Planta
Planta 133, 97-102 (1977)
9 by Springer-Verlag 1977
Nature of Light Requirement for the Flowering of Chenopodium rubrum L. (Ecotype 60 ~ 47' N) I. Pre-induction Light Period* Ramma Sawhney ** Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada
Abstract. Seedlings of the short-day plant, Chenopodium rubrum L. (Ecotype 60 ~ 47' N) were irradiated with different intensities and qualities of light for 24 h preceding a single inductive dark period (12 h). Our data shows that a relatively low intensity incandescent light (35-100 ft. c.) is not effective as the photoperiod for flowering. The above effect is not due to a requirement for a relatively high level of photosynthesis. Our results suggest a definite promotory role of a blue High Energy Reaction (HER). We could not demonstrate the involvement of a far-red HER. We suggest that ineffectiveness of far-red may have been due to establishment of rather low Phytochrome, PFR, levels, suboptimal for flowering. A certain critical level of PFR (30--40%, that presumably established by blue light) seems to be necessary for photoreactions involved in flowering of C. rubrum. There are indications in our experiments of the operation of a red radiation mediated flower inhibitory photoreaction. Key words: Chenopodium rubrum - Flowering High Energy Reaction - Photosynthesis - Phytochrome.
Introduction The flowering of short-day plants is known to require a high intensity white light photoperiod prior to in* This paper constitutes a part of a Ph.D. thesis submitted to the University of Western Ontario, London, Ontario ** Present address. Department of Biology, University of Saskatchewan, Saskatoon STN OWO, Canada
ductive darkness. It has generally been postulated that the high intensity light is necessary to sustain optimum photosynthesis for flower induction (Hamner, 1940; Parker and Borthwick, 1940; Liverman and Bonner, 1953; Carr, 1957; Cumming, 1967). However, a review of the literature reveals numerous findings which are in conflict with the above hypothesis. For instance, 1) the flower inhibitory effect of low intensity white light could be overcome in Kalancho~ blossfeldiana (Schwabe, 1954) and in Xanthium strumarium (Salisbury, 1965) by prolonging the following period of darkness-a response difficult to relate to photosynthesis, 2) Biloxi soybean plants maintained in COz depleted atmosphere were inhibited from flowering but their flowering response was unaffected when photosynthesis was significantly reduced by removing all but one leaf (Parker and Borthwick, 1940), 3) while sugar application overcame the effect of low intensity light on the flowering of Xanthium strumarium (Liverman and Bonner, 1953; Cart, 1957), some non-specific reducing agents also had a similar flower stimulatory effect (Lang, 1958), 4) sugar application was not effective in promoting flowering of Lemna perpusilla exposed to low intensity light conditions (Umemura and Oota, 1965). In view of the uncertainty regarding the exact nature of the light requirement for flowering of SD plants, the present study was undertaken to investigate the contribution of 1) photosynthesis, 2) phytochrome (PvR), and 3) High Energy Reaction (HER) of photomorphogenesis, in the flowering of Chenopodium rubrum L. (ecotype 60 ~ 47' N, selection 374).
Materials and Methods
Abbreviations." SD=short day plant; H E R = H i g h Energy Reac-
tion; PvR=far-red absorbing form of phytochrome; PR=red absorbing form of phytochrome; L.I.I. =low intensity incandescent white light; H.I.I.=high intensity incandescent white light; L.I.F. = low intensity fluorescent white light; H.I.F. = high intensity fluorescent white light; DCMU=3(2, 3, dichlorophenyl) 1, 1 allmethyl urea
Chenopodium rubrum seeds (ecotype 60 ~ 4T N, selection 374) were used in all the experiments. This ecotype normally requires at least one period of darkness of 8-9 h interrupting continuous light to induce any flowering. The seeds were sown on seven layers of Whatman filter papers, 4.25 cm in diameter, moistened with distilled water contained in
98 6-cm Petri dishes. The plants were subjected to alternating temperatures of 32.5 ~ C for 12 h and 10~ C for 12 h, for 41/2 days, Continuous cool white fluorescent light was imposed during the gerntination period and was of 600 ft. c. intensity at 32.5 ~ C and 450 ft. c. at 10~ C. After the germination period the temperature was kept constant at 20 ~ C and H o @ a n d ' s No. 1 nutrient solution, modified by providing 10 ppm iron as Sequestrene (sodium ferric ethylenediaminetetraacetate), was supplied to the plants. The seedlings were then irradiated with different light intensities and qualities for 24 h, and transferred to darkness for 12 h (standard inductive dark conditions). The plants were returned to incandescent white light (800850 ft. c.) after the inductive darkness and maintained there until assayed 10 days later. Each treatment had three replicate dishes, ten plants from each dish were sampled at random and the percentage of plants that showed visible floral primordia was recorded. Flowering was determined using a binocular dissecting microscope. The means of the results of replicate dishes were then calculated. The data was analyzed for standard error. Chemical treatments involved dissolving the chemicals in the Hoagland's nutrient solution, which was then washed on the filter paper so that the chemicals could be taken up by the roots, The p H of all the solutions was adjusted to 5.6-5,8. The range of effective concentrations of various chemicals used was determined in a series of preliminary experiments. Each final experiment was repeated at least twice to ensure the validity of the results.
Light Sources No. 1. Incandescent white light: General Electric 300W incandescent lamps (for 800-850 ft. c. irradiation). For 100 ft. c. intensity, distance from the above source was increased and also cheesecloth was used to reduce the intensity to the required level. General Electric 7.5W incandescent lamps were used for 35 ft. c. irradiation. No. 2. Fluorescent white light: Sylvania 20W, F20 TI2/CW, cool white fluorescent lamps (for 650 ft. c. irradiation). For 35 or 100 ft. c. the seedlings were maintained at a greater distance from the same light source as above and also some cheesecloth was interpolated between the light source and the seedlings, to get the required intensity. No. 3. Fluorescent blue light: Sylvania 20W, F20 T12-B Fluorescent (for 650 ft. c.). For 100 ft. c. half the number of lamps were used and also the plants were maintained farther from the light source. No. 4. Broad band blue light (lOft. c.) : This source was prepared by wrapping two layers of blue cinemoid (number 19) on 20W Sylvania cool white fluorescent lamps. Two such lamps were used to provide 10 ft. c. blue light intensity at the plant level. No. 5. Broad band blue light (35ft. c. and lOO fi. e.) : For 35 ft. c. intensity of blue light two layers of blue cinemoid filter were wrapped around fluorescent blue tubes described above (No. 3) and for 100 ft. c., one layer of blue cinemoid was used. No. 6. Broad band red and far red irradiation: Red light (20-ft. c,) was obtained by filtering light from red fluorescent lamps (General Electric F30 T12 R-RS) through 2 mm of window glass plus one layer of No. 14 ruby cinemoid filter (supplied by Standard Electric and Engineering Co., 755 Yonge St., Toronto, Ontario). Far red (2 ft. c.) was obtained by passing light from incandescent tubes (Westinghouse, Lumiline 60W, 115-125V) through 2 mm of infrared absorbing glass KG1, 2 m m + 3.15 m m far red plastic No. V58015.
