ADENINE INCORPORATION AND CELL DIVISION IN SHOOT APICES BY F. A. L. CLOWES

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1 ADENINE INCORPORATION AND CELL DIVISION IN SHOOT APICES BY F. A. L. CLOWES Department of Botany, University of Oxford {Received lo January 1958) (With Plate 2) SUMMARY Incorporation of '^C labelled adenine into DNA has been used to investigate the rates of division among cells of shoot meristems. This technique shows that all the cells of a growing apex, including the axial cells of the outermost layers, synthesize DNA. There is no quiescent region such as is found in root meristems. This conclusion is at variance with some of the recent views on the behaviour of shoot meristems. INTRODUCTION Ever since the discovery of the tetrahedral apical cell in shoot and root meristems of pteridophytes, anatomists have believed that a group of cells at the tip of the shoot apex and near the stele pole of the root apex could be regarded as the equivalent in seed plants of the tetrahedral apical cell. The word promeristem has sometimes been used for this group of initial cells. It could be thought of as the minimum number of cells which give rise to all future parts of the root or shoot. The identification of the promeristem with the cells at the tip of the shoot apex and near the pole of the stele of the root apex has recently been questioned. Buvat (1952), in an account of the shoot meristems of several dicotyledons, held that the axial cells of the tunica and corpus were inactive except in the initiation of a floral axis. These cells which had until then been regarded as the initial cells constituted a meristeme d'attente. The histogenetic region of the apex was away from the tip in an anneau initial and a medullary meristem. The evidence for this view was the apparent absence of mitoses and the cytological appearance of the cells. Buvat (1953) holds that monocotyledonous apices are similar, and Lance (1952,1953) has supported his view with evidence from other species. The absence of histogenetic activity in vegetative apices fits well with Gregoire's theory of the nature of floral apices and with Plantefol's phyllotaxis theory. But almost no worker outside France supports these theories. The main criticisms of the French school have been summarized by Gifford (1954) and Wardlaw () In the root apex the evidence for questioning the classical identification of the initials is based on the cell arrangement and comparison of rates of synthesis of desoxyribose nucleic acid (DNA). In some roots it is possible to determine the plane and frequency of cell division from the cell pattern. In Zea I was able to show that most of the cells, hitherto regarded as initials, in fact did not divide or divided only very infrequently i6

Shoot apices 17 (Clowes, 1954). The promeristem could be regarded as made up of the cells on the surface of the quiescent region at the pole of the meristem.

2 Shoot apices 17 (Clowes, 1954). The promeristem could be regarded as made up of the cells on the surface of the quiescent region at the pole of the meristem. The identification of such a quiescent centre was confirmed by determining comparative rates of DNA synthesis and using the belief that cells usually synthesize DNA only in preparation for division (Clowes, 1956a). A similar position is beheved to exist in the roots of other plants except those with single apical cells (Clowes, 19^66, 19^8^). Work on protein synthesis is also consistent with the existence of a quiescent centre to the meristem (Clowes, 1958^). It is not held that the cells of the quiescent centre are incapable of division, for there is evidence to the contrary (Clowes, 1953, 19^4)- But it is held that, because of their position within the meristem, these cells divide rarely or never in the normal ontogeny of the root. It might be held that these views on root meristems fit well with the French school on shoot meristems. This paper describes the results of applying to shoot meristems the techniques used in identifying the quiescent centre in roots. METHODS The technique used on roots was to feed them with adenine, a constituent of both ribose and desoxyribose nucleic acids. The RNA was removed by hydrolysis after feeding. The adenine was labelled with a '^C atom at position 8, and high resolution autoradiography was used to trace its incorporation into DNA (Clowes, 19566). A number of modifications have had to be introduced to make the technique suitable for shoot apices. In the shoot apices used the number of nuclei synthesizing DNA in 24 hours is much smaller than in roots and so adenine has been fed to them over periods of from i to 8 days. The aquatic plants ValUsneria spiralis, Elodea canadensis, and Cabomba Carolina were simply immersed in adenine sulphate hemihydrate solution of activity 200 pc/litre and strength 25 mg/litre. Land plants do not take up adenine through the cut surface of stems in sufficient quantities for it to be detected in the apices. Therefore, adenine of higher specific activity was injected into the stem just below the apex by a hypodermic needle. About 0.02 ml of solution containing 2 nc and 0.08 mg was supplied in this way to each shoot apex of Coleus bhtmei plants grown in pots in a greenhouse. The only other modification of the previous techniques was the substitution of a polyester wax, 400 polyethylene glycol distearate, for paraffin in embedding the apices for microtoming (Steedman, 1957). The lower melting point of this wax enables distortion of the cells to be reduced. It was found unnecessary to use amylopectin to stick the sections and photographic emulsion on to the slides. Gelatine could be used with advantage either as a dried film or as Haupt's adhesive. In both cases water and not formalin was used to float the ribbons of sections on to the slide. RESULTS All the plants investigated have domed apices and all provided good autoradiographs. The silver grains were conspicuously grouped over some of the nuclei, but some occurred also over the cytoplasm. The reason for this is probably that acid hydrolysis does not remove the RNA completely. In this respect ribonuclease is no better than in hydrochloric acid. Plate I a is a dark ground photograph of the autoradiograph overlying a median section of a shoot apex of Coleus injected with adenine 8 days before it was killed. In it nearly all B N.P.

