Ed. Robert B. Withrow
PHOTOPERIODISM IN SEEDS AND BUDS
P. F. WAREING
Department of Botany, University College of Wales,
Aberystwyth, Wales cm2
Although only a small fraction of the total work on photoperiodism has been devoted to the effects of day length on dormancy, nevertheless it is clear that such effects are very widespread in higher plants, particularly in woody species. The seedlings of many woody species respond to short days by ceasing active extension growth and forming resting buds, whereas under long-day conditions the onset of dormancy is delayed or entirely suppressed (for the relevant literature, see Wareing. 1956). Moreover, in certain species, such as Fagus sylvatica, Betula pubescens, Larix europaea, and Pinus spp., seedlings that have been rendered dormant by previous short-day treatment can be induced to resume growth by transferring them to long-day conditions or continuous illumination. Even leafless dormant seedlings of these last species expand their buds under long days, whereas under short days they remain dormant. There is every indication that this is a real photoperiodic effect (Wareing, 1953). Thus, in these species we have photoperiodic sensitivity even in the very young leaf primordia present in the resting buds. When this fact was first established it seemed very unusual to find photoperiodic sensitivity in embryonic tissue, since it was generally held that, in the photoperiodic control of flowering in herbaceous species, it is only the fully expanded leaves which are sensitive to daylength conditions. More recently, however, it has been shown that, in Xanthium, it is the half-expanded leaves which are most sensitive (Khudairi and Hamner, 1954). The difference between woody and herbaceous species with respect to the sensitivity of the immature leaves would thus appear to be one of degree only.
SEED GERMINATION IN VARIOUS SPECTRAL REGIONS
The observation that the buds of birch seedlings show photoperiodic responses led us to investigate whether the seeds of this species show photoperiodic effects, and this was found to be so (Black and Wareing, 1954, 1955). The behavior of the seeds is markedly affected by temperature. At 15°C, the seeds show definite photoperiodic responses, so that a high germination is obtained under long days, whereas under short days germination is low. It is found that 8 long-day cycles are required to give maximum germination. At temperatures of 20-25°C, the response is radically modified and 50% germination will now occur in response to a single light exposure of 8 to 12 hr. At the higher temperature, therefore, the photoperiodic behavior is lost and germination occurs in response to a single light-exposure, as with lettuce seed. It is found that the most effective spectral region for stimulation of germination of birch seed lies in the red, and the effect of a single exposure to red radiation can be completely nullified if it is followed by infrared irradiation.
Photoperiodic effects in seeds have also been reported by Isikawa (1954) and Bünning et al. (1955), who found not only seeds in which germination is promoted by long days, but others which give a higher germination under short days than under long days. We have investigated (Black, 1957; Black and Wareing, 1957) the responses in such a "short-day" seed, as a corollary to our studies in birch seed, and for this purpose the seed of Nemophila insignis was selected, since it has long been known to be a light-inhibited seed (Lehmann, 1909). This seed is also known to be very temperature-sensitive, and germinates equally in light and dark at temperatures of 19°C or lower, whereas it is completely inhibited in both light and darkness at temperatures of 26°C and above. At 21-22°C, however, the germination is affected by the light conditions, and with white fluorescent tubes the response is markedly photoperiodic, a high germination percentage being obtained under short days, whereas germination is strongly inhibited under long days. The response is affected by the duration of both the light and the dark periods, but particularly by the latter. We have investigated the response of this seed to various spectral regions.
