Vernalization and Photoperiodism: A Symposium (1948)
RELATION TO VERNALIZATION AND PHOTOPERIODISM
by KARL C. HAMNER
United States Plant, Soil and Nutrition Laboratory, Ithaca, N. Y.
In recent years a great many investigations have been concerned with the internal changes in plants which lead to the differentiation of flower primordia and the development of flowers. Much interest has centered in the earliest changes which take place in the plant which mark the initial transition from the vegetative to the flowering condition. This present discussion will deal primarily with these early changes and with several theories which postulate the sequence of events leading to the differentiation of flower primordia. No attempt will be made to give an adequate review of the literature since it has been carefully covered in other chapters. Selected references will be made to illustrate particular points.
Many years ago, SACHS postulated the existence of "organ-forming substances" in plants. He produced no definite evidence for the existence of flower-forming substances. GARNER and ALLARD (12) working with Cosmos sulphureous, a typical short-day plant, demonstrated the localization of the flowering responses to photoperiod when one part of the plant was exposed to short day and the other part to long day. They demonstrated that a portion of the plant exposed to continuous darkness exhibited some response when an adjacent portion of the plant was exposed to short day, indicating some transmission of the short-day stimulus to the darkened portion of the plant. KNOTT (19) working with spinach, a long-day plant, found that exposure of the bud to long-day conditions did not induce flowering if the leaves were exposed to short day. On the other hand, exposure of the buds to short day did not inhibit flowering if the leaves were exposed to long day. KNOTT concludes, "the part played by the foliage of spinach in hastening the response to a photoperiod favorable to reproductive growth may be in the production of some substance, or stimulus, which is transported to the growing point." Most of the credit for the recognition of the green leaves as the organs for the perception of the photoperiodic stimulus, however, must go to two Soviet botanists,
CAILAHJAN and MOSKOV, both of whom, apparently working independently, performed conclusive experiments which indicated that the green leaves first perceive the photoperiodical stimulus which is then transmitted to the growing point. Since then, many investigators have obtained similar results in photoperiodic studies.
Perception of the Photoperiodic Stimulus:— There can be little question that the green leaves are the organs which perceive the photoperiodic stimulus. This has been shown for both long-day and short-day plants (5, 10, 17, 19, 25).The young expanding leaves seem to be in sensitive, while the sensitivity of the fully-expanded foliage leaves seems to depend somewhat on the age of the leaf, the youngest being the most sensitive, and the oldest, mature leaves being relatively insensitive (2, 13, 26, 32, 34, 37). Individual plants become more sensitive to photoperiodic treatment as they grow older, but it may be that this increase in sensitivity is related to the fact that older plants have a greater number of foliage leaves to receive the stimulus.
In short-day plants, of course, the leaves respond to exposure to short-day conditions. Evidence is accumulating that an effective short day must contain a photoperiod of a certain minimum intensity and duration of illumination followed by a period of complete darkness of a certain minimum duration. HAMNER (15) has concluded that an effective short day for Xanthium pennsylvanicum must include a photoperiod of approximately 30 minutes or more (the length required is dependent on light intensity) followed by a dark period of more than 8 1/2 hours. The above sequence is not reversible with Xanthium; the photoperiod must precede the dark period. With Biloxi soybean (1, 14, 15) an effective short day also must include a photoperiod of over a certain minimum intensity and duration of illumination (the minimum length is about one hour and the maximum length is about 20 hours) and a period of complete darkness of more than 10 1/4 hours. Two or more (usually three or more) of these short days must occur in direct succession if flowering is to result. MOSKOV (28) with Perilla ocymoides concludes that this plant in order to be stimulated to flower must have uninterrupted dark periods of more than 8 hours and must have light periods of more than three hours. He (29) noted that short-day plants must be exposed to a cyclic alternation of light and darkness although he emphasized the importance of the length of the dark period. CAILAHJAN (7) also concludes that short-day plants respond to a definite cyclic alternation of light and darkness. Until evidence to the contrary is forthcoming, it seems desirable at this time to conclude that the specific length and character of both the photoperiod and the dark period determine the results of photoperiodic induction in all short-day plants, and it appears that determinative reactions take place during both phases of the cycle and also that there is an interaction among them (14).
Long-day plants seem to have no requirements with respect to darkness; the initiation of floral primordia takes place in continuous light as well as in the long day (14, 33). A certain minimum intensity of illumination is apparently necessary in order to stimulate flowering, but if the plant is illuminated continuously, it will flower provided it is intermittently exposed to fairly intense light. NAYLOR (33) has shown that continuous illumination does not stimulate flowering in beet unless the illumination intensity is above 700 foot candles. With dill continuous light increased in effectiveness with increasing intensities up to 300 foot candles. Either of these plants will flower if exposed to natural light during the day and to low intensities of light (less than 5 foot candles) at night. It may be concluded that the only effect which dark periods have on long-day plants is to inhibit or delay flowering.