R. Sawhney: Light Requirement for Flowering. I
No. 7. BCJ Light: Six General Electric 60W BCJ incandescent filament photographic ruby lamps were used to give 35 ft. c. intensity of BCJ light at plant level, For lower intensities of BCJ light, cheesecloth was interpolated between the light source and the seedlings. Foot-candle unit has been used as an approximate expression of the light intensity emitted by any particular light source. We realize that for any specific photoreaction the total energy emitted by the source (whether expressed as foot-candles or ~twatt/cm2) is irrelevant. Therefore, energy emitted in specific wavebands (blue, red, or far-red) of the spectrum (expressed in Figs. 1, 2, and 3 as ~watt/cm 2) has been used as the basis of all our interpretations. The light intensities (in foot candles) were measured with a Weston illumination meter, model 756. The spectral intensity distribution of all the light sources was recorded with an ISCO SR spectroradiometer. The respective spectral intensity distribution curves are presented in Figures 1, 2, and 3.
Experiments and Results
1. Photosynthetic Requirement during Pre-induction Light Period Our preliminary experiments showed that the flowering of C. rubrum was inhibited by irradiation with relatively low intensity incandescent (L.I.I. 35 ft. c . - Fig. 1) white light (24 h), in contrast to a high level of flowering with a high intensity incandescent (H.I.I.-850ft. c.-Fig. 3) white light (24h), given prior to inductive darkness (12 h) (Table I). To test if a high level of photosynthesis is directly involved in the positive flowering response in high intensity white light, we treated the seedlings with a photosynthetic inhibitor DCMU (3 (2, 3, dichlorophenyl) 1, 1 dimethyl urea) (Wessels and Van der Veen, 1956). DCMU is known to inhibit the formation of NADPHz and ATP, thereby inhibiting the production of energy substrates. DCMU was supplied to the seedlings at the beginning of the high intensity photoperiod and washed off with Hoagland's solution at the end, 24 h later. 3.0
.
.
.
.
5.1~
watts
a t '~50 n m
42~u watts
at 725 nrr
o
2~
/
U y ~ - - T ~ T - - a - - i - - r - -
400
/,50
500
i
i.
J
I
...
550 600 650 Wavetength (rim)
i
i
700
,
'T
750
Fig. 1. Spectral energy distribution cmves of low intensity incandescent white light (35 ft. c.) , - - A , low intensity fluorescent white light (35 ft. c.) m - - . , low intensity BCJ light (10 ft. c.) o - - o
R. Sawhney: Light Requirement for Flowering. I
99 Table 1. Flowering response of C, rubrum seedlings maintained in different light conditions for 24 h preceding a single inductive dark period (12 h) Light treatment
% Flowering Mean a
3
400
450
500
550 600 650 Wavelength (nm)
700
750
Fig. 2. Spectral energy distribution curves of broad band blue light 100 ft. c. (5.4 btwatt/cm2 at 437 rim) e - - e , 35 ft. c. (2.1 gwatt/cm ~ at 437 nm) o--o, 10 ft. c. (0.9 lawatt/cm2 at 437 nm) ~--c~, and red light, 20 ft. c. (1.17 pwatt/cm 2 at 660 rim) zx--zx 25
i
f
i
~
i
I
r
/ 2o
High intensity incandescent (850 ft. c.) High intensity+DCMU (10 -6 M) High intensity+DCMU (5x 10-7 M) Medium intensity incandescent (100 ft. c.) Low intensity incandescent (35 ft. c.) High intensity fluorescent (650 ft. c.) Medium intensity fluorescent (100 ft. c.) Low intensity fluorescent (35 ft. c.) High intensity blue fluorescent (650 ft. c.) Medium intensity blue fluorescent (100 ft. c.) Broad band blue (100 ft. c.) (5.4 gwatt/cm2 at 437nm) Broad band blue (35 ft. c.) (2.1 lawatt/cm2 at 437 nm) Broad band red (20 ft. c.) (1.17 lawatt/cm2 at 660 nm) Broad band far-red (2 ft. c.) (8.4 pwatt/cm 2 at 725 nm) BCJ (35 ft. c.) (12 gwatt/cm 2 at 725 nm)
86.6 (3.3) 96.6 (3.3) 90.0 (5.7) 3.3 (3.3) 0.0 (0.0) 86.6 (3.3) 76.66 (3.3) 70.0 (5.7) 100.0 (0.0) 86.6 (3.3) 90.0 (0.0) 50.0 (5.7) 0.0 (0.0) 0.0 (0.0) 6.6 (3.3)
,,,...,..// a Mean of three replicates. Figures in brackets indicate standard error
k
~
400
450
/
500
550 600 650 Wovetength (nm)
2. Phytochrome (PFR) Requirement during Pre-induction Light Period
700
750
Fig. 3. Spectral energy distribution cmves of high intensity incandescent white light (800 ft. c.) ~,--A, high intensity fluorescent white light (650 ft. c.) 9 9 high intensity fluorescent blue light (650 ft. c.) e - - o , BCJ light (35ft. c.) x - - x , and far-red 2ft. c. or 8,28 gwatt/cmz at 725nm o - - o
There was no inhibition o f flowering with D C M U ( 1 0 - 6 M and 5x 1 0 - T M ) (Table l). The concentrations o f D C M U used have been shown to inhibit photosynthesis completely in C. rubrum (Chia, A h Sing, 1971). It appears that inhibition o f p h o t o s y n t h e sis for 24 h prior to inductive darkness, does not interfere with the light or dark processes involved in the induction o f flowering in C. rubrum. Therefore, the lack o f flowering with a L.I.I. p h o t o p e r i o d can not be explained on the basis o f low photosynthesis.
A n u m b e r o f studies suggest that for the flowering o f short-day plants, a high level of PeR m a y be required during the light period that precedes inductive darkness (see Evans, 1971). Despite the fact that in our experiments, due to a similarity in the red/far ratios o f L.I.1. and H.I.I. lights (o.7 and 0.8 respectively), these would tend to maintain somewhat similar PeR levels in the seedlings, recent studies suggest that intensity of light can shift the level of PeR established at photoequilibrium (Borthwick et al., 1969; Kendrick, 1972). Thus, it is possible that different levels o f PFR were maintained in the seedlings irradiated with L.I.I. and H.I.I. white lights. We irradiated the seedlings with low intensity fluorescent ( L . I . F . 35 ft. c . - F i g . 1) and high intensity fluorescent ( H . I . F . - 6 5 0 ft. c . - F i g . 3) white lights prior to darkness. These lights should establish a relatively high level of PeR in the seedlings due to their high red/farred ratio (8.0). Exposure o f seedlings to L.I.F. light prior to inductive darkness sustained a high, t h o u g h not optimal, level of flowering and H.I.F. light led to an optimal level o f flowering (Table 1). However, when b r o a d b a n d red light ( r e d / f a r - r e d - 8 . 0 ) was provided during the p h o t o p e r i o d preceding inductive darkness,
100
there was no flower initiation. It suggests that high PeR (presumably established by broad band red light) during the photoperiod is not sufficient for photoperiod reactions of flowering in C. rubrum.
