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Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules

Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules Abstract An ability to form symbiotic associations with rhizobia and to utilize atmospheric nitrogen makes legumes ecologically successful. High iron content in legume grains, partially relocated from root nodules, is another-nutritional-advantage of this group of plants. The ferritin complex is the major cell iron storage and detoxification unit and has been recognized as a marker of many stress-induced responses. The possible participation of ferritin in nodule formation and functioning was investigated here. Correlation of increased accumulation of both ferritin polypeptide and mRNA with actual in situ localization of ferritin allowed ferritin synthesis in the developing, indeterminate-type root nodules to be related to differentiating bacteroid tissue. This kind of tissue, in contrast to the determinate-type nodules, is present in lupin nodules at almost all stages of their development. Interestingly, it was found that, in this type of nodule, senescence starting in the decaying zones induces ferritin accumulation in younger, still active, tissues. Based on the presented data, and in correlation with previous results, some aspects of the regulation of expression of lupin ferritin genes are also discussed. Biological nitrogen fixation, ferritin, indeterminate-type nodule, iron, oxidative stress, plant–microbe interactions Introduction Iron-containing proteins play a key role in such crucial processes as respiration, photosynthesis and DNA or hormone syntheses. Symbiotic nitrogen fixation, however, is a phenomenon particularly dependent on iron-containing proteins. Free-living bacteria turning into microsymbionts (bacteroids) triple their cytochrome accumulation and synthesize nitrogenase. The latter may contain more than 30 atoms of iron and can constitute more than 10% of total bacterial proteins (Verma and Long, 1983; Sangwan and O'Brian, 1992). To protect this enzyme from oxygen-induced inactivation and to supply bacteroids with enough oxygen for their respiration, a plant fills the cytoplasm of bacteroid tissue cells with another iron-containing protein, leghaemoglobin (Lb). Lb may account for up to 30% of all soluble proteins of the infected cell, which makes a nodule one of the most iron-loaded organs of a plant (Appleby, 1984; Kuzma et al., 1993). Although cellular iron is mostly bound in proteins and their cofactors, it may be released to the cytoplasm in various stress situations. The physico-chemical properties of iron ions (small ionic radius, high reactivity) and the fact that they catalyse the formation of hydroxyl radical (Fenton reaction), the most potent oxidizing agent known in nature, make iron responsible for the damage to nucleic acids, proteins, and lipids (Guerinot and Yi, 1994). The nodules are better equipped with all kinds of antioxidant systems (i.e. ascorbate-glutathione pathway or superoxide dismutases) than the parent root, which ensures effective detoxification of the superoxide radical and excess H2O2 (Becana et al., 2000; Matamoros et al., 2003). However, during senescence, a rise in proteases activities, a decline in the antioxidant pool, and increased ROS accumulation promote nodule decay (Evans et al., 1999; Alesandrini et al., 2003). Protein degradation and membrane disruption lead to the significant increase of free iron concentration (Becana and Klucas, 1992; Mathieu et al., 1998; Hernandez-Jimenez et al., 2002). Such ‘unattended’ iron accelerates the spread of nodule tissue disruption and nitrogen fixation termination. The most effective way to limit iron noxiousness seems to be its sequestration. Organisms from bacteria to animals possess a protein cage in which iron may be kept harmless, but available for all vital processes, and this is ferritin (Theil, 1987). Ferritins are 24-subunit protein spherical complexes, capable of the reversible transfer of iron between liquid and solid phases (Chasteen and Harrison, 1999; Theil, 2003). Iron is then concentrated inside the protein cavity (up to 4500 iron atoms) as a hydrated ferric oxide (Liu and Theil, 2005). Not only iron sequestration, but also removal of dioxygen from the cytoplasm (mineral core formation) make ferritin an effective part of the antioxidant system (Zhao et al., 2003). It was suggested earlier that both the rapidity of senescence and the extent of stress tolerance of symbiotic interactions may be, to a great extent, defined by the structure and developmental pattern of the nodules (indeterminate- versus determinate-type of nodule) (Sprent, 1980; Hirsch, 1992). In our research on iron management in symbiotic nitrogen fixation, yellow lupin was used as a plant model. Possession of long-lasting meristems as well as clearly divided developmental zones makes the general anatomy of lupinoid nodules similar to those of an indeterminate type (Fig. 1) (Golinowski et al., 1987, 1992; Lotocka et al., 2000). In addition, because of the special evolutionary position of lupins with those of the earliest branching lineages within the Papilionoideae subfamily, this plant system appeared very attractive to study (Kass and Wink, 1995; Strozycki and Legocki, 1995; Strozycki et al., 2000). Previously, three lupin ferritin genes were identified, which, similarly to those in maize and Arabidopsis, were differently regulated in response to iron and ABA (Fobis-Loisy et al., 1995; Petit et al., 2001a; Strozycki et al., 2003). Here, for the first time, are presented the analyses of both RNA and protein samples, as well as tissue fragments originating from the same plants. This allowed the changes in ferritin gene expression during development and senescence of a nodule to be correlated with specific tissues of this organ. Characteristic, developmental distribution of ferritin in indeterminate type lupin nodules may indicate that its function is more complex than just being an iron storage unit. Fig. 1. View largeDownload slide Lupinus luteus root nodules. (A) Young nodules: because of very early division of laterally-positioned nodule meristem and its growth in all directions, the lupin nodule escapes from its cylindrical shape. (B) As a result of the activity of lateral meristems, the initially spherical nodules grow laterally encircling the root (Golinowski et al., 1987). (C) Cross-sections through nodules at different stages of development. (D) Schematic diagram of the mature nodule. Fig. 1. View largeDownload slide Lupinus luteus root nodules. (A) Young nodules: because of very early division of laterally-positioned nodule meristem and its growth in all directions, the lupin nodule escapes from its cylindrical shape. (B) As a result of the activity of lateral meristems, the initially spherical nodules grow laterally encircling the root (Golinowski et al., 1987). (C) Cross-sections through nodules at different stages of development. (D) Schematic diagram of the mature nodule. Elucidation of possible ways of iron management in the regulation of symbiotic interactions seems to be essential for understanding legume plant development. In addition, the data proving that iron accumulated in seed ferritin comes in large part from the nodule, partially explain the high iron content of legume seeds (Burton et al., 1998). This, together with growing evidence on the nutritional importance of ferritin iron, substantiates the studies on legume ferritin (Theil, 2004). Materials and methods Plant material Yellow lupin (Lupinus luteus