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Effects of carbon dioxide and pH on some phytochrome-mediated responses in plants Bassi, Pawan Kumar 1976

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EFFECTS OF CARBON DIOXIDE AND pH ON SOME PHYTOCHROME-MEDIATED RESPONSES IN PLANTS by PAWAN KUMAR BASS I B.Sc. (Hons. School), Panjab University, Chandigarh, India, 1971 M.Sc. (Hons. School), Panjab University, Chandigarh, India, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DEPARTMENT OF BOTANY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1976 C ^ ) Pawan Kumar Eassi, 1976 DOCTOR OF PHILOSOPHY in In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f< an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e tha the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia,/ 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date I n mmO'Xij oh my l a t e . Yno^eAboh. E.B. Txz.qu.nna., who P'\o\>i.de.d guidance, and height -in mofiz than just 4cx.ence, AMV to my )dan.zntd, tohoiz lovz and i,acAA.{jtcz made, - c t a l t poi'h-LbZ.2., tktb theAi& th dddtnatoA. ABSTRACT This investigation was initiated to study the effect of CC^ on phytochrome-mediated morphogenesis in flowering and seed germination. Removal of CfX, by flushing the plant environment with Cu\,-free a i r inhibited the red light interruption effects on flowering in Xanthium pennsylvanicum and on seed germination in Lactuca sativa cv Grand Rapids. Further experiments were done to investigate the involvement of CO^ exchange 14 in the effects of night interruptions on flowering in Xanthium. CC^ feeding t r i a l s showed that red light given for 5 minutes caused a net increase in ^C activity in the ethanol soluble fraction when ^CO^ was fed during the light treatment. There was no effect of red light on the extent of ^CG^ fixation in the dark period immediately following red l i g h t . The types of free amino acids recovered after paper chromatography were essentially the same after ^CC^ feedings in darkness, red l i g h t , and far red light following red light. However, there was a considerable increase in 1 4C activity in most of the amino acids in leaves given red light interruption, and the amount further increased when far red lig h t was 14 given following the red light. The extent of C label in tyrosine, valine and leucine was essentially the same in a l l the three treatments. In CO^-exchange experiments using the IRGA, brief red or far red light treatments were applied to Xanthium plants under inductive dark periods and the subsequent flowering response was assessed according to bud morphology. The occurrence of flowering depended on the timing, wavelength and intensity of the light treatments, and on the CCL i i concentration during the light treatments. CC^ exchange was measured during the night interruptions in single attached leaves. CC^ exchange was influenced by the conditions during the night interruptions, but there was no apparent correlation between the pattern of CC^ exchange observed and the subsequent flowering response. It appears that the action of during night interruptions is not associated with the exchange of during the night interruption. In an attempt to investigate other possible roles of CC^, experiments were done with light sensitive lettuce and Amaranthus retroflexus L. seeds. These experiments pertained to changes in pH of the incubation medium and CC^ concentration simultaneously. Germination was strongly promoted at pH 4.0 but the promotion diminished with increases in pH and did not occur at pH 7.5. The response of germination to red i r r a d i -ation was suppressed by CO2 removal and enhanced by C 0 2 enrichment in ai r or atmospheres. There was a close similarity between the pH effects on percentage germination and pH dependence of the CX^/HCOg" equilibrium. Transfer experiments, in which lettuce seeds were exchanged between buffers of pH 4.0 and pH 3.0, showed that the red/far red photo-transformation of phytochrome v/as independent of pH. Low pH, however, was required for onset of germination following red irradiation. There-after, pH between 4.0 and 8.0 did not limit the progress of germination. It i s postulated that following red irradiation, a product develops which is distinguishable from the P^r form of phytochrome. The product is stable at pH 8.0 and a t pH 4.0 i t acts to promote germination. i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS x INTRODUCTION 1 1. Phytochrome-mediated photomorphogenesis 1 2. Phytochrome and flowering in short day plants 4 (i) Promotive effect of P f r preceding inductive dark period: the high P^r reaction 4 ( i i ) The "night break" phenomenon in short day plants: low P f r reaction 5 3. Phytochrome and seed germination 7 (i) Light effects on seed germination 8 ( i i ) Interactions between light and other factors affecting seed germination 10 (a) Interaction between light and temperature 11 (b) Interaction between light and growth regulators 12 4. Mechanism of phytochrome (P^ r) action 13 (i) Pf r as an enzyme 13 ( i i ) Pf^s a regulator of gene activation ,14 ( i i i ) P f r and changes in membrane properties 15 5. High-energy reactions 16 6. Objectives 18 MATERIALS AND METHODS 21 1. Studies on the photoperiodic control of flowering in Xanthium  pennsylvanicum L. 21 (i) Night interruption experiments 21 14 ( n ) CO2 feeding experiments 23 ( i i i ) CO2 exchange experiments 25 2. Studies on phytochrome-mediated seed germination 28 i v Page RESULTS AND DISCUSSION 1. Studies on the photoperiodic control of flowering in Xanthium  pennsylvanicum L. (i) Night interruption experiments 38 14 ( n ) C 0 2 feeding experiments ( i i i ) COg exchange experiments 2. Studies on phytochrome-mediated seed germination (i) Regulation by CO2 and pH of phytochrome-mediated seed germination in Lactuca sativa and Amaranthus retroflexus ( i i ) pH and CO2 transfer experiments GENERAL DISCUSSION LITERATURE CITED APPENDIX 38 38 43 50 61 62 70 80 93 m V LIST OF TABLES Table Page Effect of removal of in the middle of a dark inductive period on the flowering response of Xanthium pennsylvanicum 41 Effect of red light interruption on the total 1 4C a c t i v i t y 14 nr recovered in ethanol-soluble fraction on CO2 fixation 45 Effect of red light interruption on the total a c t i v i t y 14 „r recovered in ethanol-soluble fraction by dark CO2 fixation 46 Effect of different treatments on the activity in different amino acids synthesized during short term feeding experiments 49 Stomatal resistance of the lower surface of the leaf under different conditions used for measurement of COg exchange 60 Effect of CO2 removal during red light irradiation on lettuce seed germination 63 Effect of pH and 1 hr-long gas treatments on lettuce seed germination 71 Effects of night interruptions and CO2 exchange by Xanthium pennsylvanicum L. leaves 83 LIST OF FIGURES 1 : Spectral characteristics of red and far red light used 14 in CO2 feeding experiments. 2: Closed gas exchange system used to measure CO2 exchange patterns of the Xanthium leaves under different light treatments. 3: Spectral distribution of the 3 red light intensities used in the CO2 exchange experiments, 4: Germination box used for red light treatment in seed germination experiments. 5: Germination box used for far red light treatment in seed germination experiments. 6: The transmittance pattern of the red and the far red plastic f i l t e r s obtained with a Coleman 124 Spectrophoto-meter. 7: Transmittance patterns of the CBS 650 red and 730 far red f i l t e r s , obtained with a Coleman 124 Spectrophotometer. 8: Transfer assemblies used for the pH transfer experiments. 9: Effect of CO2 removal during red light interruption on devel-opment of fl o r a l buds in Xanthium pennsylvanicum. CO2 was removed for 35 min with the red light interruption being given for 5 min in the middle of the CO2 removal period. 0: Transverse sections of apical buds of Xanthium pennsylvanicum 20 days after the SD photoperiodic treatment. Fig.10A shows a bud section for the dark controls. Red light interruptions were given in the middle of the dark period under normal air (Fig.lOB) and under C0 2-free conditions (Fig.10.C). 1: Effect of CO2 addition during red light interruption given after 4 hours of darkness on development of fl o r a l buds in Xanthium pennsylvanicum. CO2 was added for 35 min with the red light interruption being given for 5 min in the middle vi i Pagj. of the removal period. A l l the plants given a red light interruption after 8 hours of darkness under either normal air or 5% CC^, remained vegetative. 42 Fig.12: Trace drawing of the amino acids stained with ninhydrin on chromatogram of the ethanol-soluble extracts of leaves 14 from different treatments. was fed during a 5 min red light interruption in the middle of the dark period followed by either 5 min of darkness or 5 min of far red light before the leaves were k i l l e d . In the dark controls, 1 A the leaves were allowed to f i x C 0 2 for 10 minutes. 48 Fig.13: Changes in CO2 concentration within the closed gas exchange system during the following night interruption treatments: ( '• ) 5 min low intensity red l i g h t ; ( ) 5 min far red light; ( _ ) 5 min low intensity red light followed by 5 min far red light. The interruptions were begun at the 5 min point on the time axis (+), which was 8 hr after the start of the dark period. Fig.14: Effects of night interruptions after 4 (A), 8 (B), or 12 (C) hr of darkness on the rate of CO2 exchange. The inter-ruptions were begun at the 5 min point on the time axis (+) and were the same as in Fig. 9. The i n i t i a l CO2 concen-tration was 350 ppm. Fig.15: Effects of a 5 min red light interruption on the rate of CO2 exchange by leaves exposed to i n i t i a l CO2 concentrations of 20 ppm (A) or 350 ppm (B). The interruptions were begun at the 5 min point on the time axis ( 4 - ) , which was 8 hr after the start of the dark period. The changes in CO2 exchange caused by night interruptions (Table 8) were determined from the decrease in CO2 release plus CO2 uptake associated with the interruptions, as illustrated by the hatched area on curve A. 51 53 55 Fig.16: Effect of a 10 sec interruption with high intensity red light on the net rate of CO2 release. The interruption was given at 8 hr after the start of the dark period and the i n i t i a l CO2 concentration was 350 ppm. Fig.17: Changes in CO2 concentration within the closed gas exchange system during 30 and 60 sec of high intensity red light night interruption. Fig.18: Net rates of CO2 exchange during 5 min night interruptions with low ( e • ) , medium ( A • ) , or high ( •- • ) intensity red light. The i n i t i a l CO2 concentration was 350 ppm and the interruptions were begun (time = 0) after 8 hr of darkness. Fig.19: Effects of pH and CO2 concentration on lettuce seed germin-ation following red irradiation. Seeds were in normal a i r (• • •) or in C0 2-free a i r ( o ) , and they were incubated in citrate-phosphate (• o ) , citrate ( • ) , or phosphate (•) buffers. For the reaction: C 0 2 + HgO^— *HC0 3 " + H + » t n e dashed line indicates the percentage of total carbon in the form of CO2 at different pH levels (pK =6 . 1 ) . Fig.20: Effects of pH and CO2 concentration on seed germination in Amaranthus retroflexus L. following red irradiation. Seeds were in normal a i r ('•) or in C0 2-free a i r ( o ) and they were incubated in citrate-phosphate buffers in a l l cases except pH 7.5 and 8.0 where phosphate buffer was used. The dashed line represents the ^/HCOg" equilibrium as in Fig.l< Fig.21: Transfer experiments showing the effects of the timing of pH 4.0 or 8.0 and red irradiation on percentage germination of lettuce seeds. Vertical arrows indicate the times when 5 min red irradiation was applied. Fig.22: Transfer experiments showing the effects of duration of incubation and time of pH 4.0 treatment on percentage germin ation of lettuce seeds. Vertical arrows indicate the times when 5 min red irradiation was applied. 23: Transfer experiments showing the effects of the duration of pH 4.0 treatment on percentage germination in lettuce seeds following red irradiation (arrows). .24: Effects of delay between 5 min red and far red irradiation treatments on percentage germination of lettuce seeds. The seeds were incubated in citrate-phosphate buffers either at pH 4.0 throughout ( A ) , or in phosphate buffer at pH 8.0 until the far red treatment, then in pH 4.0 ( o ) . Percentage germination was measured at 24 hr following the far red treatments. .25: A model for the sequence of events in phytochrome-mediated seed germination. X ACKNOWLEDGEMENTS It is with sadness of heart that I express my deep sense of gratitude to my late Professor E. Bruce Tregunna for his help, guidance and support until his sudden death on September 13, 1975. I am also very grateful to Dr. Peter A. J o l l i f f e for his interest, assistance and advice which helped bring this project to a successful completion. Indeed, the long discussions with him were enjoyable and rewarding. Dr. A.N. Purohit provided a very stimulating atmosphere in the laboratory and I sincerely appreciate his help in the i n i t i a l stages of this project. I also wish to express my thanks to Dr. Paul G. Harrison, Dr. Paul J. Harrison, Dr. Jack Maze, Dr. R.F. Scagel, and Dr. G.H.N. Towers, for their help and advice. Technical assistance by Mr. Mel Davies and Mr. Ken J e f f r i e s , and financial assistance from the Killam Foundation in the form of a pre-doctoral fellowship, are gratefully acknowledged. I am grateful to a l l my friends for providing a very congenial atmosphere in and outside the laboratory. Above a l l , I owe a lot to Nisha whose support and encouragement have been a constant source of inspiration a l l these years. 1 INTRODUCTION 1. Phytochrome-mediated photomorphogenesis Radiant energy is perhaps the most important environmental factor regulating the growth and development of plants. Plants obtain energy from sunlight and transform i t by the process of photosynthesis into chemical potential energy. Apart from the provision of energy, light performs many other important functions in plants. The term "photomorphogenesis" refers to the non-photosynthetic effects of radiant energy on plant growth and development. Most photomorphogenetic responses of plants have been shown to be under the control of the phytochrome pigment system (120, 164). Some examples of major phytochrome responses are: germination of some seeds (60), hypocotyl elongation (60, 38), plumular hook opening (82), photoperiodic control of flowering (11, 36), anthocyanin synthesis (117, 160), chloroplast orientation in algae (61), chlorophyll synthesis (138), cotyledon expansion (118), formation of leaf primordia (122), and closure of Mimosa pudica leaflets (45). These responses are characterized by red/far red* r e v e r s i b i l i t y , location of response maxima at or near 660 nm and 730 nm, and by saturation at low energies. The presence of the pigment phytochrome was f i r s t demon-strated in etiolated corn coleoptiles (21). Since 1959 much work has been * for convenience of expression,the terms "red light" and "far red li g h t " will be used in this thesis to refer to wavelengths of radiant energy which cause the characteristic phytochrome responses. Although the use of these terms is common in publications on photomorphogenesis, i t should be recognized that far red wavelengths do not f a l l within the visible spectrum. 2 done to isolate and characterize the pigment. Phytochrome is a blue chromoprotein (159, 161, 162) and the chromophore is a b i l i t r i e n consisting of a linear tetrapyrrole ring; i t s structure has been f u l l y characterized (see 164). Phytochrome is known to be concentrated in the growing regions of etiolated plants. Evidence from studies using polarized light (62, 63) indicates that phytochrome is probably located in various membranes in the c e l l . Phytochrome exists in two interconvertible forms designated by P r and P f r (or P g 6 0 and P 7 3 q ) for the red-absorbing and far red-absorbing forms respectively. The reversible reaction is represented as: p 6 6 0 n m ^ p _ ^observed response r„ 730 nm ,'Tr Darkness The phototransformation of phytochrome is never complete due to some overlap in the absorption spectra of the two forms. Red light (ca_. 