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Cytochemical localization of peroxidase during the development of root nodules of Pisum sativum L.
Botanisch Laboratorium, Rijksuniversiteit Leiden, Nonnensteeg 3, Leiden. The Netherlands
Cytochemical Localization of Peroxidase during the Development of Root Nodules of Pisum sati1.mm 1. H. OOSTROM, F. E. TREURNIET and A. M. MENNES With 12 figures Received September 3, 1974
Summary Isoenzymes of peroxidase and IAA oxidase were separated electrophoretically from the soluble and cell-wall fractions of mature root cortex and root nodules of Pisum sativum L. It was shown that all IAA-oxidase isoenzymes exhibited peroxidase activity. In cytochemical localization studies of peroxidase in roots and root nodules the absence of activity specifically in the cytoplasm of root-nodule bacteroid cells was demonstrated. This result is interpreted to mean that hyperauxiny of root nodules may be due to low or even no IAA-oxidase activity in the bacteroid cells. In early nodulation the peroxidase activity in the infected area of the root cortex was not suppressed but had even increased. The results of preliminary experiments with isolated rhizobial auxin support the suggestion that nevertheless IAA is the auxin involved in the induction of the first cell divisions in the formation of nodules. Key words: fAA oxidase, peroxidase, root nodules.
Introduction It is generally believed that changes in hormone balance are part of the response of plant cells to infections (SEQUEIRA, 1973). These changes can be ascribed to excretion of hormones in diseased tissues by the invaders, but an alteration of the hormone metabolism of host cells as a reaction to the infection cannot be excluded (SEQUEIRA, 1963). Mature root nodules of leguminous plants contain relatively high amounts of indole-3-acetic acid (IAA) (PATE, 1958; DULLAART, 1967). THIMANN (1936) presented evidence that this hyperauxiny in root nodules may be due to auxin produced in the bacteroid tissue of the nodule. According to DULLAART (1970) at least a substantial part of the large amount of IAA present in the root nodules of Lupinus luteus was of host origin and MENNES (1973 b) found a lower specific activity of IAA oxidase extracted from these root nodules than from nonnodulated roots.
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It is generally accepted that, at least in higher plants, IAA is degraded by enzymes with peroxidase activity (RAY, 1958). TRucHET and COULOMB (1971) and TRucHET (1972) have cytochemically localized peroxidase activity in cells of pea root nodules, but a cytochemical study of peroxidase activity during the formation of these nodules has never been reported, although such a study might point to changes in IAA metabolism during the development of nodules. After we had found that all IAA-oxidase isoenzymes extracted from mature non-infected root-cortex tissue and from mature root nodules indeed exhibited peroxidase activity, we studied the cytochemical localization of peroxidase in fresh and fixed sections of mature non-infected and infected root-cortex tissue and of root nodules. The results of this study show that in particular during bacteroid-cell formation a drastic reduction in cytoplasmic peroxidase activity occurs. This and other results will be discussed in relation to the hyperauxiny in root nodules, and the suggested role of auxin in triggering root nodule formation (LIBBENGA et aI., 1973). Also preliminary experiments on the production of auxin by Rhizobium in vitro and the effect of the rhizobial auxin on the initiation of cell division in root-cortex explants will be described.
Materials and Methods Plant Cultures Plants (Pisum sativum L. cv. RONDO) used for histochemical investigations were cultured as described in LIBBENGA et al. (1973) and inoculated with Rhizobium leguminosarum, strain PRE (d. LIBBENGA and HARKES, 1973). Sterile conditions were maintained till the 10th day of culture, when the cotton plugs had to be removed to allow free growth of the seedlings. Care was taken to maintain a constant water level of 3 cm in the tubes during the rest of the culture period. For preparation of enzyme extracts non-infected plants were cultured in plastic basins as described in LIBBENGA et a!. (1973). Infected plants were cultured as described above.
Preparation
0/ enzyme extracts
a) Root cortex: 7-d-old root-cortex cylinders (1 to 5 cm below the insertion of the cotyledons) isolated following LIBBENGA et a!. (1973) were frozen in liquid nitrogen and ground in a mortar with double-distilled water (1 ml per g. fr.wt.) for 15 min. The brei was centrifuged for 1 hr at 30,000 X g and the supernatant filtered through Whatman No. 1 filter paper. The filtrate is referred to as the soluble (S) fraction, and the residue combined with the 30,000 X g pellet as the cell-wall-bound (CW) fraction. Fraction S was centrifuged for 16 hr at 100,000 X g. For extraction of membrane-bound peroxidase enzymes the 100,000 X g pellet was resuspended in 0.02 M Tris-HCI buffer (pH 8.5) and treated for 30 minutes with either 6 M urea, 1.5 % Triton X-100 in 0.5 M KCl, 0.5 M KCI or 0.5 Ufo sodium deoxycholate (DOC) in 0.5 M KC!. We found that the DOC-treatment resulted in an increase of the peroxidase activity of the resuspended pellet and in the complete solubilization of the enzyme. In all other cases there was no change or only a decrease of the peroxidase activity. After the DOC treatment the suspension was centrifuged for 16 hr at 100,000 X g. The supernatant was dialysed against 0.02 M Tris-HCI buffer (pH 8.5) to remove excess DOC and KCI and combined with fraction S; it was
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then concentrated to a small volume, dialysed against 0.004 M Tris-HCl buffer (pH 8.5) Prior to electrophoresis the fraction was dialysed against 0.004 M and stored at - 20 acetate buffer (pH 4.6). The CW fraction was extracted with 0.5 M CaCl 2 for 24 hr and centrifuged for 1 hr at 30,000 X g, followed by 16 hr at 100,000 X g. After removal of the CaCl 2 by dialysis, the supernatant was concentrated to a small volume, dialysed against 0.004 M Tris-HCl buffer (pH 8.5) and stored at - 20 ac. Prior to electrophoresis the fraction was dialysed against 0.004 M acetate buffer (pH 4.6). The pellet of the CW fraction was resuspended in double-distilled water and dialysed against excess ethylenediaminetetracetate (EDTA). After centrifugation for 1 hr at 30,000 X g almost no peroxidase activity was found in the supernatant. Therefore the pellet was discarded. b) Root nodules: Root nodules were collected from 21-d-old plants and immediately frozen in liquid nitrogen. The procedure for enzyme extraction was similar to that used for the cortex but now 5 ml of double-distilled water was added per g. fr.wt. of nodules, and the solutions used for extraction contained 0.3 M sucrose to prevent disruption of bacteroids by osmotic shock.
ac.