L. cv. Ventus) was used for all analyses. The seeds were sterilized with sodium hypochloride or chlorox, washed and germinated for 3 d at 25 °C in the dark. When needed for the analyses of symbiotic interactions, the roots of 3-d-old seedlings were inoculated with Bradyrhizobium bv. genistearum, previously cultivated for 5–7 d at 28 °C. The inoculated plants were transferred to perlite-filled pots in a culture room. They were grown in a 16/8 h light/dark photoperiod at 24/26 °C during the day, and 21/23 °C at night, fertilized with nitrogen-free medium every three days, except for the first week when 0.01 mM nitrogen was applied. A detailed description of growth conditions and medium contents was previously described by Strozycki (2003). The tissues (i.e. root segments, root nodules, stems, cotyledons, flowers, pods, seeds) were collected at the designated time and immediately frozen in liquid nitrogen. The time points for sample collecting were designated based on previous observations of lupin development. Individual nodule tissue samples were harvested up to 91 d post Bradyrhizobium inoculation (dpi). The early stages of nodule formation were not analysed. The soluble proteins and total RNA isolated from the collected tissues were analysed. Isolation and analysis of protein and RNA fractions The soluble proteins and fractions of total RNA were isolated ‘in parallel’ from separate fragments of the same tissue samples, as described previously by Strozycki and Legocki (1995); the ammonium sulphate precipitation for the proteins was omitted. The samples (35 μg of proteins or 10 μg of total RNA) were separated in the required gels [15% SDS-PAGE (Laemmli, 1970) or 2% agarose, containing formaldehyde (Sambrook et al., 1989)], and transferred onto proper membranes (Hybond-C extra or Hybond-N; Amersham). The ferritin and leghaemoglobin polypeptides were identified by western-type hybridizations, using the appropriate polyclonal antibodies (Sikorski et al., 1995; Strozycki et al., 2003) and the biotin/streptavidin-AP system (Amersham). To display the possible participation of each ferritin class in the overall ferritin content, specific (3′-end) molecular probes generated in PCR reactions were used in northern hybridizations. Full-length cDNAs of lupin ferritins were used as a mixed probe in order to show the general (global alterations) in ferritin mRNA level. For more details see Strozycki et al. (2003). In making quantitative assumptions, it has to be considered that, in lupin, continually growing collar-shaped nodules, meristem, and young tissues are located laterally within the mature nodule (in the tip of the ‘cone’) (Fig. 1). Their share in nodule volume decreases with time (Lotocka et al., 1995). Therefore, in matured nodules, their volumes make proportionally small quantitative contributions to the overall picture. In situ immunochemical protein detection Fragments of nodules (collected from the same plants as for protein and RNA isolations) c. 3 mm thick, were fixed in 4% paraformaldehyde in PBS buffer (10 mM Na2HPO4/NaH2PO4 pH 7.5, 150 mM NaCl) for 18 h at –400 hPa air pressure, and then washed in PBS (3×15 min.). The fixed tissue was dehydrated by successive changes of graded ethanol and xylene. Afterwards, paraffin chips (Histosec; Merck) were added to saturation, first at 37 °C and then at 60 °C. Finally, the tissue fragments were infiltrated with pure paraffin at 60 °C and embedded in the paraffin blocks. The paraffin blocks were cut into 3 μm sections with a Leica RM2165 microtome. After mounting on microscopic slides, the sections were deparaffinized with xylene, and then gradually rehydrated in an ethanol/xylene mixture, ethanol, and PBS. Next, the sections were subjected to the immunodetection procedure, which was carried out using the biotin/streptavidin-Alkaline Phosphatase system (Amersham), as recommended by the supplier. The sera raised against ferritin or leghaemoglobin polypeptides were used at the dilutions of 1:10 000. The sections were examined using a Nikon SMZ-10A microscope under bright-field optics and photographed with the Nikon H III and FDX-35 camera system. Results and discussion In respect to nodule senescence characteristics and stress resistance, lupins comprise a very special group among legumes. In bean, soybean, and alfalfa nodules, for example, damage of symbiosome membranes takes place before bacteroid disintegration (Pladys and Rigaud, 1985). Yet in white lupin, peribacteroid membranes are being degraded at very advanced stages of senescence, and the process of bacteroid disintegration starts inside symbiosomes (Hernandez-Jimenez et al., 2002). Furthermore, the experiments with stress-induced root nodule senescence showed a higher tolerance of lupin nodule tissues to darkness or nitrate than in other legumes (Matamoros et al., 1999; Hernandez-Jimenez et al., 2002). Our previous analyses concerning possible ferritin involvement in symbiotic interactions were limited to three major time points: before nitrogen fixation onset (5 dpi), fully active nodules (25–35 dpi), and senescing nodules (50–70 dpi) (Strozycki et al., 2003). The results suggested that the overall level of ferritin synthesis in lupin nodules is linearly correlated with nodule age. It was in agreement with the general belief that ferritin synthesis in nodules is associated mainly with the senescence of this organ. Ferritin accumulation during the development of lupin indeterminate-type root nodule Immunodetection with ferritin-specific antibodies demonstrated significant amounts of ferritin polypeptides throughout the course of lupin nodule development. In a more detailed experiment presented in this paper, two major periods of ferritin accumulation were distinguished in growing nodules (Figs 2, 3). The first temporary increase, around 14 d post-inoculation (14 dpi), correlated with massive (apo)leghaemoglobin synthesis, may be, at least partially, explained by the properties of haem synthesized to form functional leghaemoglobin (O'Brian, 2000). Haem and its precursors catalyse the formation of reactive oxygen species and, unlike ‘free’ iron, they readily enter the hydrophobic domain of biological membranes (Mock and Grimm, 1997; Balla et al., 1993). It has been suggested that haem binding to ferritin influences the rate of reductive iron release (Moore et al., 1992). Moreover, it has also been demonstrated for animal systems that haem itself might directly enhance ferritin synthesis by increasing RNA translation (Lin et al., 1990; Goessling et al., 1992). Establishment of a physical oxygen barrier and oxygen gradient in the nodule most probably results in a minimized synthesis of ferritin (Iannetta et al., 1995; Karlowski et al., 2000). The second substantial increase in ferritin accumulation (around 24 dpi) was correlated with the highest leghaemoglobin level (Figs 2, 3; Strozycki and Legocki, 1995), and may also be due to an increase in processes relating to respiration and nitrogen reduction. Interestingly, in soybean (determinate-type nodules), ferritin polypeptide accumulation correlated with leghaemoglobin synthesis only during the first phase of the symbiotic interaction (Ragland and Thiel, 1993). Fig. 2. View largeDownload slide Developmental regulation of ferritin accumulation in lupin nodules. Immunodetection of ferritin (anti-ferritin antibody: Ab-Fer) and leghaemoglobin (anti-leghaemoglobin antibody: Ab-Lb) proteins. dpi (numbers on top), protein preparation from roots of 3-d-old seedlings (r), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kD). 