nodules were studied by electron microscopy, gas chromatography, and laser flow microfluorometry, respectively. Bacteroids in the nodule tips were small (1to ...
Plant Physiol. (1978) 62, 526-530
Development of Bacteroids in Alfalfa (Medicago sativa) Nodules Received for publication March 21, 1978 and in revised form May 15, 1978
ALAN S. PAAU AND JOE R. COWLES Department of Biology, University of Houston, Houston, Texas 77004 DAN RAVEED' Charles F. Kettering Research Laboratory, 150 East South College Street, Yellow Springs, Ohio 45387 MATERIALS AND METHODS The morphology, acetylene reduction capability, and nucleic acid content Alfalfa plants (Medicago sativa var. Buffalo) were grown in a of bacterolds In different regions of alfalfa (Medcago sadva var. Buffalo) greenhouse and inoculated with cultures of Rhizobium meliloti F28 nodules were studied by electron microscopy, gas chromatography, and I week after germination. The nodules, typically 3 to 4 mm in laser flow microfluorometry, respectively. Bacteroids in the nodule tips length and 1 mm in diameter, were harvested 6 to 8 weeks after were small (1 to 2.5 micrometers in length), had low nucleic acid content, inoculation and either fixed for microscopic examination or secand contained distinct central nucleolds. These bacteroids were compara- tioned into three regions based primarily on their color charactertively inactive in acetylene reduction in situ. Bacteroids in the middle istics. Morphologically, the tip portion of a 6- to 8-week-old alfalfa regions of alfalfa nodules were greatly enlarged (5 to 7 micrometers in nodule can be visually distinguished by its white coloration. This length), had relatively high nucleic acid content, and did not possess central is followed by a pink middle region and fmally a greenish brown nucleolds. The bacteroids were very active in acetylene reduction. Bacte- basal region next to the plant root. The nodule sections were roids in the basal nodule region also were enlarged and without distinct pooled for various analyses. nucleoid regions, but had relatively low nucleic acid content and low in situ For light microscopy, whole nodules were fixed for 2 days in acetylene-reducing activity. five changes of formalin-acetic acid-ethanol (1:1:13) and dehydrated in a graded series of ethanol. The samples were then embedded in Paraplast, sectioned, and stained with Bengal rose and light green (2). For electron microscopy, the whole nodules were fixed in 2% glutaraldehyde for 24 hr at room temperature, postfixed in 1% The establishment of symbiotic nitrogen fixation between Rhi- OS04 for 1 hr, and dehydrated in a graded series of acetone. The zobium spp. and leguminous plants includes the successful devel- samples were then embedded in Spurr's medium (13), sectioned opment of free-living rhizobia to bacteroids. Bacteroids, especially and stained with 4% uranyl acetate and lead citrate (12). those of alfalfa and clover, have been shown to differ from the The FMF2 analysis was conducted on bacteroids of the three free-living rhizobia in several parameters including size, shape, nodule sections. The nodule sections were homogenized and bacand nucleic acid content (7, 10). The details of the subtle devel- teroids isolated by differential centrifugation as previously deopment or transformation of bacteroids have been difficult to scribed (4, 5). The isolated bacteroids were fixed overnight at 0 to study partly because the bacteroid populations isolated from 4 C in 64% ethanol and stained for 30 min in 0.005% ethidium legume nodules are usually heterogeneous and contain bacteroids bromide-1.12% sodium citrate (10). The architecture and operarepresenting various stages of development. tional principles of the FMF are similar to that described by Recent efforts to obtain comparatively more homogeneous bac- Steinkamp et al. (14). The intensity of the EB fluorescence emitted teroid populations, with respect to cell density and size, have been from each bacteroid in a population after laser irradiation was reported (3, 15). Attempts to determine which developmental stage used to measure the relative nucleic acid content. The intensity of each of these fractionated populations represents, are still difficult. the EB signal was amplified, recorded, and subsequently displayed Microscopic examination of nodules reveals the predominant on a 256-channel pulse-height analyzer. The intensity of the location of certain bacteroids with respect to size, shape, and fluorescence signals quantitatively reflects the nucleic acid content morphological form but it is difficult to relate these morphological of the bacteroids. The channel number in which a signal is parameters to changes in such physiological or biochemical pa- recorded depends on the signal intensity and is directly proporrameters as enzyme activities and nucleic acid content (1, 6, 10, tional to the nucleic acid content. A bacteroid that emitted a signal 11), which also are known to accompany the developmental recorded at channel 100 had approximately twice the nucleic acid process. This communication reports an effort to relate the mor- content of a bacteroid whose signal is recorded at channel 50 (9). phological, physiological, and biochemical changes in alfalfa bac- The reported profiles are histograms of the analyses of individual teroids through the combined use of electron microscopy, gas bacteroids within a population. Bacteroid size determined by the chromatography, and laser flow microfluorometry. Alfalfa nod- FMF was based on light-scattering signals. Again, the larger the ules were chosen for this study because the nodules are nonspher- bacteroid, the more intense the scattering signal and the higher ical and propagate by nodule elongation and the bacteroids are the channel number the signal is recorded. In addition, the meaheterogeneous in size. The term "bacteroids" in this report refers surement of bacteroid size was only on particles (bacteroids) which to all of the rhizobia that are enclosed in the nodules regardless of emitted EB fluorescence. The FMF was gated on the EB fluorescence (9) such that light-scattering signals which originated from the size, shape, or location. unstained particles such as cell wall debris, were not recorded. ABSTRACT
' Present address: Department of Pathology, Indiana University School of Medicine, 100 W. Michigan St., Indianapolis, Indiana 46206. 526
2Abbreviations: FMF: flow microfluorometry; EB: ethidium bromide.