It is of interest to compare the responses of long-day with short-day plants with respect to the influence of darkness. With long-day plants, a long, dark period seems to inhibit flowering while with short-day plants, a long, dark period seems to stimulate flowering. In either case, if the dark period is interrupted with a short period of illumination, it is noneffective; in long-day plants, it does not inhibit flowering and with short-day plants, it does not stimulate flowering. The dark period may also be rendered ineffective by very low intensities of illumination. In both plants rather high intensities of illumination are required for the stimulation of flowering. While it is true that certain long-day plants seem to be stimulated to flower by the use of low intensities of illumination at night, it seems apparent that this illumination has merely served to shorten the dark periods.
With short-day plants the changes which occur during the dark period and which lead to initiation of flowers should receive more careful study. With Xanthium, for example, dark periods of 8 1/2 hours duration are ineffective in stimulating flowering whereas dark periods of 9 1/2 hours are almost as effective as dark periods considerably longer. It would appear, therefore, that changes occur in the leaves after a period of 8 1/2 hours of darkness which result in the production of a stimulus for flowering. Whatever these changes are, in some short-day plants they seem to be of a rather permanent or irreversible nature (this is indicated in the work with Xanthium pennsylvanicum and Perilla ocymoides). Such chemical investigations as have been made to date have not given a clear indication as to just what these changes are. Apparently during the entire dark period a progressive change is taking place, and this change passes a certain critical point after a very definite length of time. Whatever this change is, it is dependent upon a previous exposure to bright light and the complete absence of light while the actual changes are taking place.
Transmission of the Photoperiodic Stimulus:—The evidence to date indicates that the stimulus is transmitted from the leaf to the stem rather slowly. In Xanthium (16), one short day is sufficient to cause the initiation of flowers. If plants receive two short days and are then defoliated, no flowers result. On the other hand, if a small portion of a mature leaf remains attached to the plant, flowering will result. Thus, the stimulus from only a small portion of one leaf is sufficient to cause flowering but not enough stimulus is received from all of the leaves during the first two days of treatment. CAILAHJAN (8) has also concluded that the transfer of the stimulus is slow. One of his arguments for a special flower hormone is based upon his conclusion that the transmission of the stimulus is much slower than the translocation of organic nutrients and growth hormones. Both MOSKOV (27) and CAILAHJAN (6, 8) conclude that the transfer of the flower-producing substances from the leaves is through living cells. Cooling the petioles (3, 8) greatly reduces or completely stops the transmission of the stimulus also indicating its passage through living cells.
The leaves of some short-day plants apparently continue to supply a stimulus even after the short-day treatment has been discontinued, while with other plants such is not the case. CAILAHJAN (6) with Perilla found that large leaves of a plant growing under short-day conditions would induce flowering in a plant continuously exposed to long day when grafted to it, even though the grafted leaf was placed on long day. LONG (22) obtained similar results with Xanthium. CAILAHJAN concluded that the flower hormone accumulated in the leaves of plants exposed to short day and that it was used up in flower bud and flower formation. HAMNER and BONNER (16) obtained evidence with Xanthium that the continuation of the supply of the stimulus subsequent to a short-day treatment was not due to simple storage of the stimulus during actual exposure to short day but to a continuing generation of the stimulus after short-day treatment was discontinued. MOSKOV (31) with Perilla reaches similar conclusions. Other short-day plants do not seem to continue to supply the stimulus after the short-day treatment has been discontinued. With Biloxi soybeans, LONG (22) found that two periods of flowering would result after exposure to two short-day induction treatments occurring about two weeks apart. BOTVINOVSKII (4) obtained similar results with hemp. Thus, a given induction treatment resulted in a certain flowering response and the effect then disappeared.
There is some evidence that the stimulus for flower initiation is used up in the actual process of flower formation and floral development. HAMNER and BONNER (16) with two-branched Xanthium plants found that the stimulus was received with greater force by the receptor branch (maintained on long day) when all buds of the donor branch (exposed to short day) were removed. It would be of interest to determine whether or not the stimulus would be stored provided there were no buds available to use it up.