20 μg of soluble proteins were separated by PAGE. Fig. 2. View largeDownload slide Developmental regulation of ferritin accumulation in lupin nodules. Immunodetection of ferritin (anti-ferritin antibody: Ab-Fer) and leghaemoglobin (anti-leghaemoglobin antibody: Ab-Lb) proteins. dpi (numbers on top), protein preparation from roots of 3-d-old seedlings (r), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kD). 20 μg of soluble proteins were separated by PAGE. Fig. 3. View largeDownload slide Expression of lupin ferritin genes during root nodule development and senescence. Northern hybridizations of total RNA isolated from nodules of different ages (numbers: dpi), with DNA probes: for mixed full-length cDNAs of all three lupin ferritins (Llfer), for 3' end ferritin class-specific probes (LlFer1, LlFer2, and LlFer3), and for mixed full-length cDNAs of lupin leghaemoglobins (Lllb). The last panel: ethidium bromide-stained fragment of an agarose gel. 25 μg of total RNA preparations were separated in denaturing agarose gels. Fig. 3. View largeDownload slide Expression of lupin ferritin genes during root nodule development and senescence. Northern hybridizations of total RNA isolated from nodules of different ages (numbers: dpi), with DNA probes: for mixed full-length cDNAs of all three lupin ferritins (Llfer), for 3' end ferritin class-specific probes (LlFer1, LlFer2, and LlFer3), and for mixed full-length cDNAs of lupin leghaemoglobins (Lllb). The last panel: ethidium bromide-stained fragment of an agarose gel. 25 μg of total RNA preparations were separated in denaturing agarose gels. In white lupin, ferritin was localized in plastids (including amyloplasts) at all nodule stages and in bacteroids as well (Lucas et al., 1998). Intense immunolabelling was observed in the plastids of the infected cells of young nodules, but it decreased with nodule age. These results are generally in agreement with our observations as far as ‘pre-senescing’ nodules are concerned. Our in situ immunodetection experiments revealed that, ferritin accumulation was indeed localized in the infected meristem, and mainly within young, differentiating bacteroid tissue. The central part of a 14-d-old yellow lupin nodule consists entirely of this type of tissue and thus stained uniformly with both ferritin- and leghaemoglobin-specific antibodies (Fig. 4A, D). Next, at the time of maximum nodule activity and leghaemoglobin synthesis (Fig. 4E), low levels of ferritin polypeptides were detected in differentiated bacteroid tissue (Fig. 4B). For most sections of fully developed nodules, marked amounts of ferritin were localized in the sites of remaining meristems (Fig. 4C). At the same time, together with the expansion of differentiated bacteroid tissue, leghaemoglobin detection was extended as well (Fig. 4E, F, H). The discovery of a distinct layer of cells characterized by higher ferritin content on some sections of mature nodules (c. 45 dpi; Fig. 4G) suggests that, in fact, in indeterminate lupin nodules the elevated synthesis of ferritin polypeptides may be restricted to an area located between the meristem and the differentiated bacteroid tissue. This layer is most probably a remainder of differentiating bacteroid tissue and/or the equivalent of interzone II/III of alfalfa (Vasse et al., 1990). The latter resemblance may apply only to its physiological function since, in lupin, in contrast to cylindrical indeterminate nodules, the cells of this zone do not exhibit dramatic starch accumulation (Golinowski et al., 1987). Fig. 4. View largeDownload slide Immunolocalization of ferritin and leghaemoglobin polypeptides in the tissues of developing lupin nodules. Age of the nodule (dpi); antibodies used for protein detection: Ab-Fer (anti-ferritin), Ab-Lb (anti-leghaemoglobin). Markers in the right upper corner define 1 mm scale, nodule meristem (m), differentiating bacteroid tissue (b1), differentiated bacteroid tissue (b2), arrows indicate specific zone of cells with increased ‘ferritin staining’ (G). Note that the dense clustering and indeterminate nature of lupin nodule development results in the sections that contain fragments of overlapping nodules. (B) and (E) as well as (C) and (F) come from the same nodules. Fig. 4. View largeDownload slide Immunolocalization of ferritin and leghaemoglobin polypeptides in the tissues of developing lupin nodules. Age of the nodule (dpi); antibodies used for protein detection: Ab-Fer (anti-ferritin), Ab-Lb (anti-leghaemoglobin). Markers in the right upper corner define 1 mm scale, nodule meristem (m), differentiating bacteroid tissue (b1), differentiated bacteroid tissue (b2), arrows indicate specific zone of cells with increased ‘ferritin staining’ (G). Note that the dense clustering and indeterminate nature of lupin nodule development results in the sections that contain fragments of overlapping nodules. (B) and (E) as well as (C) and (F) come from the same nodules. Ferritin and lupin nodule senescence The first symptoms of lupin nodule tissue decay, visible at the light microscope level (c. 45–55 dpi; Fig. 1), occurred just before the signs of flower formation, and were always followed by local bacteroid tissue degradation. Most probably, the successive generation of free radicals and degradation of bacteroid membranes participated in a ‘spread’ of nodule tissue destruction (Becana and Klucas, 1992; Moreau et al., 1996). Senescence was also the period when the highest accumulation of ferritin mRNA and corresponding polypeptides were observed in lupin nodules (Figs 2, 3). Based on strong ferritin immunostaining in leucoplasts or amyloplasts of cells of the nodule cortex, it was earlier suggested for white lupin that the increase in ferritin content during nodule senescence was exclusively due to this tissue (Lucas et al., 1998). In our more ‘macroscopic’ view, this could be true for the late stages of nodule decay. It was found that the most intense staining at this stage, however, concentrated in the remaining ‘young’ zones, close to meristems that were still active. Interestingly, in the senescing nodules, intense staining also extended over the cells of differentiated bacteroid tissue (Fig. 5A, B). At the same time, strong leghaemoglobin staining remained throughout the unaffected bacteroid tissue (Fig. 5C). It is concluded that, at the time when the predominant part of the nodule is still active, the quantitative increase in ferritin accumulation (detected by protein blots) comes from its elevated accumulation in bacteroid tissue. This indicates that the signals related to iron and/or oxidative stress penetrate the still functioning cells adjoining the senescent zone that induces ferritin synthesis. This may also suggest that ferritin may be a part of the ‘second-line’, defence system and participate in prolongation of functioning of indeterminate-type nodule, compared with an early decline in nitrogen fixation in soybean nodules (Dalton et al., 1991; Mohammadi and Karr, 2001). In addition, our observations of the increase in ferritin immunostaining of the nodule cortex, as well as nodule vascular bundles and root stele (Fig. 5B, D), correlated with the symptoms of bacteroid tissue senescence, fully support the results on iron translocation from the senescing nodule to other parts of a plant (Burton et al., 1998). Fig. 5. View largeDownload slide Ferritin and leghaemoglobin polypeptides in senescing lupin nodules. Individual sections described as for Fig. 4. Senescent zone (sc), degraded zone (dg), inner cortex (ic), arrows indicate phloem strands in the nodule vascular