APICAL DOMINANCE IN FUCUS VESICULOSUS

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1 APICAL DOMINANCE IN FUCUS VESICULOSUS BY BETTY MOSS Department of Botany, University of Newcastle upon Tyne (Received 2 December 1964) SUMMARY Apical tips of Fucus vesiculosus L. were grown in sterile sea water. The apical cell region was excised surgically and this resulted in a decreased rate of growth of the segnient and the regeneration of new branches. Treatment of cultured tips, whose apical cell region had been excised, with four auxins and two anti-auxins revealed a striking effect. The latter treatment induced up to three or four times the number of branches regenerated in control experiments. Extraction and chromatographic procedures carried out on apical tips from November to Januars' showed that a mixture of stimulatory and inhibitor)' growth substances was present. It is concluded that growth regulators play a part in determining apical dominance in Fucus. INTRODUCTION The apical meristem of Fucus is quite distinct from that of any other group of plants. It consists of a large apical cell, sunken at the base of a groove, and surrounded on its four sides and the base by small meristematic cells. These are pigmented and like the tissues around them are probably photosynthetic. Behind them, there is no differentiation of distinct vascular tissues, so that the apical meristem of Fucus may not be so dependent on the fiow of nutrients from below as is the apical meristem of vascular plants. This independence of the apical meristem from the rest of the thallus accounts for the fact that apices of F. vesiculosus can be cultured in sterile sea water without the addition of other nutrients, provided that light and temperature requirements are adequate (Moss, 1964). When narrow segments from the middle part of a thallus are cultured, wound healing callus quickly develops over both cut surfaces. From this callus new branches develop in profusion, each of them with a functioning apical meristem (Moss, 1964). On the other hand, detached apices kept in culture continue to grow and to differentiate wings and midrib behind them as do the apices of intact thalli. A wound healing layer forms over the cut surface, but there is rarely any regeneration of a new branch from it. Here the presence of the apical cell is inhibiting the development of new apices from the cut end of the thallus, demonstrating apical dominance in Fucus. The following experiments, using culture methods described already (Moss, 1964), were designed to study this phenomenon further, and to determine if, in a relatively simple thallus like Fucus, apical dominance is as complex a problem as in the vascular plants. RESULTS Surgical experiments The contrast in appearance at the end of 6 months' culture, between i mm segments which had been taken from the apices of young thalli with the apical meristem intact and those from the middle region of the same thalli, are shown in Plate 11, Nos. i and 4. The apical segments lengthen and dichotomize as if they were still attached to the thallus. 387

2 388 BETTY MOSS If at the end of 6 months in culture, the apices of the cultured apices are then cut off, new branches will gradually regenerate from both the upper and lower cut surfaces (Plate II, No. 2), over the following few months. This demonstrates that in these segments it was the presence of the original apical meristem which was inhibitmg the regeneration of branches from the cut surface. Once this inhibiting region is removed (Plate II, No. 3), branches regenerate in a similar way to that from a segment from the middle region of a thallus which originally contained no apical meristem. As the apical cell is sunken in a groove, and surrounded on its sides and its base by small meristematic cells, it was impossible either to destroy it, or to remove it, without damaging some of its adjacent cells. Hence in the following experiments where 'apices minus apical cell' are referred to, some of the surrounding meristematic cells as well as the apical cell have, in fact, been removed. This was done by making a small V-shaped incision in the apical groove. 150H Months in culture A -A Fig. I. Percentage increase in fresh weight of apical segments of Fucus kept in culture and also the number of branches regenerated from these segments. O, Apices intact; x, apices minus apical cells. A = apices intact; A = apices n:iinus apical cells. At different times of the year, cultures were started to compare the rate of growth and the regeneration of new branches from intact apices and from apices minus their apical cell. For each experiment, 200 apices each i mm wide were cut from young thalli which had not yet dichotomized. One hundred apices were kept intact, while the apical cell was dissected out from the other 100. They were then cultured, ten segments per flask in 15 ml of sterile sea water. The cultures were kept at 12 C and given 8 hours' illumination each day. The medium was changed each month, when the fresh weight of the segments was recorded. At the end of 4 months, the number of regenerated branches was counted, and the dry weight of the samples determined. Whether the results are expressed on a basis of dry weight or of fresh weight, the rate of growth of the segments minus their apical cells was always much lower than that of intact apices. Fig. i shows the percentage increase in fresh weight of apices started in culture in December It also shows the numbers of branches regenerated from intact apices and from those minus their apical cells. Although intact apices when kept 4