R. Sawhney: Light Requirement for Flowering. I Table 2. Flowering response of C. rubrum seedlings irradiated with low intensity white incandescent, white fluorescent and BCJ light (35 ft. c. intensity, respectively) with or without supplemental low intensity blue light (10 ft. c.) Light treatment
% Flowering Mean a
3. Blue HER Requirement during Pre-induction Light Period Fluorescent lamps emit a higher level of energy in the blue region of the spectrum as compared to incandescent lamps (Figs. 1 and 3). We investigated if blue wavelenghts have a role in the photoperiod reactions of flowering in C. rubrum. Different groups of seedlings were irradiated with different intensities of broad band blue light (Fig. 2) or blue fluorescent light (Fig. 3) for 24 h prior to inductive darkness (12 h). There was a high degree of flowering when optimum intensity of blue was used as the irradiation during the photoperiod (Table 1). Blue light seems to play a stimulatory role in the flowering of C. rubruin and a sufficiently high energy of blue, by itself, satisfies the light requirement preceding dark induction of flowering. Since the High Energy Reaction of photomorphogenesis (Mohr, 1964; Borthwick et al., 1969) has its action maxima in the blue and far-red it is possible that the flower promoting, intensity dependent blue light reaction, manifested in our experiments, may be the HER.
4. Far-red HER Requirement during Pre-induction Light Period We investigated if far-red HER participated in the photoperiod reactions involved in the flowering of C. rubrum. The quality and intensity of far-red used (Fig. 3) saturates the H E R for betacyanin synthesis in C. rubrum (Wagner and Cumming, 1970). There was no flowering in response to a single inductive dark period when far-red radiation was used prior to inductive darkness (Table 1). The lack of flowering when far-red serves as the photoperiod has been reported in several SD plants and has been interpreted to be due to establishment of suboptimal levels of PeR (red/far-red r a t i o - 0.0002). In another experiment we used BCJ light which has a high level of energy in the far-red (Fig. 3) and a higher red/far-red ratio (0.04) as compared to far-red (0.0002). Very low (6.6%) level of flowering occurred as a result of BCJ irradiation before inductive darkness. Thus, we could not obtain any evidence for the involvement of a far-red H E R in the flowering of C. rubruin.
Low intensity incandescent (35 ft. c.) Low intensity fluorescent (35 ft. c.) BCJ (35 ft. c.) Low intensity blue (10 ft. c.) Low intensity incandescent + blue Low intensity fluorescent + blue B CJ + blue
o (o.o) 70.0 6.6 6.6 3.3 93.3
(5.7) (3.3) (3.3) (3.3) (3.3)
30.0 (o.o)
Mean of three replicates. Figures in brackets indicate standard error
5. Low Intensity Blue Light, Reaction during the Pre-induction Light Period Examining the spectral energy distribution curves of all the light sources we used in our experiments (Figs. 1, 2, 3), we noted that all the light qualities which were ineffective as the photoperiod for flower induction (L.I.I., red, far-red, BCJ), did not have any energy in the blue region of the light spectrum. Also, L.I.F. light allowed high degree of flowering and it emits some energy in the blue (Fig. 1). Considering that Expt. 3 also indicated a flower promotory role of some blue mediated photoreaction system, we studied the effect of supplementing low energy blue light (0.9 gwatt/cm 2 at 437 n m - F i g . 2) to the light sources which were other-wise ineffective (L.I.I. and BCJ) or suboptimal (L.I.F.) for flowering. The addition of blue light stimulated the flowering in seedlings maintained in BCJ light and L.I.F. light (Table 2). There was no promotory effect of supplemental blue light on the flowering of seedlings irradiated with L.I.I. white light. It seems some factor other than energy in the blue may also be limiting flowering in L.I.I. light conditions. Various theoretical possibilities will be projected in the discussion.
Discussion
I. Photosynthetic Requirement for Flowering Our findings suggest that lack of flowering with L.I.L white light does not reflect a necessity for a high level of photosynthesis for flower induction in C. rubruin. Bavrina et al. (1969) also showed that D C M U application has no effect on the flowering of Xanthium
R. Sawhney: Light Requirement for Flowering. 1
strumarium, a SD plant. Furthermore, SD plants Perilla nankinensis and Panicum miliaceum were still able to form flowers in CO2 depleted atmosphere. It is known that SD plants require a definite period of illumination in order to respond to photoperiodic induction, at least in some SD plants this period can be reduced from 2-5 h to several minutes or even seconds every day (Harder and Grimmer, 1947; Lang, 1958). Certainly a minimum critical level of photosynthetic energy input is necessary for the mere survival of the plants but it seems photosynthesis is generally not the limiting factor for flowering in SD plants maintained in low intensity light conditions.
2. Phytochrome (PER) Requirement for Flowering A most recent and highly favoured hypothesis is that relatively high intensity light may establish lower levels of active PFR during the irradiation, as compared to relatively low intensity light of the same quality (Borthwick et al., 1969 and Kendrick and Spruit, 1972). This mechanism is postulated to prevent PFR photodestruction, since the rate of photodestruction of PFR has been shown to depend on PFR/PR ratio (Kendrick, 1972). If we base our interpretations on the above postulate, it can be suggested that there is a relatively lower level of PvR in C. rubrum seedlings maintained in H.I.I. white light as compared to those irradiated with L.I.I. white light. Therefore, the lack of flowering with L.I.I. photoperiod could not be due to suboptimal Pv~. On the other hand, it is possible that due to a high rate of photodestruction of PFR in the low intensity incandescent light, the level of active PFR may, ultimately, be reduced to suboptimal for photoperiod reactions or the following dark inductive reactions. A high PVR requirement during the early part of inductive darkness for optimum flowering of SD plants, has been suggested by numerous workers (see King, 1971). Thus a high level of destruction of PvR in L.I.I. and red photoperiods may prevent flowering of C. rubrum. However, the above explanation does not reconcile with our findings that L.I.F. which, due to its high red/far-red ratio, should maintain a high level of PeR during the light exposure, sustains a high level of flowering. Thus, light conditions (L.I.F.) that are theoretically favourable for PFR decay are still effective for flowering of C. rubrum. Thus, the differences in flowering response of C. rubrum to different intensities of incandescent white light can not be explained on the basis of PVR decay kinetics. The general conclusion that high PeR during the photoperiod is optimum for flowering of SD plants is based primarily on the finding that red photoperiod
101
leads to high induction of flowering in some SD plants, (see Evans, 1971). In C. rubrum, red (Expt. 2) or far red (Expt. 4) photoperiod was ineffective, but blue photoperiod leads to a high level of flowering (Expt. 3). Blue irradiation has been shown to establish only 30-40% PF• at photoequilibrium (Butler et al., 1964). Thus, 30-40% PFR may be considered optimum for flowering in C. rubrum. The differential flowering pattern in different SD plants, in response to varied light qualities may be due to differences in optimal PFR requirement as well as different PFR decay or synthesis kinetics or the existence of stable and unstable, or active and inactive pools of PFR, in varying proportions in different plants. Direct measurements of stable or unstable pools of PFR, PFR decay kinetics and synthesis rates in different plants, maintained in different light conditions, are desireable before any meaningful conclusions can be drawn regarding the PFR requirement for the flowering of SD plants.