527
DEVELOPMENT OF ALFALFA BACTEROIDS
Plant Physiol. Vol. 62, 1978
by a dark electron-dense material at the bacteroid periphery (Fig. lb). The bacteroids outside the infection threads in the tip region are small and still possess a distinct central nucleoid region but do not contain large electron-empty granules. The bacteroids are enclosed by membrane envelopes and probably are newly released from the infection threads. Despite differences in location and the presence or absence of electron-empty granules, bacteroids in the tip region are characteristically small and possess a distinct central nucleoid region. The middle region of a 6- to 8-week-old alfalfa nodule usually represents about 50% of the nodule length. The infected plant cells in this region are heavily filled with bacteroids (Fig. 2a). Infection threads, which are sometimes seen in this region are, in comparison to the tip region, quantitatively negligible. Most of the bacteroids in the middle region are 4 to 6 ,um in length, 1 to 1.5 ,um in diameter, and devoid of electron-empty granules and distinct central fibrillar nucleoids (Fig. 2b). The remainder of a 6- to 8-week-old alfalfa nodule (20-25%) is made up of a greenish brown basal region and the bacteroids in this region are devoid of electron-empty granules and distinct fibrillar nucleoids (Fig. 3). Some of the bacteroids in this region, however, contain dark electron-dense granules and the surrounding envelope appears to be loosened, leaving large spaces between the envelopes and the bacteroid walls (Fig. 3b). In addition, the plant cell cytoplasm in this region very often becomes electrondense (Fig. 3a). FMF Analyses. Measurements of electron micrographs reveal that bacteroids in the middle and basal regions of an alfalfa nodule are larger than those in the tip region. This size difference
Nitrogenase activity in the nodule sections was estimated by in situ acetylene reduction (8) using a Perkin-Elmer gas chromatograph 900 equipped with a flame ionization detector. Samples of approximately equal weights (50-100 mg) representing each of the three nodule sections were incubated at 25 C in 40-ml Kimax culture tubes which were held airtight by serum stoppers. The gaseous phase in the tubes contained by volume, 65% helium, 25% oxygen, and 10o acetylene. Leghemoglobin concentration in the three sections was determined spectrophotometrically using human hemoglobin as a standard. After the sections were homogenized and the bacteroids collected, the cytosol from the nodule sections was centrifuged at 10,000g for 20 min. A 2.5-ml sample of the cytosol was added to 0.1 g of sodium-dithionite in 0.5 ml of 5 N KOH and the absorbance (A5m) determined. Cell numbers were determined either with a Petroff-Hausser bacteria chamber or with a fluorescence counter equipped in the FMF.
