PHYTOCHROME AND SEED GERMINATION

Transcription

1 New Phytol. (197) 71, PHYTOCHROME AND SEED GERMNATON BY M. HOLDSWORTH University of Otago, New Zealand {Received l^june 1971) SUMMARY Both tobacco 'Virginia Gold' and Plantago hirtella seed germinate on exposure to red light. During the first day or so after sowing, the response can be stopped by a following exposure to deep-red, indicating phj'tochrome action. However, the effects of successive exposures to red are additive through intervening exposures to deep-red. Moreover, after several days in the dark, both red and deep-red induce germination. NTRODUCTON The existence of phytochrome, proposed 5 years ago to account for the contrary photoperiodic effects of different hues of red light, was soon used to explain practically all lightgrowth effects (Borthwick, Hendricks and Parker, 195). The prediction has recently been fulfilled in the isolation of a pigment with the required photochemical characteristics, but in the meantime most light-dependant growth effects have proved too complex to be explained by simple phytochrome action. The only apparent exception is the promotion of seed germination, the light-dependant growth effect from which Borthwick originally gained the idea of a chameleon-like pigment. Borthwick argued from the spectral sensitivity of Grand Rapids lettuce seed (Flint and McAlister, 195) but the light sensitivity of that example is an artefact (Berrie, 1966) manifest only in a narrow and unnatural temperature range. Examples of seeds needing illumination at ordinary temperatures are, indeed, uncommon but tobacco (Gardner, 191) and Plantago hirtella appear genuine. MATERALS AND METHODS T obacco seed (cv. Virginia Gold) was supplied by the Tobacco Research nstitute, Motueka. Plantago hirtella is a South American plantain naturalized in New Zealand. T he stock used was derived from Wanganui Forest, Westland. Seed, in lots of 1, was sown on filter paper in petri dishes. The dishes were kept before and after illumination in double black-cloth bags and those in honey-tins painted black inside. Orange-red ('Red') light was taken from two W Philips' No. 17 fluorescent lamps; green from a single No. 15 screened with green Perspex; and blue from two No. 18 screened with blue. As these lamps flicker before striking, short exposures were measured With a mechanical shutter. Deep-red light was from a 5 W reflector bulb, through a dish of water, and then through red and royal-blue Perspex (the greener shades of blue Perspex arc opaque to deep-red). The dishes were exposed. m from the lamps. The tobacco fests were done at C, the plantain tests at 5. The need for light is no less at lower temperatures but, of course, experiments would take longer. At ^ C, tobacco germinates 15

2 6 M. HOLDSWORTH in 7 days in the light, and in 5 days after a saturating exposure to light given on the fourth day or later. For plantain the corresponding times are 4 and days. Germination is, however, rather slower following earlier or less illumination and in the tests, one day's grace was allowed before counting. RESULTS Tobacco n complete darkness, tobacco did not germinate at all, not even old seed years after harvest. Red, deep-red, blue and green light could all induce germination but, at Table i. Tobacco: percentage germination in response to a single exposure to red light {averages usually of two experiments, but some of only one and of ten for minutes at 4 hours) sowing to end of Duration of exposure (minutes) exposure i i i i 4 8 i6 hours o 1 4 hours o 61 8 hours hours o days days days days days 6 7 days 57 8 days 7 least immediately after sowing, red was much more active than the others: in the order green, blue, deep-red. After sowing, sensitivity to a brief illumination increased with time (Table i and ). With non-saturating exposures to red, the probits increased practically linearly for days, then more slowly to become stationary after the fifth day. Sensitivity to green and blue ran a parallel course, but to deep-red it seemed to increase more rapidly until by the fourth day it equalled the sensitivity to red. The effects of the Table. Tobacco: percentage germination in response to a single exposure to deepred light {averages of two experiments usually) Duration of exposure (seconds or minutes) sowing (days) is 5s 15 s s im m 4m 8m 16 m 144m 88m 8,S ^ '^! ' ^ 9 - * o i» two reds cannot, however, really be compared. At any time after sowing, a longer exposure to red, blue or green increased germination and, for red, the probits are hyperbolic to the dosage. To deep-red, any exposure of more than a few seconds seemed to cause the same proportion to germinate on any particular day. So if exposures of less than a minute were to be given on the first day or two after sowing, the seed would appear more sensitive to deep-red than to any other colour. Although deep-red, by itself, made tobacco seed germinate, it reduced germination