660 nm) converts about 80% of the pigment to P f while far red light (ca_. 730 nm) converts 99% of P f to P r (20). Both forms show considerable absorption in blue and ultraviolet zones of the spectrum,and blue light maintains 30-40% of the total phytochrome in P f form (20, 41). P r^ is considered to be the physiologically active form of phytochrome, and a close correlation between the optically detectable P f r form and the physiological responses has been shown (66, 69, 102, 144). There are, however, some striking exceptions to this correlation. Red light reduces phototropic sensitivity of maize coleoptiles without any detection of 3 phytochrome spectrophotometrically (15). Similarly, in peas i t is possible to show red/far red reversibility of stem elongation (see 67) in the absence of any spectrophotometrically assayable phytochrome. These apparent "paradoxes" have been explained on the basis that phytochrome may consist of two distinct populations: an "active" fraction which is smaller in size and responsible for metabolic and developmental responses to light, and a "bulk" fraction responsible for the observed in vivo spectrophotometric change ( 6 7 ) . Indeed, two kinetically distinguishable populations of P^r have been shown to exist in etiolated seedlings (6, 155). Besides i t s phototransformation to the stable P r form, a large portion of P^r is destroyed to a non-detectable form, on exposure to light (19, 101). It has been postulated that most of the phytochrome con-stitutes a large destructible fraction and a small fraction is the stable component (18). In the dark, two transformational processes are known to occur: dark destruction of P^r into a non-detectable or irreversible form, and dark reversion of to P^  (67). "Inverse reversion", the dark transformation of P r to P^r, has been suggested to explain results from some studies with Chenopodium rubrum seedlings, Pharbitis nil plants, a dark germinating variety of lettuce seeds, and Amaranthus and Cucumis seeds (7, 26, 28, 87, 90, 165). As mentioned earlier, phytochrome has been shown to control several responses in plants. These responses have been reviewed in detail by a number of workers (e.g. 115, 120, 164). This thesis is primarily concerned with the effects of C0 2 on phytochrome-mediated flowering and seed germination, although a general effect of C0 2 on a l l photomorphogenetic 4 responses under phytochrome control is not yet ruled out. The following sections will review the significant findings of previous investigations relevant to phytochrome control of flowering and seed germination. 2. Phytochrome and flowering in short day plants Flowering plants can broadly be classified into three main groups depending upon their response to daylength. Short day (SD) plants flower in response to dark periods longer than a c r i t i c a l length, long day (LD) plants require daily dark periods shorter than a c r i t i c a l length, and day-neutral plants are not sensitive to variation in daylength for the flowering response. Two types of phytochrome action can be distinguished in the photo-periodic control of flowering in SD plants; the light reaction has a requirement for the P^r form of phytochrome, whereas the dark reaction proceeds when P^r is low. It is presumed that the P^r form of phytochrome starts reverting to the P r form when plants are transferred from ligh t to darkness. Thus, when a normally inductive long dark period is interrupted with a brief exposure to red l i g h t , phytochrome is converted back to P r and flowering is inhibited. (i) Promotive effect of preceding inductive dark period: the high P^r reaction. The promotive effect of P^r was f i r s t shown in Pharbitis n i l seedlings when a short exposure to far red light before the seedlings were transferred to dark suppressed the flowering response (129). This suppression could 5 be reversed by a brief exposure to red light immediately following the far red, suggesting that the presence of P f r at the close of the daily photoperiod had a flower-promoting function. Takimoto and Naito (167) reported that Pharbitis nil seedlings grown in continuous light for 1-2 days could be induced to flower when exposed to a short day cycle. When seedlings were germinated in darkness and kept in darkness for 2-4 days followed by continuous illumination, however, they failed to i n i t i a t e f l o r a l buds. In other experiments, seedlings exposed to far red light followed by a dark period were inhibited whereas ones exposed to red light initiated f l o r a l buds, thereby suggesting the involvement of phytochrome. Flowering in Xanthium pennsylvanicum has also been shown to be inhibited by far red light given at the beginning of the dark period (10). A number of other workers have reported that in Xanthium. plants exposed to red light or lig h t with a high ratio of red/far red (maintaining high P^r) preceding darkness, there was a high level of flowering. Far red lig h t or light of low red/far red ratio (maintaining low P f r ) , however, was somewhat inhibitory (26, 89, 97, 112, 147, 166, 180). Thus, i t can be concluded that during the light period preceding an inductive dark period, a high level of the P f r form of phytochrome is optimal for flowering of SD plants. ( i i ) The "night-break" phenomenon in short day plants: low P f reactions For plants grown in sunlight, at the close of the photoperiod most of the phytochrome is present in the P f r form. A brief saturating illumination with red light has no effect on the flowering response when given at the end of the day or during the f i r s t few hours of darkness. 6 After several hours, however, such a red light interruption completely n u l l i f i e s the inductive effect of the dark period. This so-called "night break" phenomenon was discovered in Xanthium pennsylvanicum when Hamner and Bonner (59) showed that a 1 minute red light interruption in the middle of an inductive 9-hr dark period completely prevented flowering. The red light effect could be completely reversed by far red l i g h t (46). Many different aspects of the interruption effects in Xanthium have since been studied (see e.g. 31, 35, 98, 140, 146, 147, 148). The reversibile night break effect has since been demonstrated in many other SD plants including Amaranthus caudatus (31), Glycine max cv Biloxi (31), Sorghum  vulgare (95), Chrysanthemum morifolium (24) and Salvia occidental is (111). In some SD plants, the night break reaction is irreversibly completed within less than 5 minutes of establishing P^r in the middle of the 16-hr inductive dark period (8, 51, 52). In Xanthium, however, the r e v e r s i b i l i t y could be maintained for up to 40 minutes (31). Similar results have also been reported for Chenopodium rubrum (24) and Chrysanthemum morifolium (86). In their experiments on the kinetics of the interruption effects in Xanthium, Salisbury and Bonner (148) reported that a constant dose of red light is required to bring about maximum inhibition of flowering at any given time after the f i r s t 2 or 3 hours of the dark period. The maximum response was obtained with about 6 seconds of red light i f a very high -2 light intensity (approximately 1800 yW cm ) was used for interruption. These experiments indicate that an uninterrupted period of darkness is of fundamental importance for flowering in SD plants. It has also been shown experimentally that P f r reverts back to P p in the dark (11, 12). Based on these observations i t was postulated that the dark reversion of 7 P^r to P r could constitute the primary timing factor in the photoperiodic control of flowering, and the time taken for P^r reversion would determine the c r i t i c a l day length. Further experimentation has indicated that phytochrome reverts to P form in considerably less time than the c r i t i c a l dark length (37, 149). Other evidence has also suggested that phytochrome reversion alone cannot account for the time measurement controlling the c r i t i c a l night length in flowering of SD plants (136, 146, 149). Although the mechanism for photoperiodic timing remains unknown, i t has been suggested that flor a l induction involves an endogenous rhythm having phases of different sensitivity to light. P^r either promotes or inhibits flowering depending upon the status of the endogenous os c i l l a t i n g circadian timer. Phytochrome may also be involved in the phasing of the rhythm i t s e l f (see e.g. 175). 3. Phytochrome and seed germination Seed germination was one of the f i r s t developmental processes recog-nized to be photoregulated. The f i r s t report on the beneficial effect of light on seed germination was published in 1860 by Caspary (23) who demonstrated that seeds of Bulliarda aquatica would not germinate in dark-ness. In 1908, Kinzel lis t e d 672 species showing positive photoblastism, i.e. an induction or stimulation of germination by light (see 164). A turning point in the history of photoblastism came in 1935-1937 when Flin t and McAlister demonstrated the opposing effects of red and far red light in the germination of lettuce seeds (43, 44). Their work formed the basis of the discovery of red/far red photoreversibility of germination, 8 and ultimately of phytochrome i t s e l f (13). (i) Light effects on seed germination Seeds are "positively photoblastic" when germination is stimulated by light and are "negatively photoblastic" i f light inhibits germination. "Non-photoblastic" seeds germinate in darkness as well as in l i g h t . In a l l the three categories, evidence has been obtained that germination can be under phytochrome control. Many non-photoblastic seeds can be made light-sensitive by external conditions of stress (39). When such seeds are put in mannitol, coumarin, or at temperatures above 30C, a short exposure to red light becomes necessary for germination (9, 84, 107, 141 , 142). In some cases, e.g., tomato cv Ace or St. Pierre, the germination is inhibited by a short exposure to far red light and this inhibition can be reversed by red light (34, 106). These experiments on "imposed photo-blastism" seem to indicate that the processes of germination in photoblastic and non-photoblastic seeds are basically similar. Similarly, in some negatively photoblastic seeds, e.g. Lamium amplexicaule, cucumber, Nemo- phila insignis and Amaranthus caudatus (78, 178, 143, 85), inhibition of germination can be overcome by short exposures to red light. Thus, positive and negative photoblastism may represent two facets of a single phenomenon having the same photoreceptor. In positively photoblastic seeds, the amount of light required for germination varies among different species. In lettuce, for example, a very short exposure to low intensity irradiance is sufficient to induce 100% germination, whereas in Epilobium prolonged exposures of several hours may be required. Attempts have been made to classify light sensitive seeds according to this quantitative requirement (77), but the distinctions 9 such classifications involve are very a r t i f i c i a l . For example, in some seeds ordinarily requiring continuous light, the requirement can be f u l -f i l l e d by short exposures to intermittent irradiation (74). Many workers have attempted to explain the variations in light requirements of different seeds. Differences in the phytochrome content in dark and light germinating strains of Oryzopsis miliacea have been observed (131). Differences in seedcoat thickness may also lead to d i f f e r -ences in the sensitivity of different seeds to the same dose of a particular irradiation (83). In most light-sensitive seeds, the effect of a specific light treatment varies depending upon the time i t is given after start of imbibition in darkness. The relationship between time of imbibition and photosensitivity is different for different seeds (see e.g. 73, 92, 173). Even in the same cultivar of lettuce, e.g. Lactuca sativa cv Grand Rapids, different workers have observed different responses in photosensitivity with time (see 164). The basic trend, however, is consistent; a peak in photosensitivity followed by a steady decline with increased time of imbibition in darkness. Differ-ent hypotheses have been advanced to explain this phenomenon. It has been postulated that the increase in sensitivity could be due to a decrease in inhibitor level within the seeds (17), or due to the synthesis of a substance with which P f r interacts to produce an active complex (60, 93). A third, and perhaps more plausible explanation, is that the photosensit-i v i t y could increase because of a gradual synthesis of P r or a release of P r from an inactive complex (131). A significant increase in the content of phytochrome has actually been observed in some seeds during the course of dark imbibition (87, 104, 105). 10 Germination in some seeds appears to be a photoperiodic phenomenon. For example, in Epilobium cephalostigma and Tsuga canadensis, the germination rate under "SD cycles" is more than under continuous light (72, 135). Similarly, Black and Wareing (5) reported that in Betula pubescens under certain conditions, continuous irradiation gave more germination than did shorter photoperiods. These responses, however, are different from the photoperiodic control of flowering (175). The relationship between light duration and percentage germination appears to be purely quantitative and a c r i t i c a l photoperiod cannot be defined. ( i i ) Interactions between light and other factors affecting seed germin-ation. Several factors have been shown to influence the response of seed germination to red and far red irradiation in different species. The behaviour of seeds can be determined by the environment of the mother plant (40, 48). McCullough (109) pointed out that the light requirement can be eliminated in the seed of Arabidopsis thalliana by growing the parent plants in light with a high ratio of red:far red. Very recently, similar experiments have also been done with lettuce where the rate of dark germination was shown to be enhanced i f the seed matured in complete darkness on the parent plant (80). It was concluded that germination in darkness in seeds which matured in total darkness was independent of phytochrome, which was probably a l l present in the P r state. Maturation of seeds in light induces a kind of dormancy which can be partly or entirely overcome by the presence of P f r in the mature seeds. 11 (a) Interaction between light and temperature Temperature has a marked effect on the germination of photosensitive seeds. It is important to consider some of these interactions, partic-ularly in lettuce, to obtain a better understanding of the factors affecting seed germination. In lettuce, cv Grand Rapids, no germination occurs at high (ca. 30-35C) temperatures whatever the light conditions; at inter-mediate ( C J K 20-30C), the seeds are light sensitive; and at lower tem-peratures, the seeds germinate even in darkness (164). The inhibition of germination by high temperatures irrespective of the light conditions is known as "thermodormancy". It was mentioned earlier that negatively photoblastic seeds can be made light sensitive under osmotic stress. Similar experiments have been done with lettuce seeds germinating at lower temperatures, and there was a reduction in germination under osmotic stress. Even in "thermodormant" seeds, the dormancy can be broken by the application of germination-stimulatory substances, such as gibberellic acid (49). These observations indicate that both light- and temperature-sensitive processes are involved in germination and that one or the other may be dominant in a particular system. The response pattern to temper-ature variation varies among different photosensitive seeds. Thus, Lepidium virginicum seeds are photosensitive even at lower temperatures, but at higher temperatures their behaviour is the same as in lettuce (171). Seeds of Amaranthus retroflexus have a complex light and temperature requirement for optimal germination. Germination is stimulated by brief light treatments at a l l temperatures between 20-40C, whereas continuous illumination is promotive only between 25-35C. In fact, at lower temper-atures, continuous light is strongly inhibitory to germination (79). 