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Hemoglobins in the nitrogen-fixing root nodules of actinorhizal plants
JOHN D. TJEPKEMA' Harvard Forest, Harvard Utziversity, Peteraham, MA, U.S.A. 01366
Received December 1, 1982
TJEPKEMA, J . D. 1983. Hemoglobins in the nitrogen-fixing root nodules of actinorhizal plants. Can. J . Bot. 61: 2924-2929. The occurrence of high concentrations of hemoglobins in the root nodules of Casuarina cunningharniana Miq. and Myrica
gale L. has been confirmed using spectrophotometry of nodule segments. In an atmosphere of N2, absorption bands were observed at approximately 560 nm, while in 02 , bands were found at 540 and 580 nm. These bands were rapidly reversed when O2 and N2 were interchanged in the gas phase. In the presence of CO, absorption bands at 540 and 570 nm were observed. Lower concentrations of hemoglobins were present in nodules of Cornptoniaperegrina (L.) Coult., Alnus rubra Bong., and Elaeagnus angustifolia L. Zero to trace amounts were found in Ceanothus arnericarzus L. and Datisca glornerata (Presl.) Baill. Preparation of a crude extract from nodules of C . cunningharniana indicated that the hemoglobin is in a soluble form. Other workers may have failed to observe hemoglobins in actinorhizal nodules because of the low concentrations in many nodules or because hemoglobin is restricted to the tissue containing the active endophyte, and this tissue occupies a relatively small fraction of the nodule volume.
TJEPKEMA, J . D. 1983. Hemoglobins in the nitrogen-fixing root nodules of actinorhizal plants. Can. J . Bot. 61: 2924-2929. La prCsence d'hkmoglobine en concentrations ClevCes dans les nodules racinaires du Casilarir~a cunnirlgharniana Miq. et du
Myrica gale L. a CtC confirmCe par la spectrophotomktrie de segments de nodules. Dans une atmosphtre de N2, des bandes d'absorption se rencontrent i environ 560 nm, tandis que dans une atmosphtre de 0 2 , on trouve des bandes d'absorption a 540 et 580 nm. Ces bandes se remplacent mutuellement et rapidement lorsque 1102 et le N2 sont changCs l'un pour l'autre dans la phase gazeuse. En prksence de CO, on observe des bandes d'absorption B 540 et 570 nm. Des concentrations plus faibles d'hkmoglobine se rencontrent dans les nodules du Cornptoniaperegrina (L.) Coult., de 1'Alnus rilbra Bong. et de 1'Elaeagnus angustifolia L. On en rencontre des traces ou des quantitCs nulles chez le Ceanothus atnericanus L. et le Datisca glornerata (Presl.) Baill. La prkparation d'un extrait brut de nodules de C . curlnirlghatniana a montrC que I'hCmoglobine est prCsente sous une forme soluble. Si d'autres chercheurs n'ont pas rCussi a observer la prCsence d'hkmoglobines dans les nodules actinorhiziens, c'est peut-Ctre i cause de la faible concentration de ces substances dans plusieurs nodules, ou parce que 1'hCmoglobine est restreinte au tissu qui contient l'endophyte actif et que ce tissu occupe une fraction relativement petite du volume du nodule.
[Traduit par le journal]
Introduction Hemoglobins occur in all effective legume nodules
and have been extensively studied. There is much evi- dence that they are important for oxygen transport at the low oxygen concentrations present in legume nodules (2). In contrast, only a few workers have examined actinorhizal nodules for the presence of hemoglobins. Egle and Munding (10) were not able to extract hemoglobins from actinorhizal nodules but did find
slices of Alnus incana (L.) Moench subsp. rugosa (Duroi) Clausen, Elaeagnus commutata Bernh., Shepherdia canadensis (L.) Nutt., and Hippophae rhamnoides L. Other workers have reported the absence of hemoglo- bins from certain actinorhizal nodules but have not provided any experimental details. Smith found no hemoglobin in Alnus (15) or in M. gale (cited by Bond (7)) while Becking was not able to find hemoglobins in several species of Alnus (5). Bond reported no visible
elevated levels of total heme that were about half of the hemoglobin color in any of the actinorhizal nodules that concentrations in legume nodules. Davenport (9) con- he examined, with the possible exception of Casuarina firmed these observations, finding that the total heme con- (8). Bond also noted that anthocyanins are sometimes centration in Casuarina cunningharniana Miq. nodules present in actinorhizal nodules and might be confused was equal to that in pea nodules. More importantly, Davenport observed absorption bands in whole nodule clusters of C . cunninghamiana that corresponded to the deoxygenated, oxygenated, and carbon monoxide forms of hemoglobin. Davenport also reported that bound hemoglobins occur in Alnus glutinosa (L.) Gaertn. and Myrica gale L., although no experimental details were given. In contrast with the results of Davenport, Moore (13) found no hemoglobin absorption bands in nodule
'Present address: Department of Botany and Plant Pathology, University of Maine at Orono, Orono, ME, U.S.A. 04469.
with hemoglobins (7). In the present study, segments of actinorhizal nodules
were examined for hemoglobin by spectrophotometry. High concentrations were observed in two of the seven species examined, lower concentrations in three species, and zero to trace amounts in the remaining two species.