Earlier workers reported that the seed of Phacelia tanacetifolia, a species related to Nemophila, is inhibited by both blue and red light (Meischke, 1936; Resühr, 1939). For this purpose we used a set of 10 Schott interference filters (having a band pass of 10-14 mµ at half-maximum transmission) so chosen as to cover the range 405 to 760 mµ, at intervals of approximately 50 mm between peaks. The source used was a 500-watt tungsten filament lamp. In order to eliminate the possibility of the transmittance of an appreciable quantity of infrared radiation, copper sulfate gelatin filters were used in conjunction with the blue interference filters. It was arranged that the energy level at the position of the seeds was the same (50 or 80 µw/2) for each spectral region. It was found that Nemophila seed is inhibited by blue light (i.e., with filters peaking at 452, 483, and 496 mµ), but only slightly inhibited under the filters peaking at 542, 547, 596, and 651 mm.The inhibition is very strong in the far red, at 710 mµ, and somewhat less at 760 mµ.
The possibility that the blue region was inhibitory because of "stray" far-red radiation seems to be excluded by the observation that similar inhibition could be obtained by using as a source blue fluorescent tubes in conjunction with blue Perspex (B.705) and 1-cm screen of M/3 copper chloride. The latter would effectively remove any small component of far-red radiation emitted by the tubes. In spite of this, the seeds were strongly inhibited by light from this source, which covered the band from 400 to 520 mm. Thus, seed of Nemophila is inhibited not only by a far-red region, but also by blue light. These results agree very well with those of Resühr (1939) for Phacelia tanacetifolia. The inhibitory effect of far red is considerably greater than that of blue. Thus, a 4-hr daily photoperiod of far red brings about almost complete inhibition of Nemophila seed, whereas 16- to 20-hr photoperiods are required with blue radiation even at "saturating" intensities.
In view of the markedly inhibitory effect of blue in Nemophila it was decided to reinvestigate the reported effects of blue in lettuce seed. Flint and McAlister (1935) reported inhibitory effects of long periods of irradiation with blue light on lettuce seed. They exposed the seeds first to a short period of red light, sufficient to induce 50% germination, and then exposed them for 48 hr to various spectral regions. They found inhibition in the blue region and published a detailed action spectrum for this effect. Borthwick et at. (1954), apparently using short periods of irradiation, also reported both stimulation and inhibition of germination by blue alone, but the effects were not great. They found that maximum sensitivity for the promotive effect occurred at 12-20 hr of imbibition, and maximum sensitivity for inhibition after more than 48 hr from sowing. Also with short periods of irradiation Evenari and Stein (1957) confirmed that the promotive effect of blue light increases progressively during the first 16 hr of imbibition. With one type of filter they obtained inhibition during the first hours of imbibition, and this was followed by a promotive effect with longer periods of imbibition.
We have carried out experiments to determine how the responses of the seeds to blue light vary both with the imbibition period and with the duration of exposure. It was found that although a short period of irradiation with blue inhibits during the first 2-3 hr of imbibition, a longer exposure of 1-2 hr during this period is markedly stimulatory. As the period of imbibition increases, short periods of irradiation become promotive, whereas longer periods (1-2 hr) of irradiation become less promotive and ultimately become slightly inhibitory, after about 10 hr of imbibition (Fig. 1). Short periods of irradiation do not become inhibitory, at least up to 20 hr of imbibition.
|Fig. 1. Effect of irradiation with blue for various periods at different times during the period of imbibition. Blue fluorescent source. Intensity 100 mw/2. The germination percentages represent differences from "dark" control (taken as 0 ). Exposure periods (minutes) : A. 10: B, 30; C, 60; D, 120.|
Having determined the time at which the inhibitory effect becomes predominant, we investigated the interaction between the promotive effects of red light and the inhibitory effects of blue. Borthwick et al. (1954) were able to obtain only slight reversal of the promotive effect of red when the latter was followed by blue. The period of blue irradiation was not stated, but was presumably short. When we used 1 1/2 min of red (at 100 mw/2), followed by various periods of blue (at 100 mw/2) ranging from 1/2 hr to 4 hr, some reversal was obtained with periods of 1-2 hr, but much more effective reversal was obtained with 4 hr of blue. In a further experiment, in which we used 1 1/2 min of red and 4 hr of blue, the effects of a succession of irradiations with red and blue were investigated. It was found that repeated photoreversal can be obtained (Table I), and that the response of the seed is determined by the nature of the last irradiation, as in the interaction between red and far red (Borthwick et al., 1954). This would appear to be the first case in which successful reversal of the effects of red by blue have been obtained.