12 (b) Interaction between light and growth regulators Much work has been done on the effects of growth promoting and inhibiting substances on germination of light-sensitive seeds. It is not intended to review a l l the relevant!iterature here, but some previous work is pertinent to the understanding of the mechanism of P^r action. Cytokinins promote the germination of light-sensitive lettuce seeds when given in combination with sub-threshold amounts of red light (99). Gibber-e l l i n s , on the other hand, induce germination of Grand Rapids lettuce even in darkness, but a synergistic action of gibberellins with sub-threshold levels of light has also been reported (70, 81). In fact, using this approach in studies with chemical growth regulators, i t has been demonstrated that P a c t i o n occurs very rapidly after red light is given (3, 4). Thus, in experiments where the time of P^r a v a i l a b i l i t y was controlled by reconverting i t to P r with far red light at different times after red irradiation, i t was shown that the presence of P^r for as short a time as five minutes was sufficient to induce germination in combination with sub-threshold levels of gibberellic acid. Based on these observations, Smith (164) has concluded that the photoconversion of phytochrome, the production or release of growth hormones, and the maintenance of optimal temperatures, are three c r i t i c a l requirements for germination. Under different conditions, e.g. the application of high concentrations of gibberellins, or the exposure of seeds to low temperatures, the requirement for P f can be eliminated. 13 4. Mechanism of phytochrome (Pf r) action Any plausible hypothesis on the mechanism of phytochrome action must explain both the long-term developmental effects of red light,-(e.g. seed germination or photoperiodic control of flowering) and the very rapid effects of red li g h t , (e.g. leaflet opening and chloroplast movement in algae). It is generally assumed that there is a single primary action of P^r and that the many responses of phytochrome in plants are different manifestations of the same primary action. Mohr ejt al_(l25), however, have suggested that phytochrome may be involved in qualitatively different primary reactions in different tissues and species. (i) Pf r as an enzyme Based on the observation that light of low energy saturates phyto-chrome responses, Hendricks and Borthwick (125) suggested that P^r is the active form and P r the inactive form of an enzyme. The proteinaceous nature of phytochrome and the higher reactivity of P^, as compared to P r lend some support to this hypothesis. The primary reaction which this "enzyme" may catalyze remains unknown, but the reaction must be fundamental to metabolism and capable of regulating the rates of many different meta-bolic pathways. The enzymatic nature of phytochrome could explain the rapidity of phytochrome action i f the substrate which the enzyme catal-yzes is readily available. 14 ( i i ) P f r as a regulator of gene activation Mohr (119) postulated that phytochrome acts through the activation or repression of specific genes. The differential status of the genome of the cells is then responsible for subsequent changes in enzyme levels and for consequent different responses in the plant. Support for this hypothesis comes from the following observations: there is a lag of at least a few hours for the i n i t i a t i o n of a number of phytochrome responses; RNA and protein levels increase following illumination; and the photores-ponses are inhibited by inhibition of nucleic acid and protein syntheses (9 7, 119 , 123, 156). However, rapid phytochrome responses such as the rotation of chloroplasts in Mougeotia (62), leaflet closing in Mimosa  pudica (45, 47), nyctinastic leaf closure in Albizzia j u l i b r i s s i n and other legumes (68) cannot be explained on the basis of gene activation as the primary site of P^r action. Even in other responses where' the phenomena themselves are not displayed readily, a rapid action of phytochrome has been shown. Thus, in the photoperiodic control of flowering in Pharbitis  n i l (51) and Kalanchoe1 blossfeldiana (52), far red r e v e r s i b i l i t y of a red light break in the middle of a long dark period is lost within 5 minutes of red light. A similar example is the red light-induced germination of light-sensitive lettuce seeds. It has been shown that even 5 minutes of P^r a v a i l a b i l i t y in the presence of a sub-threshold level of gibberellic acid is sufficient to induce germination (3, 4). Evidently, Mohr's hypothesis of phytochrome-mediated gene activation is inadequate to account for these observations on the rapid actions of phytochrome. 15 ( i i i ) P f r and changes in membrane properties It has been suggested that phytochrome action is i n i t i a l l y on membrane permeability and that other changes follow sequentially (46, 65). Evidence for this hypothesis comes from studies on the adhesion of mung bean root tips to a negatively charged glass surface following illumination with red light. The adhesion caused by red light is reversed by far red light (168). This effect has come to be known as the "Tanada Effect" after T. Tanada who f i r s t observed this response (168). It has been suggested that the basis of the adhesion is the electrical potential existing between the outer surface and the interior of the root, and that the potential difference is rapidly changed due to "phytochrome activation". Jaffe (76) measured the potential difference between the root apex and base, and found that the potential across the root changes in response to red li g h t , and that the change is substantially reversed by far red lig h t . These results have since been confirmed by Newman and Briggs (133) who found that exposure of etiolated Avena coleoptiles to red light produced a pronounced increase in the positive potential of the coleoptile, while far red light reversed the change. They postulated that the transformation of phytochrome affects ion pumps in the membrane. Similarly, the rapid flux of ions (especially K+) in the ce l l s of the pulvini of Albizzia pinnules has been suggested to be involved in the pinnule closing and opening movements (55). Haupt e_t al_. (63), in their experiments with polarized microbeans, have shown that the orientation of the phytochrome molecules in the plasma-lemma of Mougeotia is rotated through 90° on conversion from P r to P^r and vice versa. Thus, red light polarized parallel to the cell surface 16 converts P r to P^r, whereas far red light has to be polarized at the normal plane for i t to reverse the red light effect. It has been postu-lated that changes in the orientation of phytochrome molecules maybe involved in the activation or inhibition of certain carriers responsible for membrane transport (163). 5. High-energy reactions (HER) As mentioned earlier, many phytochrome responses are saturated at low intensities of light. In addition, long exposures of high intensity radiation have also been shown to control a number of photomorphogenetic responses. Thus, the term "High-Energy Reaction", commonly abbreviated as HER, pertains to photomorphogenetic responses requiring radiation of higher intensity and duration than required to saturate low-energy phyto-chrome responses. First reported, in cotyledonary expansion of mustard seedlings (117), the HER has subsequently been implicated in many develop-mental phenomena including seed germination (121), stem and leaf expansion (119, 124), anthocyanin synthesis (117), and changes in the level of specific enzymes (35, 134). The action spectrum for the HER involved in the inhibition of hypocotyl elongation in lettuce seedlings shows a sharp peak at 717 nm, and a complex series of peaks in the blue and near ultra-violet zones (see 164). This contrasts with the low energy phytochrome responses which have peaks at 660 (R) and 730 nm (FR). Since i t s discovery, there has been considerable controversy over the pigments involved in the HER (14, 38, 47, 58, 60, 113, 174, 176), but now i t is generally accepted that for the blue and far red zones of 17 the action spectrum, two separate photoreceptors may be involved. Carotenoids, which are probably involved in phototropism, and P^r, which is involved in low-energy phytochrome actions, have been suggested to be the li k e l y candidates for the blue and far red effects of the HER, respectively. The effect of high-energy radiation is not always the same as that of 1 energy red light. High-energy radiation, for example, inhibits lettuce seed germination in contrast to low-energy red light which promotes germination (120), whereas both high- and low-energy radiation promote anythocyanin synthesis (117). This thesis i s primarily concerned with low-energy phytochrome systems but an attempt will be made to relate the observations obtained in these investigations to the HER system. 18 6. Objectives In previous studies of the photoperiodic responses controlled by phytochrome, some attempts have been made to study the role of photo-synthesis during the light period. It has been shown that the photoinduc-tion of flowering in LD plants is more dependent on photosynthesis than is the induction in SD plants (36, 179). This has been confirmed by the inhibitions of flowering in the LD specues, Anagallis arvensis, by DCMU* and in the LD species, Sinapis alba, by the exclusion of CO2 from the air (88, 164). Moreover, continuous application of a high level of CO^ induced flowering under SD conditions, in the LD plant Silene armeria. possibly as a result of enhanced photosynthesis (139). On the other hand, in SD plants i t has been shown that even the etiolated seedlings of Pharbitis can be photo-induced in the complete absence of photosynthesis (53). The induction of flowering in Pharbitis in the presence of a very short photoperiod of 1 or 2 minute duration (54), and the ineffectiveness of DCMU in inhibiting f l o r a l induction in Chenopodium seedlings (153, 154), provide further confirmation. Information on the involvement of dark C0£ fixation in photoperiodic control of flowering, however, is rather scanty. There are some reports which indicate that the removal of CO2 during the dark period inhibited flowering of short-day plants in some experiments (98, 127) but not in others (22, 96). At the outset of my research, Purohit and Tregunna (139) had just reported on the effects of CO2 on photoperiodic responses of some plants. High CO2 concentration was shown to inhibit flowering in several SD plants under inductive conditions, and to induce flowering in several *DCMU [3-(3,4-dichlorophe.nyl ) - l , 1-dimethyl ureali s an.inhibitor of photo-synthesis 19 LD plants under non-inductive conditions. They postulated that the photoperiodic responses of plants may depend on the extent of photosynthesis during the light period preceding darkness. An alternative po s s i b i l i t y , however, is that the CCV, effects on flowering were due to an interaction with phytochrome. This involvement of phytochrome in the photoperiodic control of flowering is best demonstrated by night inter-ruption experiments. The present work was therefore initiated to study the relationship between C0 2 exchange and phytochrome using night inter-ruption experiments for the control of flowering. Most previous work on night interruption effects and flowering has been done on Xanthium pennsyl vanicum (148), a qualitative SD plant. The presence of a terminal inflorescence and a clear demarcation between different stages of induction in Xanthium (145) were additional factors favouring the selection of this plant material for the present investi-gations. The f i r s t series of experiments was designed to test whether COg is essential for the inhibition of flowering in Xanthium by a red lig h t night interruption. Xanthium plants were exposed to different CC^ concentrations during different kinds of night interruptions to study their flowering responses. Following the discovery of a CC^ requirement, a second series of experiments was carried out to test whether CC^ fixation i s responsible for the effect during the red light night interruption. The approach taken was to determine whether the pattern of leaf C0 2 exchange during or after red and far red night interruptions was correlated with the effects of the interruptions on flowering. 20 The directness of the relationship between CO2 and red light in the photoperiodic control of flowering can be questioned. It can be argued that CO^ is involved with other factors regulating time measure-ment in photoperiodism, and the effect of CO2 may be independent of phyto-chrome transformation. It was important, therefore, to test the involve-ment of CO2 in other non-periodic phytochrome-mediated responses. Thus, a third series of experiments was done to study the interaction of CO2 in the induction of germination in light-sensitive seeds. It was also possible that atmospheric CO2 levels could have acted indirectly by affecting general tissue characteristics, such as tissue pH. Experiments were done, therefore, to examine the effects of pH and CO2 on the response of seed germination to red and far red light. This was the fourth and final series of experiments done, and i t included tests in which the timing of pH and CO2 treatments was varied to determine whether or not their effects were directly linked with the effects of light treatments. The results of these investigations and their possible interpretations will be considered in this thesis. 