3 i8 F. A. L. CLOWFS the cells show up because t)f their nuclear autoradiograph. The cells whicli synthesize DNA are those in the dome of the apex, the leaf primordia, and the stem tor 700 n or more from the tip. Furtlier from the apex, outside the photograph, the cells do not synthesize DNA except for those in the procambium. The silver grains are densest over the dome of the apex and over the procambium because there are more nuclei per unit volume there. Between the two youngest leaf primordia silver grains are slightly denser on the Hanks of the apex than near the axis for the same reason. The cells on the flanks are smaller than those near the axis (Popham, 1952, p. 169). But all the apical nuclei in the section are covered by groups of silver grains. There is no quiescent region. At the level of the foliar buttresses the grouping of the silver grains over the nuceli is much clearer than in the apical dome because the nuclei occupy a smaller fraction of the cell volume and the cross fire of P particles does not make the nuclear autoradiographs touch each other. Flowering apices of Coleus resemble vegetative apices in all the respects described above. Plate zb is a transmitted light photograph of the autoradiograph overlying a shoot apex of Vallisneria fed with adenine for 2 days before killing. Here most, but not all, the nuclei are labelled. Other apices from the same experiment had a similar ratio of labelled to unlabelled nuclei in the meristem, but the unlabelled nuclei were not confined to a particular region. The nuclear autoradiographs are clearer at the tip than in Plate 2rt because there is no cross fire between adjacent nuclei since not all the nuclei have incorporated adenine during the 2 days of feeding. At least half of the plants had labelled nuclei in the axial cells of the outermost layer of the tunica as in Plate zb. Flodea and Cabomba apices were essentially similar to Coleus and Vallisneria in the distribution of labelled nuclei. Feeding with adenine for 4 days or more led to nuclear labelling of all the meristematic cells. Feeding for a shorter time usually left some nuclei unlabelled within the meristem, but the unlabelled nuclei were never restricted to a particular region in any experiment. D1SCUS.SION The use of this technique depends on the fact that cells known to be meristematic have nuclear autoradiographs because they synthesize DNA, whereas cells known to be nonmeristematic do not. Within the conditions of the experiments there seems to be no turnover of adenine in the nuclei which it is not possible to ascribe to net synthesis of DNA. The reason why the frequency of mitotic figures used by Buvat and Lance is a bad indication of meristematic activity is that the time a nucleus spends in mitosis is a small and variable fraction of the time spent in interphase. The present technique is a better guide to meristematic activity because the time taken to produce a visible label can be varied to suit the frequency of mitosis. Newman (1956) has also used this timing principle in direct observations on living shoot apices and his conclusions are also different from those of Buvat and Lance. Probably most anatomists believe that cells divide more frequently in the flanks of a shoot apex than at the tip (Wardlaw, 1957). But there is no evidence for a quiescent region in shoot meristems as there is in roots. The present work shows that all the cells of the apical dome are meristematic. This fits in with the views on the histogenetie importance of the apical cells derived from knowledge of the behaviour of chimerical meristems (Clowes, 1957). In polyploid chimeras induced by