TABLE I. Lettuce, var.
Crawl Rapids, Photoreversal of
Promotion and Inhibition of Germination by Red and Blue
|R - B||33.0|
|B - R||75.5|
|R - B - R||86.1|
|R - H - R - B||49,0|
|aR, 1 1/2 min red at 100 mw/2 from fluorescent source; B, 4 hr blue at 100 mw/2 from
fluorescent source. Treatments were commenced 26 hr after sowing.
With the series of interference filters, we attempted to determine the effective spectral region for the inhibition of germination by blue, and it was found that the greatest inhibition was obtained with the filter peaking at 452 mµ.
The effects of blue have also been investigated in birch. We have never successfully induced any germination of birch by exposure to blue alone, but there is a marked interaction between blue and red. At a temperature of 21-22°C, birch seed was exposed to 8 hr of red light to induce germination, and this treatment was either preceded or followed by blue at 80 µw 2, with the blue fluorescent source described above. It was found that 24 hr of blue given after the red markedly inhibited germination (Table 11). On the other hand, blue given before the red very strongly promoted germination. Further experiments indicated that relatively long periods (about 12 hr) of exposure to blue are necessary to obtain maximum stimulation or inhibition. Experiments with the interference filters indicated that the most effective spectral region for these effects lies in the region of 452 mµ.
TABLE II. Seed of Betula
pubescens, Effect of Various
of Blue Irradiation Before and After Exposure to Red
|1.||8 hr red only||32.6|
|2.||8 hr red, preceded by blue: (a) 1 1/2 hr||37.8|
|3.||8 hr red, preceded by blue: (b) 3 hr||52.5|
|4.||8 hr red, preceded by blue: (c) 12 hr||67.0|
|5.||8 hr red, preceded by blue: (d) 24 hr||70.6|
|6.||8 hr red, followed by blue: (a) 12 hr||16.5|
|7.||8 hr red, followed by blue: (b) 24 hr||13.1|
|8.||8 hr red, followed by blue: (c) 48 hr||19.2|
|Source, blue fluorescent tubes with blue Perspex and copper chloride screen. Intensity, 80 µw/2.|
In order to explain the promotive and inhibitory effects of blue in lettuce seed, Borthwick et al. (1954) have postulated that the photoreceptors for the red and far-red responses must have absorption regions in the blue which overlap. This hypothesis would explain why the response of the seed is so dependent upon the duration of exposure and the period of imbibition, since these two variables can be envisaged as affecting the equilibrium between the promotive and inhibitory processes. The Beltsville group was unable to obtain any appreciable reversal of the effects of red by blue, however. It is now clear that, in order to obtain photoreversal, the period of irradiation by blue must be about 4 hr, whereas much shorter periods are effective with far red.
The greater period of exposure required with blue may result from the fact that, even at long imbibition periods, there may still be some promotive effect of blue and that the balance is only decisively toward inhibition when longer periods of irradiation are used. Further, it is known that light transmission by the seed coat of lettuce is very much less for blue than for far red (Evenari, 1956). and it is possible that shorter periods of irradiation with blue would be more effective at higher intensities than that used (100 µw/2) in the present experiments.
There would no longer seem to be any doubt that the inhibitory effects of far red and blue are operating through the same photoreceptor. This conclusion is in full agreement with the fact that blue and far red are frequently found to act similarly in internode elongation and in the flowering of certain species (see Wassink and Stolwijk, 1956). The promotive effects of blue on lettuce seed germination are best explained on the hypothesis that the photoreceptor for red also has an absorption in the blue, as suggested by Borthwick et al.