21 MATERIALS AND METHODS 1. Studies on the photoperiodic control of flowering in Xanthium pennsyl- vanicum L. (i) Night interruption experiments Seeds of Xanthium pennsylvanicum plants used in the experiments on the interacting effects of C0 2 and red light, were germinated in moist mica-peat in metal trays in a controlled environment room. The plants were grown at 25 C with a 16 hr photoperiod extending from 8.00 a.m. to 12.00 midnight. The radiant flux density during growth was 60 x 1.0^  uW m (400-700 nm) and this was supplied by two 400 W mercury lamps (General Electric H33). After the unfolding of the f i r s t two leaves, the plants were transplanted to 12 cm diameter pots in pairs in mica-peat. The plants were watered daily and received 100 ml per pot of 20:20:20 nutrient solution each week. At the 5-to 6-leaf stage, three groups of 8 plants each were transferred to SD photoperiod (8 hr of ligh t alter-nating with 16 hr of darkness from 4.00 p.m. until 8.00 a.m.) in three 22x22x65 cm Plexiglas chambers. The chambers were illuminated from above through a 6 cm thick water layer with cool white fluorescent tubes. The temperature in the chambers was 23 t 1 C The plants in these chambers were exposed to 8 SD cycles. In two of these chambers, the middle of each dark period was interrupted for 5 minutes with red l i g h t . The light was supplied by a bank of cool white fluorescent tubes f i l t e r e d through a CBS (Carolina Biological Supplies) 650 red f i l t e r . The intensity 4 -2 of red light at the surface was about 12x10 uW m at 650 nm. Atmospheric a i r was pumped through one of those two, chambers during the light treatment 22 and the other was flushed with medical grade CO^-free a i r for 35 minutes. The gas flushes were begun 15 minutes before and ended 15 minutes after the red light treatments. The flow rate of CC^-free a i r was approximately 5 1 min"^. After the CO^-free treatment was over, atmospheric air was circulated through the chamber for 10 minutes. After 8 such cycles, the plants from a l l the treatments were trans-ferred to non-inductive 16 hr photoperiods in normal a i r . The shoot apices of the plants were dissected 10 or 20 days after the start of the light treatments, and they were classified for their stages of develop-ment according to Salisbury's scheme (145 ). Apices of 2 replicate plants from each treatment were fixed in FAA* for anatomical analysis. After dehydration through a series of alcohol concentrations, the apices were embedded in paraffin. Longitudinal sections of the apices (10 un thick) were cut with the help of a rotary microtome. The sections were stained with safranin fast green stain and mounted on slides (for details, see 77). In experiments using high CO2 concentrations, 5% CO2 was circulated through the chamber for 35 minutes with the red light interruption being given in the middle of the C 0 2 flush. After the C 0 2 treatment, the chamber was flushed with atmospheric a i r for 15 minutes. Red light inter-ruptions were given for 5 minutes after 4 or 8 hours of darkness, depending upon the experiment. The shoot apices of the plants were dissected after 10 days. Other details of the experiment were the same as described for previous experiments. * FAA is Formalin: Acetic Acid:Absolute Alcohol (5 ml:5 ml:90 ml). ( i i ) '^ CCX? feeding experiments 14 In each fjrx, feeding experiment, plants were transferred to a dark room at 4.00 p.m. The sixth leaf was detached from a plant after 8 hours of darkness and enclosed in a small Plexiglas chamber (12 x 12 x 2 cm) with the cut petiole tip immersed in water in a small cuvette attached to the base plate of the chamber. The chamber had a detachable 14 base to f a c i l i t a t e faster k i l l i n g of the leaf after the COg feeding was over. The base plate was held in place using silicone grease to make the system airtight. There was a serum stopper f i t t e d in the side 14 of the chamber for CO^ injections. For the generation of COg, 1 ml of r^SO^ was introduced into a 5 ml syringe through a serum stopper f i t t e d 14 on the top. The h^SO^ was mixed with 200 uCi of NaH CO3. The syringe 14 was shaken gently to release the dissolved CO2 which was transferred 14 and injected into the chamber using another syringe. During the CO2 feedings, the air in the chamber was circulated by a leak-proof pump. The pump was connected in a closed loop with the chamber and the flow rate was maintained at approximately 1 l i t r e min" 1. 4 -2 Red light, at an intensity of approximately 12 x 10 pW m at 650 nm, was obtained by passing light from 4 GroLux (Sylvania) 20 W fluores-cent lamps through a 30 x 30 cm CBS 650 red f i l t e r . Far red ligh t , at 4 - 2 an intensity of 20 x 10 yW m at 750 nm, was obtained by f i l t e r i n g light from a 500 W incandescent lamp through a 30 x 30 cm CBS 750 far red f i l t e r . The red and far red light beams were passed through 6 cm of water before they were incident on the leaf chamber. The spectral characteristics of the light treatments used (Fig. 1) were determined by measurements with an ISC0 spectroradiometer (Instrumentation Special-i t i e s Co., Model SR). All of the procedures were done in a fume hood. 4 A Fig. 1: Spectral characteristics of red and far red l i g h t used in 1 4 C 0 2 feeding experiments. 25 At the end of each experiment, the pump was disconnected and the fume hood door closed for exhaustion of contaminated a i r . At the end of 14 a CGV, feeding, the plant material was immediately immersed in boiling 80% ethanol and extracted for one hour. A few drops of .2 N formic acid were introduced to bleach the samples during boiling, and the extract was concentrated to dryness at 40°C using a flash evaporator. One ml of 80% ethanol was added to the dried residue. A 50 pi aliquot of each extract was added to vials containing 15 ml of s c i n t i l l a t i o n f l u i d (PPO*: P0P0P**: Toluene; 3 gm: 100 mg: 1000 ml), and the relative amount of activity in each extract was measured using a liquid s c i n t i l l a t i o n counter (nuclear-Chicago Unilux-II A). Separation of the amino acids obtained in the ethanol soluble fraction was done by one-dimensional descending paper chromatography in butanol: acetic acid: water (4:1:1). Standards were also run for Aspartate, Asparagine, Arginine, 3-Alanine, Alanine, Glutamate, Glutamine, Leucine, Proline and Valine. The chromatograms were run for 9.hours, dried in a i r in a fume hood, and sprayed with Ninhydrin reagent (.5% sol in Butanol). The chromatograms were then placed in an oven at 80°C for 5 minutes for f u l l development of colour. The individual spots were counted by cutting the paper strips and putting them directly in vials containing 15 ml of the liquid s c i n t i l l a t i o n f l u i d . ( i i i ) C0 2 exchange experiments Single Xanthium plants were grown in 12 cm pots as described e a r l i e r . For each experiment, individual plants were given a SD treatment by * PPO is 2,5 Diphenyl-oxazole ** P0P0P is p-bis-[2-(5-phenyloxazoyl)]benzene 26 transferring them to a dark room at 23±1 C at 4.00 p.m. After 4, 8 or 12 hr of darkness, depending on the experiment, the sixth leaf of an intact plant was enclosed in a transparent Plexiglas chamber (12 x 12 x 1.5 cm). Except when red or far red light treatments were applied, a dim green safelight was the only illumination during the gas exchange experiments. The leaf chamber was connected in a closed gas exchange system in series with a flowmeter, an a i r pump, and an infra-red CO2 analyzer (Beckman IR 215). A data logger (Digitec 1208) recorded the output of the CO^ analyzer. Fig. 2 is a schematic representation of the system used. The volume of the closed system was 365 ml and the air was circulated through the system at 2 l i t r e s min"^. Preliminary tests with pulse injections of CO2 into the centre of the chamber revealed a lag of 6.5 s until maximum response was detected by the CO2 analyzer. In most experiments, the i n i t i a l CO2 concentration in the closed system was 350 ppm (yl 1"^). When lower i n i t i a l CO2 concentrations were required, the system was flushed with C^-free a i r until the desired concentration was reached. Leaf CO2 exchange was measured during red or far red light night interruptions and for 5 min periods preceding and following the inter-ruptions. Three different light intensities v/ere used in these experiments. 4 - 2 Low intensity (12 x 10 uW m at 650 nm) was obtained from the same source as in the ^COg feeding experiments. For medium (25 x 10^ yW m~^  nm"^  4 - 2 - 2 at 650 nm), and high (125 x 10 yW m nm at 650 nm) light intensities, a 300 W incandescent lamp was used and the light was f i l t e r e d through a CBS 650 red f i l t e r . An interference f i l t e r , which cut off wavelengths above 700 nm, was used to attenuate that light to achieve the medium Closed gas exchange system used to measure C0 2 exchange patterns of the Xanthium leaves under different light treatments. L E A F C H A M B E R D A T A L O G G E R I R G A VuMP F L O W M E T E R intensity. Far red light, at an intensity of 20 x IO'* yW nf" nm"' at 750 nm, was obtained in the same way as in the earlier experiments. Fig. 3 represents the spectral distribution of the 3 red light inten-s i t i e s obtained with an ISCO spectroradiometer (Instrumentation Special-i t i e s Co., Model SR). All the light beams were passed through a 6 cm water jacket placed between the light source and the chamber. Stomatal diffusion resistance was measured with a diffusion resistance porometer (Lambda Instruments Corp. L 1-60 with L 1-15 S sensor), immediately after a night interruption. Stomatal resistance measurements in daylight were made at noon under a light intensity of 60 x 10 6 uw m"2 (400-700 nm). All of the experiments and measurements were repeated 3 times using different plants. Because the effects of different treatments on C0 2 exchange were very consistent, most of the results presented here are from individual experimental t r i a l s . The effects of different night interruptions on flowering was again determined by dissecting the buds of plants 10 days after a night interruption was given. 2. Studies on phytochrome-mediated seed germination Light-sensitive lettuce (Lactuca sativa L. cv Grand Rapids) seeds used in these investigations were obtained from the Carolina Biological Supply Co. Amaranthus retroflexus L. seeds were from a f i e l d collection made by Dr. P.A. J o l l i f f e at Clearbrook, B.C., in August 1971. Before use the seeds were stored in darkness at about 23°C. 29A Fig. 3: Spectral distribution of the 3 red light intensities used in the CO- exchange experiments. W A V E L E N G T H - M y 30 In the i n i t i a l experiments, lettuce seeds were placed in batches of 50 in 4 cm diameter Petri plates lined with two layers of Whatman No. 1 f i l t e r paper. A 2.5 ml volume of d i s t i l l e d water was provided in each Petri plate for imbibition. One set of 4 Petri plates was kept continuously in darkness. In a second set, uncovered seeds were placed in a red light box, described below, and irradiated for 5 minutes after 15 minutes of imbibition in darkness. The source and intensity of red irradiation were the same as described in experiment 1. Atmospheric ai r was circulated through the chamber by a pump. The third set of Petri plates was exposed to C^-free air for 25 minutes as described in the experiment on interruption effects on flowering. After 5 min in the C02~free a i r , the seeds were exposed to far red light for 5 min and then maintained in CO^-free air for another 15 minutes. The a i r was partially humidified by bubbling i t through water contained in a flask. After the light and CX^-free a i r treatments, the Petri plates were kept in atmospheric air in darkness. Germination counts were taken after 24 and 48 hours from the start of imbibition. In later experiments on the effects of CO2 and pH on seed germination, the lettuce or Amaranthus retroflexus seeds were incubated in batches of 50 in covered 100 x 15 mm Petri plates lined with 2 layers of Whatman No. 1 f i l t e r paper. Four replicate plates were used in a l l cases. Three different kinds of buffers were used. Citrate-phosphate (0.05 M c i t r i c acid, 0.1 M Na2HP04), citrate (0.05 M c i t r i c acid; 0.5 M Na Citrate), and phosphate (0.2 M Na2HP04; 0.2 M Nah^PO^ buffers were prepared to provide pH values in the ranges 4.0 to 7.0, 4.0 to 6.0, and 6.0 to 8.0 respectively (see the Appendix for details). Before using them, the pH 31 of a l l the buffers was checked with a pH meter. For the red and far red treatments, special wooden boxes were constructed (Figs. 4, 5). Each box was f i t t e d with 2 fans for air circulation to minimize the increase in temperature under the light sources. The far red light box also had a 6 cm water jacket between the l i g h t source and the f i l t e r s , and a continuous flow of cold water 6 -2 was maintained through the jacket. Red irradiation at 7.3 x 10 yW m~ between 585 and 700 nm was obtained by f i l t e r i n g the output from two 40 W fluorescent lamps (Grolux, Sylvania) through a red plastic f i l t e r . For far red radiation, a blue f i l t e r was inserted between two plastic red f i l t e r s . Far red radiation at 94 x 10 yW m between 695 nm and 3.0 ym was provided by passing the output from sixteen 40 W tungsten filament lamps through the f i l t e r s . The spectral characteristics of red and the far red f i l t e r s (Fig. 6) were obtained with a Coleman 124 spectrophoto-meter. The transmittance patterns of the plastic f i l t e r s closely resembled the patterns obtained from the CBS f i l t e r s used earlier (Fig. 7). For the red treatment, the lamps were 0.4 m from the seeds, and for the far red treatments, the lamps were 0.6 m from the seeds. The exposure time was 5 minutes for individual irradiations. Except where otherwise stated, the red light was given 30 minutes after the start of imbibition. Aside from the red and far red treatments, a dim green safe light was the only illumination the seeds received until the time when percentage germination was assayed, and the safelight was used only during the buffer transfers. The temperature about the seeds during these germin-ation experiments was 23-1 C. In experiments v/here COp-free a i r treatments were given for 24 hrs, 32A Fig. 4: Germination box used for red light treatment in seed germination experiments. 20cm •Chamber-Lamp - D o o r F R O N T V I E W S I D E V I E W 3 3 A Fig. 5: Germination box used for far red light treatment in seed germination experiments. I 1 2 0 cm C h a m b e r Lamp W a t e r J a c k e t Door F R O N T VI E W S I D E V I E W CO 34A Fig. 6: The transmittance patterns of the red and the far red plastic f i l t e r s obtained with a Coleman 124 Spectrophotometer. WAVELENGTH-nm. CO -p. 35A Fig. 7: Transmittance patterns of the CBS 650 red and 730 far red f i l t e r s , obtained with a Coleman 124 Spectrophotometer. 36 the C0 2 was removed by enclosing a small vial containing 1.0 ml of 1M KOH within the Petri dishes. For the 1 hr long gas treatments, uncovered Petri dishes were enclosed in separate Plexiglas chambers (20 x 20 x 4 cm). Different gas mixtures were circulated through the chambers at a rate of 0.5 1 min"^. Before entering the chamber, the gases were humidified by bubbling the gas streams through d i s t i l l e d water. After the 1 hr treatments, laboratory air was circulated through the chambers for 10 min, and then the chambers were completely sealed. To f a c i l i t a t e the pH transfer experiments, transfer assemblies were prepared by removing the floors from the bottom parts of several small plastic Petri plates (Fig. 8). The floors were replaced by a fine nylon mesh which was placed in the "cover" of the Petri plate containing the buffer solution. This assembly was then covered with another Petri plate. Seeds placed on the mesh could be easily transferred by moving the assembly from one Petri plate to another under dim green safelight. Between buffers, the seeds were blotted and then rinsed thrice with a wash of the forthcoming buffer solution. Measurements of buffer pH at the end of different germination and transfer experiments indicates that during these investigations the pH remained within 0.25 pH units of the i n i t i a l value. The basic procedures used for germination experiments on Amaranthus  retroflexus seeds were similar to those used with lettuce. The seeds were imbibed in different buffer solutions at 23±1 C for 24 hrs in darkness, irradiated with red light for 5 minutes, and thereafter incu-bated in darkness. After illumination, the Amaranthus seeds were incubated at 37-1 C for 48 hrs and percentage germination was then determined. Fig. 8: Transfer assemblies used for the pH transfer experiments. RESULTS AND DISCUSSION 1. Studies on the photoperiodic control of flowering in Xanthium  pennsylvanicum L. (i) Night interruption experiments An i n i t i a l series of experiments was done to test whether C0 2 removal influenced the flowering response of Xanthium pennsylvanicum to treatments of red light given in the middle of an inductive dark period. Removal of C0 2 by flushing the chamber with medical grade C0 2-free a i r for 35 minutes in the middle of the dark period prevented the effect of red light interruptions (Fig. 9). Plants exposed to short days reached stages 4.5 and 7.5 after 10 and 20 days respectively. Those exposed to the same number of SD cycles with a red ligh t inter-ruption in the middle of the dark period in normal a i r , remained vege-tative. In contrast to t h i s , when the interruption was given in the absence of C0 2, i t was ineffective in inhibiting the flowering response. The morphogenetic responses to light and C0 2 treatments were deter-mined by observing transverse sections of the apical buds (Fig. 10). Buds from plants in the dark controls, and from plants given red light treatments in the absence of C0 2, showed differentiation of f l o r a l primordia (Figs. 10A and IOC). Plants receiving red light in the presence of atmospheric C0 2 remained vegetative (Fig. 10B). The development of flor a l primordia in plants receiving a red light interruption during the C0 2~free flush was slower than in the dark controls. This may have been caused by the incomplete removal of C0 2 during the C0 2-free flush. It is unlikely that the C0 2 concentration during the C0 2-free flush was zero because C0 2 was undoubtedly being produced by plant respiration. The results of this experiment, however, 39A Fig. 9: Effect of C0 2 removal during red ligh t interruption on development of flor a l buds in Xanthium pennsylvanicum. C0 2 was removed for 35 min with the red light interruption being given for 5 min in the middle of the C0 2 removal period. T I M E - h rs 8 — i — 12 16 2 0 ? 