4 THE NEW PHYTOLOGIST, 58, i PLATE 2 CLOWES SHOOT APICES (Facing page 19)

5 Shoot apices 19 colchicine, polyploidy is caused by the frustration of anaphase and therefore the cell which initiates the polyploid germ layer must be dividing at the time of the application of colchicine. The initiating cells can populate the whole of their germ layers with polyploid cells only if all the cells in each of the tunica layers divide. Similarly, the reversion of an unstable periclinal chimera to a non-chimerical shoot can be explained only on the assumption that all the cells of each tunica layer do divide. ACKNOWLEDGMENTS I am indebted to Miss W. M. Jones for technical assistance and to the Royal Society of London for financial assistance. REFERENCES BUVAT, R. (1952). Structure, Evolution et fonctionnement du meristeme apical de quelques dicotyledones. Ann. Sci. nat. Botanique, 2nd Ser., 13, 198. BUVAT, R. (1953). L'apex de Triticuni vidgare; modalites de reprise des mitoses Iors de la germination et du fonctionnement v^gijtatif. C.R. Acad. Sci., Paris, 236, CLOWES, F. A. L. (1953). The cytogenerative centre in roots with broad columellas. Nezv Phytot., 52, 48. CLOWES, F. A. L. (1954). The promeristem and the minimal constructional centre in grass root apices. New PhytoL, 53, 108. CLOWES, F. A. L. (1956(2). Nucleic acids in root apical meristems of Zea. Netv Phytot., 55, 29. CLOWES, F. A. L. (19566). Localization of nucleic acid synthesis in root meristems. J. exp. Bat., 7, 307. CLOWES, F. A. L. (1957). Chimeras and meristems. Heredity, 11, 141. CLOWES, F. A. L. (1958a). Development of quiescent centres in root meristems. Ne2v Phytot., 57. CLOWES, F. A. L. (19586). Protein synthesis in root meristems. J. exp. Bat., in the press. GIFFORD, E. M., JR. (1954). The shoot apex in angiosperms. Bot. Rev., 20, 477. LANCE, A. (1952). Sur la structure et le fonctionnement du point vegetatif de Vicia faba L. Anii. Sci. nat. Botanique, 2nd Ser., 13, 301. LANCE, A. (1953). Sur l'absence d'initiales apicales et la configuration de l'anneau initial chez Vicia faba 1^. C.R. Acad. Sci., Paris, 236, 510. NEWMAN, L V. (1956). Pattern in meristems of vascular plants, i. Cell partition in living apices and in the cambial zones in relation to the concepts of initial cells and apical cells. Phytomorphotogy, 6, i. PoPHAM, R. A. (1952). Developmental plant anatomy. Columbus. STEEDMAN, H. F. (1957). Polyester wax. A new ribboning embedding medium for histology. Nature, 179, WARDL.^W, C. W. (1957). The reactivity of the apical meristem as ascertained by cytological and other techniques. Nezi) PhytoL, 56, 221. EXPLANATION OF PLATE 2 Plate 2. Autoradiographs of median sections of b. Vallisneria spiratis fed 2 days before fixation, apices of plants fed with [8 "C] adenine. The sec- Photographed by transmitted light. Silver grains tions have been hydrolysed to remove RNA and the black. ;5i2. silver grains are grouped over nuclei. c. Outline of section underlymg the autoradiograph a. Coteus btumei fed 8 days befoire fixation. Photo- in b. Nuclei with dense silver grains in the autographed using a paraboloid dark-ground condenser to radiograph are drawn all black, and nuclei without a show the silver grains at low magnification. The conspicuous autoradiograph are drawn with white apparent size of the silver grains (white) is exag- centres. L, young leaf; P, procambium; Ti and Tj, gerated by their diffraction halos first and second layer of tunica.

Primary Plant Body: Embryogenesis and the Seedling

BIOL 221 Concepts of Botany Primary Plant Body: Embryogenesis and the Seedling (Photo Atlas: Figures 1.29, 9.147, 9.148, 9.149, 9.150, 9.1, 9.2) A. Introduction Plants are composed of fewer cell types,