Materials and methods Nodules of Cornptoniaperegrina (L.) Coult. were collected
from the field, while Pisurn sativurn L. cv. Sugar Snap was from a garden. All other plants were grown indoors using nitrogen-free nutrient solution. Alnus rubra Bong. was inocu-
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lated with Frankia strain AR13 (6) and grown in an aeroponics tank in the greenhouse (2 1). All other genera were inoculated with crushed nodules. Myricn gale L. was maintained in water culture in a growth chamber, Casuaritzn cuntlinglzntnintln Miq. was kept in an aeroponics tank in the greenhouse, and Elaeagnus nngusrifolin L. , Cennorhus ntnericnnus L. , and Dariscn glornerafn (Presl.) Bail1 were in pots in the green- house.
All nodules were from actively growing plants known to have acetylene-reduction activity and were selected for maxi- mum lobe diameter and for appearance of recent or current nodule growth. A single lobe or group of branched lobes was cut longitudinally to give a nodule segment 0 .6 to 1.4 mm thick and 2.2 to 4 .5 mm long.
A Beckman model DU spectrophotometer was used as a source of monochromatic light (cell compartment and detector removed), while transmitted light was measured with a separate photomultiplier photometer (Photovolt model 520-M). The spectral slit width was a maximum of 1.1 nm. An aperture of 0.8 mm diameter was cut in a piece of black electrical tape which was pressed against a piece of glass microscope slide and sealed to the surface of the entrance window of the photomultiplier housing.
A gas-tight chamber was formed by cementing rubber tubing (6 mm inner diameter, 3 mm length) to the black tape. The opposite end of the tubing was covered with a piece of glass held in place with rubber bands and sealed to the rubber with silicone stopcock grease. A small piece of wet filter paper was kept in the chamber and water-saturated gases were flushed through the chamber via hypodermic needles (27 gauge) inserted through the walls of the chamber.
A nodule segment was placed in the chamber with the cut surface centered on the aperture in the black plastic tape. Frequently, the tissue just below the point of branching of the two youngest nodule lobes was directly over the aperture. In all cases the tissue covering the aperture was judged to contain mature endophyte that was fixing nitrogen. The nodule slice was held in place by the natural adhesion between the cut surface of the nodule and the black tape or by the addition of a thin film of water between the cut surface and the tape.
Crude extracts of hemoglobin from C . cunninghnrnintza were prepared in a buffer of 0.1 M potassium phosphate (pH 7.4), 1.0 mM EDTA, 5% (w/v) polyvinylpyrrolidone (40 000 molecular weight, pharmaceutical grade, Sigma, St. Louis), and 0.2% Na2S204. Nodule roots and woody material from the nodule centers were removed, and approximately 1.5 g of trimmed nodules and 4 mL of buffer were ground at ice temperature in a mortar with a pestle under a stream of N2. The more finely ground material was removed to a centrifuge tube with a pipette, leaving behind most of the coarse debris.
Results
TJEPKEMA
Absorption spectra for C. cunninghamiana and all other nodule segments were recorded at 5-nm intervals, with 0 2 , N2, or air in the nodule chamber (Fig. 1). In 0 2 , absomtion bands occurred at about 540 and 580nm. whereas in N2 there was a diffuse absorption band at about 560nm. These spectra were rapidly reversed when O2 and N2 were interchanged in the nodule
WAVELENGTH Inml
FIG. 1. Absorption spectra for a nodule segment of Casunrina cunninghnrninnn, equilibrated in N2, air, or 02. The segment was 0 .6 mm thick.
chamber. These results confirm the report of Davenport (9) and can be explained only by the presence of a hemoglobin. There was little or no indication of the presence of the oxidized form of hemoglobin, which has an absorption band in the region of 630 nm. The absor- bance by the nodule segment was much greater at 510 nm than at 650 nm, which is due to greater light scattering at the shorter wavelengths and the possible presence of pigments other than hemoglobin that absorb more strongly at the shorter wavelengths.