4 N U M B E R O F C Y C L E S S T A G E O F I N D U C T I O N a f rer l O D a y s 2 0 D a y s 4.5 7.5 8 0 0 - C O 2 2.5 4.5 39A Fig. 9: Effect of C0 2 removal during red light interruption on development of flor a l buds in Xanthium pennsylvanicum. C0 2 was removed for 35 min with the red light interruption being given for 5 min in the middle of the C0 2 removal period. 39 s l A clearly indicate that there is a requirement for CC^ for red light to be effective in inhibiting the i n i t i a t i o n of floral buds in Xanthium  pennsylvanicum under otherwise inductive conditions. Previous studies on the role of CO^  in photoperiodism are few in number and have not always been conclusive. There have been conflicting reports on the requirement of carbon dioxide in the dark period for flo r a l induction in long and short day plants. The absence of carbon dioxide in the inductive dark period has been shown to inhibit the development of floral buds in Xanthium pennsylvanicum (98). A similar requirement for CCy in the dark period has also been reported for other plants (126, 57). On the other hand, Campbell and Leopold (22) found no effect of removal of carbon dioxide in the dark period on the flowering of Xanthium. Fredericq (50) made similar observations on KalanchoS, a SD plant, and on Hyoscyamus and Sinapis, LD plants. The slower development of flor a l buds in the experiments reported here could be explained on the basis of a simultaneous inhibitory effect of CO2 removal. But circulation of CO^-free air in the chamber for the same duration of time in an uninterrupted dark period had no effect on the development of fl o r a l buds (Table 1). The effects of red light night interruptions, given after 4 or 8 hr of darkness, were not influenced by exposure of the plants to high (5%) CO2 concentration (Fig. 11). The relative ineffectiveness of red light interruption after 4 hours of darkness is in agreement with reported observations on the kinetics of interruption effects (148). Interactions between carbon dioxide and red light interruption effects have been studied or suggested by other workers. Kirkland and TABLE 1 Effect of removal of C0 2 in the middle of a dark inductive period on the flowering response of Xanthium pennsylvanicum. , i i £ • Stage of Treatment Number of induction short days ( A f t e r 1 4 d a y s ) A 8 6.8 B 8 6.3 A: SD control B: C0 2 was removed from the chamber for 35 minutes in the middle of the dark period by flushing the chamber with C0 2-free a i r with a flow rate of approximately 2500 ml min - 1. 42A Fig. 11: Effect of C 0 2 addition during red light interruption given after 4 hours of darkness, on development of fl o r a l buds in Xanthium pennsylvanicum. was added for 35 min with the red light interruption being given for 5 min in the middle of the C0 2 removal period. All the plants given a red ligh t interruption after 8 hours of darkness under either normal air or 5% C0 9 remained vegetative. T I M E - h rs N U M B E R O F C Y C L E S r • 0 »< 4 8 , 1 -j -i 12 16 2 0 24 t J R m R •»«>fr-«"^*r"r-ij»7'rT"w»^>T>»?!f *rmvw->^-'f.*r-y^.'rvrr^i!rv ^w-r^**wr.-"T'MTTwr-'fl'*fl' S T A G E O F I N D U C T I O N o f f e r 10 Days 6-2 6-0 6-0 +co2 Light MM Dark 43 Posner (91) have recently demonstrated that DGMU can supplement far red light in nullifying the inhibitory effect of red light on flowering. They concluded that photosynthetic as well as phytochrome pigments are involved in the photoperiodic regulation of flo r a l development, indir-ectly suggesting the involvement of C0 2 in bringing about the interrup-tion effects. Moshkov ejt a]_. (127) reported that C0 2 is essential during the light interruption period in Brassica carinata, a LD plant, for causing induction in otherwise non-inductive conditions. C0 2 was not essential, however, in Peri 11a ocymoides, a SD plant. They suggested that CG"2 fixation during red light interruption is c r i t i c a l only for induction in LD plants under non-inductive conditions. The results reported here for Xanthium are contradictory to their hypothesis that nyctophylic (SD) plants dp not require C0 2 for a light interruption to be effective. ( i i ) ^ C 0 2 feeding experiments It was of interest to test whether C0 2 fixation is responsible for the C0 2 requirement during red light night interruption in Xanthium. The approach taken was to determine i f leaf C0 2 exchange during the night interruptions was greatly altered by light and C0 2 treatments which produced different flowering responses. Two previous studies have considered the effect of the night interruptions on gas exchange. Leopold and Guernsey (100) found that red light decreased net 0 2 uptake in the LD plant, Hordeum vulgare. Sen (157) reported that ^ C 0 2 which had been fixed during the preceding darkness was released during red 44 light night interruptions, and a faster rate of release was observed with far red light following the red light. Because these previous studies used interruptions which were much longer (>30 minutes) than are required to control flowering, the relevance of the gas exchange observations to the effects of light on flowering is not certain. In the present study, the night interruptions were kept quite short (<5 minutes) in an attempt to find clearcut links between C0 2 exchange and flowering. The role of C0 2 was investigated by measuring the relative ^ C 0 2 fixation rates during and after night interruptions, 14 and leaf extracts were obtained and analyzed for products of C0 2 fixation. Net fixation of 1 4 C 0 2 was obtained both in darkness and in red light given after 8 hours of darkness (Table 2). But the amount of recovered in the ethanol soluble fraction was higher in red light than in darkness. In the earlier experiments on the requirement of C0 2 for the red light response, the CC^-free treatment was continued for 15 min after the red light interruption. It was therefore possible that the presence of C0 2 may be important during the post-illumination time period. Experi 14 ments were done to test i f red light affects the uptake of C0 2 in the dark period immediately following light treatment. In the post-illumination dark period there was slightly less radio-ac t i v i t y in the ethanol soluble fraction compared to the dark controls (Table 3). A reduction in the rate of ^ C 0 2 fixation during the dark period following a night interruption has also been shown by earlier 14 workers (98, 126). The absence of an effect of red light on C0 2 45 TABLE 2 Effect of red light interruption on the total 1 4C activity recovered 14 in ethanol soluble fraction after C0 9 fixation. Treatment C Activity (Cpm) Dark Red 3216 39112 This leaf was exposed to CQ^ for 5 minutes in both treatments. The results are the average of three t r i a l s . TABLE 3 Effect of red light interruption on the total 1 x activity recovered 14 in ethanol soluble fraction by dark CCL fixation. Treatment 1 4C Activity (Cpm) Dark 633 control Dark after Q^Q red light The leaf was exposed to C0 2 for 5 minutes in both treatments. For 14 the red light treatment, the C0 2 was released in the chamber immediately after the exposure of the leaf to 5 minutes of red light. The extract from each leaf was concentrated to 1 ml and 50 ul sample was taken from each extract for analysis. 47 exchange in the post-illumination dark period rules out the possibility that the red light interruption effects are due to increased carboxy-lation in darkness immediately following red light. 14 14 • The distribution of C activity among amino acid products of CC^ fixation obtained during red light alone and red followed by far red lig h t , was determined with paper chromatography and liquid s c i n t i l l a t i o n counting. Fig. 12 is a trace drawing of the amino acids recovered after ninhydrin staining of paper chromatograms of the ethanol-soluble extracts run in butanolracetic acid:water. Similar patterns were obtained with red light alone, with red followed by far red l i g h t , and with the dark controls. Paper strips were cut separately for a l l values and used directly for liquid s c i n t i l l a t i o n counting. In comparison with the dark controls, the red light night interruptions caused a considerable increase in a l l the amino acids except tyrosine, valine and leucine (Table 4). 14 When red light was followed by far red l i g h t , the amount of C recovered in most of the amino acids was higher s t i l l . Again, the effect was not as marked in proline, tyrosine, valine and leucine. There have been earlier reports on the effects of night interruptions on the distribution of different compounds in the leaves. Mitrakos ejt a l . (116) found that the ninhydrin reacting compounds of ethanol extracts from etiolated corn leaf tissue decreased after a short,red light treat-ment. A general decrease in the free amino acid pool with red light has also been reported in other etiolated tissues (see 115). The findings reported here using green Xanthium leaves are in contrast to these reports and the difference could be due to the difference in the type of tissue used. It does not seem that general conclusions can be drawn from this ISA Fig. 12: Trace drawing of the amino acids stained with ninhydrin on chromatogram of the ethanol-soluble extracts of leaves from 14 different treatments. C0 2 was fed during a 5 min red light interruption in the middle of the dark period followed by either 5 min of darkness or 5 min of far red light before the leaves were k i l l e d . In the dark controls, the leaves were allowed 14 to f i x C0 o for 10 minutes. 4 8 T R E A T M E N T TABLE 4 Effect of different treatments on the C activity in different amino acids synthesized during short term feeding experiments. C Activity (Cpm)* Amino acid Rf Value D R + D R + FR Asn .13 . 289 340 500 Arg .17 292 669 1480 Asp .22 340 1020 1770 Ala .28 322 590 2123 B.Ala ,31 295 591 1882 Pro .37 296 700 782 Tyr .45 283 311 332 Val .53 301 331 360 Leu .65 293 334 401 * A l l the observations are mean of two samples. D: Dark R: Red FR: Far red 50 data regarding the metabolic aspects of phytochrome action. The 14 increased incorporation of C into the amino acid fraction reported here may well be the result of photosynthetic carbon fixation following red light. Further experiments need to be done to explain the increased activity in amino acid fraction after far red light following red light. There should be negligible photosynthesis in this wavelength. Mitrakos and Margaris (114), in studies with corn leaves and lettuce seeds, 14 showed that red light enhances the incorporation of leucine- C into proteins. It may be that far red light immediately following red light prevents incorporation of amino acids into proteins leading to an increase in the pool of free amino acids. ( i i i ) C0 2 exchange experiments The patterns of leaf C0 2 exchange during the night interruption experiments were observed by following the changes in C0 2 concentration within a closed gas exchange system. In darkness, leaf respiration increased the C0 2 concentration at a rate which was virtually constant between 200 and 400 ppm C0 2 (Fig. 13). During a 5 minute red light interruption, the net rate of C0 2 production decreased for the f i r s t minute and was succeeded by net C0 2 fixation for the next 4 minutes. Following the interruption, respiration quickly resumed i t s original rate in darkness. Far red light, given either as an interruption of darkness or after red light, had l i t t l e effect on C0 2 exchange. It should be noted that the red and far red light treatments used here would be sufficient to determine the occurrence of flowering in Xanthium after the inductive 8 hr photoperiod. In some of the earlier work with Changes in CG^ concentration within the closed gas exchange system during the following night interruption treatments: ( ) 5 min low intensity red light ; ( ) 5 min far red light; ( •) 5 min low intensity red light followed by 5 min far red light. The interruptions were begun at the 5 min point on the time axis (+), which was 8 hr after the start of the dark period. 52 Kalanchoe" diagramontiana, far red light has been shown to increase CO^ output from the leaves (130) due to decarboxylation of acids. But Kalanchoe" ut i l i z e s Crassulacean acid metabolism for its carbon uptake and the data for this plant cannot be directly compared with Xanthium, a Cg plant. The effectiveness of a red light interruption in inhibiting f l o r a l induction in Xanthium varies according to the time i t is given after the start of the dark period (148). It is most effective in the middle of a 16 hr dark period whereas at 4 and 12 hours i t has a marginal effect. It was decided to study the effects of red and far red interruptions given 4, 8 or 12 hours after the start of the dark period on CO2 exchange by the leaves. The results obtained from closed system gas exchange experiments have been presented as rates of CO2 exchange (Fig. 14). In a l l cases the initial (time 0) CO2 concentration in the closed system was close to 250 ppm. There was considerable variation in individual readings obtained for net CO2 exchange rate, but the general effects of red and far red light on CO2 exchange were similar to the previous experiment. Similar patterns of C0 2 exchange were observed when the night interruptions were given after 4 or 12 hours of darkness instead of 8 hours. The apparent differences in net CO2 fixation rates between 4, 8 or 12 hour interruptions, reflect experimental va r i a b i l i t y . No consistent differences were observed on replications of this experiment. These results demon-strate no clear relationship between the rate of CO2 exchange and the effectiveness of red or far red light in bringing about the photoperiodic response. As shown by the earlier experiments, the flowering response to a red 53A Fig. 14: Effects of night interruptions after 4 (A), 8 (B) or 12 (C) hr of darkness on the rate of C0 2 exchange. The interruptions were begun at the 5 min point on the time axis (+) and were the same as in Fig.13. The i n i t i a l C0 2 concentration v/as 2 50 ppm. ( ) 5 min low intensity red l i g h t ; ( ) 5 min far red li g h t ; ( ) 5 min low intensity red light followed by 5 min far red light. 