bundles (D). Dashed-line rectangle (B) defines the area which is shown as an enlargement in (D). Some background (higher on samples from older plants) comes from unspecific staining of cell walls, especially the lignified ones. Fig. 5. View largeDownload slide Ferritin and leghaemoglobin polypeptides in senescing lupin nodules. Individual sections described as for Fig. 4. Senescent zone (sc), degraded zone (dg), inner cortex (ic), arrows indicate phloem strands in the nodule vascular bundles (D). Dashed-line rectangle (B) defines the area which is shown as an enlargement in (D). Some background (higher on samples from older plants) comes from unspecific staining of cell walls, especially the lignified ones. Regulation of ferritin synthesis In contrast to animal systems, in plants iron regulates ferritin synthesis mainly at the transcriptional level (Lescure et al., 1991; Wei and Thiel, 2000; Petit et al., 2001b). In mature soybean nodules, however, the contribution of post-transcriptional mechanisms in ferritin synthesis was demonstrated (Ragland and Thiel, 1993). Post-translational regulation of ferritin turnover was also suggested (Kimata and Thiel, 1994). The results on ferritin polypeptide and mRNA accumulation during lupin nodule development revealed an unexpected picture. Ferritin levels in lupin nodules of different ages reflected nearly exactly the corresponding RNA accumulation pattern (Figs 2, 3). This similarity includes the correlation of the temporary increase in ferritin mRNA accumulation nearly when leghaemoglobin synthesis starts, then at the time of its highest mRNA levels, as well as in senescing nodules. This picture may suggest that, in lupin indeterminate-type nodules, unlike soybean determinate-type nodules, ferritin synthesis is regulated generally on a transcriptional level. The reduced ferritin mRNA accumulation, together with the highest levels of ferritin protein in the oldest nodules, could be the result of a high stability of the ferritin polypeptide. To complete the picture of ferritin expression regulation in lupin, its protein and mRNA accumulation were also investigated in organs other than nodules (3–94-d-old plants; Figs 6, 7). In roots, low but marked levels of both ferritin message and protein were detected, while in the leaves, elevated mRNA levels were followed by high polypeptide accumulation. Furthermore, both ferritin mRNA and protein contents which were low in the young stem, rose with its age. Yet in pods, both ferritin polypeptide and its mRNA were relatively abundant. Transcription seems to be the main level of ferritin synthesis regulation in these organs. In the flowers, however, where ferritin mRNA was the most abundant of all organs tested, protein accumulation was disproportionately lower. Moreover, in cotyledons (background level for the oldest) and young seeds (ferritin was detected in mature seeds; data not presented), with a distinct presence of ferritin mRNA, ferritin polypeptide was undetectable. These observations suggest that, at least in some lupin organs, post-transcriptional regulation processes participate in the control of ferritin levels. These results support those presented for maize mutants, where involvement of the second (to transcriptional; iron controlled) factor acting translationally in leaves was also postulated, but it was in contrast with those for pea, where ferritin was almost undetectable in vegetative organs such as roots and leaves (Lobreaux and Briat, 1991; Motta et al., 2001). Fig. 6. View largeDownload slide Ferritin accumulation in different organs of lupin plants. Immunodetection of ferritin polypeptides (Ab-Fer) in fractions of soluble proteins. Age of plants (numbers on top: dpi), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kDa). Amounts of proteins separated as in Fig. 2. Fig. 6. View largeDownload slide Ferritin accumulation in different organs of lupin plants. Immunodetection of ferritin polypeptides (Ab-Fer) in fractions of soluble proteins. Age of plants (numbers on top: dpi), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kDa). Amounts of proteins separated as in Fig. 2. Fig. 7. View largeDownload slide Identification of ferritin mRNAs in organs of lupin plants. Probes indicated as for Fig. 3. The last panel: ethidium bromide-stained fragment of an agarose gel. Amounts of RNAs separated as in Fig. 3. Fig. 7. View largeDownload slide Identification of ferritin mRNAs in organs of lupin plants. Probes indicated as for Fig. 3. The last panel: ethidium bromide-stained fragment of an agarose gel. Amounts of RNAs separated as in Fig. 3. Using 3′-end specific probes, it was possible to show the most probable contribution of mRNA of each ferritin class to the ferritin complex synthesis (Fig. 3). It was characteristic that only two (of the three) ferritin mRNAs (Llfer2 and 3) were clearly detectable during lupin nodule development and in most of the tissues analysed. Llfer1 mRNA was observed clearly above the background levels in lupin flowers only, suggesting that the demand for this subunit is generally low. The Llfer1 subunit may take part in stabilization of native ferritin 24-mers, as was suggested for the soybean H-2 subunit (Masuda et al., 2001). Interestingly, the Llfer2 mRNA, the only ferritin mRNA synthesized in response to ABA, seemed to be the most abundant in mature and senescing nodules (Strozycki et al., 2003). This convergence may support the model suggesting that the tissue changes in ascorbate, nitrogen, and carbon levels induce signalling, leading to increased synthesis and transport of the ABA to the nodule, and this way orchestrates nodule senescence (Puppo et al., 2005). Perspectives It seems clear that during indeterminate-type lupin nodule formation, ferritin synthesis is associated mainly with differentiating bacteroid tissue, and correlates with the expression of leghaemoglobin genes. One could speculate on a more complex function for ferritins than just acting as an iron-storage complex (changes in ion status, changes in oxidative states, etc). In animal systems for example, ferritin polypeptides directly regulate the activity of the globin gene promoter (Wu and Noguchi, 1991; Broyles et al., 2001). The senescence processes initiated in the oldest nodule zones reactivate ferritin synthesis in differentiated bacteroid tissue of the adjoining, younger, and still active zones. The latter indicates that in indeterminate-type nodules, ferritin may be an element of the senescence delay mechanism. Such a delay could prolong the time of nitrogen fixation and allow for more effective remobilization of iron to the floral parts of the plant. Abbreviations Abbreviations ABA abscisic acid ROS reactive oxygen species The authors wish to thank Ms I Femiak, Ms A Kasperska, and Ms K Szczesniak-Kolaczkowska for their excellent technical assistance. Special thanks belong to Dr EC Theil (Center for BioIron at CHORI, Oakland, CA, USA) for constructive advice and comments. This work was partially supported by the grant 6-P04B-001-13 from the Polish State Committee for Scientific Research and grants from Centre Franco-Polonais de Biotechnologie des Plantes and grant CRP/POL03-02 from ICGEB. 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Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules

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Oxford University Press
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© The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]
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0022-0957