3. High Energy Reaction (HER) Requirement for Flowering Our results show that blue light is effective as the photoperiod for flowering of C. rubrum. Reports that blue light may influence PFR levels in etiolated seedlings, due to its absorption by both forms of phytochrome (Butler et al., 1964) have led some workers to suggest that blue light action on flowering may be mediated through phytochrome (Hillman, 1965). However, in our experiments a general and consistent stimulation of flowering is observed when dim blue light (10 ft. c.) supplements some light sources which by themselves should establish low (BCJ light) or high (L.I.F.) PFR levels in plants (Expt. 5). In addition, blue light effect on flowering shows intensity dependence in our experiments (Expt. 3). Since phytochrome system is known to saturate at relatively low energy levels the above data argues against PFR as the pigment involved. The existence of a distinct pigment for blue light action on flowering has also been suggested earlier (Zeevaart and Marushige, 1967). We suggest that HER may be the photoreaction underlying the blue light action on flowering of C. rubrum. We suggest that lack of flowering in far-red or BCJ may be due to suboptimal PFR ( 1 - 2 1 % Evans et al., 1965) and the action of far-red HER may have been masked. Considering that H.I.I. bulbs emit a much greater energy in the blue and far-red as compared to L.I.I. bulbs, it seems possible that differences in HER may be the cause of the differences in flowering response. To our knowledge HER has never been implicated in the flowering of SD plants. It is interesting that L.I.F., even with almost no
102
energy in the far-red and relatively low level of energy in the blue (0.5 gwatt/cm 2 at 437 rim-Fig. 1) still sustains 70% flowering (Expt. 2). Supplementing L.I.I. with energy in the blue (0.9 p.watt/cm 2 at 437 n m - F i g . 2) still does not stimulate flowering. A comparison of the spectral distribution of L.I.I. + blue and L.I.F. (Fig. 1) reveals that the former light source emits a relatively higher level of energy in the red (600-700 nm) as compared to the latter. This raises the question whether red radiation could be actively inhibitory to flowering of C. rubrum. On the other hand, H.I.I. emits a high level of energy in the red and yet leads to optimum flowering (Expt. 1). We propose, that there may be an interaction between some promotory (HER) and inhibitory (red driven) photoreactions which affect the flowering response of C. rubrum. The above proposal adequately explains our results. Thus, H.I.I. and H.I.F. lights may be effective for flowering due to high blue or far-red HER. L.I.F. light may be effective due to a low level of red inhibition and some positive action of blue. Red, L.I.I., L.I.I.+blue light may be inhibitory to flowering due to a relatively high red and/or a relatively low blue. In light conditions with low red energy (far-red and BCJ) flowering may be limited by suboptimal, PFR- However, further work with different combinations of red, far-red and blue lights is required to affirm our proposal. In conclusion, we suggest that relatively low level of flowering in C. rubrum seedlings irradiated with relatively L.I.I. white light, prior to inductive darkness, may not be due to suboptimal photosynthesis or PFR but may be due to unfavourable balance between a blue/far-red promotory photoreaction and a flower inhibitory red reaction. This red reaction may or may not be mediated through PfR. The effect of red light distinct from PFR action has been suggested previously (Hoshizaki and Hamner, 1969; Andreae and Hopkins, 1973). The above study was supported by NRC grant to Dr. B.G, Cureruing of Western Ontario, London, Ontario. The author is grateful to him for support and helpful discussions. The author is indebted to Dr. V.K. Sawhney and Mrs. S. Andreae for very stimulating discussions and encouragement at all the stages of the above work.
References Andreae, S., Hopkins, W.G.: Interaction of abscieie acid and red light during induction of flowering of Chenopodium rubrum. Plant Physiol. 51, Suppl. 158, 29 (1973) Bavrina, T.V., Aksenova, N.P., Konstantinova, T.N. : The participation of photosynthesis in photoperiodism. Transtated from Fiziologiya Rastenii 16, 381-391 (1969) Borthwick, H.A., Hendricks, S.B., Schneider, M.J., Taylorsen, H.B., Toole, V.K.: The high energy light action controlling plant responses and development. Proc. nat. Acad. Sei. (Wash.) 64, 479-486 (1969)
R. Sawhney: Light Requirement for Flowering. I Butler, W.L., Hendricks, S.B., Siegelman, H.W.: Action spectra of phytochrome in vitro. Photochem. Photobiol. 3, 521-528 (t964) Carr, D_J.: On the nature of photoperiodic induction IV. Preliminary experiments on the effect of light following the inductive long dark period in Xanthium pennsylvanicum. Physiol. Plant. 10, 149-265 (1957) Chia Looi, Ah Sing: Ph.D. thesis, University of New Brunswick, Fredericton, N.B. Canada (1971) Cumming, B.G. : Circadian rhythmic flowering responses in Chenopodium rubrum: effects of glucose and sucrose. Canad. J. Bot. 45, 2173-2193 (1967) Evans, L.T. : Flower induction and the florigen concept. Ann. Rev. Plant Physiol. 22, 365-394 (1971) Evans, L.T., Hendricks, S.B., Borthwick, H.A. : The role of light in suppressing hypocotyl elongation in lettuce and petunia. Planta 64, 201-218 (1965) Hamner, K.C. : Interrelation of light and darkness in photoperiodic induction. Bot. Gaz. 101, 658-687 (1940) Harder, R., Grimmer, G. : Uber die untere kritische Tagesl/inge bei der Kurztagspflanze KalanchoO blossfeldiana. Planta 35, 8889 (1947) Hillman, W.S. : Red light, blue light and copper ion in the photoperiod control of flowering in Lemna perpusilla 6746. Plant and Cell Physiol. 6, 499-506 (1965) Hoshizaki, T., Hamner, K.C.: Interactions in plant photoperiodism. Photochem. Photobiol. 10, 87-96 (1969) Kendrick, R.E.: Aspects of phytochrome decay in etiolated seedlings under continuous illumination. Planta 102, 286-293 (1972) Kendrick, R.E., Spruit, C.J.P. : Light maintains high levels of phytochrome intermediates. Nature, New Biol. 237, 281-282 (1972) King, R.W.: Time measurement in the photoperiodic control of flowering. P h . D . Thesis. London-Canada: Univ. Western Ontario 1971 Lang, A.: Induction or reproductive growth in plants. Fourth international congress of biochemistry. VI, Biochem. of morphogenesis, 126 (1958) Liverman, J.L., Bonner, J. : Biochemistry of the photoperiodic response. The high intensity light reaction. Bot. Gaz. 115, 121-218 (1953) Mohr, H. : The control of plant growth and development by light. Biol. Rev. 39, 87-112 (1964) Parker, M.W., Borthwick, H.A. : Floral initiation in Biloxi Soybean as influenced by photosynthetic activity during the induction period. Bot. Gaz. 102, 256-268 (1940) Salisbury, F.B. : Time measurement and the light period in flowering. Planta 66, 1-26 (1965) Schwabe, W.W.: The effects of light intensity on the flowering of Kalancho~ blossfeMiana in relation to the critical day length. Physiol. Plant. 7, 745-752 (t954) Umemura, K., Oota, Y. : Effect of nucleic acid and protein antimetabolites on frond and flower production in Lemna gibba 43. Plant and Cell Physiol. 6, 73-85 (1965) Wagner, E., Cumming, B.G.: Betacyanin accumulation, chlorophyll content and flower intiation in Chenopodium rubrum as related to endogenous rhythmicity and phytochrome action. Canad. J. Bot. 48, 1-18 (1970) Wessels, J.S.C., Van der Veen, R. : The action of some derivatives of phenylurethane and of 3-phenyl-l,l-dimethyl urea on the photosynthetic reaction. Biochim. Biophys. Acta 19, 548-549 (1956) Zeevart, J.A.D., Marushige, K. : Biochemical approaches. In: Physiology of flowering in Pharbitis nil, 121-138, S. Imamura (Ed.), Jap, Soc. Plant Physiologists, Tokyo, Japan (1967)
Received 10. February; accepted 16. August 1976
Planta 133, 97-102 (1977)
9 by Springer-Verlag 1977
Nature of Light Requirement for the Flowering of Chenopodium rubrum L. (Ecotype 60 ~ 47' N) I. Pre-induction Light Period* Ramma Sawhney ** Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada
Abstract. Seedlings of the short-day plant, Chenopodium rubrum L. (Ecotype 60 ~ 47' N) were irradiated with different intensities and qualities of light for 24 h preceding a single inductive dark period (12 h). Our data shows that a relatively low intensity incandescent light (35-100 ft. c.) is not effective as the photoperiod for flowering. The above effect is not due to a requirement for a relatively high level of photosynthesis. Our results suggest a definite promotory role of a blue High Energy Reaction (HER). We could not demonstrate the involvement of a far-red HER. We suggest that ineffectiveness of far-red may have been due to establishment of rather low Phytochrome, PFR, levels, suboptimal for flowering. A certain critical level of PFR (30--40%, that presumably established by blue light) seems to be necessary for photoreactions involved in flowering of C. rubrum. There are indications in our experiments of the operation of a red radiation mediated flower inhibitory photoreaction. Key words: Chenopodium rubrum - Flowering High Energy Reaction - Photosynthesis - Phytochrome.