The effects of blue clearly merit further attention not only because of their importance for the study of dormancy, but also because elucidation of the phenomena in seeds may have an important bearing on the interpretation of the effects of blue on flowering.
POSSIBLE ROLE OF INHIBITORS IN DORMANCY
We turn now from a consideration of the photoreactions involved in seed photoperiodism to what would seem to be another important factor in determining the overall response. The first piece of work to be described was carried out on birch seed by Dr. M. Black at Manchester (1957).
Now, the light requirement of birch seed is a property only of the intact seed, since embryos from which the pericarp and endosperm have been dissected will germinate equally well in both light and dark and show no apparent photoperiodic effects at all. This indicates that the presence of the pericarp or endosperm must have an inhibitory effect on the growth of the embryo, and that light is necessary to enable the embryo to overcome this inhibitory effect.
Two possible ways in which this inhibitory effect might arise would appear to be that either (1) the pericarp or endosperm contains an inhibitory substance which holds the embryo dormant, or (2) these seed coverings might interfere with gaseous exchange, particularly with oxygen uptake by the embryo. Both these types of mechanism are known to play a role in the dormancy of certain seeds. In order to test the first possibility, birch seeds were extracted with 80% aqueous methanol, the extract was concentrated and then chromatographed on paper, with 80% aqueous isopropanol and 1% ammonia as a running solvent. After drying. the chromatogram was cut up into 10 equal strips, each of which was placed in a small petri dish and moistened with water. Ten isolated birch embryos were then planted on each of the filter paper strips and observations made on the growth inhibitory activity of the different regions of the chromatogram. It was found that there was a powerful growth inhibitor present on the chromatogram in the region Rf 0.7-0.9. The same inhibitory zone was found in a sample of birch achenes which were entirely lacking in the embryos. Thus, it would seem that the inhibitor is located primarily in the pericarp. The presence of a growth inhibitor in the pericarp does not, of course, necessarily imply that it constitutes the sole basis of the inhibitory effect of the pericarp on the embryo, and further experiments were carried out in an endeavor to obtain decisive evidence on this question.
First, it was found that when birch embryos were planted on the inhibitory zone and then exposed to different photoperiodic treatments a high proportion of embryos germinated when maintained under long days, but gave only a low germination percentage under short days. That is to say, whereas the isolated embryos planted on filter paper moistened only with water germinated equally well in both light and dark, when they are planted on filter paper containing the inhibitor their photoperiodic behavior is restored. In a further experiment, intact seeds were slowly leached in water in darkness for 3 weeks. They were then sown and held under long-day or short-day conditions, and their germination was compared with that of unleached seeds. It was found that in the leached seeds germination was high not only under long days but also under short days. The two foregoing experiments thus indicate that (1) the photoperiodic behavior of the intact seeds can be largely restored if isolated embryos are planted on the inhibitor, and (2) if the inhibitor is leached out of intact seeds, their photoperiodic behavior is largely lost. These results are, therefore, consistent with the hypothesis that the inhibitory effect of the pericarp arises primarily from the presence of the growth inhibitor. It was shown that at the low light intensities used, there is very little photodestruction of the inhibitor on the filter paper, and it would seem that the effect of light is primarily on the embryo, which is thereby stimulated to overcome the effect of the inhibitor.