RESULTS Electron Microscopic Studies. A typical 6- to 8-week-old alfalfa nodule is 3 to 4 mm in length and 1 mm in diameter. The tip region of the alfalfa nodule represents about 20% of the nodule length and contains cells which usually are not completely filled with bacteroids (Fig. la). Infection threads are easily seen in this region (Fig. la) and the bacteroids enclosed within the infection threads are typically 1 to 2 ,um in length. In addition, these bacteroids have electron-empty granules (possibly poly-,B-hydroxybutyrate) and a distinct central fibrillar nucleoid surrounded lo%
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FIG. 3. Electron micrographs of alfalfa nodule in basal region. a: One of the plant cells contained electron-dense cytoplasm and the envelopes enclosing the bacteroids were loosened (x 14,000); b: bacteroids contained electron-dense granules (x 29,800). Arrows point to electron-dense granules. 528
Plant Physiol. Vol. 62, 1978
DEVELOPMENT OF ALFALFA BACTEROIDS
is confirmed by light-scattering analyses of bacteroids isolated from the three nodule sections using the FMF (Fig. 4). The average amount of scattered light by bacteroids from the middle and the basal regions is 3- to 4-fold higher than those from the tip region. The bacteroids in the middle and basal nodule region then are considerably larger than the bacteroids in the tip region. The relative number of bacteroids with light-scattering signals distributed in channels 100 and higher is 5, 35, and 45% of the total population in the tip, middle, and basal regions, respectively. This again demonstrates that the middle and basal sections have a significant number of large bacteroids and differ from the tip region in this respect. Bacteroids from the three nodule regions also were compared with respect to nucleic content using the FMF. Isolated bacteroids were treated with EB which stains primarily double-stranded nucleic acids. The intensity of the EB fluorescence of each bacteroid in the population was analyzed and recorded on a multiple channel analyzer. The greatest accumulation of signals emitted from bacteroids of the nodule tip region is around channel 30 (Fig. 5a). Small peaks also can be seen around channels 60 and 120. The maximum accumulation of signals from bacteroids in the middle nodule section was channel 100 (Fig. 5b) which reflects a 3- to 4-fold higher nucleic acid content than those in the tip region. These results along with the electron microscopic and light-scattering data demonstrate that the middle region contains predominantly enlarged bacteroids with high nucleic acid content. Bacteroids in the basal region are very heterogeneous with respect to nucleic acid content (Fig. 5c). The distribution profile failed to show a distinct peak but instead formed a continuum ranging from channels 30 to 100. Although bacteroids in the nodule basal region are similar in size to bacteroids in the middle section, they
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CHANNEL NUMBER (nucleic acid content) FIG. 5. Distributions of bacteroids isolated from tip, middle, and basal nodule regions by EB fluorescence according to nucleic acid content. Channel number is directly proportional to EB fluorescent intensity and bacteroid nucleic acid content. Signals accumulated between channels I and 20 are background fluorescence. a: Tip region; b: middle region; c: basal region.
have, in general, a much lower nucleic acid content. Acetylene Reduction. The acetylene reduction capacity of the three nodule sections was estimated by gas chromatography. The rate of acetylene reduction (1-hr incubation) by the middle section was 1.5-fold higher than that of the whole nodule and 8- and 20fold higher than the tip and basal regions, respectively (Fig. 6). The production of ethylene in the tip and basal sections either stopped or proceeded very slowly after 1 to 2 hr, while the middle sections and whole nodules continued to reduce acetylene for up to 24 hr. In some experiments, the three nodule sections, after ethylene measurements, were homogenized for determination of leghemoglobin content and bacteroid numbers (Table I). The middle sections contained more leghemoglobin and bacteroids than the other two sections. Even so, the middle sections reduced significantly more acetylene/bacteroid than the tip and basal sections (Table I).
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100 200 CHANNEL NUMBER (relative cell size ) FIG. 4. Distributions of bacteroids isolated from tip, middle, and basal nodule regions by light-scattering signals according to bacteroid size. Channel number is directly proportional to intensity of light-scattering signal which is positively related to cell size. a: Tip region; b: middle region; c: basal region.
DISCUSSION A hypothetical scheme illustrating our present views on the transformation and development of bacteroids in alfalfa nodules is shown in Figure 7. After or upon invasion of the plant tissue, the rhizobia become enclosed within the infection threads. Bacteroids at this stage of development are similar to the free-living organisms in size, nucleic acid content, and possess electron-empty granules and fibrillar nucleoids. Except for the lack of electronempty granules, bacteroids that are newly released from the infection threads, in general, are similar in size and ultrastructure to bacteroids in the infection threads, but are enclosed in envelopes. Bacteroids representing these two initial stages of development are most abundant in the tip regions of the alfalfa nodules
Plant Physiol. Vol. 62, 1978
PAAU, COWLES, AND RAVEED
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TIME (min) FIG. 6. In situ acetylene-reducing activity of tip, middle, and basal nodule regions. (0-0): middle; (EI-tJ): whole nodule; (@-4): A): base. tip; (A Table I: Comparison of acetylene reduction and leghemoglobin content in whole alfalfa nodules and the tip, middle and basal sections.