Introduction The flowering of short-day plants is known to require a high intensity white light photoperiod prior to in* This paper constitutes a part of a Ph.D. thesis submitted to the University of Western Ontario, London, Ontario ** Present address. Department of Biology, University of Saskatchewan, Saskatoon STN OWO, Canada
ductive darkness. It has generally been postulated that the high intensity light is necessary to sustain optimum photosynthesis for flower induction (Hamner, 1940; Parker and Borthwick, 1940; Liverman and Bonner, 1953; Carr, 1957; Cumming, 1967). However, a review of the literature reveals numerous findings which are in conflict with the above hypothesis. For instance, 1) the flower inhibitory effect of low intensity white light could be overcome in Kalancho~ blossfeldiana (Schwabe, 1954) and in Xanthium strumarium (Salisbury, 1965) by prolonging the following period of darkness-a response difficult to relate to photosynthesis, 2) Biloxi soybean plants maintained in COz depleted atmosphere were inhibited from flowering but their flowering response was unaffected when photosynthesis was significantly reduced by removing all but one leaf (Parker and Borthwick, 1940), 3) while sugar application overcame the effect of low intensity light on the flowering of Xanthium strumarium (Liverman and Bonner, 1953; Cart, 1957), some non-specific reducing agents also had a similar flower stimulatory effect (Lang, 1958), 4) sugar application was not effective in promoting flowering of Lemna perpusilla exposed to low intensity light conditions (Umemura and Oota, 1965). In view of the uncertainty regarding the exact nature of the light requirement for flowering of SD plants, the present study was undertaken to investigate the contribution of 1) photosynthesis, 2) phytochrome (PvR), and 3) High Energy Reaction (HER) of photomorphogenesis, in the flowering of Chenopodium rubrum L. (ecotype 60 ~ 47' N, selection 374).
Materials and Methods
Abbreviations." SD=short day plant; H E R = H i g h Energy Reac-
tion; PvR=far-red absorbing form of phytochrome; PR=red absorbing form of phytochrome; L.I.I. =low intensity incandescent white light; H.I.I.=high intensity incandescent white light; L.I.F. = low intensity fluorescent white light; H.I.F. = high intensity fluorescent white light; DCMU=3(2, 3, dichlorophenyl) 1, 1 allmethyl urea
Chenopodium rubrum seeds (ecotype 60 ~ 4T N, selection 374) were used in all the experiments. This ecotype normally requires at least one period of darkness of 8-9 h interrupting continuous light to induce any flowering. The seeds were sown on seven layers of Whatman filter papers, 4.25 cm in diameter, moistened with distilled water contained in
98 6-cm Petri dishes. The plants were subjected to alternating temperatures of 32.5 ~ C for 12 h and 10~ C for 12 h, for 41/2 days, Continuous cool white fluorescent light was imposed during the gerntination period and was of 600 ft. c. intensity at 32.5 ~ C and 450 ft. c. at 10~ C. After the germination period the temperature was kept constant at 20 ~ C and H o @ a n d ' s No. 1 nutrient solution, modified by providing 10 ppm iron as Sequestrene (sodium ferric ethylenediaminetetraacetate), was supplied to the plants. The seedlings were then irradiated with different light intensities and qualities for 24 h, and transferred to darkness for 12 h (standard inductive dark conditions). The plants were returned to incandescent white light (800850 ft. c.) after the inductive darkness and maintained there until assayed 10 days later. Each treatment had three replicate dishes, ten plants from each dish were sampled at random and the percentage of plants that showed visible floral primordia was recorded. Flowering was determined using a binocular dissecting microscope. The means of the results of replicate dishes were then calculated. The data was analyzed for standard error. Chemical treatments involved dissolving the chemicals in the Hoagland's nutrient solution, which was then washed on the filter paper so that the chemicals could be taken up by the roots, The p H of all the solutions was adjusted to 5.6-5,8. The range of effective concentrations of various chemicals used was determined in a series of preliminary experiments. Each final experiment was repeated at least twice to ensure the validity of the results.
Light Sources No. 1. Incandescent white light: General Electric 300W incandescent lamps (for 800-850 ft. c. irradiation). For 100 ft. c. intensity, distance from the above source was increased and also cheesecloth was used to reduce the intensity to the required level. General Electric 7.5W incandescent lamps were used for 35 ft. c. irradiation. No. 2. Fluorescent white light: Sylvania 20W, F20 TI2/CW, cool white fluorescent lamps (for 650 ft. c. irradiation). For 35 or 100 ft. c. the seedlings were maintained at a greater distance from the same light source as above and also some cheesecloth was interpolated between the light source and the seedlings, to get the required intensity. No. 3. Fluorescent blue light: Sylvania 20W, F20 T12-B Fluorescent (for 650 ft. c.). For 100 ft. c. half the number of lamps were used and also the plants were maintained farther from the light source. No. 4. Broad band blue light (lOft. c.) : This source was prepared by wrapping two layers of blue cinemoid (number 19) on 20W Sylvania cool white fluorescent lamps. Two such lamps were used to provide 10 ft. c. blue light intensity at the plant level. No. 5. Broad band blue light (35ft. c. and lOO fi. e.) : For 35 ft. c. intensity of blue light two layers of blue cinemoid filter were wrapped around fluorescent blue tubes described above (No. 3) and for 100 ft. c., one layer of blue cinemoid was used. No. 6. Broad band red and far red irradiation: Red light (20-ft. c,) was obtained by filtering light from red fluorescent lamps (General Electric F30 T12 R-RS) through 2 mm of window glass plus one layer of No. 14 ruby cinemoid filter (supplied by Standard Electric and Engineering Co., 755 Yonge St., Toronto, Ontario). Far red (2 ft. c.) was obtained by passing light from incandescent tubes (Westinghouse, Lumiline 60W, 115-125V) through 2 mm of infrared absorbing glass KG1, 2 m m + 3.15 m m far red plastic No. V58015.
