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Kinetics and photocontrol of hypocotyl elongation in etiolated mustard (Sinapis alba L.) Kristie, David Nickolos 1983

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\ KINETICS AND PHOTOCONTROL OF HYPOCOTYL ELONGATION IN ETIOLATED MUSTARD (SINAPIS ALBA L.) by DAVID NICKOLOS KRISTIE B.Sc.(Agr.),the University Of Guelph,l976 M.Sc.,the University Of Alberta,1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF PH.D. in THE FACULTY OF GRADUATE STUDIES Plant Science Department Faculty of Agriculture We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1983 © David Nickolos K r i s t i e , 1983 In presenting t h i s thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Plant Science The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: i i Abstract The kinetics of hypocotyl elongation in darkness, and the responses of elongation to brief and prolonged i r r a d i a t i o n s were investigated in white mustard ( Sinapis alba L.) seedlings and in other species. A growth measuring apparatus consisting of a linear displacement tranducer coupled to a signal d i f f e r e n t i a t o r was developed to provide continuous high resolution measurements of the rate of hypocotyl elongation. In darkness, the growth rate of Sinapis as well as other species often underwent sustained and highly regular o s c i l l a t i o n s with a period of 3-30 minutes. A long period o s c i l l a t i o n (>50 min) generated by nutational bending was also observed in the growth rate traces of many seedlings. The e f f e c t s of monochromatic l i g h t were tested at a number of wavelengths between 380 and 780 nm waveband. Based on differences in the kinetics and spectral dependence of the responses to l i g h t , f i v e d i s t i n c t types of photoresponses could be distinguished in Sinapis. (1) Brief or prolonged i r r a d i a t i o n s with 380-500 nm l i g h t i nhibited elongation after a lag of c_a. 1 minute. (2) Phototropic bending was induced by low fluence rates of blue l i g h t , which did not cause a rapid i n h i b i t i o n of growth, indicating that these two photoresponses were d i s t i n c t . (3) Brief 550-710 nm l i g h t pulses caused a 40-50% i n h i b i t i o n of growth after about a 5 minute lag. (4) Prolonged 660-710 nm i r r a d i a t i o n s caused a fluence rate-dependent i n h i b i t i o n , with k i n e t i c s d i s t i n c t from the e f f e c t s of brief l i g h t pulses. (5) Brief or prolonged i r r a d i a t i o n s with i i i 720-780 nm l i g h t caused a small t r a n s i e n t i n h i b i t i o n f o l l o w i n g the i r r a d i a t i o n , or had no e f f e c t . The i n h i b i t o r y e f f e c t s of b r i e f or prolonged i r r a d i a t i o n s with red or f a r red l i g h t were r a p i d l y r e v e r s e d by a longwave f a r red l i g h t p u l s e . The i n h i b i t o r y e f f e c t s of 710 nm l i g h t were l o s t a f t e r a red l i g h t pretreatment. I t was concluded that the r a p i d e f f e c t s of UV-blue l i g h t were mediated by a photoreceptor d i s t i n c t from phytochrome. Hypocotyl e l o n g a t i o n was also, r a p i d l y and r e v e r s i b l y c o n t r o l l e d by a phytochrome t h r e s h o l d r e a c t i o n , with a t h r e s h o l d l e v e l of ca. 5% P f r r e q u i r e d f o r i n h i b i t i o n . The phytochrome t h r e s h o l d r e a c t i o n was a p r e r e q u i s i t e to the response to continuous i r r a d i a t i o n s . i v Table of Contents Abstract i i L i s t of Tables y L i s t of Figures . .yi L i s t of Abbreviations v i i i Acknowledgement ix I . PREFACE 1 II . INTRODUCTION 4 2.1 Light And Plant Development 4 2.2 The Phytochrome System 6 2.3 Photoresponses 18 2.3.1 Low Energy Responses 19 2.3.2 The High Irradiance Responses 27 2*4 High Resolution Measurement Of Plant Growth 35 III . MATERIALS AND METHODS 37 3.1 Plant Materials 37 3.2 Growth Measurement 38 3.3 Cuvette And Seedling Environment 43 3.4 Technical Problems 44 3.5 Light Treatments 47 IV. RESULTS AND DISCUSSION 51 4.1 Growth In Darkness 