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10.1093/jxb/erm152
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17890761
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Abstract

Abstract An ability to form symbiotic associations with rhizobia and to utilize atmospheric nitrogen makes legumes ecologically successful. High iron content in legume grains, partially relocated from root nodules, is another-nutritional-advantage of this group of plants. The ferritin complex is the major cell iron storage and detoxification unit and has been recognized as a marker of many stress-induced responses. The possible participation of ferritin in nodule formation and functioning was investigated here. Correlation of increased accumulation of both ferritin polypeptide and mRNA with actual in situ localization of ferritin allowed ferritin synthesis in the developing, indeterminate-type root nodules to be related to differentiating bacteroid tissue. This kind of tissue, in contrast to the determinate-type nodules, is present in lupin nodules at almost all stages of their development. Interestingly, it was found that, in this type of nodule, senescence starting in the decaying zones induces ferritin accumulation in younger, still active, tissues. Based on the presented data, and in correlation with previous results, some aspects of the regulation of expression of lupin ferritin genes are also discussed. Biological nitrogen fixation, ferritin, indeterminate-type nodule, iron, oxidative stress, plant–microbe interactions Introduction Iron-containing proteins play a key role in such crucial processes as respiration, photosynthesis and DNA or hormone syntheses. Symbiotic nitrogen fixation, however, is a phenomenon particularly dependent on iron-containing proteins. Free-living bacteria turning into microsymbionts (bacteroids) triple their cytochrome accumulation and synthesize nitrogenase. The latter may contain more than 30 atoms of iron and can constitute more than 10% of total bacterial proteins (Verma and Long, 1983; Sangwan and O'Brian, 1992). To protect this enzyme from oxygen-induced inactivation and to supply bacteroids with enough oxygen for their respiration, a plant fills the cytoplasm of bacteroid tissue cells with another iron-containing protein, leghaemoglobin (Lb). Lb may account for up to 30% of all soluble proteins of the infected cell, which makes a nodule one of the most iron-loaded organs of a plant (Appleby, 1984; Kuzma et al., 1993). Although cellular iron is mostly bound in proteins and their cofactors, it may be released to the cytoplasm in various stress situations. The physico-chemical properties of iron ions (small ionic radius, high reactivity) and the fact that they catalyse the formation of hydroxyl radical (Fenton reaction), the most potent oxidizing agent known in nature, make iron responsible for the damage to nucleic acids, proteins, and lipids (Guerinot and Yi, 1994). The nodules are better equipped with all kinds of antioxidant systems (i.e. ascorbate-glutathione pathway or superoxide dismutases) than the parent root, which ensures effective detoxification of the superoxide radical and excess H2O2 (Becana et al., 2000; Matamoros et al., 2003). However, during senescence, a rise in proteases activities, a decline in the antioxidant pool, and increased ROS accumulation promote nodule decay (Evans et al., 1999; Alesandrini et al., 2003). Protein degradation and membrane disruption lead to the significant increase of free iron concentration (Becana and Klucas, 1992; Mathieu et al., 1998; Hernandez-Jimenez et al., 2002). Such ‘unattended’ iron accelerates the spread of nodule tissue disruption and nitrogen fixation termination. The most effective way to limit iron noxiousness seems to be its sequestration. Organisms from bacteria to animals possess a protein cage in which iron may be kept harmless, but available for all vital processes, and this is ferritin (Theil, 1987). Ferritins are 24-subunit protein spherical complexes, capable of the reversible transfer of iron between liquid and solid phases (Chasteen and Harrison, 1999; Theil, 2003). Iron is then concentrated inside the protein cavity (up to 4500 iron atoms) as a hydrated ferric oxide (Liu and Theil, 2005). Not only iron sequestration, but also removal of dioxygen from the cytoplasm (mineral core formation) make ferritin an effective part of the antioxidant system (Zhao et al., 2003). It was suggested earlier that both the rapidity of senescence and the extent of stress tolerance of symbiotic interactions may be, to a great extent, defined by the structure and developmental pattern of the nodules (indeterminate- versus determinate-type of nodule) (Sprent, 1980; Hirsch, 1992). In our research on iron management in symbiotic nitrogen fixation, yellow lupin was used as a plant model. Possession of long-lasting meristems as well as clearly divided developmental zones makes the general anatomy of lupinoid nodules similar to those of an indeterminate type (Fig. 1) (Golinowski et al., 1987, 1992; Lotocka et al., 2000). In addition, because of the special evolutionary position of lupins with those of the earliest branching lineages within the Papilionoideae subfamily, this plant system appeared very attractive to study (Kass and Wink, 1995; Strozycki and Legocki, 1995; Strozycki et al., 2000). Previously, three lupin ferritin genes were identified, which, similarly to those in maize and Arabidopsis, were differently regulated in response to iron and ABA (Fobis-Loisy et al., 1995; Petit et al., 2001a; Strozycki et al., 2003). Here, for the first time, are presented the analyses of both RNA and protein samples, as well as tissue fragments originating from the same plants. This allowed the changes in ferritin gene expression during development and senescence of a nodule to be correlated with specific tissues of this organ. Characteristic, developmental distribution of ferritin in indeterminate type lupin nodules may indicate that its function is more complex than just being an iron storage unit. Fig. 1. View largeDownload slide Lupinus luteus root nodules. (A) Young nodules: because of very early division of laterally-positioned nodule meristem and its growth in all directions, the lupin nodule escapes from its cylindrical shape. (B) As a result of the activity of lateral meristems, the initially spherical nodules grow laterally encircling the root (Golinowski et al., 1987). (C) Cross-sections through nodules at different stages of development. (D) Schematic diagram of the mature nodule. Fig. 1. View largeDownload slide Lupinus luteus root nodules. (A) Young nodules: because of very early division of laterally-positioned nodule meristem and its growth in all directions, the lupin nodule escapes from its cylindrical shape. (B) As a result of the activity of lateral meristems, the initially spherical nodules grow laterally encircling the root (Golinowski et al., 1987). (C) Cross-sections through nodules at different stages of development. (D) Schematic diagram of the mature nodule. Elucidation of possible ways of iron management in the regulation of symbiotic interactions seems to be essential for understanding legume plant development. In addition, the data proving that iron accumulated in seed ferritin comes in large part from the nodule, partially explain the high iron content of legume seeds (Burton et al., 1998). This, together with growing evidence on the nutritional importance of ferritin iron, substantiates the studies on legume ferritin (Theil, 2004). Materials and methods Plant material Yellow lupin (Lupinus