54 light interruption also depends on C0 2 concentration during the inter-ruption. In measurements of C0 2 exchange during the interruptions, leaf gas exchange was found to be modified by low C0 2 concentration as well as by red light (Fig. 15). At an i n i t i a l C0 2 concentration of 20 ppm, the rate of respiration in darkness was relatively high. This could have been due to the steepness of the C0 2 concentration gradient driving C0 2 efflux and possibly due to lower stomatal resistance as well (42). During the red light night interruption at low C0 2 concentration, respir-ation was apparently suppressed but net C0 2 fixation did not occur. The speed with which the rate of C0 2 exchange responded to the onset and completion of the red light treatment was not affected by the C0 2 concen-tration. The decrease in respiratory production of C0 2 during red light is a manifestation of certain carboxylation reactions occurring simul-taneously. Thus, at low C0 2 concentration when red light was ineffective in inhibiting flowering, C0 2 fixation was not completely inhibited. The rate of dark respiration continued to be higher under low C0 2 concen-tration after the light treatment. The decrease in rate of C0 2 production during red light is undoubtedly related to increased C0 2 fixation, and 14 this interpretation is borne out by the results from the C0 2 feedings described earlier. The results in Fig. 15 therefore suggest that at low C0 2 concentration appreciable C0 2 fixation occurred during the red light interruption even though net fixation was not observed. The effect of a red light night interruption on flowering is energy dependent. A very brief night interruption will control the occurrence of flowering i f the light intensity used is sufficiently high. Longer interruptions are required when lower intensities are used (148). The Fig. 15: Effects of a 5 min red light interruption on the rate of CG^ exchange by leaves exposed to i n i t i a l CG^ concentrations of 20 ppm (A) or 350 ppm (B). The interruptions were begun at the 5 min point on the time axis ( + ), which was 8 hr after the start of the dark period. The changes in CO2 exchange caused by night interruption (Table 8) were determined from the decrease in CO2 release plus C 0 2 uptake associated with the interruptions, as illustrated by the hatched area on curve A. 56 amount of C0 2 fixation during low and high intensity interruptions may therefore provide evidence of a requirement for carboxylation for red ligh t effects. The effect of short, high intensity night interruptions on leaf C0 2 exchange, however, were quite minor (Figs. 16 and 17). Such interruptions caused transient depressions in the rate of C0 2 release, and the size of the depression was increased by extending the interruption. It may be mentioned that these high intensity interruptions were effective in inhibiting the flowering response. Thus, the occurrence of net fixation was not essential for a red light night interruption to be effective in inhibiting flowering. C0 2 exchange was measured during longer interruptions involving different intensities of red l i g h t . Low, medium and high intensities of -2 -1 red light (12, 25 and 125 yW m nm ) did not cause large differences in rates of C0 2 exchange, especially during the f i r s t 2 minutes of a 5 minute interruption (Fig. 18). At low and medium intensities, similar and relatively constant rates of net C0 2 fixation were maintained after the f i r s t 2 minutes of the interruption. At the high light intensity, net C0 2 fixation gradually increased during that period possibly because of decreas-ing stomatal resistance. Red light can induce stomatal opening in leaves (16, 110, 108). Comparisons of the effects on C0 2 exchange of different intensities and lengths of red light treatments could; therefore, be complicated by variations in stomatal resistance to C0 2 exchange. Measurements of stomatal resistance (Table 5), however, suggest that this is not a serious complication in the present studies. Stomatal resistance following 5 min of low or medium intensity red light or 1 min of high intensity red light 57A Fig. 16: Effect of a 10 sec interruption with high intensity red light on the net rate of CO2 release. The interruption was given at 8 hr after the start of the dark period and the i n i t i a l C0 ? concentration was 350 ppm. RATE OF CO2 RELEASE (ppm mi iv) _ M 1 1 r~ 1 \ J i-I o CD O J L )8A Fig. 17: Changes in C0 2 concentration within the closed gas exchange system during 30 and 60 sec of high intensity red ligh t night interruption. 58 370 r 59A Fig. 18: Net rates of C0 2 exchange during 5 min night interruptions with low ( • -••)> medium ( A • A ) , or high ( a • ) intensity red light. The i n i t i a l C0 2 concentration was 350 ppm and the interruptions were begun (time = 0) after 8 hr of darkness. TABLE 5 Stomatal resistance of the lower surface of the leaf under different conditions used for measurement of CG^ exchange. Light Interruption Stomatal Intensity Duration Resistance** Condition (yw cm - 2 my-1) (min) (sec cm-1) Daylight - - 6.8 Dark* - - 20.0 Red Light Interruption 12 5 19.4 25 5 17.0 125 1 22.0 125 5 13.0 * After 8 hours of dark ** Mean of three readings 61 were similar to the resistance in the darkened leaves. Only after more than 1 min in high intensity red light, stomatal resistance decreased and began to approach the daylight controls. These results show that the main effects of light treatments on flowering and gas exchange in the present investigations were not due to variations in the stomatal resistance. 2. Studies on phytochrome-mediated seed germination Photoperiodic responses are very complex and depend upon many factors including l i g h t , temperature, carbon dioxide, growth hormones and circadian rhythms. The relationship reported in the preceding section between the effects of red light interruptions and the presence of C0 2, may not necessarily represent a direct C0 2 requirement for phytochrome action. It may be that by removing C0 2 during a night interruption, the effect of one of these other factors is altered to cause the response. If the influence of C0 2 on flowering is closely linked to phytochrome, then C0 2 could be expected to influence other phytochrome-dependent phenomena. It was important, therefore, to test the response to C0 2 in other phytochrome-mediated processes. It was decided to study the germination of light sensitive seeds, one of the classical phytochrome-mediated responses. The action spectra for promotion and inhibition of Grand Rapids lettuce seeds(13) were markedly similar to ones for the control of flowering of Xanthium (137), clearly indicating that the light reaction controlling seed germination was identical with the one controlling the 62 flowering of long and short day plants. The absence of endogenous rhythms, the relatively quick response time, and the elimination of any interference from photosynthetic C0 2 fixation, were some of the additional factors in the choice of seed germination for further inves-tigations. (i) Regulation by C0 2 and pH of phytochrome-mediated seed germination in Lactuca sativa and Amaranthus retroflexus In an i n i t i a l experiment, lettuce seeds were germinated in d i s t i l l e d water, and in some cases C0 2 was removed from the germination chamber for the f i r s t 30 minutes of imbibition. Red light was given to some seed samples for a period of 5 minutes starting after 15 minutes of imbibition. In darkness, seed germination was negligible after 24 hr of imbibition but improved when the seeds were assayed after 48 hr (Table 6). Seed germination in a i r was promoted by red light as expected. After flushing the chamber with C0 2-free a i r , germination of red-irradiated seeds was considerably lower after both 24 and 48 hours of imbibition. The difference between the effects of co 2 treatments, however, was more pronounced after 24 hours. The values of percentage germination in Table 6 were a l l different at the 1% level of significance. As in the flowering experiments, the C0 2 treatments used were external treatments and the lack of complete inhibition of germination by treatment C0 2-free a i r could have been caused by incomplete removal of C0 2 from the immediate v i c i n i t y of the seed embryos. However, the results obtained clearly demonstrate the involvement of C0 2 in red li g h t TABLE 6 Effect of C0 2 removal during red light irradiation on lettuce seed germination. (hr) Percent Germination* Imbibition Continuous Dark Red Irradiation Time ; Air Air C02~Free Air 24 0.8 40.4 17.2 48 26.0 82.3 67.3 * Mean values from two experiments. 64 effects also extends to seed germination. This is remarkable because flowering and seed germination represent two quite different phytochrome-mediated responses. It is therefore possible that C0 2 may be generally involved in phytochrome-mediated morphogenesis. As will be reviewed in the general discussion, the data from the earlier C0 2 exchange studies in Xanthium did not support the hypothesis that the C0 2 requirement for inhibition of flowering by red light is associated with C0 2 fixation. It was possible that the C0 2 effects on flowering and seed germination could have arisen from general changes in tissue characteristics, such as pH changes, which might have occurred at subnormal C0 2 concentrations. In vitro experiments had shown that the photoreversibility of isolated phytochrome pigment is restricted to a narrow range from pH 6.5 to 7.5 (115). I speculated that the removal of C0 2 may change the pH of the tissues or their environment, and thereby check the photoreversibility of phytochrome. There are inherent d i f f i c u l t i e s in measuring the pH of intact c e l l s . Several.attempts to study the effect of C0 2 on the pH of crude extracts of Xanthium leaves were inconclusive. The approach taken to test this possible mode of C0 2 action was then to control the pH in the medium surrounding the tissue. It was not practical to do such experiments with Xanthium leaves. It was, however, feasible to control the pH about germinating seeds by using pH buffers. Experiments were therefore done to study the relationship between lig h t , pH, and C0 2 effects using such a system. It was possible to vary the timing of pH and C0 2 treatments to determine whether their effects were directly linked with the effects 65 of irradiation. The seeds were incubated in citrate-phosphate buffer from pH 4 to 7. At a l l pH levels used, either far red irradiation or continuous darkness resulted in very low (<2%) germination in seeds assayed 24 hr after the start of imbibition. Red irradiation given for 5 minutes after 30 minutes of imbibition promoted seed germination in buffer at pH 4 (Fig. 19). The promotion declined with increasing pH and only 10% of the seeds germinated at pH 7. At any pH, the red light promotion was completely reversed by far red light. Conversely, in seeds i n i t i a l l y exposed to far red ligh t , a subsequent red light treatment promoted germination at pH 4 and not at pH 8. These results, therefore, corres-pond to the classical observations on phytochrome-mediated lettuce seed germination (13) with the addition of an interaction with p H . At this stage in the project, i t was possible that this pH response of germination was a pecularity of lettuce seeds in citrate-phosphate buffers. It was important to test whether similar results would be obtained when other buffers or seeds were used. Over the pH ranges where they buffer effectively, either citrate (pH 4 to 6) or phosphate (pH 6 to 8) buffers alone,had comparable effects to the citrate-phosphate buffers (Fig. 19). In acetate buffer, the seeds did not germinate but gradually swelled in size with the testa eventually splitting open. Germination in A. retroflexus seed is also under phytochrome control (172), and the seed has both a low temperature and a red light requirement for germination. It was decided to test this seed for germination res-ponse to pH. The effect of pH on this seed (Fig. 20) was markedly similar to the effect on lettuce. The maximum rate of germination observed with 6 6 A Fig. 19: Effects of pH and CO,, concentration on lettuce seed germination following red irradiation. Seeds were in normal a i r ( © . • a ) or in C0 2-free a i r ( o ) , and they were incubated in cit r a t e -phosphate (•.O), citrate ( • ) , or phosphate (•) buffers. For the reaction: C0 2 + H20 ^HCOg" + H+, the dashed line indicates the percentage of total carbon in the form of C0 2 at different pH levels (pK =6.1). 67A Fig. 20: Effects of pH and C0 2 concentration on seed germination in Amaranthus retroflexus L. following red irradiation. Seeds were in normal a i r ( •) or in C0 2~free air ( o ) and they were incubated in citrate-phosphate buffers in a l l cases except pH 7.5 and 8.0 where phosphate buffer was used. The dashed line represents the C0?/HC03~ equilibrium as in Fig. 19. 68 A. retroflexus was relatively less than with lettuce, and this d i f f e r -ence was probably the result of differences in seed v i a b i l i t y , germin-ation rate and assay time. The percentage germination in water controls was essentially the same as in citrate-phosphate buffer at pH 4. The equilibrium between dissolved C0 2 and bicarbonate ion (HCO^-) is pH dependent: C0 2 + H 2C\ p H - HC03" + H + It was noticed that there was a close similarity between the effects of pH on germination and on the carbon dioxide-bicarbonate equilibrium in both lettuce and Amaranthus seeds (Figs. 19 and 20). It was possible that the buffer treatments may have acted by regulating the form or level of inorganic carbon about the seeds during germination. That i s , the presence of C0 2 or the absence of HCO^- could have been required for the red light response. These alternatives were tested using a C0 2 absorber to remove C0 2 from the atmosphere about the seeds at different pH levels. The effect of C0 2 a v a i l a b i l i t y in the medium on relative amounts of C0 2 and HCO^ " depending on pH, can be diagramatically repres-ented as follows: C0 2 - Air J pH C0 o - Solution — ^ HC0o~ Solution C0 2 -''Seed Since pH determines the ratio of the CO^HCO^- (pK. = 6.1) and not their absolute amounts, depletion of atmospheric C0 2 should lead to a marked decrease in dissolved C0 o at low (<< pH 6.1) pH levels and in HC0^~ 69 content at higher (>> pH '6.1) pH levels. If the lack of germination at high pH values is due to inhibition by HC03~, this inhibition should be reversed when atmospheric C0 2 is removed. Continuous removal of C0 2 by absorption with KOH reduced germination at a l l pH levels (Fig. 19). There was no increase in germination at pH 6.5 or above, where carbon is mainly in the form of HCO^". This experi-ment suggests that the presence of molecular C0 2 may be required for phytochrome-mediated seed germination and that bicarbonate does not act as an inhibitor. Similarly, in A. retroflexus, removal of atmospheric C0 2 suppressed the germination response to red light at a l l pH levels (Fig. 20). This provides additional evidence that the effect of pH on seed germination may be general for light sensitive seeds and that pH may be acting via regulation of the C02/HC03~ equilibrium. In these experiments, the link between pH and C0 2 effects was c i r -cumstantial. It was considered worthwhile to attempt to measure the changes in free carbon dioxide about the seeds in different pH buffers. Seeds were imbibed in batches of 100 in a Plexiglas chamber. The C0 2 content of the chamber air was detected by connecting the chamber to an infra-red gas analyzer through a closed system. Another approach was to use mass spectrometry to measure the amount of total C in 5 ml of the buffer solution at different pH values. Unfortunately, in both cases the changes in the CO-, content were too low to be detected, 70 ( i i ) pH and CO2 transfer experiments In the experiments described so far, CO2 or pH treatments were given continuously throughout the incubation period. In the normal course of germination, the presence of the P^r form of phytochrome triggers the process and radicle. elongation then ensues, causing the testa to break open. If there is a similarity between the CO2 requirement for red light effects on flowering and seed germination, the CO2 effect should be on the i n i t i a l stages of phytochrome action during or after the photo-reactions. Thus, i t was of interest to determine whether the effect of (X^-free air or pH treatments was on the induction of germination or on the reactions following induction. Several experiments were done in which lettuce seeds were incubated in buffers and irradiated as before except that different gases were circulated about the seeds during the f i r s t hour of incubation. The results from these experiments should not be directly compared with the other results since different germination chambers were used and slight drying of the seeds by the circulating gases caused a small reduction in the level of germination. In a l l cases, seed germination decreased as the pH increased (Table 7). Whenever appreciable germination occurred, the use of CX^-free a i r for 1 hour resulted in lower germination than in 5% CO2 in a i r . Simil-arly, when C^-free ^ was circulated around the seeds for 1 hour, there was a marked decrease in germination. The suppression of red lig h t -promoted germination by removal of O2 has also been reported earlier in literature (54, 71). The latter workers interpreted this to be due to the requirement for oxidation reactions for P^r action. It i s possible, TABLE 7 Effects of pH and 1 hr-long gas treatments on lettuce seed germination. Percent Germination PH C02-Free Air Gas Treatment3 5% C0 2 N 2 3% C0 2 in N 2 4.0 73.5 82.5 48.5 76.5 5.0 56.0 68.0 20.5 42.5 6.0 42.5 59.5 - 9.5 36.0 7.0 3.0 2.5 6.0 5.3 aFor the gas treatments, gases were circulated in the germination chamber for 60 minutes from the start of imbibition, and red radiation was given for 5 minutes beginning at 30 minutes after the start of imbibition. The pH treatments were continuous until germination was measured at 24 h following the start of imbibition. 