Measurement of enzyme activities
Peroxidase activity was determined by measuring optical density (OD) at 470 nm with 1-min-intervals after addition of 1 ml enzyme solution to a reaction mixture of 1 ml 100 mM guaiacol, 1 ml 0.1 % H 20 2 , and 8 ml 0.1 M acetate buffer (pH 5.5). IAA-oxidase activity was measured at 253 nm (MENNES, 1973 b). Separation of isoenzymes by electrophoresis
Electrophoresis was performed on 6 % acrylamide gel slabs (8 X 16 cm, 2 mm thick) provided with 5 starting slots (10 X 0.5 X 1.5 mm). The gels were prepared by mixing equal volumes of 24 Ofo acrylamide plus 0.64 Ofo methylene-bis-acrylamide, 0.2 Ofo ammonium persulphate, 2 Ofo ,8-dimethylaminoproprionitrile (DMAP), and double-distilled water. After polymerization the gel slabs were equilibrated overnight in electrode buffer (0.02 M acetate, pH 4.6). Preelectrophoresis was performed for 1 hr at 350 V. After applying the samples in 0.004 M acetate buffer (pH 4.6) an initial potential of 100 V was given for 15 min, after which electrophoresis was completed at 350 V (± 20 rnA) for 225 min. The peroxidase activity of the applied samples of the fractions of root cortex and nodules was the same, the activity per volume of the CW fractions being 10 times that of the S fractions. A better separation of the peroxidase isoenzymes, isolated at pH 8.5, could be obtained at pH 4.6 (acetate buffer), although this pH shift caused some loss of protein and peroxidase activity. Peroxidase-isoenzyme patterns were developed by immersing the gels immediately after electrophoresis in a solution of 0.012 Ofo H 20 2 in 0.1 M acetate buffer (pH 5.5) containing either 3,3'-diaminobenzidine (DAB; 0.5 mg/ml) or 8-aminoquinoline (AQ; 1.4 mg/ml) Incubation of the gel slabs at different pH values (5.0, 5.5, 6.0, 6.5, and 7.0 resp.) showea that all peroxidase isoenzymes had their highest activity at pH 5.5, with either AQ or DAB as the electron donor. Without the addition of H 2 0 2 we did not find any oxidation at all of either electron donor when incubated for 1 hr. After reaction with AQ (for 1 hr) or DAB (for 15 min) the gels were fixed in 7 Ofo acetic acid and scanned in a densitometer (Vitatron TLD-I00 «flying spot») at 365 nm. For the determination of IAA-oxidase isoenzymes the gels were frozen in isopentane which was cooled with liquid nitrogen, and cut into serial 2-mm-slices over their whole length. Each slide was incubated with IAA in addition to the cofactors of the enzyme (Mn2+ and 2,4-dichlorophenol (DCP)) at pH 5.5 (0.1 M phosphate buffer), and the degradation of IAA was measured after 24 hr using the Salkowski method (MENNES, 1973 a).
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Cytochemical localization 0/ peroxidase Root nodules from 21-d-old plants and parts of the main root located between 1 and 5 cm below the insertion of the cotyledons of 7-d-old plants were cut into 250-,um-thick transverse hand sections. The sections were pre-incubated for 1 hr in 50 mM Tris plus 50 mM sodium acetate (pH 5.5) containing either benzidine dihydromloride at 1 mg/ml, DAB at 2 mg/ml, or AQ at 0.7 mg/ml. Then H 20 2 was added to a final concentration of 0.012 Ufo. Controls were incubated either without H 20 2 or with potassium cyanide (0.01 M) or sodium azide (0.01 M or 0.1 M). After incubation for 20 min the sections were rinsed in distilled water, fixed in 2.5 Ufo glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) and post fixed in 1 Ufo osmium tetroxide in the same buffer. The fixed sections were dehydrated in an alcohol series followed by propylene-oxide, and embedded in Epon-812. Selected 250-,um-sections were cut into approximately radial (root-cortex) or tangential (nodules) l-,um-thick sections and examined by normal-light and phase-contrast microscopy. Rhizobium culture Rhizobium leguminosarum, strain PRE, was cultured at 25°C in the medium described by VAN EGERAAT (1972) with or without the addition of tryptophan (0.1 Ufo). Isolation and assay 0/ rhizobial auxin Extraction of the culture medium of Rhizobium in its growing phase was performed according to the method described by DULLAART (1967). The acid ether-soluble fraction was purified by paper mromatography and examined spectrofluorometrically as described by DULLAART (19'67). It was tested for its capacity to replace the auxin in the induction of cell divisons in root-cortex explants of pea as used by LIBBENGA et al. (1973).
Results and Conclusions fAA-oxidase and peroxidase isoenzyme patterns in root cortex and in nodules The combined results of several electropherograms of the 4 different fractions are shown in Figs. 1 and 2. As can be seen there was a great difference in reactivity between the two electron donors. However, it must be mentioned that, when using AQ (Fig. 2), the fixation in 7 % acetic acid caused a partial disappearance of the formed colour. Preliminary experiments with more concentrated enzyme fractions showed that all isoenzymes found with DAB were also visible with AQ. It is clear from Fig. 1 that peroxidase isoenzymes with high reactivity also showed a clear IAA-oxidase activity. Moving from the origin to the cathode, the first two IAA-oxidase activity peaks in the Sand CW fraction of the root cortex were correlated with the two shoulders in the scanning pattern of peroxidase activity. Since we found no IAA oxidases that did not show peroxidase activity, we have Fig. 1: Electropherograms of peroxidase and IAA-oxidase isoenzymes in polyacrylamide gels. Peroxidase activity (--) is shown as a densitometer trace at 365 nm of the gel after staining for enzyme activity with DAB-H2 0 2 • The numbers indicate the cathodic and anodic peroxidase isoenzymes whim were visible. After cutting the frozen gels into 2-mm-slices, the IAA-oxidase activity of eam slice was assayed with the Salkowski reagent and is given as decrease in OD 530 • The curve of IAA-oxidase activity (..... ) was obtained by interpolation between the separate measurements. Z. P/lanzenphysiol. Bd. 74. S. 451-463. 1975.