On the other hand, it was found that it is not necessary to remove the pericarp from the embryo completely in order to abolish its light requirement. Slitting the pericarp and endosperm on one side, or even simply pricking, is sufficient to bring about an appreciable germination in the dark. This observation is difficult to interpret on the "inhibitor hypothesis" and suggests rather that the inhibitory effect of the pericarp is due to interference with gaseous exchange. Further evidence in support of this view is seen in the fact that pricking the pericarp is even more effective in stimulating germination if the seeds are subsequently maintained in an atmosphere of high oxygen content instead of in air. However, a high oxygen tension is ineffective if the pericarp is maintained intact. These results strongly suggest that interference with oxygen uptake also constitutes an important part of the inhibitory effect of the pericarp. Experiments to determine the minimum oxygen requirements for the growth of birch embryos indicated that they will germinate even in commercial nitrogen which has been passed through alkaline pyrogallol, and which must have had an extremely low oxygen content. Therefore, it seems unlikely that the oxygen requirement would not be met in an intact seed maintained in an atmosphere of 70% oxygen. Thus interference with oxygen uptake by the pericarp does not seem adequate to explain all the phenomena. Now, from a study of dormancy in Xanthium seeds it appears that both interference with oxygen uptake by the testa and the occurrence of a growth inhibitor in the embryo play important roles, and that oxygen is necessary for the breakdown of the inhibitor, before germination can occur (Wareing and Foda, 1957). A similar hypothesis would seem to be best adapted to explain the dormancy effects in birch seed. Some evidence in support of this hypothesis is seen from the results of an experiment in which leached and unleached seeds were first scratched and then exposed to various oxygen concentrations. It was found that leached seeds gave an appreciably higher germination at low oxygen tensions than did unleached seeds.
We have found that gibberellic acid is effective in breaking the dormancy of birch seed, as with lettuce seed (Lona, 1956; Kahn et al., 1957). Moreover, the dormancy of birch and Xanthium seed has several other features in common with that of lettuce seed. For example, light-requiring lettuce seed may be induced to germinate if maintained in an atmosphere of pure oxygen (Borthwick and Robbins, 1928), or if the pericarp is split or pricked (Evenari and Neumann, 1952); this suggests that oxygen effects are important also in this seed.
These observations raise the question as to whether inhibitors also play a role in the dormancy of lettuce seed. The presence of inhibitors in lettuce seed has been demonstrated (Shuck, 1935; Wareing and Foda, 1957; Poljakoff-Mayber et al., 1956), and one of these is a water-soluble substance occurring at about the same position on chromatograms as the main Xanthium inhibitor (Wareing and Foda, 1957). It has not been possible, however, to determine whether the inhibitors play any role in the dormancy of lettuce seed, which is, for this purpose, more difficult material than Xanthium seed. Nevertheless, the close parallel between the dormancy phenomena in the seeds of these two species (both members of the family Compositeae) strongly suggests that the underlying mechanism is the same in both cases. It is tempting, therefore, to postulate that the dormancy of lettuce seed involves a growth inhibitor, the effect of which is in some way overcome by light, as in birch seed. One difficulty for this hypothesis is that we have recently found that certain light-requiring varieties of lettuce seed contain no detectable amounts of the water-soluble inhibitor found in Grand Rapids. It must be remembered, however, that the light requirement of lettuce seed is very small, and this may arise from the fact that the level of inhibitor present is also very low.
The responses of birch seeds also have certain features in common with those of birch buds (Wareing, 1957). It seems unlikely that dormancy in the buds is due primarily to interference with oxygen exchange, since frequently it is observed that the terminal bud formed in response to short days is lax and by no means tightly enclosed by bud scales. Moreover, it is clear that interference with oxygen exchange by the bud scales cannot be the primary factor inducing the formation of resting buds, since until such a bud is formed there is no interference with oxygen exchange by the shoot apical region. On the other hand, some evidence that bud dormancy may be due to the presence of growth inhibitors has been put forward by Hemberg (1949) and others. It is possible, therefore, that the formation of resting buds in response to short days may be due to the production of greater amounts of a growth inhibitor under short days than under long days. We have carried out investigations to test this hypothesis using primarily Acer pseudoplatanus (Phillips and Wareing, 1958).