3 Phytochrome and germination when given after red (Tables and 4). During the first day, 1 seconds deep-red almost stopped response. With the passage of time after sowing, however, its abilit\' to counteract red declined, and response to exposures given after the third day could not be stopped however long the following exposure. rrespective of the duration of the exposure to red, maximum reduction of germination was attained, on any day, by about 1 seconds deepred. When deep-red was given before red on the first or second days, it neither increased nor decreased the effect of the red. On the third day and after, it appeared to add to the effect of red. Table. Tobacco: percentage germination in response to deep-red light, following an exposure to red which ought to have caused half to germinate {average of two experiments only except on first day) sovifing (days) 4 Red exposure min min 15 s 7 s S SO Duration 5 s of deep-red 1[second or minutes) s m 4 m During the first day, if saturating exposures to red were alternated with saturating exposures to deep-red, the effect of each appeared to be completely reversible by the other. For example, if 16 minutes of red was alternated with 8 seconds of deep-red, the germination was either complete or negligible, according to the final exposure. f, however, 8 seconds of deep-red was alternated with only i minute red, germination, although always small if the last exposure was deep-red, gradually increased if the last exposure was red and with many alternations virtually all the seed germinated. On the other hand, the effects of successive exposures to deep-red were not additive through alternative periods in red. f, say, on the first day 8 minutes red was alternated with seconds deepred, the final germination was never less than about 7%. Table 4. Tobacco: percentage germination in response to deepred light, following an exposure to red which ought to have caused all to germinate {single tests except on first andfourth days, which are averages of pairs) 8 n 7 S sowing 6 hours 8 hours day days days 4 days Red exposure h h min 16 min min s Duration S of deep-red (seconds or 5 s 15 s m minute Newly harvested seed, at least from plants outdoors, did not germinate at all in the dark. With keeping, however, the need for light declined and after a year's storage more ftari half were able to germinate without exposure. From greenhouse plants a few seed occasionally germinated in darkness even when fresh. n the case of fresh outdoor seed a few minutes exposure to red light later than about S hours after sowing would produce some germination, and, although the responses were

4 O8 M. HOLDSWORTH much less consistent than those of tobacco, the collective results of many experiments leave no doubt that germination increased both with the length of exposure and its time after sowing (Table 5). Response to blue or green light was always much less, an hour's exposure even as late as a week after sowing, causing only a small percentage to germinate. None germinated in response to deep-red, however long continued. Deep-red given before red had no apparent effect on germination. Given immediately following red, it reduced it often to zero during the first day after sowing (Table 6). Even so, the effect of the red had not been completely erased. For example, in one experi- Table 5. Plantain: new seed ripened outdoors; percentage germination in response to a single exposure to red light {averages of pairs from a single batch of seed the absolute values varied from one batch to another) sowing when exposed (days) 4 5 i 5 8 i Duration of exposure (minutes) ment, minutes red by itself caused 1% to germinate, but none germinated if the red was followed by minutes deep-red. A second exposure then to minutes red raised the percentage to 17, a further alternation to 5 and practically every seed could be made to germinate by continuing. Conversely constructed experiments showed that the effects of deep-red also, are carried over through intervening periods in red light. For example, 4 minutes red caused 98% germination and 15 seconds of deep-red reduced this only to 91, but a second alternation of 4 minutes red and 15 seconds deep-red brought the percentage down to 9. The addition of deep-red exposures was, however, never so consistent Table 6. Plantain: new seed ripened outdoors; percentage germination in response to deep-red light given after an exposure to red more than sufficient to make all germinate {single tests with a single batch of seed; the trends of parts of the table having been confirmed with other batches) sowing (days) 4 Exposure to red m 16 m 8 m 4 m S OO 9 OO OO Exposure to deep-red (seconds or minutes) 5 s 1 s 15 s s m 16 m as that of red for subsequent alternations usually did not lower the germination further, sometimes indeed it rose again. As mentioned earlier a small percentage of some batches of greenhouse-ripened plantain seed would germinate in darkness. The rest could, of course, be made to germinate by sufficient exposure to red. A following exposure to deep-red would reduce the germination again, but in no test could it be brought below the dark germination of the same batch. With both stored seed and greenhouse seed (even batches which would not germinate at all in darkness) short exposure to deep-red promoted germination if given alone- As towards red, the seed became more sensitive to deep-red with storage in the dark