When the data from Fig. 1 were plotted directly in the units read from the photometer, the absorption bands were more distinct, and the difference between the O2 and N2 plots was greater (Fig. 2). This occurred because the absorbance plots in Fig. 1 are logarithmic and also because the photometer was less sensitive in the red than in the blue. All data in this paper have been plotted in photometer units, because this makes it easier to detect weak absorption bands, especially in the nodules with low hemoglobin concentrations. In these plots the absorption bands appear as minima in the photometer readings, since minimum light is transmitted at these wavelengths. Thus the absorption bands for oxygenated
l , , , f , C , l
500 520 540 560 580 600 620 640 WAVELENGTH lnml
FIG. 2. Raw data from Fig. 1 plotted directly in photometer units.
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2926 CAN. J . BOT. VOL. 61, 1983
hemoglobin are the minima that occur at 540 and 580 nm in Fig. 2.
Using this type of spectrum, the absorption bands characteristic of the carbon monoxide complex of hemoglobin were observed at 540 and 570 nm (Fig. 3). This is similar to the result of Davenport (9).
For comparison, pea nodules were examined and absorption bands for oxygenated hemoglobin were found at about 542 and 575 nm, while the absorption band for deoxygenated hemoglobin was at 555 nm (Fig. 4). These results agree with the spectra for hemoglobins in soybean nodule segments (I).
Myrica gale was the only other actinorhizal plant in which I observed strong absorption bands for oxygenated and deoxygenated hemoglobins (Fig. 5). However, weaker absorption bands were observed in Comptonia peregrina, Alnus rubra, and Elaeagtzus angustifolia (Figs. 6-8). The absorption band of oxyhemoglobin at 580 nm was most distinct in these nodules. A range in absorption band intensity was observed and the spectra presented were selected for maximum intensity; most other spectra had weaker absorption bands.
500 520 540 560 580 600 620 640 WAVELENGTH i n m l
FIG. 3. Spectra for the nodule of Fig. 1 in N2 and CO.
I ! , I I , I 500 520 540 560 580 600
WAVELENGTH i n m l
The absorption spectra for Ceanothus americanus and Datisca glomerata show zero to trace amounts of hemoglobin (Figs. 9 and 10). The difference in absor- bance at 580 nm between measurements in O2 and N2 is consistent with the presence of a hemoglobin, but no distinct absorption bands were observed. However, in C . americanus there were distinct absorption bands in an atmosphere of N2 at 560 and 533 nm which are consistent with the presence of a cytochrome b or o as
Myr,co gale
/? '7 -02
f
l I I m I I I l 500 520 540 560 580 600
WAVELENGTH l n m l
FIG. 5. Spectra for a nodule segment (0.9 mm thick) of Myrica gale.
i Ld 500 520 540 560 580 600
WAVELENGTH i n m l
FIG. 6. Spectra for a nodule segment (0.9mm thick) of Comptonia peregrina.
U d 500 520 540 560 580 600
WAVELENGTH Inml
FIG. 4. Spectra for a nodule segment (0.9mm thick) of FIG. 7. Spectra for a nodule segment (0.7 mm thick) of Pisum sativum. Alnus rubra.
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TJEPKEMA
w 500 520 540 560 580 600
WAVELENGTH inml
FIG. 8. Spectra for a nodule segment (0.9 mm thick) of Elaeagnus angustifolia.
500 520 540 560 580 600 WAVELENGTH (nml
FIG. 9. Spectra for a nodule segment (1.4 mm thick) of Ceanothus americanus.
found in rhizobia (3). These absorption bands decreased in an atmosphere of O2 but never disappeared. It was thought that water films at the nodule surface might be restricting oxygen diffusion into the nodule, but identi- cal results were obtained when water was kept away from the nodules at all times. These cytochrome absorption bands may have been less apparent in the other actinorhizal nodules examined because they merged with the absorption band of deoxygenated hemoglobin or because the nodule lobe thickness and light path were considerably greater in the C . americanus nodules examined. Cytochrome bands were also observed in Datisca glomerata in the vicinity of 550 to 565 nm (Fig. 10) and could occasionally be seen in other species, such as Comptonia peregrina ( ~ i ~ . 6).
Contrary to the results of Davenport (9), I was able to extract a soluble hemoglobin from C . cunninghamiana. After initial centrifugation of the crude extract at 7800 X g for 10 min under N2, spectra were taken directly through the centrifuge tubes. A diffuse absorption band was observed at 560nm, which is consistent with the presence of deoxygenated hemoglobin. After shaking the supernatant with CO, absorption bands were ob- served at 535 and 565 nm, which correspond to the CO complex of hemoglobin. The extract was then centri-
A 500 520 540 560 580 600
WAVELENGTH inml
FIG. 10. Spectra for a nodule segment (0.6 mm thick) of Datisca glomernta.