luteus L. cv. Ventus) was used for all analyses. The seeds were sterilized with sodium hypochloride or chlorox, washed and germinated for 3 d at 25 °C in the dark. When needed for the analyses of symbiotic interactions, the roots of 3-d-old seedlings were inoculated with Bradyrhizobium bv. genistearum, previously cultivated for 5–7 d at 28 °C. The inoculated plants were transferred to perlite-filled pots in a culture room. They were grown in a 16/8 h light/dark photoperiod at 24/26 °C during the day, and 21/23 °C at night, fertilized with nitrogen-free medium every three days, except for the first week when 0.01 mM nitrogen was applied. A detailed description of growth conditions and medium contents was previously described by Strozycki (2003). The tissues (i.e. root segments, root nodules, stems, cotyledons, flowers, pods, seeds) were collected at the designated time and immediately frozen in liquid nitrogen. The time points for sample collecting were designated based on previous observations of lupin development. Individual nodule tissue samples were harvested up to 91 d post Bradyrhizobium inoculation (dpi). The early stages of nodule formation were not analysed. The soluble proteins and total RNA isolated from the collected tissues were analysed. Isolation and analysis of protein and RNA fractions The soluble proteins and fractions of total RNA were isolated ‘in parallel’ from separate fragments of the same tissue samples, as described previously by Strozycki and Legocki (1995); the ammonium sulphate precipitation for the proteins was omitted. The samples (35 μg of proteins or 10 μg of total RNA) were separated in the required gels [15% SDS-PAGE (Laemmli, 1970) or 2% agarose, containing formaldehyde (Sambrook et al., 1989)], and transferred onto proper membranes (Hybond-C extra or Hybond-N; Amersham). The ferritin and leghaemoglobin polypeptides were identified by western-type hybridizations, using the appropriate polyclonal antibodies (Sikorski et al., 1995; Strozycki et al., 2003) and the biotin/streptavidin-AP system (Amersham). To display the possible participation of each ferritin class in the overall ferritin content, specific (3′-end) molecular probes generated in PCR reactions were used in northern hybridizations. Full-length cDNAs of lupin ferritins were used as a mixed probe in order to show the general (global alterations) in ferritin mRNA level. For more details see Strozycki et al. (2003). In making quantitative assumptions, it has to be considered that, in lupin, continually growing collar-shaped nodules, meristem, and young tissues are located laterally within the mature nodule (in the tip of the ‘cone’) (Fig. 1). Their share in nodule volume decreases with time (Lotocka et al., 1995). Therefore, in matured nodules, their volumes make proportionally small quantitative contributions to the overall picture. In situ immunochemical protein detection Fragments of nodules (collected from the same plants as for protein and RNA isolations) c. 3 mm thick, were fixed in 4% paraformaldehyde in PBS buffer (10 mM Na2HPO4/NaH2PO4 pH 7.5, 150 mM NaCl) for 18 h at –400 hPa air pressure, and then washed in PBS (3×15 min.). The fixed tissue was dehydrated by successive changes of graded ethanol and xylene. Afterwards, paraffin chips (Histosec; Merck) were added to saturation, first at 37 °C and then at 60 °C. Finally, the tissue fragments were infiltrated with pure paraffin at 60 °C and embedded in the paraffin blocks. The paraffin blocks were cut into 3 μm sections with a Leica RM2165 microtome. After mounting on microscopic slides, the sections were deparaffinized with xylene, and then gradually rehydrated in an ethanol/xylene mixture, ethanol, and PBS. Next, the sections were subjected to the immunodetection procedure, which was carried out using the biotin/streptavidin-Alkaline Phosphatase system (Amersham), as recommended by the supplier. The sera raised against ferritin or leghaemoglobin polypeptides were used at the dilutions of 1:10 000. The sections were examined using a Nikon SMZ-10A microscope under bright-field optics and photographed with the Nikon H III and FDX-35 camera system. Results and discussion In respect to nodule senescence characteristics and stress resistance, lupins comprise a very special group among legumes. In bean, soybean, and alfalfa nodules, for example, damage of symbiosome membranes takes place before bacteroid disintegration (Pladys and Rigaud, 1985). Yet in white lupin, peribacteroid membranes are being degraded at very advanced stages of senescence, and the process of bacteroid disintegration starts inside symbiosomes (Hernandez-Jimenez et al., 2002). Furthermore, the experiments with stress-induced root nodule senescence showed a higher tolerance of lupin nodule tissues to darkness or nitrate than in other legumes (Matamoros et al., 1999; Hernandez-Jimenez et al., 2002). Our previous analyses concerning possible ferritin involvement in symbiotic interactions were limited to three major time points: before nitrogen fixation onset (5 dpi), fully active nodules (25–35 dpi), and senescing nodules (50–70 dpi) (Strozycki et al., 2003). The results suggested that the overall level of ferritin synthesis in lupin nodules is linearly correlated with nodule age. It was in agreement with the general belief that ferritin synthesis in nodules is associated mainly with the senescence of this organ. Ferritin accumulation during the development of lupin indeterminate-type root nodule Immunodetection with ferritin-specific antibodies demonstrated significant amounts of ferritin polypeptides throughout the course of lupin nodule development. In a more detailed experiment presented in this paper, two major periods of ferritin accumulation were distinguished in growing nodules (Figs 2, 3). The first temporary increase, around 14 d post-inoculation (14 dpi), correlated with massive (apo)leghaemoglobin synthesis, may be, at least partially, explained by the properties of haem synthesized to form functional leghaemoglobin (O'Brian, 2000). Haem and its precursors catalyse the formation of reactive oxygen species and, unlike ‘free’ iron, they readily enter the hydrophobic domain of biological membranes (Mock and Grimm, 1997; Balla et al., 1993). It has been suggested that haem binding to ferritin influences the rate of reductive iron release (Moore et al., 1992). Moreover, it has also been demonstrated for animal systems that haem itself might directly enhance ferritin synthesis by increasing RNA translation (Lin et al., 1990; Goessling et al., 1992). Establishment of a physical oxygen barrier and oxygen gradient in the nodule most probably results in a minimized synthesis of ferritin (Iannetta et al., 1995; Karlowski et al., 2000). The second substantial increase in ferritin accumulation (around 24 dpi) was correlated with the highest leghaemoglobin level (Figs 2, 3; Strozycki and Legocki, 1995), and may also be due to an increase in processes relating to respiration and nitrogen reduction. Interestingly, in soybean (determinate-type nodules), ferritin polypeptide accumulation correlated with leghaemoglobin synthesis only during the first phase of the symbiotic interaction (Ragland and Thiel, 1993). Fig. 2. View largeDownload slide Developmental regulation of ferritin accumulation in lupin nodules. Immunodetection of ferritin (anti-ferritin antibody: Ab-Fer) and leghaemoglobin (anti-leghaemoglobin antibody: Ab-Lb) proteins. dpi (numbers on top), protein preparation from roots of 3-d-old seedlings (r), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kD). 