51 4.2 Light Pulse Experiments 76 4.2.1 Eff e c t s Of Blue Light Pulses 76 4.2.2 E f f e c t s Of Red Light Pulses 81 4.2.3 R-FR R e v e r s i b i l i t y 87 4.2.4 380-780 nm. Light Pulses 91 4.2.5 380-500 nm Waveband 93 4.2.6 550-710 nm Waveband 93 4.2.7 720-780 nm Waveband 102 4.2.8 Eff e c t s Of Broadband Green Light 106 4.2.9 Phytochrome And The Response To Blue Light ..107 4.3 Effects Of Prolonged Irradiations And Repeated Light Pulses 112 4.3.1 UV-blue Waveband 112 4.3.2 The Rapid Blue Light Response And Phototropic Bending 113 4.3.3 Phytochrome And The Response To Continuous Irradiations 121 4.3.4 720-760 nm Waveband 123 4.3.5 660-710 nm Waveband .- 124 V. GENERAL DISCUSSION ....154 VI. CONCLUSIONS 168 BIBLIOGRAPHY 170 V L i s t of Tables 1. Charact e r i s t i c s of standard l i g h t treatments using PTR interference f i l t e r s 48 2. Occurrence of SPOs and LPOs in dark-grown seedlings... 61 3. Latent period and inhib i t o r y e f f e c t s of 10 minute l i g h t pulses in the 380 to 760 nm waveband 94 v i L i s t o f F i g u r e s 1 . S u m m a r y o f t h e t r a n s f o r m a t i o n s o f t h e p h y t o c h r o m e s y s t e m i n v i v o 9 2 . M o d i f i e d s c h e m e o f p h y t o c h r o m e t r a n s f o r m a t i o n s i n v i v o 1 5 3 . G r o w t h m e a s u r i n g a p p a r a t u s . . 3 9 4 . C u v e t t e , s e e d l i n g a n d c o l l a r a s s e m b l y 41 5 . M e t h o d s o f r e c o r d i n g t r a n s d u c e r o u t p u t 4 2 6 . S y s t e m s t a b i l i t y a n d r e s p o n s e t o t e m p e r a t u r e s h i f t s 4 6 7 . T r a n s m i s s i o n s p e c t r a o f C B S 6 5 0 r e d a n d 7 3 0 f a r r e d f i l t e r s a n d d a r k y e l l o w g r e e n R o s c o l u x NO 9 1 a c e t a t e f i l t e r 4 9 8 . R e p r e s e n t a t i v e t r a c e s s h o w i n g A ) S P O s , a n d L P O s , a n d B ) l a r g e a m p l i t u d e L P O s i n t h e g r o w t h r a t e t r a c e s o f t h r e e d i f f e r e n t S i n a p i s s e e d l i n g s 5 2 9 . S h o r t p e r i o d o s c i l l a t i o n s i n t h e g r o w t h r a t e o f S i n a p i s 5 3 1 0 . V a r i a t i o n s i n t h e p e r i o d , a m p l i t u d e a n d o c c u r r e n c e o f S P O s i n i n d i v i d u a l s e e d l i n g s 5 5 1 1 . E f f e c t s o f i n c r e a s i n g t e n s i o n o n t h e g r o w t h r a t e o f S i n a p i s 5 6 1 2 . E f f e c t s o f 5 ° C t e m p e r a t u r e s h i f t o n S P O s i n S i n a p i s . . . 5 8 1 3 . E f f e c t s o f a 2 5 t o 1 5 C t e m p e r a t u r e s h i f t o n t h e g r o w t h r a t e o f S i n a p i s 5 9 1 4 . R e p r e s e n t a t i v e t r a c e s s h o w i n g t h e g r o w t h o f A ) o a t , B ) r a d i s h a n d C ) c u c u m b e r s e e d l i n g s 6 3 1 5 . E x a m p l e s o f l a r g e a m p l i t u d e L P O s i n t h e g r o w t h r a t e t r a c e s o f e t i o l a t e d A ) s u n f l o w e r a n d B ) r a d i s h s e e d l i n g s 6 4 1 6 . L a r g e a m p l i t u d e L P O s i n t h e g r o w t h r a t e t r a c e o f r a d i s h m e a s u r e d u s i n g t h e c o l l a r a s s e m b l y / 6 6 1 7 . G r o w t h r a t e m e a s u r e m e n t s o f S i n a p i s m a d e a t t h e h y p o c o t y l h o o k o r u s i n g t h e c o l l a r a s s e m b l y 6 9 1 8 . E f f e c t s o f r o o t e x c i s i o n o r d e c a p i t a t i o n o n S P O s i n S i n a p i s 7 4 1 9 . E f f e c t s o f a 5 m i n u t e B 4 5 0 l i g h t p u l s e o n t h e g r o w t h r a t e o f A ) c u c u m b e r , B ) r a d i s h , a n d C ) S i n a p i s s e e d l i n g s 7 8 2 0 . E f f e c t s o f a b r i e f R 6 6 0 l i g h t p u l s e o n t h e g r o w t h r a t e o f A ) r a d i s h a n d B ) S i n a p i s s e e d l i n g s 8 2 2 1 . E f f e c t s o f 10 m i n u t e R 6 7 0 l i g h t p u l s e o n t h e g r o w t h r a t e o f S i n a p i s 8 4 2 2 . R e v e r s a l o f r e d l i g h t i n d u c e d i n h i b i t i o n b y F R 7 4 0 l i g h t p u l s e s 8 9 2 3 . E f f e c t s o f v a r y i n g p e r i o d s o f d a r k n e s s b e t w e e n R 6 6 0 a n d F R 7 4 0 l i g h t p u l