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Cytochemical Localization of Peroxidase
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good reasons to believe that peroxidase-positive sites in tissue sections, as revealed by cytochemical methods, probably represent sites of potential IAA-oxidase activity. Cytochemical localization of peroxidases a) Specificity and penetration of the electron donors
Of the electron donors used (benzidine, DAB and AQ) AQ gave cytochemically the best results with respect to penetration and specificity (cf. MENNES et al., 1974). b) Localization of peroxidase activity Non-infected root cortex: The cortex cells showed strongly reacting cell walls and a relatively weak reaction of the thin layer of cytoplasm. An activity in specific organelles in the cytoplasIp could not be detected. Infected root cortex: Cells penetrated by the infection thread and adjacent cells showed positive particles in the vacuoles in addition to the strongly reacting cell walls and weakly reacting cytoplasm (Fig. 3). The wall and the matrix of the infection thread reacted positively although the wall only weakly, whereas the bacteria in the matrix were negative (Fig. 4). The dividing cells in the inner cortex ahead of the infection thread showed the same activities as the cells of the infected area, including the positive particles in their vacuoles. Inhibition of peroxidase reacting with AQ by 0.1 M sodium azide showed that this reaction was completely inhibited in normal root-cortex cells, but hardly inhibited in the infected area. This may be an indication of the presence of a high concentration of peroxidase in this area, or there may be a different type of peroxidase not sensitive to inhibition by azide. Root nodules a) Nodule cortex (Fig. 5): Here we found posltlve cell walls, slightly active cytoplasm and many positive particles in the vacuoles. b) Meristematic zone (Fig. 5): Activity was mainly restricted to the cytoplasm. Cell walls were very weakly active except for the middle lamella which sometimes reacted strongly, particulary in the intercellular regions. In the vacuoles positive particles were absent. Activity along the tonoplast was sometimes stronger than in the rest of the cytoplasm. c) Younger part of the infection zone (Fig. 6): In this region the activity in the walls of the host cells, especially in the intercellular regions had increased as
Fig. 2: Electropherogram of peroxidase isoenzymes in polyacrylamide gels. Peroxidase activity is shown as a densitometer trace at 365 nm of the gel after staining for enzyme activity with AQ-H20 2 • The numbering of the visible peaks is according to Fig. 1. Note the lower number of peaks and the lower activity as compared with Fig. 1. Z. PJlanzenphysiol. Bd. 74. S. 451-463. 1975.
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compared to (b). Also in some cells the reaction along the tonoplast was very strong. The infection-thread matrix was positive; bacteria and wall negative. d) Older part of the infection zone (Figs. 7 and 8): The wall of the infection thread was positive now (Fig. 7). The activity of the cell walls had strongly increased. In infected cells (Fig. 7) the activity of the cytoplasm had decreased. In non-infected cells (Fig. 8), showing a large vacuole and the presence of amyloplasts, there was a strong increase in peroxidase activity along the tonoplast. Positive particles were present in the vacuoles. e) Bacteroid zone (Figs. 9, 10 and 12): In this region the differences between noninfected and infected cells noted in (d) were even more pronounced. In bacteroid cells only the wall reacted positively while there was no activity at all in the cytoplasm (Fig. 9). Non-infected cells containing big starch grains in a still active cytoplasm also showed very active walls but, moreover, many positive particles in the vacuole and a strong reaction along the tonoplast (Fig. 10). The differences in peroxidase activity between the two cell types in this zone was striking (Fig. 12). f) Degeneration zone (Fig. 11): In the degenerating bacteroid cells the cytoplasm was almost negative, but the activity had reappeared along the tonoplast and especially in the vacuole in the form of positive particles. Isolation and assay of rhizobial auxin Our preliminary experiments have shown that, only when tryptophan had been added 1)0 the Rhizobium culture medium, and acid ether-soluble extract of this medium in addition to a cytokinin, induced cell divisions in pea-root cortex explants. Spectrofluorometric examination of this fraction showed the clear presence of IAA. Discussion
Our results are contradictory with those of TRUCHET and COULOMB (1971) and TRucHET (1972) with respect to the complete absence of cytoplasmic peroxidase activity we found in bacteroid cells. The positive reactions which they found may Fig. 3: Radial l-,um-section of a 7-d-old infected pea root incubated in AQ-H2 0 2 • Note the positive particles in the vacuoles of the cells in the infected area. Arrows indicate the infection thread. The partially negative reaction of the dividing cells in the inner cortex (left) is due to non-penetration of the AQ. Fig. 4: Infection thread in a 7-d-old infected pea root penetrating a cortex-cell wall. The matrix reacted very strongly, whilst the bacteria (B) were completely negative. The infectionthread wall (IW) reacted rather weakly as compared with the cortex-cell wall (CW). Fig. 5: Tangential l-,um-section through the meristematic zone of a 21-d-old nodule incubated in AQ-H2 0 2 • The nodule-cortex cells (left) reacted strongly and showed positive particles in the vacuoles. The meristematic cells (right) showed a very low peroxidase activity. Z. P/lanzenphysiol. Bd. 74. S. 451--463. 1975.
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and A.