20 μg of soluble proteins were separated by PAGE. Fig. 2. View largeDownload slide Developmental regulation of ferritin accumulation in lupin nodules. Immunodetection of ferritin (anti-ferritin antibody: Ab-Fer) and leghaemoglobin (anti-leghaemoglobin antibody: Ab-Lb) proteins. dpi (numbers on top), protein preparation from roots of 3-d-old seedlings (r), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kD). 20 μg of soluble proteins were separated by PAGE. Fig. 3. View largeDownload slide Expression of lupin ferritin genes during root nodule development and senescence. Northern hybridizations of total RNA isolated from nodules of different ages (numbers: dpi), with DNA probes: for mixed full-length cDNAs of all three lupin ferritins (Llfer), for 3' end ferritin class-specific probes (LlFer1, LlFer2, and LlFer3), and for mixed full-length cDNAs of lupin leghaemoglobins (Lllb). The last panel: ethidium bromide-stained fragment of an agarose gel. 25 μg of total RNA preparations were separated in denaturing agarose gels. Fig. 3. View largeDownload slide Expression of lupin ferritin genes during root nodule development and senescence. Northern hybridizations of total RNA isolated from nodules of different ages (numbers: dpi), with DNA probes: for mixed full-length cDNAs of all three lupin ferritins (Llfer), for 3' end ferritin class-specific probes (LlFer1, LlFer2, and LlFer3), and for mixed full-length cDNAs of lupin leghaemoglobins (Lllb). The last panel: ethidium bromide-stained fragment of an agarose gel. 25 μg of total RNA preparations were separated in denaturing agarose gels. In white lupin, ferritin was localized in plastids (including amyloplasts) at all nodule stages and in bacteroids as well (Lucas et al., 1998). Intense immunolabelling was observed in the plastids of the infected cells of young nodules, but it decreased with nodule age. These results are generally in agreement with our observations as far as ‘pre-senescing’ nodules are concerned. Our in situ immunodetection experiments revealed that, ferritin accumulation was indeed localized in the infected meristem, and mainly within young, differentiating bacteroid tissue. The central part of a 14-d-old yellow lupin nodule consists entirely of this type of tissue and thus stained uniformly with both ferritin- and leghaemoglobin-specific antibodies (Fig. 4A, D). Next, at the time of maximum nodule activity and leghaemoglobin synthesis (Fig. 4E), low levels of ferritin polypeptides were detected in differentiated bacteroid tissue (Fig. 4B). For most sections of fully developed nodules, marked amounts of ferritin were localized in the sites of remaining meristems (Fig. 4C). At the same time, together with the expansion of differentiated bacteroid tissue, leghaemoglobin detection was extended as well (Fig. 4E, F, H). The discovery of a distinct layer of cells characterized by higher ferritin content on some sections of mature nodules (c. 45 dpi; Fig. 4G) suggests that, in fact, in indeterminate lupin nodules the elevated synthesis of ferritin polypeptides may be restricted to an area located between the meristem and the differentiated bacteroid tissue. This layer is most probably a remainder of differentiating bacteroid tissue and/or the equivalent of interzone II/III of alfalfa (Vasse et al., 1990). The latter resemblance may apply only to its physiological function since, in lupin, in contrast to cylindrical indeterminate nodules, the cells of this zone do not exhibit dramatic starch accumulation (Golinowski et al., 1987). Fig. 4. View largeDownload slide Immunolocalization of ferritin and leghaemoglobin polypeptides in the tissues of developing lupin nodules. Age of the nodule (dpi); antibodies used for protein detection: Ab-Fer (anti-ferritin), Ab-Lb (anti-leghaemoglobin). Markers in the right upper corner define 1 mm scale, nodule meristem (m), differentiating bacteroid tissue (b1), differentiated bacteroid tissue (b2), arrows indicate specific zone of cells with increased ‘ferritin staining’ (G). Note that the dense clustering and indeterminate nature of lupin nodule development results in the sections that contain fragments of overlapping nodules. (B) and (E) as well as (C) and (F) come from the same nodules. Fig. 4. View largeDownload slide Immunolocalization of ferritin and leghaemoglobin polypeptides in the tissues of developing lupin nodules. Age of the nodule (dpi); antibodies used for protein detection: Ab-Fer (anti-ferritin), Ab-Lb (anti-leghaemoglobin). Markers in the right upper corner define 1 mm scale, nodule meristem (m), differentiating bacteroid tissue (b1), differentiated bacteroid tissue (b2), arrows indicate specific zone of cells with increased ‘ferritin staining’ (G). Note that the dense clustering and indeterminate nature of lupin nodule development results in the sections that contain fragments of overlapping nodules. (B) and (E) as well as (C) and (F) come from the same nodules. Ferritin and lupin nodule senescence The first symptoms of lupin nodule tissue decay, visible at the light microscope level (c. 45–55 dpi; Fig. 1), occurred just before the signs of flower formation, and were always followed by local bacteroid tissue degradation. Most probably, the successive generation of free radicals and degradation of bacteroid membranes participated in a ‘spread’ of nodule tissue destruction (Becana and Klucas, 1992; Moreau et al., 1996). Senescence was also the period when the highest accumulation of ferritin mRNA and corresponding polypeptides were observed in lupin nodules (Figs 2, 3). Based on strong ferritin immunostaining in leucoplasts or amyloplasts of cells of the nodule cortex, it was earlier suggested for white lupin that the increase in ferritin content during nodule senescence was exclusively due to this tissue (Lucas et al., 1998). In our more ‘macroscopic’ view, this could be true for the late stages of nodule decay. It was found that the most intense staining at this stage, however, concentrated in the remaining ‘young’ zones, close to meristems that were still active. Interestingly, in the senescing nodules, intense staining also extended over the cells of differentiated bacteroid tissue (Fig. 5A, B). At the same time, strong leghaemoglobin staining remained throughout the unaffected bacteroid tissue (Fig. 5C). It is concluded that, at the time when the predominant part of the nodule is still active, the quantitative increase in ferritin accumulation (detected by protein blots) comes from its elevated accumulation in bacteroid tissue. This indicates that the signals related to iron and/or oxidative stress penetrate the still functioning cells adjoining the senescent zone that induces ferritin synthesis. This may also suggest that ferritin may be a part of the ‘second-line’, defence system and participate in prolongation of functioning of indeterminate-type nodule, compared with an early decline in nitrogen fixation in soybean nodules (Dalton et al., 1991; Mohammadi and Karr, 2001). In addition, our observations of the increase in ferritin immunostaining of the nodule cortex, as well as nodule vascular bundles and root stele (Fig. 5B, D), correlated with the symptoms of bacteroid tissue senescence, fully support the results on iron translocation from the senescing nodule to other parts of a plant (Burton et al., 1998). Fig. 5. View largeDownload slide Ferritin and leghaemoglobin polypeptides in senescing lupin nodules. Individual sections described as for Fig. 4. Senescent zone (sc), degraded zone (dg), inner cortex (ic), arrows indicate phloem strands in the