5 Phytochrome and germination 19 after sowing. f, however, a very long exposure (more than 4 hours) to deep-red was begun as the seed was sown, none at all germinated in addition to that expected in darkness. Moreover, the long initial exposure also reduced germination in response to a later short exposure to the same colour. For example, minutes deep-red given 5 days after sowing caused 5% to germinate, but a preceding long exposure to deep-red reduced this to 11. DSCUSSON Morphogenetic effects of blue light have sometimes been quoted as evidence that phytochrome is not the sensor but as most organic pigments are blackish when concentrated they all, presumably, absorb all colours. Both tobacco and plantain seeds germinate in blue or green light, but both are much more sensitive to red. Both seeds can be prevented from responding to red if subsequently exposed to deep-red, so phytochrome is very probably the primary detector of illumination. All the same, the evidence is ambiguous: (i) the effects of red and deep-red may be complementary but only in certain circumstances; () the 'reversal' is more apparent than real though the seeds react only to the last sort of red they experience, all previous experiences of the other kind were not wholly eliminated. The first of these contingencies is familiar in the photoperiodism of short-day plants (e.g. Takimoto and Hamner, 1965) in which deep-red counteracts an immediately preceding red 'night-break', but long exposure to deep-red (and exposures given a long time after the red) themselves act as night-breaks. Tobacco seed germinates in response to deepred just as well as to red, if exposed a long time after sowing; plantain seed likewise if exposed a long time after harvesting. According to Duke and Wickliff (1969), growth of the mesocotyl of Zea is inhibited by both red and deep-red, even though deep-red prevents inhibition by previous red. There are some puzzling features of the positive response of tobacco seed to deep-red. in particular, it is curious that unlike red, green or blue, no amount of deep-red could, cause 1% germination immediately after sowing. On the contrary, during the first day, there seemed to be only about 5% of the seed that could show the positive response, however long an exposure was given. Yet for those 5%, very little (less than 1 seconds) sufficed. The sensitivity, in fact, did not really change as the days passed, it was the proportion of susceptible seed which increased. As mentioned, C was adopted as the test temperature for tobacco but the response to a short exposure (to red or deep-red) on each of the first or 4 days is considerably greater at 5 C. Undoubtedly the higher temperature hastens the sensitization. n plantain it appeared that high temperature in the greenhouse during ripening of the seed also hastened the sensitization (to deep-red) ^ that it occurred during ripening itself instead of during storage. f!k' second contingency, the summation of the effects of repeated exposures to red through intervening periods in deep-red has also been recorded before in Blaauw, olaauw-jansen and van Leeuwen (1968) investigation of the effects of light on the growth ot Oat mesocotyls. Most attempts to show the 'reversal' of red effects by deep-red, whether on growth, on germination, or on flowering, have deliberately used saturating dosages of th colours in order to demonstrate that reversal can be complete. Blaauw et al., nowcverj were concerned to measure quantitative effects of graded doses of red light and "'^'r Table i {he. cit. p. 91) shows quite clearly the addition of the effects of successive ^^ exposures through intervening periods in deep-red. f tobacco and Plantago hirtella

6 no M. HOLDSWORTH are at all typical, it appears very likely that seed photoblasty has some, if not all, of the features that make phytochromatic explanations of photoperiodism so tortuous. Biinning has repeatedly (e.g. i96) emphasized that in photoperiodism a plant reacts to light in the daytime oppositely from what it does at night. t is now known that it may also react conversely to red or deep-red light as the night itself wears on (e.g. Takimoto and Hamner, 1965). n seed germination too, responses to deep-red appear to reverse as the period in darkness is extended. So far, attempts to explain the time factors in photoperiodism in terms of phytochrome have been unconvincing. Similar difficulties obviously present themselves in considering the part played by phytochrome in germination. REFERENCES BERRE, A. M. M. (1966). The effect of temperature and light on the germination of lettuce seeds. Physiologia PL, 19, 49. BLAAUW, O. H., BLAAUW-JANSEN, G., LEEUWEN, W. J. VAN (1968). An irreversible red-light-induced growth response in Arena. Planta, 8, 87. BoRTHWicK, H. A., HENDRCKS, S. B., P.\RKER, M. W. (195). The reaction controlling floral initiation. Proc. natn. Acad. Sci. U.S.A., 8, 99. Bt'NNiNG, E. (i96). Circadian rhythms and the time measurement in photoperiodism. Cold Spring Harb. Symp. quant. BioL, 5, 49. DUKE, S. C, WCKLFF, J. L. (1969). Zea shoot development in response to red light interruption of the dark-growth period. PL PhysioL, Lancaster, 44, 17. FLNT, L. H., MCALSTER, E. E). (195). Wavelength of radiation in the visible spectrum inhibiting the germination of light sensitive lettuce seed. Smithson. misc. Collns, 94, i. GARD.MHR, W. A. (191). Effect of light on germination of light sensitive seeds. Bot. Gas., 71, 49. T.wiMOTo, A., HAMNER, K. C. (1965). Effect of far-red light and its interaction with red light in the photoperiodic response of Pharbitis nil. PL Physiol., Lancaster, 4, 859.

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