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be ascribed to enzymes different from peroxidase because, as we have reported (MENNES et aI., 1974), DAB does not react exclusively with peroxidase. The absence or inactivity of the cytoplasmic peroxidases (found in this study probably to be potential IAA oxidases) may, at least partiy, contribute to the accumulation of IAA in root nodules. Unlike in root nodules, an apparent increase instead of a decrease in peroxidase activity was found in the infected areas of the root cortex. This increase is mainly due to the appearance of strongly active particles in the vacuoles of infected and adjacent cells. Since it was shown in Fig. 1 that the peroxidases present throughout the cell probably are potential IAA oxidases, one may wonder whether enough rhizobial IAA from the infection thread reaches the target cells in the inner cortex (LIBBENGA et aI., 1973) without being degraded. The possibility that a rhizobial auxin different from IAA is involved in early nodulation cannot be excluded. However, our preliminary experiments with the rhizobial auxin strongly indicate that IAA may be the active auxin involved in the induction of cell divisions in spite of the high IAA-oxidase activity in the root-cortex cells. This indicates that IAA oxidase is carefully regulated in vivo as was suggested by MENNES (1973 b). Acknowledgement The authors are indebted to Dr. K. R. LIBBENGA and Dr. R. ]. BOGERS for their stimulating criticism during the preparation of the paper. The comments on this paper of Dr. G. TRUCHET and Dr. L. SEQUEIRA are highly appreciated.
References DULLAART, J.: Quantitative estimation of indoleacetic acid and indolecarboxylic acid in root nodules and roots of Lupinus luteus L. Acta bot. neer!. 16,222-230 (1967). - The bioproduction of indole-3-acetic acid and related compounds in root nodules and roots of Lupinus luteus L. and by its rhizobial symbiont. Acta bot. neer!. 19, 573-615 (1970). Fig. 6: Tangential 1-,um-section through the younger part of the infection zone or, a 21-d-old nodule incubated in AQ-H2 0 2 • The cell walls reacted positively in some places (e.g. on the right-hand side of this figure). The infection threads (arrow) showed a strong positive matrix but a negative wall and negative bacteria. The cytoplasm of most cells reacted weakly. However, some cells, e.g. the presumably non-infected cell in the upper middle of this figure, showed a stronger reaction of the cytoplasm in addition to a very strong reaction along the tonoplasts. Fig. 7: Tangential section through the older part of the infection zone of the same nodule as in Fig. 6, showing an infected cell. The most striking change as compared with Fig. 6 is the strong positive reaction of both cell wall and infection-thread wall. Arrow indicates the infection thread. Fig. 8: Non-infected cell in the neighbourhood of the cell in Fig. 7. In addition to a positive reaction of the cell wall the tonoplast showed a strong reaction. Heavily stained particles were present in the large vacuole.
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Pflanzenphysiol. Bd. 74. S. 451-463. 1975.
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and A.
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VAN EGERAAT, A. W. S. M.: Pea-root exudates and their effect upon root-nodule bacteria. Doct. dissert., Agric. Univ., Wageningen, The Netherlands (1972). LIBBENGA, K. R., and P. A. A. HARKES: Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L. Planta (Berl.) 114, 17-28 (1973). LIBBENGA, K. R., F. VAN IREN, R. ]. BOGERs, and M. F. SCHRAAG-LAMERS: The role of hormones and gradients in the initiation of cortex proliferation and nodule formation in Pisum sativum L. Plant a (Berl.) 114,29-39 (1973). MENNES, A. M.: The indole-3-acetic acid oxidase of Lupinus luteus L. I. A qualitative comparison of the activity of this enzyme in root nodules and roots. Acta bot. neerl. 22, 694-705 (1973 a). - The indole-3-acetic acid oxidase of Lupinus luteus L. II. A quantitative comparison of the activity of this enzyme in root nodules and roots. Acta bot. neer!. 22, 706-729 (1973 b). MENNES, A. M., H. OOSTROM, and F. E. TREURNIET: Light and electron microscopic localization of peroxidase during the development of root nodules of Pisum sativum L. In: Plant Growth Substances 1973, proceedings of the 8th international conference on plant growth substances (Tokyo, 1973) (in press). PATE, ]. S.: Studies of the growth substances of legume nodules using paper chromatography. Aust. ]. BioI. Sci. 11,516-528 (1959). RAY, P. M.: Destruction of auxin. Ann. Rev. Plant Physiol. 9, 81-118 (1958). SEQUEIRA, L.: Growth regulators in plant disease. Ann. Rev. Phytopathol. 1,5-30 (1963). - Hormone metabolism in diseased plants. Ann. Rev. Plant Physiol. 24, 353-380 (1973). THIMANN, K. Y.: On the physiology of the formation of nodules on legume roots. Proc. Nat. Acad. Sci. (Wash.) 22, 511-514 (1936). TRucHET, G.: Mise en evidence de l'activite peroxidasique dans les differentes zones des nodules radiculaires de Pois (Pisum sativum L.). Localisation de la leghemoglobine. C. R. Acad. Sc. Paris 274, Serie D, 1290-1293 (1972). TRucHET, G., and P. COULOMB: Localisation de la phosphata~e acide et de la peroxydase dans les cellules de nodules radiculaires de Pois (Pisum sativum). Relations entre la cellule h8te et la bacterie. C. R. Acad. Sc. Paris 272, Serie D, 1499-1502 (1971). H. OOSTROM, F. E. TEURNIET and A. M. MENNES, Botanisch Laboratorium, Rijksuniversiteit Leiden, Nonnensteeg 3, Leiden, The Netherlands.
Fig. 9: Tangential 1-,um-section through the bacteroid zone of a 21-d-old nodule incubated in AQ-H202' showing a mature bacteroid cell. The most striking fact is the complete absence of cytoplasmic peroxidase activity. The cell wall still reacted positively. Fig. 10: Non-infected cell adjacent to the bacteroid cell in Fig. 9. As compared with the cell in Fig. 8 the only changes are the presence of large starch grains and more positive particles in the vacuole. Fig. 11: Tangential 1-,um-section through the degeneration zone of a 21-d-old nodule incubated in AQ-HzO z, showing a bacteroid cell in which the cytoplasmic peroxidase activity had returned in the form of positive particles in the vacuole and activity along the tonoplast. Fig. 12: Tangential 1-,um-section through the bacteroid zone of a 21-d-old nodule incubated in AQ-Hz0 2 , showing a non-infected cell surrounded by bacteroid cells. Bottom: normal light; top: phase-contrast microscopy, showing cytoplasm packed with bacteroids. Note the absence of cytoplasmic peroxidase activity in the bacteroid cells.