WAVELENGTH inmi
FIG. 11. Spectmm of CO-bound hemoglobin in the superna- tant of a crude extract from Cnsunrinn cuntzingharninnn.
fuged at 3 1 000 x g for 20 min and the supernatant was poured into a spectrophotometer cell of 1 .O-cm path length. Absorption bands corresponding to CO-bound hemoglobin were then recorded (Fig. 11). The reason why the hemoglobin was soluble rather than bound as found by Davenport is not known but may be due to the use of soluble polyvinylpyrrolidone, which absorbs phenolics.
Discussion Davenport is the only other worker to observe
hemoglobin absorption bands in actinorhizal nodules (9). The reason why others have been unsuccessful is probably because the concentration of hemoglobin is low in many actinorhizal nodules, necessitating careful observation of thick nodule segments by spectropho- tometry. In contrast, hemoglobins in legume nodules are readily observed as a red color by the naked eye. In the present study, a distinct red color was observed by eye only in Myrica gale and Casuarina cunninghamiana. Even in these nodules the color did not stand out, because it was confined to the zone of active infective
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2928 CAN. J . BOT. VOL. 61, 1983
tissue which is very small in dimensions compared with the bacteroid-containing tissue in most legume nodules. Anthocyanins may also have led to some confusion in previous work, but in my study these were usually not present. If present, they were of a more purple than red hue and occurred primarily away from the infected cells in the outer tissue layers.
No attempt was made to measure the concentration of hemoglobin. Judging from the change in photometer current between oxygenated and nonoxygenated forms, the concentration of hemoglobin is roughly comparable in Pisum sativum, Causarina cunninghamiana, and Myrica gale. This is consistent with observations of cut nodule surfaces by naked eye and with previous measure- ments by Davenport in C . cunninghamiana (9). It should also be noted that the infected cells in acti- norhizal nodules frequently occur in small groups separated by noninfected cells. Thus there are probably fewer hemoglobin-containing cells than in legume nodules, and some light may bypass the infected cells in spectrophotometric measurements.
Many of the actinorhizal species examined apparently have a rather low concentration of hemoglobin. A large number of nodule segments were examined in the hope of finding more intense absorption bands than presented in Figs. 4-10, but none were found in which the intensity was as great as in C . cunninghamiana or M. gale. The lower concentrations were not due to the inactivity of the nodules, especially in the case of Alnus rubra where the nodules had acetylene-reduction rates of 10-30 pmol C2H2 h-' g fresh weight-'. The geometry and thickness of the nodule segments were similar in all species examined.
The function of hemoglobins in actinorhizal nodules is not known. The occurrence of hemoglobin in C . cunninghamiana in a soluble form (Fig. 11) is consistent with a role in oxygen transport. Legume hemoglobins are thought to facilitate oxygen flux only at low Po, (2). Such low Po" values do not necessarily occur in acti- norhizal nodules, since Frankia, the symbiotic endo- phyte, can fix nitrogen at atmospheric Po, (20). How- ever, areas of low PO2 do in fact occur in the nodules of Myrica gale (18, 19). Also, both M. gale and C. cunning- hamiana are usually found in wet soils where the Po" is often low. Myrica gale grows in peatlands and along the shores of lakes and streams (14, 16), while the normal habitat of C . cunninghamiana, "the river she oak," is along river banks between the edge of the water and the high water mark (1 1). Both species possess nodule roots, which can increase oxygen uptake in wet soils (16, 17). Thus hemoglobins, like nodule roots, may in part repre- sent an adaptation to growth in wet soils. The partial oxygenation of hemoglobin in C . cunninghamiana in air (Fig. 1) is consistent with a function in oxygen transport.
In conclusion, the present results and those of
Davenport (9) show that hemoglobins can occur in actinorhizal nodules in high concentrations. However, the concentrations are low in some species, and hemo- globin may be totally absent from other species. The function of hemoglobin in actinorhizal nodules is not known, but a role in oxygen transport at low Po, is possible. A question of major current interest is the evolutionary origin of the globin gene in legumes (12) and Parasponia (4). This is also of great interest for the globins in actinorhizal nodules, especially if they are coded for by the plant hosts which are not closely related to each other or to legumes.
Acknowledgments This work was supported by U.S. Department of Agri-
culture competitive grant No. 78-59-2252-0- 1-055- 1 and by the Maria Moors Cabot Foundation for Botanical Research of Harvard University. I thank Martin Zimmer- mann for the loan of the photomultiplier photometer.
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