nodule vascular bundles (D). Dashed-line rectangle (B) defines the area which is shown as an enlargement in (D). Some background (higher on samples from older plants) comes from unspecific staining of cell walls, especially the lignified ones. Fig. 5. View largeDownload slide Ferritin and leghaemoglobin polypeptides in senescing lupin nodules. Individual sections described as for Fig. 4. Senescent zone (sc), degraded zone (dg), inner cortex (ic), arrows indicate phloem strands in the nodule vascular bundles (D). Dashed-line rectangle (B) defines the area which is shown as an enlargement in (D). Some background (higher on samples from older plants) comes from unspecific staining of cell walls, especially the lignified ones. Regulation of ferritin synthesis In contrast to animal systems, in plants iron regulates ferritin synthesis mainly at the transcriptional level (Lescure et al., 1991; Wei and Thiel, 2000; Petit et al., 2001b). In mature soybean nodules, however, the contribution of post-transcriptional mechanisms in ferritin synthesis was demonstrated (Ragland and Thiel, 1993). Post-translational regulation of ferritin turnover was also suggested (Kimata and Thiel, 1994). The results on ferritin polypeptide and mRNA accumulation during lupin nodule development revealed an unexpected picture. Ferritin levels in lupin nodules of different ages reflected nearly exactly the corresponding RNA accumulation pattern (Figs 2, 3). This similarity includes the correlation of the temporary increase in ferritin mRNA accumulation nearly when leghaemoglobin synthesis starts, then at the time of its highest mRNA levels, as well as in senescing nodules. This picture may suggest that, in lupin indeterminate-type nodules, unlike soybean determinate-type nodules, ferritin synthesis is regulated generally on a transcriptional level. The reduced ferritin mRNA accumulation, together with the highest levels of ferritin protein in the oldest nodules, could be the result of a high stability of the ferritin polypeptide. To complete the picture of ferritin expression regulation in lupin, its protein and mRNA accumulation were also investigated in organs other than nodules (3–94-d-old plants; Figs 6, 7). In roots, low but marked levels of both ferritin message and protein were detected, while in the leaves, elevated mRNA levels were followed by high polypeptide accumulation. Furthermore, both ferritin mRNA and protein contents which were low in the young stem, rose with its age. Yet in pods, both ferritin polypeptide and its mRNA were relatively abundant. Transcription seems to be the main level of ferritin synthesis regulation in these organs. In the flowers, however, where ferritin mRNA was the most abundant of all organs tested, protein accumulation was disproportionately lower. Moreover, in cotyledons (background level for the oldest) and young seeds (ferritin was detected in mature seeds; data not presented), with a distinct presence of ferritin mRNA, ferritin polypeptide was undetectable. These observations suggest that, at least in some lupin organs, post-transcriptional regulation processes participate in the control of ferritin levels. These results support those presented for maize mutants, where involvement of the second (to transcriptional; iron controlled) factor acting translationally in leaves was also postulated, but it was in contrast with those for pea, where ferritin was almost undetectable in vegetative organs such as roots and leaves (Lobreaux and Briat, 1991; Motta et al., 2001). Fig. 6. View largeDownload slide Ferritin accumulation in different organs of lupin plants. Immunodetection of ferritin polypeptides (Ab-Fer) in fractions of soluble proteins. Age of plants (numbers on top: dpi), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kDa). Amounts of proteins separated as in Fig. 2. Fig. 6. View largeDownload slide Ferritin accumulation in different organs of lupin plants. Immunodetection of ferritin polypeptides (Ab-Fer) in fractions of soluble proteins. Age of plants (numbers on top: dpi), purified ferritin polypeptide used as a control (fp), molecular weight markers (numbers on the left; kDa). Amounts of proteins separated as in Fig. 2. Fig. 7. View largeDownload slide Identification of ferritin mRNAs in organs of lupin plants. Probes indicated as for Fig. 3. The last panel: ethidium bromide-stained fragment of an agarose gel. Amounts of RNAs separated as in Fig. 3. Fig. 7. View largeDownload slide Identification of ferritin mRNAs in organs of lupin plants. Probes indicated as for Fig. 3. The last panel: ethidium bromide-stained fragment of an agarose gel. Amounts of RNAs separated as in Fig. 3. Using 3′-end specific probes, it was possible to show the most probable contribution of mRNA of each ferritin class to the ferritin complex synthesis (Fig. 3). It was characteristic that only two (of the three) ferritin mRNAs (Llfer2 and 3) were clearly detectable during lupin nodule development and in most of the tissues analysed. Llfer1 mRNA was observed clearly above the background levels in lupin flowers only, suggesting that the demand for this subunit is generally low. The Llfer1 subunit may take part in stabilization of native ferritin 24-mers, as was suggested for the soybean H-2 subunit (Masuda et al., 2001). Interestingly, the Llfer2 mRNA, the only ferritin mRNA synthesized in response to ABA, seemed to be the most abundant in mature and senescing nodules (Strozycki et al., 2003). This convergence may support the model suggesting that the tissue changes in ascorbate, nitrogen, and carbon levels induce signalling, leading to increased synthesis and transport of the ABA to the nodule, and this way orchestrates nodule senescence (Puppo et al., 2005). Perspectives It seems clear that during indeterminate-type lupin nodule formation, ferritin synthesis is associated mainly with differentiating bacteroid tissue, and correlates with the expression of leghaemoglobin genes. One could speculate on a more complex function for ferritins than just acting as an iron-storage complex (changes in ion status, changes in oxidative states, etc). In animal systems for example, ferritin polypeptides directly regulate the activity of the globin gene promoter (Wu and Noguchi, 1991; Broyles et al., 2001). The senescence processes initiated in the oldest nodule zones reactivate ferritin synthesis in differentiated bacteroid tissue of the adjoining, younger, and still active zones. The latter indicates that in indeterminate-type nodules, ferritin may be an element of the senescence delay mechanism. Such a delay could prolong the time of nitrogen fixation and allow for more effective remobilization of iron to the floral parts of the plant. Abbreviations Abbreviations ABA abscisic acid ROS reactive oxygen species The authors wish to thank Ms I Femiak, Ms A Kasperska, and Ms K Szczesniak-Kolaczkowska for their excellent technical assistance. Special thanks belong to Dr EC Theil (Center for BioIron at CHORI, Oakland, CA, USA) for constructive advice and comments. This work was partially supported by the grant 6-P04B-001-13 from the Polish State Committee for Scientific Research and grants from Centre Franco-Polonais de Biotechnologie des Plantes and grant CRP/POL03-02 from ICGEB. 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Journal

Journal of Experimental Botany – Oxford University Press

Published: Sep 22, 2007

Keywords: Biological nitrogen fixation ferritin indeterminate-type nodule iron oxidative stress plant–microbe interactions

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