Z. Pflanzenphysiol. Bd. 74. S. 451-463. 1975,
Cytochemical Localization of Peroxidase during the Development of Root Nodules of Pisum sati1.mm 1. H. OOSTROM, F. E. TREURNIET and A. M. MENNES With 12 figures Received September 3, 1974
Summary Isoenzymes of peroxidase and IAA oxidase were separated electrophoretically from the soluble and cell-wall fractions of mature root cortex and root nodules of Pisum sativum L. It was shown that all IAA-oxidase isoenzymes exhibited peroxidase activity. In cytochemical localization studies of peroxidase in roots and root nodules the absence of activity specifically in the cytoplasm of root-nodule bacteroid cells was demonstrated. This result is interpreted to mean that hyperauxiny of root nodules may be due to low or even no IAA-oxidase activity in the bacteroid cells. In early nodulation the peroxidase activity in the infected area of the root cortex was not suppressed but had even increased. The results of preliminary experiments with isolated rhizobial auxin support the suggestion that nevertheless IAA is the auxin involved in the induction of the first cell divisions in the formation of nodules. Key words: fAA oxidase, peroxidase, root nodules.
Introduction It is generally believed that changes in hormone balance are part of the response of plant cells to infections (SEQUEIRA, 1973). These changes can be ascribed to excretion of hormones in diseased tissues by the invaders, but an alteration of the hormone metabolism of host cells as a reaction to the infection cannot be excluded (SEQUEIRA, 1963). Mature root nodules of leguminous plants contain relatively high amounts of indole-3-acetic acid (IAA) (PATE, 1958; DULLAART, 1967). THIMANN (1936) presented evidence that this hyperauxiny in root nodules may be due to auxin produced in the bacteroid tissue of the nodule. According to DULLAART (1970) at least a substantial part of the large amount of IAA present in the root nodules of Lupinus luteus was of host origin and MENNES (1973 b) found a lower specific activity of IAA oxidase extracted from these root nodules than from nonnodulated roots.
Z. Pjlanzenphysiol. Bd. 74. S. 451-463. 1975.
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It is generally accepted that, at least in higher plants, IAA is degraded by enzymes with peroxidase activity (RAY, 1958). TRucHET and COULOMB (1971) and TRucHET (1972) have cytochemically localized peroxidase activity in cells of pea root nodules, but a cytochemical study of peroxidase activity during the formation of these nodules has never been reported, although such a study might point to changes in IAA metabolism during the development of nodules. After we had found that all IAA-oxidase isoenzymes extracted from mature non-infected root-cortex tissue and from mature root nodules indeed exhibited peroxidase activity, we studied the cytochemical localization of peroxidase in fresh and fixed sections of mature non-infected and infected root-cortex tissue and of root nodules. The results of this study show that in particular during bacteroid-cell formation a drastic reduction in cytoplasmic peroxidase activity occurs. This and other results will be discussed in relation to the hyperauxiny in root nodules, and the suggested role of auxin in triggering root nodule formation (LIBBENGA et aI., 1973). Also preliminary experiments on the production of auxin by Rhizobium in vitro and the effect of the rhizobial auxin on the initiation of cell division in root-cortex explants will be described.
Materials and Methods Plant Cultures Plants (Pisum sativum L. cv. RONDO) used for histochemical investigations were cultured as described in LIBBENGA et al. (1973) and inoculated with Rhizobium leguminosarum, strain PRE (d. LIBBENGA and HARKES, 1973). Sterile conditions were maintained till the 10th day of culture, when the cotton plugs had to be removed to allow free growth of the seedlings. Care was taken to maintain a constant water level of 3 cm in the tubes during the rest of the culture period. For preparation of enzyme extracts non-infected plants were cultured in plastic basins as described in LIBBENGA et a!. (1973). Infected plants were cultured as described above.
Preparation
0/ enzyme extracts
a) Root cortex: 7-d-old root-cortex cylinders (1 to 5 cm below the insertion of the cotyledons) isolated following LIBBENGA et a!. (1973) were frozen in liquid nitrogen and ground in a mortar with double-distilled water (1 ml per g. fr.wt.) for 15 min. The brei was centrifuged for 1 hr at 30,000 X g and the supernatant filtered through Whatman No. 1 filter paper. The filtrate is referred to as the soluble (S) fraction, and the residue combined with the 30,000 X g pellet as the cell-wall-bound (CW) fraction. Fraction S was centrifuged for 16 hr at 100,000 X g. For extraction of membrane-bound peroxidase enzymes the 100,000 X g pellet was resuspended in 0.02 M Tris-HCI buffer (pH 8.5) and treated for 30 minutes with either 6 M urea, 1.5 % Triton X-100 in 0.5 M KCl, 0.5 M KCI or 0.5 Ufo sodium deoxycholate (DOC) in 0.5 M KC!. We found that the DOC-treatment resulted in an increase of the peroxidase activity of the resuspended pellet and in the complete solubilization of the enzyme. In all other cases there was no change or only a decrease of the peroxidase activity. After the DOC treatment the suspension was centrifuged for 16 hr at 100,000 X g. The supernatant was dialysed against 0.02 M Tris-HCI buffer (pH 8.5) to remove excess DOC and KCI and combined with fraction S; it was
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then concentrated to a small volume, dialysed against 0.004 M Tris-HCl buffer (pH 8.5) Prior to electrophoresis the fraction was dialysed against 0.004 M and stored at - 20 acetate buffer (pH 4.6). The CW fraction was extracted with 0.5 M CaCl 2 for 24 hr and centrifuged for 1 hr at 30,000 X g, followed by 16 hr at 100,000 X g. After removal of the CaCl 2 by dialysis, the supernatant was concentrated to a small volume, dialysed against 0.004 M Tris-HCl buffer (pH 8.5) and stored at - 20 ac. Prior to electrophoresis the fraction was dialysed against 0.004 M acetate buffer (pH 4.6). The pellet of the CW fraction was resuspended in double-distilled water and dialysed against excess ethylenediaminetetracetate (EDTA). After centrifugation for 1 hr at 30,000 X g almost no peroxidase activity was found in the supernatant. Therefore the pellet was discarded. b) Root nodules: Root nodules were collected from 21-d-old plants and immediately frozen in liquid nitrogen. The procedure for enzyme extraction was similar to that used for the cortex but now 5 ml of double-distilled water was added per g. fr.wt. of nodules, and the solutions used for extraction contained 0.3 M sucrose to prevent disruption of bacteroids by osmotic shock.
ac.