72 however, that the effect of anaerobsis was partly due to a reduction in respiratory C0 2 production because below pH 7 .0 , much higher levels of germination occurred with 3% C0 2 in N 2 than in N 2 alone. Analysis of variance of the data summarized in Table 7 indicated that the effects of pH, C0 2 concentration and 0 2 concentration, were highly significant (<1% level). It is important that the effects of these gas treatments were detected following short treatments early in the imbibition period since i t suggests that C0 2 and 0 2 may influence events near the onset of germination. . . < The action of pH was further examined in several experiments in which lettuce seeds were transferred between pH 4 and pH 8 buffers, before or after red irradiation. Germination was negligible after 24 hr of incubation unless the seeds received both red light and pH 4 (Fig. 21 a-e). Germination was promoted by red light even i f the pH 4 treatment was restricted to the start of the incubation period including the period of ligh t treatment (Fig. 21 f ) . The effect of the pH 4 treatment, therefore, appeared to be on the induction of germination and pH 8 did not prevent germinati on following completion of induction. Transfer from pH 8 to pH 4 immediately after irradiation, however, also resulted in germination (Fig. 21 g). Experiments summarized in Fig. 21 h-j were done to test whether pH 4 is effective when given before the red light. With red irradiation at 3 hour following the start of imbibition, germination was very high at pH 4 (Fig. 21 h) and also was moderate at pH 8 (Fig. 21 i ) . These increased rates of germination probably reflect increased sensitivity to red l i g h t given later in the imbibition period (164 ). Brief treatments 73A Fig. 21: Transfer experiments showing the effects of the timing of pH 4.0 or 8.0 and red irradiation on percentage germination of lettuce seeds. Vertical arrows indicate the times when 5 min red irradiation was applied. 73 pH 4 L = i p H 8 % GERM I NATION e c 1 f E T — J g oa 1 I I I I I 1 I 1 1 1 0 3 6 9 12 15 18 21 24 TIME ( h ) 1.5 0.0 J 0-0 J 0.0 82.9 92.6 97.2 • 42.3 45.3 74 with pH 4 at 2.5 hours before the red light did not appreciably improve germination above the value observed in pH 8 (Fig. 21 j ) . It seems, therefore, that pH 4 must be given during or after red irradiation to be effective. Similar experiments demonstrated that germination would ensue following red irradiation even i f the pH 4 treatment was given many hours after the irradiation. In pH 8 (Fig. 22 a-d), there was a gradual increase in percentage germination i f the incubation period was extended beyond 24 hours. Such an increase, however, also occurred in the dark, and those low rates of germination, therefore, do not represent a response to light treatment. When the incubation period was kept constant at 24 hours, the level of germination decreased as the time interval between i r r a d i -ation and transfer to pH 4 increased (Fig. 22 e-j). This could be inter-preted in many ways. The response could be due to the delay in applying pH 4. For example, P^r may gradually be destroyed following the red light during the time before pH 4 is given, thereby becoming unavailable for the induction of germination. Another possible reason for the decrease in germination could be the decreased time available between the exposure to pH 4 and the assay of germination. These possi b i l i t i e s were tested by increasing the time of pH 4 treatment following the transfer from pH 8 to 24 hours. In a l l cases, extending the time in pH 4 to 24 hr increased the germination to almost 100% (Fig. 22 k-m). Therefore, a delay in supplying pH 4 following red light treatment does not reduce the level of germination as long as sufficient time is provided between the onset of pH 4 and the assay of germination. From these experiments, i t is evident that low pH is essential for 75A Fig. 22: Transfer experiments showing the effects of duration of incubation and time of pH 4.0 treatment on percentage germination of lettuce seeds. Vertical arrows indicate the times when 5 min red irradiation was applied. 75 a I i 0.0 b 1 ! 15.8 C 1 ' 24.5 H) j 31.1 1 i j l WW 30.3 I l kasdSfflMMB^ ^ 98.2 I 1 m 1 rj^mbevBw&iimsyjssza 100.0 1 1 1 1 ! 1 1 48 36 24 12 0 12 24 TIME Ch) 76 the promotion of lettuce germination by red light. The requirement can be f u l f i l l e d by exposure to low pH either during or after red light. The separation of the radiation and the pH effect in Fig. 22 indicates that the action of low pH is on later events in the process of germination than phytochrome phototransformation. The lack of germination after a 24 hour incubation in pH 8 (Fig. 22 a) and the rapid response to transfer to pH 4 (Fig. 22 j) support the earlier conclusion from Fig. 21 e,f, that low pH is required for the onset of germination following red light. In a l l the transfer experiments so far, pH 4 treatment was continued once the transfer from pH 8 had been made. Experiments were also done to test whether a brief exposure to pH 4 would satisfy the low pH require-ment following red irradiation. Seeds were exposed to different periods of pH 4 following red irradiation in pH 8 and then transferred back to pH 8 until germination was assayed. Similar levels of germination resulted from a l l the pH 4 treatments, even in the case where pH 4 was given for only 35 minutes following the light treatment (Fig. 23 c). It i s clear that the ini t i a t i o n of germination in red irradiated lettuce seeds requires a brief exposure to low pH, but the later course of germin-ation proceeds well in both pH 4 and pH 8, Many experiments with young seedlings and roots have shown the gradual disappearance of the far red absorbing form of phytochrome (P^ r) due to destruction or reversion to the red absorbing form (P ) in dark periods following P^r formation by red irradiation (103, 120). The disappearance of P^r in seeds, however, is less well established and in Amaranthus caudatus, some results have indicated that the original seed phytochrome is stable in time (27). An interesting finding in the 77 A Fig. 23: Transfer experiments showing the effects of the duration of pH 4.0 treatment on percentage germination in lettuce seeds following red irradiation (arrows). pH 4 i ipH 8 % G E R M I N A T I O N 0-0 91.1 C CEC • 94.4 d rE5H«r • 95.0 3 91.6 95.2 86-1 • 90-2 1 93 4 ^ ^ a s ^ ^ i ^ • loo.o _L I I I I I I I 0 9 12 15 T I M E (h) 18 21 24 78 pH transfer experiments was the observation that complete promotion by red light occurred even when pH 4 was not given until 24 hour after irradiation (Fig. 22 m). This suggested that either P^r was stable during the interval between the red irradiation and pH 4 treatment, or a product of P^r was formed and stabilized during that interval. To distinguish between these two alternatives, the effect of far red light after different time intervals following red treatment was investigated. As has been already shown (170), far red declined in i t s a b i l i t y to reverse the response to red light as the time interval between the irradiations increased (Fig. 24). The pH present during the interval did not affect the response. Since far red irradiation after a 24 hr interval in pH 8 did not prevent subsequent germination in pH 4, i t is evident that the earlier red light had produced a stable product (X) in the seed which was not photoreversible P^r, and which v/as effective in causing germination once pH 4 became available. As will be discussed later, the nature of the stable product is unknown at present. 79A Fig. 24: Effects of delay between 5 min red and far red irradiation treatments on percentage germination of lettuce seeds.. The seeds were incubated in citrate-phosphate buffers either at pH 4.0 throughout ( A ) , or in phosphate buffer at pH 3.0 until the far red treatment, then in pH 4.0 ( o ) . Percentage germin ation was measured at 24 hr following the far red treatments. 79 TIME B E T W E E N RED AND FAR RED . ( h ) GENERAL DISCUSSION This research has investigated the influence of pH and light on some aspects of phytochrome-mediated photomorphogenesis. The results clearly demonstrate that CO2 is essential for red light to be effective in two phytochrome-mediated responses: the inhibition of flowering in the SD plant, Xanthium, by red light interruptions in the middle of long dark periods, and the promotion of germination in light sensitive seeds. To the author's knowledge, there is only one earlier report which has considered the role of CO2 during a night interruption in a SD plant. In work with Peri 11 a ocymoides, i t was concluded that SD plants do not require CO2 for a night interruption to be effective (127). This contrasts with the CO2 requirement found in the present study with Xanthium. This difference could be due to several factors: physiological differences between Xanthium and Peri 11a, or differences in conditions or techniques between the experiments on the two species. It is possible, for example, that the removal of CO2 in the Peri 11 a experiments was not adequate to cause an effect. Even in the experiments reported here, the inhibition of the red light response by CO2 removal was not complete and this could have been due to incomplete removal of CO2 which was constantly replen-ished by respiration. The effect of different wavelengths of light on phytochrome responses has always been considered to be essentially photochemical. It is being reported here for the f i r s t time that the CO2 is required in addition to red light for phytochrome action in flowering and seed germination. 81 These findings raise the interesting possibility of a general CC^ requirement for phytochrome action. More recently, evidence has been obtained for the involvement of CG^ in phytochrome-mediated anthocyanin synthesis and in de-etiolation of mustard seedlings (J. Hoddinott, P. Hicklenton, personal communication). CG^ has also been shown to promote bean hypocotyl hook opening but i t is not certain whether CG^ is inter-acting directly with phytochrome (82). It is plausible that even in the experiments of Purohit and Tregunna (139) on the effects of on flower-ing, may have interacted with phytochrome. The i n i t i a l observations of a CG^ requirement for Xanthium flowering suggested the poss i b i l i t y that CO2 fixation reactions might be involved in bringing about the red light response. Circumstantial support for that pos b i l i t y was provided by the finding that the photosynthetic inhibitor DCMU could supplement far red light in nullifying the effect of red light on flowering in Lemna perpusilla (91). The evidence then available in l i t e r -ature on the requirement for CO2 during the dark period of SD plants was inconclusive (22, 57, 98, 126). Some attempts were made to study the effect of night interruptions on gas exchange (100, 157). Unfortunately, those attempts had employed much longer light treatments than are required for the effect on flowering. Thus, the relationship between CO2 exchange and phytochrome action remained uncertain. The results reported in this thesis on the effects of night interruption treatments on the CO2 exchange of Xanthium leaves will now be reviewed to assess whether or not a correlation exists between CO2 exchange during night inter ruptions and the effects of the night interruptions on flowering. Since net CO2 uptake was observed during some of the night inter-ruptions (Figs. 13, 14, 15), i t is evident that processes of C0 ? fixation 82 as well as CC^ production contributed to the CC^ exchange results. The number and relative a c t i v i t i e s of the individual exchange processes cannot be judged, however, since only net CO2 exchange was measured. There were technical d i f f i c u l t i e s in the replication of the 14 CO2 data, especially in studies of the effect of far red light on gas exchange; hence, most of the experiments involved the measurement of C 0 2 exchange with the IRGA. As an aid to the discussion of the C C ^ exchange results, Table 3 summarizes the CO2 exchange and flowering responses obtained during these investigations. The change in CO^ exchange caused by a night interruption was estimated from the decrease in net CO^ release plus any CO^ uptake observed during or immediately following a night interruption (Figs. 14, 15, 16, 17). It is clear from Table 8 that there was no consistent relationship between the effects of night interruptions on C 0 2 exchange and their subsequent effects on flowering. In several cases, treatments which had quite different effects on CC^ exchange had similar effects on flowering. Conversely, in some cases treatments with similar effects on CO2 exchange had different effects on flowering. In accord with the i n i t i a l experiment, lowering the CO2 concentration to 20 ppm prevented the inhibition of flowering by a red light night interruption. The low C 0 2 concentration, however, decreased the effect on C 0 2 exchange of a red light night interruption by only about 30%. In contrast to the low C0 2 concentration results, high intensity red light treatments inhibited flowering but involved much less change in CO2 exchange. Therefore, sufficiently high CO2 concentration must be present for red light to inhibit flowering, yet the effects of red light on flowering are not TABLE 8 Effects of night interruptions and C0 ? exchange by Xanthium pennsylvanicum L. leaves Light Treatments Initial Net Change Source Inhibition Type Duration (sec) Intensity x 10-4 (yW cm - 2 nnH) Time During Dark Period (hr) C0 2 Concentration (ppm) in C02 Exchanged* (yl C0 2 dm-2) of CO2 Exchange Data of Flowering** Red 300 12 4 350 351 Fig. 14 No Red 300 12 8 350 248 Fig. 14 Yes Red 300 12 12 350 320 Fig. 14 No Red 300 12 8 20 192 Fig. 15 No Red 300 12 8 350 299 Fig. 15 Yes Red 10 125 8 350 18 Fig. 16 Yes Red 30 25 8 350 11 Fig. 17 Yes Red 60 25 8 350 14 Fig. 17 Yes Far Red 300 20 8 350 18 Fig. 14 No. * Overall decrease in the amount of C0 2 released by the leaves during the red light night interruption and during the 2 min following the interruption. ** Based on observations of the morphology of dissected buds on the treated plants, using the scheme of Salisbury 045)to assess bud development. Flowering occurred when the 16 hr dark period was not interrupted by light. 