The transmission of the stimulus longitudinally through a stem of a short-day plant from a leaf exposed to short day to an actively growing bud is in some way inhibited or partially inhibited by the presence on the stem of mature leaves exposed to long day. CAILAHJAN and JARKOVAJA (9) with Perilla found that the removal of such leaves increased the transfer. HAMNER and BONNER (16) with Xanthium demonstrated that the receptor branch of a two-branched plant, one branch on long day and the other branch on short day, did not flower provided the young leaves were removed and the older, mature leaves remained attached to the receptor branch. With no defoliation or with complete defoliation, the receptor branch flowered. Thus, there were indications that young, developing leaves exert a promotive effect on the transmission of the stimulus which more than counterbalances the inhibitory effect of mature leaves. BORTHWICK and PARKER (1) with two-branched Biloxi soybeans, one branch on long day and the other branch on short day, found that the receptor branch initiated flowers provided its leaves were removed. HEINZE, et al., (18) with Biloxi soybeans found that defoliation of a receptor plant of graft-partners increased the response of the receptor. MOSKOV (30) found that the receptor components of grafted plants responded more satisfactorily if the leaves were placed in complete darkness rather than in long day.
The stimulus is transferred readily across a graft-union. MOSKOV (25), in his early work from which he first concluded that there was transfer of a stimulus, used two varieties of tobacco, Maryland Mammoth and Sampson. The Maryland Mammoth tobacco is a typical short-day plant, while the Sampson variety seems to be indeterminate according to GARNER and ALLARD'S classification. When both are grown under long-day conditions, the former remains vegetative while the latter produces flowers. The experiments were conducted under continuous illumination, and various types of grafts were made when the plants were at an age at which the Sampson variety was ready to initiate flower buds. Decapitated plants of Maryland Mammoth tobacco were partially defoliated, and scions of either the Sampson or Maryland Mammoth variety grafted to them. Under these conditions, the stock soon produced lateral branches, and these flowered provided the scion was of the Sampson variety and was not defoliated. The stocks did not flower when Maryland Mammoth scions were used. KUIJPER and WIERSUM (20) at about the same time obtained analogous results with soybeans. Since that time many investigators have found it possible to stimulate short-day plants to flower under long-day conditions by grafting to them other plants of the same variety which have been induced to flower by exposure to short day or by grafting to them other varieties or species which will flower in spite of long-day conditions. Most of the grafting experiments have been carried out using short-day plants growing under long-day conditions as the test object and receptor.
MOSKOV (26) obtained transfer of the stimulus from scions even though the graft union between stock and scion was notably weak. HAMNER and BONNER (16) obtained transmission of the stimulus across what they called "a diffusion contact." They separated the injured surfaces of Xanthium plants with lens paper and obtained transfer of the stimulus from a plant treated with short day to the receptor plant maintained on long day. They found no evidence of tissue contact between the injured surfaces of the two plants and assumed that only substances capable of diffusion would have been transferred from one plant to the other. WITHROW (38) repeated these experiments and found that in those cases where transmission of the stimulus occurred cells had grown through the lens paper from one plant to another although admittedly the union between the two plants was very slight.
Vernalization:— A detailed discussion of vernalization has been given in another chapter. For purposes of discussion here, vernalization will be considered to involve the treatment of certain plants with low temperature in order to induce subsequent flowering. Many plants fail to flower unless exposed to a certain period of low temperature either in the seedling stage or after they have developed a number of foliage leaves.
There is a little evidence that hormones may be involved in the vernalization process. MELCHERS (23, 24) induced the biennial form of Hyoscyamus niger to flower without a cold treatment by grafting to it the flowering plants of Hyoscyamus albus, Petunia hybrida, and Nicotiana tabacum. These results indicate that this particular plant, which is ordinarily assumed to require vernalization, may be induced to flower as the result of a stimulus received from other flowering plants without the necessity of vernalization treatment. There is little additional evidence with other plants of this nature.
PURVIS (36) has shown that excised embryos of rye grown on a culture medium may undergo vernalization. There is some evidence (35) that small fragments of embryos may also be vernalized. Vernalization of embryos, therefore, is not dependent upon substances received from the endosperm during germination. If substances of a hormone nature are involved in vernalization, they may be manufactured in the embryo itself. PURVIS has shown, however, that whole grains of rye respond more rapidly than do excised embryos and suggests that certain substances received from the endosperm speed up vernalization. CHOLODNY (10) suggests that these substances are auxins or the B vitamins or a certain combination of them.
As far as the author is aware, no one has successfully substituted for the low temperature treatment the application of pure substances or extracts of plant material. The work of MELCHERS (23, 24) with Hyoscyamus presents encouraging possibilities in this direction, but more evidence is necessary with other plants before one may be encouraged to postulate that a hormone may be found which, upon application to the plant, may induce vernalization without a low temperature treatment. LANG and MELCHERS (21) suggest that those plants which require vernalization (i.e., a low temperature treatment) do not make the flower-forming hormone prior to vernalization. Subsequent to vernalization they may produce the flower-forming hormone provided environmental conditions are favorable. Presumably such plants will flower without vernalization provided the hormone is supplied from some external source (i.e., from a graft-partner). On such a basis, vernalization simply removes some physiological restriction to the production of the flower-hormone.
