasitized and unparasitized hosts receiving inorganic N as their only N source. In these plants, asparagine became the most abundant amino compound (Fig. 6; see Supplementary Table S1 at JXB online). The parasite xylem sap contained a much wider range of, and was more concentrated in, amino compounds than the xylem sap of the host (Fig. 6). The most abundant amino compound, asparagine was significantly more concentrated in the sap of the parasite than that of the host plants (Fig. 6; see Supplementary Table S1 at JXB online). Parasites attached to the host plants inoculated with rhizobia and thus receiving fixed atmospheric N2 as the only N source did not achieve a sufficient biomass for the extraction of xylem sap via the pressure chamber method.
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Haustorial anatomy
The mature haustorium formed by R. minor on V. faba fixing atmospheric N2 as the only N source is fully differentiated. The hyaline body has developed and abounds with secondary xylem formed between the endophytic penetration peg and the parasite's root (Fig. 7). The haustorium has penetrated the root and the endophyte has degraded the host root cortex and endodermis, accessing the vascular core of the host stele and penetrating the host's xylem vessels.
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Discussion |
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The high quality of legumes as hosts for the parasitic plant R. minor has long been assumed to be a function of their ability to fix N despite an absence of evidence for the underlying reasons. In contrast, it is shown here, for the first time, that the ability to fix N does not account for the elevated growth of a hemiparasite attached to leguminous hosts. In this case, the hemiparasites attached to hosts where the only N source was via host N fixation in association with rhizobia were smaller than those attached to a host that had access to soil-borne N. This is not a completely artificial situation as Rhinanthus often grows in habitats with extremely low soil N (Cameron, 2004; Westbury, 2004) although there is likely to be free organic N in the soil substrate. Furthermore, V. faba hosts whose only N source was via N2 fixation in association with rhizobia showed no symptoms of N deficiency, mirroring the development of the V. faba host plants receiving inorganic N as the only N source and achieving similar growth rates and final size (see Supplementary Fig. S1 at JXB online). Such N-free growth media have been successfully employed to study N relations in the Australian hemiparasite Olax phyllanthi (Tennakoon and Pate, 1997). In the present study, the parasites attached to the hosts supplied with inorganic, substrate-borne N also had greater concentrations of N, a likely function of direct N uptake from the substrate. Moreover, reduced parasite growth on legumes fixing atmospheric N2 as the only N source appeared to be a function of host N content as the hosts only fixing N contained less N than those only receiving N from the substrate.
Hwangbo (2004) suggested that compatible amino acids that are easily assimilated by the parasite may represent the underlying physiological basis for this high host quality and enhanced parasite performance. Pate et al. (1994) also suggest that the amino acid composition of the parasite xylem sap may ‘reflect the compositional idiosyncrasies of the host on which it is feeding’ in the Olax–Acacia spp. association. However, as there are no direct lumen–lumen connections between Olax and its hosts, xylem-borne solutes are not transferred to the parasite by bulk mass flow. Pate et al. (1994) also show that the amino acid composition of the parasite may differ significantly from that of its host due to processing/metabolism of nitrogenous compounds in the parasite haustorium. Both of these situations have also been observed here; Rhinanthus has direct vascular continuity with its host's xylem (Cameron et al., 2006; Cameron and Seel, 2007) and as such can abstract xylem solutes via bulk mass flow driven by transpiration and cohesion (Press and Graves, 1995) and, concurrent with this, it was observed that the major amino acid in the host's xylem sap, asparagine, was also the most abundant component of the parasite's xylem sap, albeit at a lower concentration in the host. In contrast, the amino acid composition of xylem sap extracted from attached Rhinanthus also differs in composition from that of its host's xylem sap in the range of major amino acids (>2% of the total on a molar basis), with the parasite's xylem sap containing significantly more glutamine, alanine, valine, and 
-aminobutyric acid than that of its host. The difference in amino acid composition of parasite xylem sap compared with host xylem sap thus appears to support the hypothesis of Seel and Jeschke (1999) that there is amino acid synthesis and/or metabolism in the parasite tissues prior to xylem loading, potentially in the haustorium. Care must also be taken in this interpretation, however, as the parasite had access to N in the substrate in the present experiments and its roots have been shown to access this resource effectively independently of the host (Cameron et al., 2005). Furthermore, the absence of asparagine, the main amino compound present in the parasite xylem, in hosts exclusively receiving organic N from their rhizobial symbionts may influence the poor growth of the parasite on these hosts. In any case, it is clear that while compatible, host-derived organic N compounds in the form of amino acids that do not require metabolism may play a role explaining host quality in general terms, they are not unique to legumes fixing N2 as their only N source and are thus not significant factors in explaining the quality of legumes as hosts. It is therefore essential to consider alternative factors which may underlie legume host quality for R. minor.