3 THE NEW PHYTOLOGIST, 64, 3 PLATE II Growth of segments ofthallus in eulture. No. I. Apex intact, resulting in continued lengthening of segment. No. 2. After 4 months in culture, removal of apices leads to proliferation from both the upper and lower cut surfaces. No. 3. Apical cell remo\ed, resulting in proliferation from hoth the upper and lower CLit surfaces. No. 4. Segment from the middle region of thallus showing proliferation from hoth the cut surfaces. BETTY MOSS APICAL DOMINANCE IN FUCUS [facing page 388)

4 Apical dominance in Fucus 389 months in culture may increase twenty times or more in length and dichotomize, apices minus their apical cells increase only slightly in size (Fig. i) and do not dichotomize. Instead, they regenerate new branches, each one with a functional apical meristem, from both cut surfaces. So that while the intact apices quoted in Fig. i had regenerated no branches, a total of 103 branches had been regenerated from apices minus their apical cells. From these experiments, it appears that the site of inhibition to regeneration of new branches lies in the apical cell itself; if removed, regeneration proceeds. The effect of certain growth regulatory substances on the apices As before, i mm segments were taken from the tips of young thalli and cultured in sterile sea water containing growth regulatory substances in concentrations of 100, 10 and I mg 1 respectively. The effects of three auxins: 3-indolyl-acetic acid (IAA), i-naphthalene Fig. 2. Numbers of branches regenerated from segments of Fucus cultured with various growth regulating substances. Control in sea water; B = 3-indolyl-acetic acid; C = z,.\.- dichlorophenoxyacetic acid; D = i-naphthalene acetic acid; E = i-naphthoxyacetic acid; F = 2,3,5-tri-iodobenzoic acid. Concentrations employed as follows: A = intact apices in lo mg/1; -A = apices minus apical cells in 10 mg/1; AX = intact apices in i mg/1; -AX = apices minus apical cells in I mg/1. acetic and 2,4-dichlorophenoxyacetic acid; and two anti-auxins: i-naphthoxyacetic acid and 2,3-5-tri-iodobenzoic acid were studied, both on intact apices and on apices minus their apical cell. The cultures were maintained for 4 months, with the fresh weight of the segments being determined each month. The numbers of branches which had regenerated were counted at the end of the 4 months. In all, four sets of experiments were completed. With each of the five growth regulatory substances used, concentrations of 100 mg/1 led to a gradual disintegration of the segments both of those with intact apices and of