Theories Regarding the Mechanism of the Formation of a Flowering Hormone:— Generalizations with respect to the possible action of a flower-forming hormone are difficult because of the variations in response which occur in different species of plants. Various investigators have attempted to correlate the responses of all long-day plants or all short-day plants or all plants of the various classifications on the basis of a given series of reactions of a similar nature. Some postulate the presence of a single flower-forming substance of similar nature in all plants and ascribe the variations in response to given environmental treatments to the influence of these treatments in affecting the rate of synthesis of certain precursors. Critical evaluation of certain theories must await additional research with many different kinds of plants. A review of some of the theories may be of interest from the standpoint of indicating what additional information is necessary before any one theory is to be favored or discarded.
A recent paper by LANG and MELCHERS (21) gives a summary of most of their work with Hyoscyamus niger. They have studied two forms of this plant, one of which is an annual and the other a biennial. The biennial form must be exposed to a certain period of low temperature before it may be induced to flower. Subsequent to the low temperature treatment, it apparently reacts in every particular in the same manner as does the annual form. The annual form is a long-day plant. In ten-hour days it remains vegetative, whereas it flowers when the days are over ten hours and forty minutes. Removal of all of the leaves results in the prompt initiation of floral primordia. On the other hand, if one of the leaves is regrafted to the plant near the growing point, the plant does not flower unless the leaf is exposed to long day. Exposure of an intact plant to a low temperature even under short day results in flowering. Infiltration of the leaves with sugar also results in flowering.
LANG and MELCHERS consider that the annual form and the biennial form subsequent to vernalization fail to flower under short-day conditions because of the removal by the leaves of substances which would ordinarily result in flowering (i.e., a flower-forming hormone). This restrictive influence of the leaves becomes operative only during long, dark periods and apparently is related to exhaustion of carbohydrates in the leaves since the infiltration of the leaves with sugar or the treatment of the leaves with low temperature (a process which might conserve the leaf carbohydrates) removes the restrictive influence. They subscribe to the theory that flowering results from the transmission of a specific stimulus, "florigen," to the growing point. In annual Hyoscyamus they believe that this stimulus may be in some way continually supplied-perhaps from the roots or stored in the stem during exposure to light and that during long, dark periods the mature leaves remove this stimulus from the stem, and it does not have an opportunity to accumulate in the growing point and result in floral initiation. They speculate on a mechanism of response for all long-day plants based upon the results obtained with the annual Hyoscyamus niger.
The results which have been obtained with Hyoscyamus niger have not been obtained, as yet, with other plants. It was noted with several short-day plants (see above) that mature foliage leaves in some way inhibited the transfer of the flowering stimulus when they were exposed to an unfavorable day length. The removal of such leaves often results in the transmission of the stimulus from parts exposed to short day to other branches maintained continually on long day.
CHOLODNY (10) presents the hypothesis that auxin is involved in the flowering processes and the various environmental factors through their action on the synthesis and distribution of auxin determine whether or not a given plant flowers. He reviews the various experiments which indicate the possibility of a "flower-forming hormone." He believes that he could find no evidence to rule out the possibility that auxins were involved in the transition from vegetative to the flowering condition, although he indicates that it may be possible under certain conditions that it is the lack of auxin rather than the presence of auxin which causes this transition. He states, "We must form no hasty conclusion regarding special 'organ-forming substances' . . . we must first test whether some of the already known phytohormones are endowed with the faculty to induce the physiological effect under observation . . . ." Other investigators have questioned CHOLODNY'S hypothesis, and it seems to the author that experimental evidence fails to give it much support.
CHOUARD (11) believes the hormone of flower formation is distinct from auxin. He was able to induce flower formation in the long-day plant, China aster, growing under short days by applying dehydrofolliculin. He postulates that the hormone of flower formation is synthesized in the green leaves only under the action of light but believes that the hormone is produced in an inactive form and that this inactive form after it reaches a certain concentration limits the production of more of the inactive form. The production of the active form presumably takes place, in short-day plants, only during darkness by a process which is sensitive to temperature and inhibited by light. In long-day plants the light inhibition of the change to active form is presumed to be weak or completely absent.
The evidence that has accumulated for the existence of a flower forming hormone is sufficient to warrant a great deal more work in an attempt to discover just what it is. If there is a single substance, or relatively few substances of similar chemical nature, whose presence in the meristem determines the course of development from the vegetative to the flowering condition in all plants, the importance of discovering the chemical nature of this substance or substances is obvious. The possible practical importance of such a knowledge cannot be overemphasized. In view of the encouraging nature of the results to date, it seems strange that there are not more plant physiologists actively engaged in this field of research at the present time. It is hoped that this present symposium may encourage additional investigators to undertake work in this fascinating field.