Measurement of enzyme activities
Peroxidase activity was determined by measuring optical density (OD) at 470 nm with 1-min-intervals after addition of 1 ml enzyme solution to a reaction mixture of 1 ml 100 mM guaiacol, 1 ml 0.1 % H 20 2 , and 8 ml 0.1 M acetate buffer (pH 5.5). IAA-oxidase activity was measured at 253 nm (MENNES, 1973 b). Separation of isoenzymes by electrophoresis
Electrophoresis was performed on 6 % acrylamide gel slabs (8 X 16 cm, 2 mm thick) provided with 5 starting slots (10 X 0.5 X 1.5 mm). The gels were prepared by mixing equal volumes of 24 Ofo acrylamide plus 0.64 Ofo methylene-bis-acrylamide, 0.2 Ofo ammonium persulphate, 2 Ofo ,8-dimethylaminoproprionitrile (DMAP), and double-distilled water. After polymerization the gel slabs were equilibrated overnight in electrode buffer (0.02 M acetate, pH 4.6). Preelectrophoresis was performed for 1 hr at 350 V. After applying the samples in 0.004 M acetate buffer (pH 4.6) an initial potential of 100 V was given for 15 min, after which electrophoresis was completed at 350 V (± 20 rnA) for 225 min. The peroxidase activity of the applied samples of the fractions of root cortex and nodules was the same, the activity per volume of the CW fractions being 10 times that of the S fractions. A better separation of the peroxidase isoenzymes, isolated at pH 8.5, could be obtained at pH 4.6 (acetate buffer), although this pH shift caused some loss of protein and peroxidase activity. Peroxidase-isoenzyme patterns were developed by immersing the gels immediately after electrophoresis in a solution of 0.012 Ofo H 20 2 in 0.1 M acetate buffer (pH 5.5) containing either 3,3'-diaminobenzidine (DAB; 0.5 mg/ml) or 8-aminoquinoline (AQ; 1.4 mg/ml) Incubation of the gel slabs at different pH values (5.0, 5.5, 6.0, 6.5, and 7.0 resp.) showea that all peroxidase isoenzymes had their highest activity at pH 5.5, with either AQ or DAB as the electron donor. Without the addition of H 2 0 2 we did not find any oxidation at all of either electron donor when incubated for 1 hr. After reaction with AQ (for 1 hr) or DAB (for 15 min) the gels were fixed in 7 Ofo acetic acid and scanned in a densitometer (Vitatron TLD-I00 «flying spot») at 365 nm. For the determination of IAA-oxidase isoenzymes the gels were frozen in isopentane which was cooled with liquid nitrogen, and cut into serial 2-mm-slices over their whole length. Each slide was incubated with IAA in addition to the cofactors of the enzyme (Mn2+ and 2,4-dichlorophenol (DCP)) at pH 5.5 (0.1 M phosphate buffer), and the degradation of IAA was measured after 24 hr using the Salkowski method (MENNES, 1973 a).
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Cytochemical localization 0/ peroxidase Root nodules from 21-d-old plants and parts of the main root located between 1 and 5 cm below the insertion of the cotyledons of 7-d-old plants were cut into 250-,um-thick transverse hand sections. The sections were pre-incubated for 1 hr in 50 mM Tris plus 50 mM sodium acetate (pH 5.5) containing either benzidine dihydromloride at 1 mg/ml, DAB at 2 mg/ml, or AQ at 0.7 mg/ml. Then H 20 2 was added to a final concentration of 0.012 Ufo. Controls were incubated either without H 20 2 or with potassium cyanide (0.01 M) or sodium azide (0.01 M or 0.1 M). After incubation for 20 min the sections were rinsed in distilled water, fixed in 2.5 Ufo glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) and post fixed in 1 Ufo osmium tetroxide in the same buffer. The fixed sections were dehydrated in an alcohol series followed by propylene-oxide, and embedded in Epon-812. Selected 250-,um-sections were cut into approximately radial (root-cortex) or tangential (nodules) l-,um-thick sections and examined by normal-light and phase-contrast microscopy. Rhizobium culture Rhizobium leguminosarum, strain PRE, was cultured at 25°C in the medium described by VAN EGERAAT (1972) with or without the addition of tryptophan (0.1 Ufo). Isolation and assay 0/ rhizobial auxin Extraction of the culture medium of Rhizobium in its growing phase was performed according to the method described by DULLAART (1967). The acid ether-soluble fraction was purified by paper mromatography and examined spectrofluorometrically as described by DULLAART (19'67). It was tested for its capacity to replace the auxin in the induction of cell divisons in root-cortex explants of pea as used by LIBBENGA et al. (1973).
Results and Conclusions fAA-oxidase and peroxidase isoenzyme patterns in root cortex and in nodules The combined results of several electropherograms of the 4 different fractions are shown in Figs. 1 and 2. As can be seen there was a great difference in reactivity between the two electron donors. However, it must be mentioned that, when using AQ (Fig. 2), the fixation in 7 % acetic acid caused a partial disappearance of the formed colour. Preliminary experiments with more concentrated enzyme fractions showed that all isoenzymes found with DAB were also visible with AQ. It is clear from Fig. 1 that peroxidase isoenzymes with high reactivity also showed a clear IAA-oxidase activity. Moving from the origin to the cathode, the first two IAA-oxidase activity peaks in the Sand CW fraction of the root cortex were correlated with the two shoulders in the scanning pattern of peroxidase activity. Since we found no IAA oxidases that did not show peroxidase activity, we have Fig. 1: Electropherograms of peroxidase and IAA-oxidase isoenzymes in polyacrylamide gels. Peroxidase activity (--) is shown as a densitometer trace at 365 nm of the gel after staining for enzyme activity with DAB-H2 0 2 • The numbers indicate the cathodic and anodic peroxidase isoenzymes whim were visible. After cutting the frozen gels into 2-mm-slices, the IAA-oxidase activity of eam slice was assayed with the Salkowski reagent and is given as decrease in OD 530 • The curve of IAA-oxidase activity (..... ) was obtained by interpolation between the separate measurements. Z. P/lanzenphysiol. Bd. 74. S. 451-463. 1975.