R. Sawhney: Light Requirement for Flowering. I
No. 7. BCJ Light: Six General Electric 60W BCJ incandescent filament photographic ruby lamps were used to give 35 ft. c. intensity of BCJ light at plant level, For lower intensities of BCJ light, cheesecloth was interpolated between the light source and the seedlings. Foot-candle unit has been used as an approximate expression of the light intensity emitted by any particular light source. We realize that for any specific photoreaction the total energy emitted by the source (whether expressed as foot-candles or ~twatt/cm2) is irrelevant. Therefore, energy emitted in specific wavebands (blue, red, or far-red) of the spectrum (expressed in Figs. 1, 2, and 3 as ~watt/cm 2) has been used as the basis of all our interpretations. The light intensities (in foot candles) were measured with a Weston illumination meter, model 756. The spectral intensity distribution of all the light sources was recorded with an ISCO SR spectroradiometer. The respective spectral intensity distribution curves are presented in Figures 1, 2, and 3.
Experiments and Results
1. Photosynthetic Requirement during Pre-induction Light Period Our preliminary experiments showed that the flowering of C. rubrum was inhibited by irradiation with relatively low intensity incandescent (L.I.I. 35 ft. c . - Fig. 1) white light (24 h), in contrast to a high level of flowering with a high intensity incandescent (H.I.I.-850ft. c.-Fig. 3) white light (24h), given prior to inductive darkness (12 h) (Table I). To test if a high level of photosynthesis is directly involved in the positive flowering response in high intensity white light, we treated the seedlings with a photosynthetic inhibitor DCMU (3 (2, 3, dichlorophenyl) 1, 1 dimethyl urea) (Wessels and Van der Veen, 1956). DCMU is known to inhibit the formation of NADPHz and ATP, thereby inhibiting the production of energy substrates. DCMU was supplied to the seedlings at the beginning of the high intensity photoperiod and washed off with Hoagland's solution at the end, 24 h later. 3.0
.
.
.
.
5.1~
watts
a t '~50 n m
42~u watts
at 725 nrr
o
2~
/
U y ~ - - T ~ T - - a - - i - - r - -
400
/,50
500
i
i.
J
I
...
550 600 650 Wavetength (rim)
i
i
700
,
'T
750
Fig. 1. Spectral energy distribution cmves of low intensity incandescent white light (35 ft. c.) , - - A , low intensity fluorescent white light (35 ft. c.) m - - . , low intensity BCJ light (10 ft. c.) o - - o
R. Sawhney: Light Requirement for Flowering. I
99 Table 1. Flowering response of C, rubrum seedlings maintained in different light conditions for 24 h preceding a single inductive dark period (12 h) Light treatment
% Flowering Mean a
3
400
450
500
550 600 650 Wavelength (nm)
700
750
Fig. 2. Spectral energy distribution curves of broad band blue light 100 ft. c. (5.4 btwatt/cm2 at 437 rim) e - - e , 35 ft. c. (2.1 gwatt/cm ~ at 437 nm) o--o, 10 ft. c. (0.9 lawatt/cm2 at 437 nm) ~--c~, and red light, 20 ft. c. (1.17 pwatt/cm 2 at 660 rim) zx--zx 25
i
f
i
~
i
I
r
/ 2o
High intensity incandescent (850 ft. c.) High intensity+DCMU (10 -6 M) High intensity+DCMU (5x 10-7 M) Medium intensity incandescent (100 ft. c.) Low intensity incandescent (35 ft. c.) High intensity fluorescent (650 ft. c.) Medium intensity fluorescent (100 ft. c.) Low intensity fluorescent (35 ft. c.) High intensity blue fluorescent (650 ft. c.) Medium intensity blue fluorescent (100 ft. c.) Broad band blue (100 ft. c.) (5.4 gwatt/cm2 at 437nm) Broad band blue (35 ft. c.) (2.1 lawatt/cm2 at 437 nm) Broad band red (20 ft. c.) (1.17 lawatt/cm2 at 660 nm) Broad band far-red (2 ft. c.) (8.4 pwatt/cm 2 at 725 nm) BCJ (35 ft. c.) (12 gwatt/cm 2 at 725 nm)
86.6 (3.3) 96.6 (3.3) 90.0 (5.7) 3.3 (3.3) 0.0 (0.0) 86.6 (3.3) 76.66 (3.3) 70.0 (5.7) 100.0 (0.0) 86.6 (3.3) 90.0 (0.0) 50.0 (5.7) 0.0 (0.0) 0.0 (0.0) 6.6 (3.3)
,,,...,..// a Mean of three replicates. Figures in brackets indicate standard error
k
~
400
450
/
500
550 600 650 Wovetength (nm)
2. Phytochrome (PFR) Requirement during Pre-induction Light Period
700
750
Fig. 3. Spectral energy distribution cmves of high intensity incandescent white light (800 ft. c.) ~,--A, high intensity fluorescent white light (650 ft. c.) 9 9 high intensity fluorescent blue light (650 ft. c.) e - - o , BCJ light (35ft. c.) x - - x , and far-red 2ft. c. or 8,28 gwatt/cmz at 725nm o - - o
There was no inhibition o f flowering with D C M U ( 1 0 - 6 M and 5x 1 0 - T M ) (Table l). The concentrations o f D C M U used have been shown to inhibit photosynthesis completely in C. rubrum (Chia, A h Sing, 1971). It appears that inhibition o f p h o t o s y n t h e sis for 24 h prior to inductive darkness, does not interfere with the light or dark processes involved in the induction o f flowering in C. rubrum. Therefore, the lack o f flowering with a L.I.I. p h o t o p e r i o d can not be explained on the basis o f low photosynthesis.
A n u m b e r o f studies suggest that for the flowering o f short-day plants, a high level of PeR m a y be required during the light period that precedes inductive darkness (see Evans, 1971). Despite the fact that in our experiments, due to a similarity in the red/far ratios o f L.I.1. and H.I.I. lights (o.7 and 0.8 respectively), these would tend to maintain somewhat similar PeR levels in the seedlings, recent studies suggest that intensity of light can shift the level of PeR established at photoequilibrium (Borthwick et al., 1969; Kendrick, 1972). Thus, it is possible that different levels o f PFR were maintained in the seedlings irradiated with L.I.I. and H.I.I. white lights. We irradiated the seedlings with low intensity fluorescent ( L . I . F . 35 ft. c . - F i g . 1) and high intensity fluorescent ( H . I . F . - 6 5 0 ft. c . - F i g . 3) white lights prior to darkness. These lights should establish a relatively high level of PeR in the seedlings due to their high red/farred ratio (8.0). Exposure o f seedlings to L.I.F. light prior to inductive darkness sustained a high, t h o u g h not optimal, level of flowering and H.I.F. light led to an optimal level o f flowering (Table 1). However, when b r o a d b a n d red light ( r e d / f a r - r e d - 8 . 0 ) was provided during the p h o t o p e r i o d preceding inductive darkness,
100
there was no flower initiation. It suggests that high PeR (presumably established by broad band red light) during the photoperiod is not sufficient for photoperiod reactions of flowering in C. rubrum.