8 4 correlated with exchange during the interruptions. Several explanations can be advanced to account for this apparent paradox. It is possible that different experimental conditions limit the effect of, or alter the requirement for, exchange during red light night interruptions. This possibility is consistent with the results obtained, but i t represents a relatively complex explanation. It demands that the effect of, or the requirement for, CO^  exchange be modified by CO2 concentration, by light wavelength and intensity, and by timing of the night interruption. Another alternative i s that some CO2 fixation is essential for red light to inhibit flowering and this essential fixation is masked by other more active CO2 exchange processes. The essential CO2 fixation occurs at effective rates only above a threshold CO2 concentration, but the occurrence of other CO2 exchange processes prevents a correlation between CO2 exchange during the night interruptions and flowering res-ponses. The appeal of this po s s i b i l i t y is diminished by the similar CO2 exchange results obtained with some light treatments which had d i f f e r -ent effects on flowering, such as interruptions given at different times during the dark period. A more straightforward explanation of the results i s that CO^ exchange is not causally involved in the inhibition of flowering by red ligh t . Instead, the presence of CO2 above a threshold concentration is a necessary condition for the inhibition, and the role of CO2 is equivalent to an activator of the red light responses. This view i s in accord with the very small effects on CO2 exchange of high intensity red light night interruptions which inhibited flowering. In addition, there 85 was l i t t l e indication with the far red treatments of any C0 2 exchange associated with the reversal of the red light effects. This poss i b i l i t y , therefore, appears to be the most simple and direct explanation of the results. Some earlier studies have reported interactions between P^ and sucrose in the photomorphogenesis of etiolated seedlings. For example, after excision of the cotyledons, an external carbohydrate supply was required for the occurrence of red and far red light effects on e t i o l -ation (2), terminal bud expansion (56), and the elimination of the lag phase in chlorophyll synthesis (30). Similarly, sucrose has been shown to promote flowering in Chenopodium seedlings (27). It has been suggested that in cases where the photoactivated phytochrome is spatially separated from the region of the response, sucrose may be required for the trans-location of the stimulus. Very recently, Satter e_t al_. (152) have reported a requirement for sucrose for the manifestation of the P^, effect on the rhythmicity of leaflet movement in Albizzia, a process which involves K+ and CI" transfers between ce l l s (150, 152). They proposed that sucrose may act as a substrate for ATP synthesis or may supply carbon skeletons for organic ions which move with potassium. In view of these interactions with sucrose, i t may be argued that the C0 2 requirement for phytochrome action is due to i t s involvement in carbohydrate synthesis during red light treatments. The evidence obtained in the present studies, however, does not support such a hypothesis. There was negligible C0 2 fixation obtained under conditions when red light was effective in inhibiting flowering in Xanthium (Figs. 16 and 17). The observation that the presence of C0 2 promotes lettuce 86 seed germination in red light in the absence of photosynthesis, also supports the interpretation that CC^ fixation i s not causally involved. In addition, light-induced anthocyanin synthesis in etiolated seedlings has recently been shown to be independent of photosynthesis during the light treatment (32). Thus, i t seems unlikely that CO^ is acting via sucrose photosynthesis in the results reported in this thesis. It remains possible that sucrose acted as a source of CO^ via respiration in the earlier studies on the role of sucrose in phytochrome action. Similarly, in reports on the inhibition of phytochrome-mediated ion transport, flowering and seed germination by anaerobic conditions or by respiratory inhibitors (25, 54, 71, 128, 164), the inhibitory effect has been suggested to be due to the inhibition of respiratory ATP formation or the suppression of substrate production (96, 151). On the basis of results obtained in the present investigations, i t can be suggested that the inhibitory actions of such treatments may be partly due to the suppression of respiratory CO^ production. This possibility is supported by the experiments on the effect of anaerobsis on seed germination in which the suppression of germination under O^-free conditions was largely overcome by supplying high CO2 concentration (Table 7). It is of interest to consider mechanisms other than CO2 fixation by which C 0 2 may regulate photomorphogenesis. Removal of CO2 may increase tissue pH through the C^/HCO^- equilibrium: C 0 2 + H 20 p H - HC0 3 " + H+ A change in pH may in turn affect many cellu l a r functions. The role of pH was examined by experiments on seed germination in which the atmospheric 87 concentration and the pH of the incubation medium were controlled together. It was found that there was a steady decrease in red light-induced germination when the pH of the incubation medium was increased from 4 to 8. This curious response of light-induced seed germination has never been reported before. In vitro experiments have shown that the phototransformation of isolated phytochrome is pH dependent (see 115). The photoreaction proceeds best between pH 6.5and 7.5. The present CO^  and pH effects on seed germination, however, cannot be explained on the basis of pH effects on phytochrome phototransformation. Results from the transfer experiments have shown that the low pH requirement can be f u l f i l l e d many hours after the red light treatment (Fig. 22). Thus, the germination res-ponse to pH appears to be independent of phytochrome phototransformation during red light. A comment may be made here about the uncertainty of the true nature of the 00*2'and pH treatments used during these investigations. These were external treatments, and i t is therefore not known whether they acted directly or indirectly through secondary changes in tissue charac-t e r i s t i c s which occurred at different CG^ and pH levels. Hence, i t becomes d i f f i c u l t to rigorously interpret the data in terms of the mode of action of these treatments. This kind of d i f f i c u l t y is an inherent problem in studies on the effect of external factors controlling plant growth and development. It is inevitable not only in relation to CG^ and pH, but also with other factors such as temperature. It can also be suggested that in the present studies, different buffers could be acting through changes in the ionic strength of the medium at different levels of pH; but the similarity in the effects of different buffers of different ionic 88 strengths tends to negate this possibility. Moreover, the f u l l induction of germination with a brief exposure to pH 4 in the early stages of imbibition cannot be explained on the basis of an inhibitory effect of ion concentration. Nevertheless, the effects of the treatments were highly consistent and reproducible. The experiments involving the transfer of seeds between different and pH treatments indicated that the effect of C0 2 was probably on early events during germination (Table 7). Similarly, there was a require-ment for low pH for induction of germination following red light treatment. Low pH must be given either during or after red light and the low pH requirement could be f u l f i l l e d within a few minutes (Figs. 21, 23). Although germination could be induced with pH 4 treatment as long as 24 hours following the red light, .-P^  escaped photoreversibil ity by far red light during this time period (Fig. 24). The close similarity between the effects of pH on seed germination and on the C02/HC03~ equilibrium is intriguing. It is possible that the results obtained in these experiments are either due to changes in pH or due to changes in the availability of C0 2 and HCO^". As mentioned earlier, the data from experiments on C0 2 removal indicate that the results cannot be explained by an inhibitory effect of HC0o" (Figs. 19, 20). It remains possible, however, that the inhibition of germination at high pH levels is essentially a pH effect irrespective of the amount of C0 2 or HCO^- available to the seeds. It must be recognized that the link between pH responses of seed germination and the COo/HCO^- equilibrium is circumstantial and the possibility of independent effects of pH and C0 9 cannot be ruled out on the basis of data presented in this thesis. 89 Negm et al_. (132) investigated the interacting effects of pH, C0 2 and ethylene treatments on thermodormancy of lettuce seeds. They found a high percentage of germination in normally thermodormant seeds over a wide pH range when high C0 2 concentration or ethylene was supplied. Thus, the effect of C0 2 on lettuce seed thermodormancy may d i f f e r from i t s influence on seed response to red light (Table 6). The results of Negm et al_., however, did not contain data on pH alone. The data pertaining to the experiments on the interaction between pH, C0 2 and red light in germination, obtained in the present investi-gations, can be summarized by a simple diagram (Fig. 25), which represents a general sequence of events during the ini t i a t i o n of germination in li g h t sensitive lettuce seeds. The photochemical transformations of the phytochrome are independent of external pH over the level of those factors used in these studies. There is l i t t l e evidence at present for the destruction of phytochrome in seeds although such observations have been reported in other phytochrome-controlled systems (120). It is postulated that P f r gives rise to a stable product X which results in the promotion of germination when the pH is less than 7.5. The nature of X is uncertain at present, but once i t has been formed i t remains present at pH 8 for 24 hours and is not eliminated by far red radiation. The experiments involving gas treatments indicated that-C0 2 may affect the early stages of germination (Table 7), but the specific steps which are sensitive to C0 2 have not been identified. If the pH effects on germination and on the COo/HCO^" equilibrium represent a functional correlation, then C0 2 could act at the same stage in germin-ation as pH 4. 90A Fig. 25: A model for the sequence of events in phytochrome-mediated seed germination. 90 co 2 / s 660 nm p B. p r 730 nm - + r " pH<Z5) * Promotion of Germination reversion destruction 91 It appears that X is a stable product of P^r which induces germin-ation on transfer of seeds to low pH even 24 hr after red lig h t treatment. This finding can be of far-reaching significance in understanding the immediate action of P^r in phytochrome responses. In spite of a l l the work on structure and properties of phytochrome, there is a large gap in our understanding of the events which take place between the phototransfor-mation of phytochrome and the occurrence of photomorphogenetic responses. It is hoped that the findings reported in this thesis on the temporal separation of a red Tight treatment and i t s ultimate effect on germin-ation, will provide an avenue for future studies on how P^r operates. It is worthwhile to consider the possibility that CG^ may have interacted with endogenous growth hormones to cause the observed effects on phytochrome-mediated responses. Much research has been done on the role of growth regulators in seed germination and flowering in plants. There are no reports, however, on the effects of growth regulators on the "night-break" phenomenon. There is no present reason to believe that the CG^ effects observed in these studies are mediated by any specific group of endogenous hormones, particularly the gibberellins, cytokinins, or auxins. In fact, the requirement for CO^ in phytochrome-mediated de-etiolation is in marked contrast to the requirement for auxin-mediated elongation in coleoptiles. The only well-established relation-ship between and a growth hormone is the interaction with ethylene. Both synergistic and antagonistic effects of CO2 and ethylene have been shown in different systems ( 1 ). Both inhibitory and promotory effects of ethylene on flowering have been shown, but i t is known to promote seed germination (1). However, in the present studies on the 92 interacting effects of CO2 and light on flowering and seed germination, i t is not li k e l y that CO2 acted via ethylene for the following reasons. In experiments in which the CO2 requirement for phytochrome action in flower-ing and seed germination was established, the chambers were flushed with a i r . Presumably the high flow rate of the flushing gas minimized any changes in ethylene levels about the tissues. Moreover, there was no difference in the extent of germination obtained when CO2 was removed either by flushing or by absorption with KOH. Even the promotory effect of removal of CO2 in the interruption experiments cannot be explained on the basis of antagonism with ethylene, since ethylene has been shown to inhibit flowering in Xanthium pennsylvanicum and the presence of CO2 in the red light night interruption experiments also acted to inhibit flower-ing. All this evidence indicates that in the present studies, any simple interaction between CO2 and ethylene cannot account for the results obtained. The promotive effect of CO2 on seed germination has long been known. Thornton (163), for example, had reported the promotory effect of CO2 on lettuce seed germination as early as 1936. Almost simultaneously, Flint and McAllister (43, 44) independently obtained the f i r s t evidence for the involvement of phytochrome in lettuce seed germination. It is surprising, therefore, that 40 years had to pass before the interrelationship between CO2 and phytochrome action in lettuce seed germination was discovered during the course of this thesis research. LITERATURE CITED 1. Abeles, F.B. 1973. Ethylene in Plant Biology. Academic Press Inc., New York and London, pp. 302. 2. Bertsch, W.F. and W.S. Hillman. 1961. The photoinhibition of growth in etiolated stem segments. I. Growth caused by sugar in Pisum. Am. J. Bot. 48: 504-511. 3. Bewley, J.D., M. Black and M. Negbi. 1967. Immediate action of phyto-chrome in light sensitive seeds. Nature 215: 648-649. 4. Bewley, J.D., M. Negbi and M. Black. 1968. 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Biochemical approaches, In Physiology of flowering in Pharbitis n i l , 121-138, S. Imamura (ed.), Jap. Soc. Plant Physiologists, Tokyo, Japan. m APPENDIX I . Preparation of buffers used in these studies: 1) Citrate Buffer Stock solutions A: 0.1 M solution of c i t r i c acid (20.01 g in 1000 ml) B: 0.1 M solution of sodium citrate (29.41 g, CgH507Na3.2H20 in 1000 ml) x ml of A + Y ml of B, diluted to a total of 100 ml x Y pH 33.0 17.0 4.0 26.8 23.2 4.5 20.5 29.5 5.0 14.9 35.1 5.5 9.5 41.5 6.0 2) Phosphate Buffer Stock solutions A: 0.2 M solution of monobasic sodium phosphate (27.8 g. in 1000 ml) B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na^HPO^. 7H20 in 1000 ml) x ml of A + Y ml of B, diluted to a total of 200 ml x 68.5 39.0 16.0 5.3 Y pH 31.5 6.5 61.0 7.0 84.0 7.5 94.7 8.0 3) Citrate-Phosphate Buffer Stock solutions A: 0.1 M solution o f . c i t r i c acid (21.01 gm in 1000 ml) 112 B: 0.2 M solution of dibasic sodium phosphate (53.65 gm of Na^ HPO 7H20 in 1000 ml) ^ x ml of A + Y ml of B, diluted to a total of 100 ml X Y PH 30.7 19.3 4.0 27.3 22.7 4.5 24.3 25.7 5.0 21.6 28.4 5.5 17.9 32.1 6.0 14.5 35.5 6.5 6.5 43.6 7.0 The pH of. a l l the buffers was verified with a pH meter before use. 

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