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Cytochemical Localization of Peroxidase
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good reasons to believe that peroxidase-positive sites in tissue sections, as revealed by cytochemical methods, probably represent sites of potential IAA-oxidase activity. Cytochemical localization of peroxidases a) Specificity and penetration of the electron donors
Of the electron donors used (benzidine, DAB and AQ) AQ gave cytochemically the best results with respect to penetration and specificity (cf. MENNES et al., 1974). b) Localization of peroxidase activity Non-infected root cortex: The cortex cells showed strongly reacting cell walls and a relatively weak reaction of the thin layer of cytoplasm. An activity in specific organelles in the cytoplasIp could not be detected. Infected root cortex: Cells penetrated by the infection thread and adjacent cells showed positive particles in the vacuoles in addition to the strongly reacting cell walls and weakly reacting cytoplasm (Fig. 3). The wall and the matrix of the infection thread reacted positively although the wall only weakly, whereas the bacteria in the matrix were negative (Fig. 4). The dividing cells in the inner cortex ahead of the infection thread showed the same activities as the cells of the infected area, including the positive particles in their vacuoles. Inhibition of peroxidase reacting with AQ by 0.1 M sodium azide showed that this reaction was completely inhibited in normal root-cortex cells, but hardly inhibited in the infected area. This may be an indication of the presence of a high concentration of peroxidase in this area, or there may be a different type of peroxidase not sensitive to inhibition by azide. Root nodules a) Nodule cortex (Fig. 5): Here we found posltlve cell walls, slightly active cytoplasm and many positive particles in the vacuoles. b) Meristematic zone (Fig. 5): Activity was mainly restricted to the cytoplasm. Cell walls were very weakly active except for the middle lamella which sometimes reacted strongly, particulary in the intercellular regions. In the vacuoles positive particles were absent. Activity along the tonoplast was sometimes stronger than in the rest of the cytoplasm. c) Younger part of the infection zone (Fig. 6): In this region the activity in the walls of the host cells, especially in the intercellular regions had increased as
Fig. 2: Electropherogram of peroxidase isoenzymes in polyacrylamide gels. Peroxidase activity is shown as a densitometer trace at 365 nm of the gel after staining for enzyme activity with AQ-H20 2 • The numbering of the visible peaks is according to Fig. 1. Note the lower number of peaks and the lower activity as compared with Fig. 1. Z. PJlanzenphysiol. Bd. 74. S. 451-463. 1975.
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compared to (b). Also in some cells the reaction along the tonoplast was very strong. The infection-thread matrix was positive; bacteria and wall negative. d) Older part of the infection zone (Figs. 7 and 8): The wall of the infection thread was positive now (Fig. 7). The activity of the cell walls had strongly increased. In infected cells (Fig. 7) the activity of the cytoplasm had decreased. In non-infected cells (Fig. 8), showing a large vacuole and the presence of amyloplasts, there was a strong increase in peroxidase activity along the tonoplast. Positive particles were present in the vacuoles. e) Bacteroid zone (Figs. 9, 10 and 12): In this region the differences between noninfected and infected cells noted in (d) were even more pronounced. In bacteroid cells only the wall reacted positively while there was no activity at all in the cytoplasm (Fig. 9). Non-infected cells containing big starch grains in a still active cytoplasm also showed very active walls but, moreover, many positive particles in the vacuole and a strong reaction along the tonoplast (Fig. 10). The differences in peroxidase activity between the two cell types in this zone was striking (Fig. 12). f) Degeneration zone (Fig. 11): In the degenerating bacteroid cells the cytoplasm was almost negative, but the activity had reappeared along the tonoplast and especially in the vacuole in the form of positive particles. Isolation and assay of rhizobial auxin Our preliminary experiments have shown that, only when tryptophan had been added 1)0 the Rhizobium culture medium, and acid ether-soluble extract of this medium in addition to a cytokinin, induced cell divisions in pea-root cortex explants. Spectrofluorometric examination of this fraction showed the clear presence of IAA. Discussion
Our results are contradictory with those of TRUCHET and COULOMB (1971) and TRucHET (1972) with respect to the complete absence of cytoplasmic peroxidase activity we found in bacteroid cells. The positive reactions which they found may Fig. 3: Radial l-,um-section of a 7-d-old infected pea root incubated in AQ-H2 0 2 • Note the positive particles in the vacuoles of the cells in the infected area. Arrows indicate the infection thread. The partially negative reaction of the dividing cells in the inner cortex (left) is due to non-penetration of the AQ. Fig. 4: Infection thread in a 7-d-old infected pea root penetrating a cortex-cell wall. The matrix reacted very strongly, whilst the bacteria (B) were completely negative. The infectionthread wall (IW) reacted rather weakly as compared with the cortex-cell wall (CW). Fig. 5: Tangential l-,um-section through the meristematic zone of a 21-d-old nodule incubated in AQ-H2 0 2 • The nodule-cortex cells (left) reacted strongly and showed positive particles in the vacuoles. The meristematic cells (right) showed a very low peroxidase activity. Z. P/lanzenphysiol. Bd. 74. S. 451--463. 1975.