R. Sawhney: Light Requirement for Flowering. I Table 2. Flowering response of C. rubrum seedlings irradiated with low intensity white incandescent, white fluorescent and BCJ light (35 ft. c. intensity, respectively) with or without supplemental low intensity blue light (10 ft. c.) Light treatment
% Flowering Mean a
3. Blue HER Requirement during Pre-induction Light Period Fluorescent lamps emit a higher level of energy in the blue region of the spectrum as compared to incandescent lamps (Figs. 1 and 3). We investigated if blue wavelenghts have a role in the photoperiod reactions of flowering in C. rubrum. Different groups of seedlings were irradiated with different intensities of broad band blue light (Fig. 2) or blue fluorescent light (Fig. 3) for 24 h prior to inductive darkness (12 h). There was a high degree of flowering when optimum intensity of blue was used as the irradiation during the photoperiod (Table 1). Blue light seems to play a stimulatory role in the flowering of C. rubruin and a sufficiently high energy of blue, by itself, satisfies the light requirement preceding dark induction of flowering. Since the High Energy Reaction of photomorphogenesis (Mohr, 1964; Borthwick et al., 1969) has its action maxima in the blue and far-red it is possible that the flower promoting, intensity dependent blue light reaction, manifested in our experiments, may be the HER.
4. Far-red HER Requirement during Pre-induction Light Period We investigated if far-red HER participated in the photoperiod reactions involved in the flowering of C. rubrum. The quality and intensity of far-red used (Fig. 3) saturates the H E R for betacyanin synthesis in C. rubrum (Wagner and Cumming, 1970). There was no flowering in response to a single inductive dark period when far-red radiation was used prior to inductive darkness (Table 1). The lack of flowering when far-red serves as the photoperiod has been reported in several SD plants and has been interpreted to be due to establishment of suboptimal levels of PeR (red/far-red r a t i o - 0.0002). In another experiment we used BCJ light which has a high level of energy in the far-red (Fig. 3) and a higher red/far-red ratio (0.04) as compared to far-red (0.0002). Very low (6.6%) level of flowering occurred as a result of BCJ irradiation before inductive darkness. Thus, we could not obtain any evidence for the involvement of a far-red H E R in the flowering of C. rubruin.
Low intensity incandescent (35 ft. c.) Low intensity fluorescent (35 ft. c.) BCJ (35 ft. c.) Low intensity blue (10 ft. c.) Low intensity incandescent + blue Low intensity fluorescent + blue B CJ + blue
o (o.o) 70.0 6.6 6.6 3.3 93.3
(5.7) (3.3) (3.3) (3.3) (3.3)
30.0 (o.o)
Mean of three replicates. Figures in brackets indicate standard error
5. Low Intensity Blue Light, Reaction during the Pre-induction Light Period Examining the spectral energy distribution curves of all the light sources we used in our experiments (Figs. 1, 2, 3), we noted that all the light qualities which were ineffective as the photoperiod for flower induction (L.I.I., red, far-red, BCJ), did not have any energy in the blue region of the light spectrum. Also, L.I.F. light allowed high degree of flowering and it emits some energy in the blue (Fig. 1). Considering that Expt. 3 also indicated a flower promotory role of some blue mediated photoreaction system, we studied the effect of supplementing low energy blue light (0.9 gwatt/cm 2 at 437 n m - F i g . 2) to the light sources which were other-wise ineffective (L.I.I. and BCJ) or suboptimal (L.I.F.) for flowering. The addition of blue light stimulated the flowering in seedlings maintained in BCJ light and L.I.F. light (Table 2). There was no promotory effect of supplemental blue light on the flowering of seedlings irradiated with L.I.I. white light. It seems some factor other than energy in the blue may also be limiting flowering in L.I.I. light conditions. Various theoretical possibilities will be projected in the discussion.
Discussion
I. Photosynthetic Requirement for Flowering Our findings suggest that lack of flowering with L.I.L white light does not reflect a necessity for a high level of photosynthesis for flower induction in C. rubruin. Bavrina et al. (1969) also showed that D C M U application has no effect on the flowering of Xanthium
R. Sawhney: Light Requirement for Flowering. 1
strumarium, a SD plant. Furthermore, SD plants Perilla nankinensis and Panicum miliaceum were still able to form flowers in CO2 depleted atmosphere. It is known that SD plants require a definite period of illumination in order to respond to photoperiodic induction, at least in some SD plants this period can be reduced from 2-5 h to several minutes or even seconds every day (Harder and Grimmer, 1947; Lang, 1958). Certainly a minimum critical level of photosynthetic energy input is necessary for the mere survival of the plants but it seems photosynthesis is generally not the limiting factor for flowering in SD plants maintained in low intensity light conditions.
2. Phytochrome (PER) Requirement for Flowering A most recent and highly favoured hypothesis is that relatively high intensity light may establish lower levels of active PFR during the irradiation, as compared to relatively low intensity light of the same quality (Borthwick et al., 1969 and Kendrick and Spruit, 1972). This mechanism is postulated to prevent PFR photodestruction, since the rate of photodestruction of PFR has been shown to depend on PFR/PR ratio (Kendrick, 1972). If we base our interpretations on the above postulate, it can be suggested that there is a relatively lower level of PvR in C. rubrum seedlings maintained in H.I.I. white light as compared to those irradiated with L.I.I. white light. Therefore, the lack of flowering with L.I.I. photoperiod could not be due to suboptimal Pv~. On the other hand, it is possible that due to a high rate of photodestruction of PFR in the low intensity incandescent light, the level of active PFR may, ultimately, be reduced to suboptimal for photoperiod reactions or the following dark inductive reactions. A high PVR requirement during the early part of inductive darkness for optimum flowering of SD plants, has been suggested by numerous workers (see King, 1971). Thus a high level of destruction of PvR in L.I.I. and red photoperiods may prevent flowering of C. rubrum. However, the above explanation does not reconcile with our findings that L.I.F. which, due to its high red/far-red ratio, should maintain a high level of PeR during the light exposure, sustains a high level of flowering. Thus, light conditions (L.I.F.) that are theoretically favourable for PFR decay are still effective for flowering of C. rubrum. Thus, the differences in flowering response of C. rubrum to different intensities of incandescent white light can not be explained on the basis of PVR decay kinetics. The general conclusion that high PeR during the photoperiod is optimum for flowering of SD plants is based primarily on the finding that red photoperiod
101
leads to high induction of flowering in some SD plants, (see Evans, 1971). In C. rubrum, red (Expt. 2) or far red (Expt. 4) photoperiod was ineffective, but blue photoperiod leads to a high level of flowering (Expt. 3). Blue irradiation has been shown to establish only 30-40% PF• at photoequilibrium (Butler et al., 1964). Thus, 30-40% PFR may be considered optimum for flowering in C. rubrum. The differential flowering pattern in different SD plants, in response to varied light qualities may be due to differences in optimal PFR requirement as well as different PFR decay or synthesis kinetics or the existence of stable and unstable, or active and inactive pools of PFR, in varying proportions in different plants. Direct measurements of stable or unstable pools of PFR, PFR decay kinetics and synthesis rates in different plants, maintained in different light conditions, are desireable before any meaningful conclusions can be drawn regarding the PFR requirement for the flowering of SD plants.