5 390 BETTY MOSS those minus their apical cell, over the 4 month period. Concentrations of 10 mg/1 of both auxins and anti-auxins always slightly lowered the rate of growth of segments as measured by the percentage increase in fresh weight. Concentrations of i mg/1 had little effect upon the rate of growth of both intact apices and of those minus their apical cells. But there were large differences in the numbers of branches regenerated by segments in auxins and anti-auxins. One set of results for cultures started in July 1963 are recorded as a histogram in Fig. 2. The control segments cultured in sea water regenerated no branches from intact apices, while 100 segments minus their apical cells regenerated a total of eightyeight new branches. No branches were regenerated from intact apices cultured in all three auxins. Yet in concentrations of 10 mg/1 of both anti-auxins, a considerable number of intact apices regenerated branches from the cut end of the segment. The effect of i-naphthalene acetic acid at concentrations of 10 and i mg/1 was to decrease the amount of budding from the cut surfaces of apices minus their apical cell. The other two auxins were not so consistent in their behaviour; sometimes resulting in a decrease in budding and sometimes having little effect. It is known that IAA soon oxidizes in solution and as the cultures were kept for 4 months, this may well have taken place, or the IAA have been absorbed by any microorganisms appearing in the cultures. On the other hand, anti-auxins at concentrations of 10 mg/1 gave increases up to three and four times the number of the controls. Increases were obtained with anti-auxins at concentrations of i mg 1, but these were not so great as with the higher concentrations. Extraction ofgrozvth regulatory substances Young plants which had not yet dichotomized were collected for the extraction of growth regulating substances in November, January and February. On each occasion, 2000 apical segments, as had been used in the previous culture experiments, were cut from the freshly collected plants. These apical segments were macerated with a small quantity of 8o o methanol in a bottom drive blender and extracted at 15 C according to the method described by Thurman and Street (i960). After 24 hours' extraction in the dark the methanol was removed under vacuum. The aqueous residue was then adjusted to ph 3.0 with H2SO4 and shaken four times with twice its volume of ethyl acetate. The total ethyl acetate fraction was then chromatogrammed. The aqueous fraction left after the ethyl acetate extraction was adjusted to ph 5.0 with barium hydroxide before chromatography. Chromatograms were prepared of both the ethyl acetate and the aqueous fractions so that each spot contained the equivalent extraction of from six to eight apices. The chromatograms were run in wo-propanol 400 ml; ammonia 20 ml; water 40 ml. Parallel samples of IAA and tryptophan were also run. Comparable chromatograms were cut into 2.5 cm squares and these were eluted with 2 ml of distilled water and bioassayed with the Avena coleoptile straight growth test. The growth of ten coleoptiles, each originally 10 mm in length, was compared in the eluates with the growth of similar coleoptiles in distilled water over a period of 24 hours. The beakers containing the eluates and the coleoptiles were kept at 27^ C. When chromatograms of the ethyl acetate fraction were sprayed wdth Erhlich's reagent, no colour reaction was obtained corresponding with the marker spot of IAA. There was stimulation of the growth of coleoptiles in eluates from the chromatograms corresponding with the zone of IAA (Fig. 3a). But there was increased stimulation in front of and following the corresponding zone of IAA. With Erhlich's reagent the aqueous fraction always gave a slight but positive blue

6 Apical dominance in Fucus 301 colour corresponding with the position of tryptophan. In all three extracts taken from the apices there was marked stimulation of the growth of coleoptiles in the eluate corresponding with the zone of tryptophan, but this was not the zone of maximum stimulation (Fig. 3b). However, it will be necessary to carry out further tests in order to confirm the presence of tryptophan. Also, as Fig. 3 shows, there were marked zones of mhibition of the growth of coleoptiles, both in the aqueous, and ethyl acetate, fractions. AO 20 S 0- ;-2o -AO- Distance from starting line (cm) Fig. 3. The growth activity of eluates from chromatograms from: (a) ethyl acetate fraction with position of IAA marked; (b) the aqueous fraction with position of tryptophan marked. DISCUSSION Culture experiments have demonstrated clearly that even in a relatively simple thallus like that of Fucus, apical dominance occurs. A narrow segment, with the apical cell intact, will continue to grow in culture and to elongate and to differentiate into midrib and wings as if it were still attached to the thallus. It will do this in a medium of sterile sea water, for unlike the apical meristem of vascular plants, the apex of Fucus is pigmented and photosynthetic so that it can exist independently from the parent thallus. Removal of the apical cell region results in a decreased rate of growth of apical segments and they fail to lengthen and to differentiate to the same extent as do intact apices. But the intact apex when in culture rarely, if ever, regenerates a new branch from the cut surface. In this case, are all the available nutrients being channelled to the existing meristem so that there are insufficient nutrients available for the development of new meristems from the cut surface? This seems unlikely, for if the apical cell is removed, regeneration of new branches takes place from both cut surfaces. For a narrow apical segment can, through the activity of its photosynthetic cells, supply enough nutrition for the regeneration of new apices, provided that the inhibitory effect of the original apical cell is removed. Discussions on apical dominance in the vascular plants fall broadly into two categories based on either nutritional, or hormonal, control. These culture experiments with F. vesiculosus provide no evidence in favour of nutritional control but show clearly that it is the apical meristem which inhibits the organization of new meristems in adjacent tissue. What, then, is the mechanism which maintains a single apical cell surrounded by small meristematic cells at the tip of each branch of a thallus, and inhibits the organization of similar meristems in its vicinity? In these culture experiments the