It is well known that parasitic plants such as the Giant Witchweed, Striga hermonthica, can significantly influence host metabolism to favour nutrient acquisition by the parasite. Striga can induce elevated levels of ABA in host tissues (Taylor et al., 1996; Frost et al., 1997) resulting in a reduction of host stomatal conductance and the generation of a water potential gradient facilitating mass flow of solutes from host to parasite (Taylor et al., 1996). In this study, elevated levels of ABA were not observed in infected V. faba plants compared with uninfected controls, and thus it is concluded that Rhinanthus is not able to induce shifts in the metabolism of V. faba to facilitate solute capture. This lack of effect of Rhinanthus on host ABA levels has also been shown for the graminoid host Hordeum vulgare–Rhinanthus association (Jiang et al., 2004b). In common with other parasitic plants such as Striga species, R. minor contained high levels of ABA (Press and Graves, 1995). Moreover, the amount of ABA detected in the parasite was significantly higher than that of its hosts. The high host quality of the legume V. faba compared with graminoid hosts for Rhinanthus is therefore not a function of its ability to influence host ABA metabolism.
Successful differentiation of the haustorium, the organ of parasite attachment to the host vascular system, is important for parasite growth and reproduction (Cameron et al., 2006), especially at early stages of development (Jiang et al., 2007). Thus host-induced defences against the invading haustorium will significantly impact upon parasite success. In an in-depth histological study, Rümer et al. (2007) clearly demonstrate that the legume Vicia cracca showed the least response to the invasion of the parasite R. minor compared with the graminoid hosts H. vulgare and Phleum bertolonii. In contrast, forbs showed the greatest response to the parasite, with Plantago lanceolata able to induce localized cell death and Leucanthemum vulgare encapsulating the parasite endophyte in lignin (Cameron et al., 2006; Rümer et al., 2007) that prevents the parasite from abstracting almost any of the host's resources (Cameron and Seel, 2007). It has also been suggested that the rhizobial symbiont has the potential to induce legume resistance to parasitic plants. Recent studies by Mabrouk et al. (2007a, b) have shown that pea plants (Pisum sativum) colonized by R. leguminosarum (strain P.SOM) exhibit significantly greater resistance to the root holoparasite Orobanche crenata associated with the induction of the phenylpropanoid and isoflavonoid pathways (Mabrouk et al., 2007b) and the accumulation of toxic phenolic compounds (Mabrouk et al., 2007a). Histological investigation of the haustorial interface between R. minor and V. faba colonized by R. leguminosarum (strain DSM 2108) (Fig. 7) in the present study, however, shows no signs of any defence or resistance responses to infection. Moreover, the mature haustorium formed by R. minor on V. faba roots is fully differentiated, containing the nucleus-rich hyaline body, hypothesized to represent the haustorium's ‘power house’ for solute processing (Riopel and Timko, 1995), and secondary xylem connecting the host and parasite xylem stream via the haustorial penetration peg. Rümer et al. (2007) and Cameron et al. (2005) suggest that the lack of an exodermis, the first line of physical defence of the root, in the legumes may underpin the extreme susceptibility of legume roots to the invasion of the haustorium of R. minor.
The position at which Rhinanthus forms haustoria on the host's root network has been shown to be a key factor determining parasite success (Keith et al., 2004). If the parasite is too far from the host it forms haustoria at the distal root tips and is thus unable to intercept the nutrients taken up by the host's roots (Keith et al., 2004); if it is too close to the host then the parasite can be outcompeted by the host (Keith et al., 2004). Moreover, the parasite may be unable to form functional haustoria on the older, thickened, and better defended roots close to the root meristem (Cameron, 2004), although this is not considered to be the primary factor influencing vascular penetration. After excavation of the roots from the substrate, it was observed that haustoria were formed on the newer fine roots lower in the substrate column. As Rhinanthus withdraws xylem sap, along with dissolved nutrients, from its hosts via cohesion, it is unlikely that the parasite could intercept much of the organic N loaded into the xylem from any of the nodules located ‘upstream’ of the parasite. There certainly is substantial recycling of organic N from the shoots to the roots via the phloem, as shown in Lupinus albus (Layzell et al., 1981; Jeschke et al., 1995), but much of this N is likely to be utilized for nodule development (Jeschke et al., 1995).
As discussed earlier, both the legume host and the parasite will have access to soil-borne N in nature (Cameron, 2004), but the present study clearly demonstrates that the legume V. faba is a poor host for Rhinanthus in the absence of soil N. However, this situation is reversed once N is again available in the soil despite the lack of N2 fixation. Thus it is clear that the ability to fix nitrogen does not alone underpin the quality of legumes as hosts for R. minor. In contrast, the well-developed haustorium formed by the parasite coupled with the lack of defensive response of the host tissues to the invading haustorium would appear to be the primary factor influencing the improved growth and fecundity observed in R. minor attached to legume compared with graminoid hosts.
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Supplementary material |
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Supplementary material is available at JXB online.
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Acknowledgements |
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We thank Bianca Röger (Julius-von-Sachs Institut, Würzburg, Germany) for expert technical assistance and Robin Wacker (University of Würzburg, Germany) for undertaking the histology. FJ has received financial support from the State Key Basic Research and Development Plan (2007CB106802), the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry, and Beijing Normal University. We also acknowledge generous financial support from the Deutsche Forschungsgemeinschaft (award number: SFB 567, TP A6). DDC is supported by a Natural Environment Research Council (UK) independent fellowship (award number: NE/E014070/1).
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  K. Suetsugu, A. Kawakita, and M. Kato Host Range and Selectivity of the Hemiparasitic Plant Thesium chinense (Santalaceae) Ann. Bot., July 1, 2008; 102(1): 49 - 55. [Abstract] [Full Text] [PDF]
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