3. High Energy Reaction (HER) Requirement for Flowering Our results show that blue light is effective as the photoperiod for flowering of C. rubrum. Reports that blue light may influence PFR levels in etiolated seedlings, due to its absorption by both forms of phytochrome (Butler et al., 1964) have led some workers to suggest that blue light action on flowering may be mediated through phytochrome (Hillman, 1965). However, in our experiments a general and consistent stimulation of flowering is observed when dim blue light (10 ft. c.) supplements some light sources which by themselves should establish low (BCJ light) or high (L.I.F.) PFR levels in plants (Expt. 5). In addition, blue light effect on flowering shows intensity dependence in our experiments (Expt. 3). Since phytochrome system is known to saturate at relatively low energy levels the above data argues against PFR as the pigment involved. The existence of a distinct pigment for blue light action on flowering has also been suggested earlier (Zeevaart and Marushige, 1967). We suggest that HER may be the photoreaction underlying the blue light action on flowering of C. rubrum. We suggest that lack of flowering in far-red or BCJ may be due to suboptimal PFR ( 1 - 2 1 % Evans et al., 1965) and the action of far-red HER may have been masked. Considering that H.I.I. bulbs emit a much greater energy in the blue and far-red as compared to L.I.I. bulbs, it seems possible that differences in HER may be the cause of the differences in flowering response. To our knowledge HER has never been implicated in the flowering of SD plants. It is interesting that L.I.F., even with almost no
102
energy in the far-red and relatively low level of energy in the blue (0.5 gwatt/cm 2 at 437 rim-Fig. 1) still sustains 70% flowering (Expt. 2). Supplementing L.I.I. with energy in the blue (0.9 p.watt/cm 2 at 437 n m - F i g . 2) still does not stimulate flowering. A comparison of the spectral distribution of L.I.I. + blue and L.I.F. (Fig. 1) reveals that the former light source emits a relatively higher level of energy in the red (600-700 nm) as compared to the latter. This raises the question whether red radiation could be actively inhibitory to flowering of C. rubrum. On the other hand, H.I.I. emits a high level of energy in the red and yet leads to optimum flowering (Expt. 1). We propose, that there may be an interaction between some promotory (HER) and inhibitory (red driven) photoreactions which affect the flowering response of C. rubrum. The above proposal adequately explains our results. Thus, H.I.I. and H.I.F. lights may be effective for flowering due to high blue or far-red HER. L.I.F. light may be effective due to a low level of red inhibition and some positive action of blue. Red, L.I.I., L.I.I.+blue light may be inhibitory to flowering due to a relatively high red and/or a relatively low blue. In light conditions with low red energy (far-red and BCJ) flowering may be limited by suboptimal, PFR- However, further work with different combinations of red, far-red and blue lights is required to affirm our proposal. In conclusion, we suggest that relatively low level of flowering in C. rubrum seedlings irradiated with relatively L.I.I. white light, prior to inductive darkness, may not be due to suboptimal photosynthesis or PFR but may be due to unfavourable balance between a blue/far-red promotory photoreaction and a flower inhibitory red reaction. This red reaction may or may not be mediated through PfR. The effect of red light distinct from PFR action has been suggested previously (Hoshizaki and Hamner, 1969; Andreae and Hopkins, 1973). The above study was supported by NRC grant to Dr. B.G, Cureruing of Western Ontario, London, Ontario. The author is grateful to him for support and helpful discussions. The author is indebted to Dr. V.K. Sawhney and Mrs. S. Andreae for very stimulating discussions and encouragement at all the stages of the above work.
References Andreae, S., Hopkins, W.G.: Interaction of abscieie acid and red light during induction of flowering of Chenopodium rubrum. Plant Physiol. 51, Suppl. 158, 29 (1973) Bavrina, T.V., Aksenova, N.P., Konstantinova, T.N. : The participation of photosynthesis in photoperiodism. Transtated from Fiziologiya Rastenii 16, 381-391 (1969) Borthwick, H.A., Hendricks, S.B., Schneider, M.J., Taylorsen, H.B., Toole, V.K.: The high energy light action controlling plant responses and development. Proc. nat. Acad. Sei. (Wash.) 64, 479-486 (1969)
R. Sawhney: Light Requirement for Flowering. I Butler, W.L., Hendricks, S.B., Siegelman, H.W.: Action spectra of phytochrome in vitro. Photochem. Photobiol. 3, 521-528 (t964) Carr, D_J.: On the nature of photoperiodic induction IV. Preliminary experiments on the effect of light following the inductive long dark period in Xanthium pennsylvanicum. Physiol. Plant. 10, 149-265 (1957) Chia Looi, Ah Sing: Ph.D. thesis, University of New Brunswick, Fredericton, N.B. Canada (1971) Cumming, B.G. : Circadian rhythmic flowering responses in Chenopodium rubrum: effects of glucose and sucrose. Canad. J. Bot. 45, 2173-2193 (1967) Evans, L.T. : Flower induction and the florigen concept. Ann. Rev. Plant Physiol. 22, 365-394 (1971) Evans, L.T., Hendricks, S.B., Borthwick, H.A. : The role of light in suppressing hypocotyl elongation in lettuce and petunia. Planta 64, 201-218 (1965) Hamner, K.C. : Interrelation of light and darkness in photoperiodic induction. Bot. Gaz. 101, 658-687 (1940) Harder, R., Grimmer, G. : Uber die untere kritische Tagesl/inge bei der Kurztagspflanze KalanchoO blossfeldiana. Planta 35, 8889 (1947) Hillman, W.S. : Red light, blue light and copper ion in the photoperiod control of flowering in Lemna perpusilla 6746. Plant and Cell Physiol. 6, 499-506 (1965) Hoshizaki, T., Hamner, K.C.: Interactions in plant photoperiodism. Photochem. Photobiol. 10, 87-96 (1969) Kendrick, R.E.: Aspects of phytochrome decay in etiolated seedlings under continuous illumination. Planta 102, 286-293 (1972) Kendrick, R.E., Spruit, C.J.P. : Light maintains high levels of phytochrome intermediates. Nature, New Biol. 237, 281-282 (1972) King, R.W.: Time measurement in the photoperiodic control of flowering. P h . D . Thesis. London-Canada: Univ. Western Ontario 1971 Lang, A.: Induction or reproductive growth in plants. Fourth international congress of biochemistry. VI, Biochem. of morphogenesis, 126 (1958) Liverman, J.L., Bonner, J. : Biochemistry of the photoperiodic response. The high intensity light reaction. Bot. Gaz. 115, 121-218 (1953) Mohr, H. : The control of plant growth and development by light. Biol. Rev. 39, 87-112 (1964) Parker, M.W., Borthwick, H.A. : Floral initiation in Biloxi Soybean as influenced by photosynthetic activity during the induction period. Bot. Gaz. 102, 256-268 (1940) Salisbury, F.B. : Time measurement and the light period in flowering. Planta 66, 1-26 (1965) Schwabe, W.W.: The effects of light intensity on the flowering of Kalancho~ blossfeMiana in relation to the critical day length. Physiol. Plant. 7, 745-752 (t954) Umemura, K., Oota, Y. : Effect of nucleic acid and protein antimetabolites on frond and flower production in Lemna gibba 43. Plant and Cell Physiol. 6, 73-85 (1965) Wagner, E., Cumming, B.G.: Betacyanin accumulation, chlorophyll content and flower intiation in Chenopodium rubrum as related to endogenous rhythmicity and phytochrome action. Canad. J. Bot. 48, 1-18 (1970) Wessels, J.S.C., Van der Veen, R. : The action of some derivatives of phenylurethane and of 3-phenyl-l,l-dimethyl urea on the photosynthetic reaction. Biochim. Biophys. Acta 19, 548-549 (1956) Zeevart, J.A.D., Marushige, K. : Biochemical approaches. In: Physiology of flowering in Pharbitis nil, 121-138, S. Imamura (Ed.), Jap, Soc. Plant Physiologists, Tokyo, Japan (1967)
Received 10. February; accepted 16. August 1976