Preliminary experiments showed that there is an inhibitor in the leaves and buds of A. pseudoplatanus which is completely extractable with 80% aqueous methanol. After extracting the tissues with this solvent, the extracts were partitioned by paper chromatography by using a running solvent consisting of 80 parts isopropanol to 20 parts aqueous ammonia (0.88 S.G. x 1/100). After development the chromatograms were eluted in water and assayed for growth activity by using primarily the wheat coleoptile section test. The growth inhibitor was found to occur between Rf 0.6 and 0.8. A study was made of the inhibitor contents of the leaves and shoot apices of seedlings grown under long-day or short-day conditions. Samples of the leaves and shoot apices were taken from each series after 2, 5, 10, and 33 days following the commencement of the treatments. It was found that there was consistently more inhibitor present in the leaves and shoot apices of short-day seedlings than of long-day seedlings (Fig. 2). A difference between the two series was detectable even after two short-day cycles, but this became greater after five cycles (Fig. 3). We have confirmed these results several times. When the chromatograms were assayed by planting seed of lettuce var. New Market (a non-light-requiring variety) on the various zones, it was found that germination was markedly inhibited by the same region as were wheat coleoptiles, and that there were very great differences between the short-day and long-day extracts in this respect. Recent experiments with extracts of seedlings of birch grown under long and short days have given similar results.
|FIG. 3. Coleoptile section assay of the inhibitory eluates (Rf 0.55-0.88) from chromatograms of extracts of mature leaves. The extracts were prepared from plants growing under controlled photoperiodic conditions; the leaf samples were made at the start of the experiment, and after 2, 5, 10, and 33 cycles of long day or short day. Extract from 1.0 g dry weight of tissue was chromatographed in each ease. Upper curve, long day; lower curve, short day.|
|FIG. 2. Assay with wheat coleoptile sections of chromatographed extracts (each equivalent to 0.1 g dry weight of tissue) of mature leaves.|
These results do not, of course, necessarily imply a causal relationship between the production of inhibitor and the induction of dormancy. It is possible that the greater production of inhibitor under short days is the result of reduced growth. The observation that differences in inhibitor content can be detected after 2-5 days of treatment, although the short-day plants continued to expand leaves for a further 1 0 days, would seem to indicate, however, that the inhibitor differences are not primarily due to differences in growth. If the inhibitor hypothesis is substantiated, we shall have made an important step forward in the elucidation of the mechanism of photoperiodism in buds and seeds. It is not suggested that photoperiodic control of flowering necessarily involves growth inhibitors. Considerable evidence suggests that there is much in common between photoperiodism in dormancy phenomena and in the control of flowering (Wareing, 1956), and this would seem to imply that the basic light and dark processes are identical in both types of response. Nevertheless, the induction of dormancy is clearly different from the induction of flowering, and it is possible that the changes in inhibitor content observed in woody plants are secondary effects arising from earlier steps in the basic photoperiodic processes.
Photoperiodic control of dormancy is well established for both buds and seeds. Among light-sensitive seeds the germination of some species is promoted by long days, whereas in others germination is inhibited by long days and promoted by short days. The seed of Nemophila insignis behaves as short-day seed at temperatures of 21-22°C. The seed of this species is inhibited by both far red and by blue.
A reexamination of the effects of blue in Grand Rapids lettuce seed has shown that blue may be promotive or inhibitory to germination depending upon the duration of exposure and the imbibition period. With 4-hr periods of blue, it is possible to reverse the effects of a preceding exposure to red. The photoreceptors for the well-known effects of red and far red in lettuce seed appear to have absorption bands in the blue which overlap. The inhibition of germination by blue and far red appears to involve the same photoreceptor.
Studies of dormancy in birch seed seem to indicate that the inhibitory effect of the pericarp and endosperm involve both the presence of a growth inhibitor and also interference with gaseous exchange. Light is apparently necessary to enable the embryo to overcome this inhibiting effect of the pericarp and endosperm.
A study of the growth inhibitors in Acer pseudoplatanus in relation to day length conditions seems to indicate that more inhibitor is produced in the leaves under short days than under long days, suggesting that onset of dormancy of the short apices in response to short days is due to the accumulation of inhibitor in this region under such day length conditions.