Fraction
Whole nodules
Acetylene Reduction (emol C2H2 reduced * 1010 bacteroids)
Leghemoglobin (mg * g 1 fresh wt.)
59.3
3.16
4.9
1.95
Middle sections
83.3
6.35
Basal sections
16.7
1.90
Tip sections
1
The amount of acetylene reduced was determined after exposure of the nodules and sections to acetylene as described in Materials and Methods for 24 hr. The production of ethylene by the tip sections stopped in less than 2 hr in this particular experiment.
and have not yet gained the in situ capacity to reduce appreciable quantities of acetylene. After release into the host cytoplasm, the bacteroids increase in size and nucleic acid content and lose the distinct central nucleoid structures. This type of bacteroid is closely packed in the cytoplasm of plant cells which are located in the middle region of a 6- to 8-week-old nodule. These bacteroids are most active in acetylene reduction. As alfalfa nodules further develop, bacteroids in the basal region begin to lose most of their acetylene reducing activity and nucleic acid content. These bacteroids, however, retain their enlarged size and begin to accumulate electron-dense particles. The envelopes surrounding these bacteroids also begin to loosen and develop large electron-lucent spaces between the membrane envelopes and the bacteroid walls. Judging from the decrease in nucleic acid content and the loosening of the bacteroid envelopes, these bacteroids are likely to have begun deterioration and represent a terminal stage of bacteroid development.
FIG. 7. Diagrammatic representation of development and transformation of bacteroids in alfalfa nodules. IT: infection threads; en: envelope; s: electron-lucent space. Acknowledgments-We thank J. Oro and D. Schomer for their assistance in the operation of the FMF and A. Bartel and J. Hungerford for making the FMF available to us. LITERATURE CITED 1. BISSELING T, RC VAN DEN Bos, A VAN KAMMEN, M VAN DER POEG, P VAN DULN, A HOUWERS 1977 Cytofluorometrical determination of the DNA content of bacteroids and corresponding broth-cultured Rhizobium bacteria. J Gen Microbiol 101: 79-84 2. BRENCHLEY WE, HG THORNTON 1925 The relationship between the development, structure and functioning of the nodules on Viciafaba, as influenced by the presence or absence of boron in the nutrient medium. Proc R Soc Lond B98: 373-399 3. CHING TM, S HEDLKE, W NEWCOMB 1977 The isolation of bacteria, transforming bacteria and bacteroids from soybean nodules. Plant Physiol 60: 771-774 4. COWLES JR, HJ EVANS 1968 Some properties of the ribonucleotide reductase from Rhizobium meliloti. Arch Biochem Biophys 127: 770-778 5. COWLES JR, HJ EVANS, SA RussEL 1969 B12 coenzyme-dependent ribonucleotide reductase in Rhizobium species and the effects of cobalt deficiency on the activity of the enzyme. J Bacteriol 97: 1460-1465 6. DILWORTH JM, DC WILLIAMS 1967 Nucleic acid changes in bacteroids of Rhizobium lupini during nodule development. J Gen Microbiol 48: 31-36 7. GOURRET JP, H FERNANDEz-ARIAs 1974 Etude ultrastructurale et cytochimique de la differenciation des bacteroides de Rhizobium trifolii Dangeard dan les nodules de Trifolium repens L Can J Microbiol 20: 1169-1181 8. HARDY RWF, RD HOLSTEN, EK JACKSON, RC BURNS 1968 The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 43: 1185-1207 9. PAAU AS, JR COWLES, J ORO 1977 Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle. Can J Microbiol 23: 1165-1169 10. PAAu AS, D LEE, JR COWLES 1977 Comparison of nucleic acid content in populations of free-
living and symbiotic Rhizobium meliloti by flow-microfluorometry. J Bacteriol 129: 1156-1158 11. REIJNDER L, L VISSER, AMJ AALBERS, A VAN KAMMEN, A HoUWERs 1974 A comparison of DNA from free-living and endosymbiotic Rhizobium leguminosarsm (strain PRE). Biochim Biophys Acta 414: 206-216 12. REYNOLDS ES 1963 The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17: 208-212 13. SPUJRR, AR 1969 A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 41-43 14. STEINKAMP JA, MJ FULWYLER, JR COULTER, RD HIEBERT, JL HORNEY PR MULLANEY 1973 A new multiparameter separator for microscopic particles and biological cells. Rev Sci Instrum 44: 1301-13 10 15. SUTTON WD, P MAHONEY 1977 Preparation and separation of Rhizobium bacteroids by zonal sedimentation through sucrose gradient. Plant Physiol 60: 800-802