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be ascribed to enzymes different from peroxidase because, as we have reported (MENNES et aI., 1974), DAB does not react exclusively with peroxidase. The absence or inactivity of the cytoplasmic peroxidases (found in this study probably to be potential IAA oxidases) may, at least partiy, contribute to the accumulation of IAA in root nodules. Unlike in root nodules, an apparent increase instead of a decrease in peroxidase activity was found in the infected areas of the root cortex. This increase is mainly due to the appearance of strongly active particles in the vacuoles of infected and adjacent cells. Since it was shown in Fig. 1 that the peroxidases present throughout the cell probably are potential IAA oxidases, one may wonder whether enough rhizobial IAA from the infection thread reaches the target cells in the inner cortex (LIBBENGA et aI., 1973) without being degraded. The possibility that a rhizobial auxin different from IAA is involved in early nodulation cannot be excluded. However, our preliminary experiments with the rhizobial auxin strongly indicate that IAA may be the active auxin involved in the induction of cell divisions in spite of the high IAA-oxidase activity in the root-cortex cells. This indicates that IAA oxidase is carefully regulated in vivo as was suggested by MENNES (1973 b). Acknowledgement The authors are indebted to Dr. K. R. LIBBENGA and Dr. R. ]. BOGERS for their stimulating criticism during the preparation of the paper. The comments on this paper of Dr. G. TRUCHET and Dr. L. SEQUEIRA are highly appreciated.
References DULLAART, J.: Quantitative estimation of indoleacetic acid and indolecarboxylic acid in root nodules and roots of Lupinus luteus L. Acta bot. neer!. 16,222-230 (1967). - The bioproduction of indole-3-acetic acid and related compounds in root nodules and roots of Lupinus luteus L. and by its rhizobial symbiont. Acta bot. neer!. 19, 573-615 (1970). Fig. 6: Tangential 1-,um-section through the younger part of the infection zone or, a 21-d-old nodule incubated in AQ-H2 0 2 • The cell walls reacted positively in some places (e.g. on the right-hand side of this figure). The infection threads (arrow) showed a strong positive matrix but a negative wall and negative bacteria. The cytoplasm of most cells reacted weakly. However, some cells, e.g. the presumably non-infected cell in the upper middle of this figure, showed a stronger reaction of the cytoplasm in addition to a very strong reaction along the tonoplasts. Fig. 7: Tangential section through the older part of the infection zone of the same nodule as in Fig. 6, showing an infected cell. The most striking change as compared with Fig. 6 is the strong positive reaction of both cell wall and infection-thread wall. Arrow indicates the infection thread. Fig. 8: Non-infected cell in the neighbourhood of the cell in Fig. 7. In addition to a positive reaction of the cell wall the tonoplast showed a strong reaction. Heavily stained particles were present in the large vacuole.
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VAN EGERAAT, A. W. S. M.: Pea-root exudates and their effect upon root-nodule bacteria. Doct. dissert., Agric. Univ., Wageningen, The Netherlands (1972). LIBBENGA, K. R., and P. A. A. HARKES: Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L. Planta (Berl.) 114, 17-28 (1973). LIBBENGA, K. R., F. VAN IREN, R. ]. BOGERs, and M. F. SCHRAAG-LAMERS: The role of hormones and gradients in the initiation of cortex proliferation and nodule formation in Pisum sativum L. Plant a (Berl.) 114,29-39 (1973). MENNES, A. M.: The indole-3-acetic acid oxidase of Lupinus luteus L. I. A qualitative comparison of the activity of this enzyme in root nodules and roots. Acta bot. neerl. 22, 694-705 (1973 a). - The indole-3-acetic acid oxidase of Lupinus luteus L. II. A quantitative comparison of the activity of this enzyme in root nodules and roots. Acta bot. neer!. 22, 706-729 (1973 b). MENNES, A. M., H. OOSTROM, and F. E. TREURNIET: Light and electron microscopic localization of peroxidase during the development of root nodules of Pisum sativum L. In: Plant Growth Substances 1973, proceedings of the 8th international conference on plant growth substances (Tokyo, 1973) (in press). PATE, ]. S.: Studies of the growth substances of legume nodules using paper chromatography. Aust. ]. BioI. Sci. 11,516-528 (1959). RAY, P. M.: Destruction of auxin. Ann. Rev. Plant Physiol. 9, 81-118 (1958). SEQUEIRA, L.: Growth regulators in plant disease. Ann. Rev. Phytopathol. 1,5-30 (1963). - Hormone metabolism in diseased plants. Ann. Rev. Plant Physiol. 24, 353-380 (1973). THIMANN, K. Y.: On the physiology of the formation of nodules on legume roots. Proc. Nat. Acad. Sci. (Wash.) 22, 511-514 (1936). TRucHET, G.: Mise en evidence de l'activite peroxidasique dans les differentes zones des nodules radiculaires de Pois (Pisum sativum L.). Localisation de la leghemoglobine. C. R. Acad. Sc. Paris 274, Serie D, 1290-1293 (1972). TRucHET, G., and P. COULOMB: Localisation de la phosphata~e acide et de la peroxydase dans les cellules de nodules radiculaires de Pois (Pisum sativum). Relations entre la cellule h8te et la bacterie. C. R. Acad. Sc. Paris 272, Serie D, 1499-1502 (1971). H. OOSTROM, F. E. TEURNIET and A. M. MENNES, Botanisch Laboratorium, Rijksuniversiteit Leiden, Nonnensteeg 3, Leiden, The Netherlands.
Fig. 9: Tangential 1-,um-section through the bacteroid zone of a 21-d-old nodule incubated in AQ-H202' showing a mature bacteroid cell. The most striking fact is the complete absence of cytoplasmic peroxidase activity. The cell wall still reacted positively. Fig. 10: Non-infected cell adjacent to the bacteroid cell in Fig. 9. As compared with the cell in Fig. 8 the only changes are the presence of large starch grains and more positive particles in the vacuole. Fig. 11: Tangential 1-,um-section through the degeneration zone of a 21-d-old nodule incubated in AQ-HzO z, showing a bacteroid cell in which the cytoplasmic peroxidase activity had returned in the form of positive particles in the vacuole and activity along the tonoplast. Fig. 12: Tangential 1-,um-section through the bacteroid zone of a 21-d-old nodule incubated in AQ-Hz0 2 , showing a non-infected cell surrounded by bacteroid cells. Bottom: normal light; top: phase-contrast microscopy, showing cytoplasm packed with bacteroids. Note the absence of cytoplasmic peroxidase activity in the bacteroid cells.
Z. Pflanzenphysiol. Bd. 74. S. 451-463. 1975,