7 392 BETTY MOSS effect of anti-auxins in the medium was very striking on apices minus their apical cell. In concentrations of io mg/1 increases up to three and four times the number of branches regenerated in control experiments were obtained. Each of these branches has an apical meristem, so that high concentrations of anti-auxins in the medium have stimulated the organization of apical meristems from the wound healing callus. Also, from a number of intact apices in the higher concentrations of anti-auxins, a few branches were regenerated from the wounded end. So that in these instances the normal inhibitory effect of the apical cell has been removed by the presence of anti-auxin. The results with auxins were neither so consistent nor so striking as with the anti-auxins. But the cultures were maintained for 4 months and it may be that oxidation of the growth substance or absorption by microbes took place. There is no evidence to show that these growth substances actually penetrate through the mucilaginous covering of Fucus but, if this did not occur, then it would be expected that they would exert an effect on the cut surfaces of the segments where wounded tissues are directly in contact with the culture medium. In those experiments where auxins did have a significant effect, they brought about a decrease in the number of branches regenerated, i.e. their effect was the same as in higher plants, where frequently auxins have been shown to inhibit bud formation from wound callus. Extract and separation by chromatography of the growth regulatory substances in the apical meristems of F. vesiculosus are of a preliminary nature and further work is required before definite conclusions can be drawn. They illustrate that during the 3 months of November, December and January when extractions were prepared, the apical meristem contains a mixture of both growth stimulatory and growth inhibitory substances. With the techniques used IAA was not detected but tryptophan was always present. The zone corresponding with tryptophan, however, was not the only area of the chromatograms which stimulated the growth of Avena coleoptiles. These zones of marked stimulation and marked inhibition need further examination. Recently Mowatt (1964) has demonstrated the presence of gibberellin-like substances in whole thalli of F. vesiculosus but extracts prepared from the apical meristems have not yet been tested for these compounds. The evidence obtained from these culture experiments illustrates that apical dominance is as marked a phenomenon in the thalloid F. vesiculosus as it is in the vascular plants. In Fucus the single apical cell, surrounded by meristematic cells, inhibits the origin of new meristems in its vicinity. Growth regulatory substances undoubtedly play a role in this and it may be that in Fucus we have a mechanism depending on a balance between growth stimulators and grov^^h inhibitors as has been suggested for vascular plants. When the proportion of growth stimulators is decreased, as by the addition of antiauxin in cultures, then proliferation of new apical meristems is encouraged. Such a condition may arise when the thallus is growing in natural conditions on the seashore only once, or at most, a few times each year. Then the apical cell divides and one of the daughter cells so formed goes on to initiate the development of a new branch. REFERENCES Moss, B. L. (1964). Wound healing and regeneration in Fucus vesiculosus. Quatrieme Congrees International des Algiies Marines, Biarritz, 1961, p MoWAT, J. A. (1964). Auxins and gibberellins in marine algae. Quatrieme Congrees International des Algues Marines, Biarritz, 1961, p THURMAN, D. A. & STREET, H. E. (i960). The auxin activity extractable from exised tomato roots by cold 80 per cent methanol. J. exp. Bot., 32, 188.

Useful Propagation Terms. Propagation The application of specific biological principles and concepts in the multiplication of plants.

Useful Propagation Terms. Propagation The application of specific biological principles and concepts in the multiplication of plants. Useful Propagation Terms Propagation The application of specific biological principles and concepts in the multiplication of plants. Adventitious Typically describes new organs such as roots that develop