s e s 9 0 2 4 . R e p r e s e n t a t i v e t r a c e s s h o w i n g t h e e f f e c t s o f 10 m i n u t e s A ) 3 8 0 , B ) 4 0 0 , C ) 4 5 0 , a n d D ) 5 0 0 n m l i g h t p u l s e s 9 5 v i i 2 5. R e p r e s e n t a t i v e t r a c e s s h o w i n g t h e e f f e c t s o f 10 m i n u t e A) 550, B) 600, C) 700 and D) 710 nm l i g h t p u l s e s 96 26. E f f e c t s o f 10 m i n u t e A) 700 and B) 710 nm l i g h t p u l s e s 97 27. N u m e r i c a l l y a v e r a g e d a n d n o r m a l i z e d c u r v e s s h o w i n g e f f e c t s o f 660, 700 a n d 710 nm l i g h t p u l s e s 100 28. E f f e c t s o f 10 m i n u t e l o n g w a v e FR l i g h t p u l s e s 101 29. M o d e l s h o w i n g h y p o t h e t i c a l l o g a r i t h m i c d e c l i n e s i n t h e P f r / P t o t r a t i o f o l l o w i n g b r i e f 6 6 0 , 700, 710 and 720 nm l i g h t p u l s e s 104 30. E f f e c t s o f s i m u l t a n e o u s a n d / o r s e q u e n t i a l i r r a d i a t i o n s w i t h B450, R660 and l o n g w a v e FR 111 31 . E f f e c t s o f p r o l o n g e d A) B450 o r B) UV380 i r r a d i a t i o n s , a n d r e c o v e r y i n A) d a r k n e s s o r B) a f t e r l o n g w a v e FR 114 32. E f f e c t s o f c o n t i n u o u s B450 i r r a d i a t i o n s a t A) .0016, B) .16 a n d C) .016 Wm"2 116 33. I n d u c t i o n o f p h o t o t r o p i c b e n d i n g by r e p e a t e d FR760 l i g h t p u l s e s 119 34. E f f e c t s o f 1 h o u r l o n g w a v e FR i r r a d i a t i o n s 125 35. E f f e c t s o f p r o l o n g e d A) 660 o r B) 670 nm i r r a d i a t i o n s a n d B) s u b s e q u e n t FR7 1 0 i r r a d i a t i o n 126 36. E f f e c t s o f b r i e f p r o l o n g e d FR700 a n d FR710 l i g h t t r e a t m e n t s 127 37. E f f e c t s o f 3 h o u r FR710 i r r a d i a t i o n s a n d r e c o v e r y i n d a r k n e s s 131 38. E f f e c t s o f FR740 l i g h t p u l s e s f o l l o w i n g p r o l o n g e d A) 660 and B) 710 nm i r r a d i a t i o n s 132 39. F l u e n c e r a t e d e p e n d e n c e o f t h e r e s p o n s e t o p r o l o n g e d A) FR710 and B) R660 i r r a d i a t i o n s 136 40. R e s p o n s e t o i n c r e a s i n g f l u e n c e r a t e s o f A) FR710 and B) R660 i r r a d i a t i o n s 139 41 . E f f e c t s o f s i m u l t a n e o u s i r r a d i a t i o n s w i t h FR710 and FR740 1 40 42. E f f e c t s o f h o u r l y , 6 m i n u t e FR710 l i g h t p u l s e s 143 43. E f f e c t s o f 1 m i n u t e 710 l i g h t p u l s e s r e p e a t e d a t 10 m i n u t e i n t e r v a l s 144 44. E f f e c t s o f h o u r l y , 6 m i n u t e R660 l i g h t p u l s e s 149 45. E f f e c t s o f c o n t i n u o u s FR710 o r B450 i r r a d i a t i o n s f o l l o w i n g a 1 h o u r R660 p r e t r e a t m e n t 151 46. H y p o t h e t i c a l scheme o f p h y t o c h r o m e a c t i o n 166 v i i i L i s t Of Abbreviations B blue l i g h t BAP blue l i g h t absorbing photoreceptor FR far red l i g h t HIR high irradiance responses LFR low fluence-response LOG 1ipoxygenase LPO long period o s c i l l a t i o n Pf r far red absorbing form of phytochrome Pr red absorbing form of phytochrome Ptot t o t a l phytochrome R red l i g h t SPO short period o s c i l l a t i o n UV u l t r a v i o l e t l i g h t VLFR very low fluence-response i x Acknowledgement I wish to thank my thesis supervisor, Dr. Peter J o l l i f f e for his encouragement, and helpful c r i t i c i s m at a l l stages during t h i s work. I am also grateful to the members of my supervisory committee, Dr. A. Glass, Dr. V.C. Runeckles, and Dr. G. Eaton, for helping me bring t h i s project to a successful completion. The expert technical assistance provided by Peter Garnett and Brian MacMillan is also g r a t e f u l l y acknowledged. Above a l l I wish to thank Margaret K r i s t i e for her invaluable \ assistance in preparing t h i s manuscript, as well as for the support and i n s p i r a t i o n she has provided. 1 I. PREFACE The regulation of plant growth and development by l i g h t , acting through mechanisms not d i r e c t l y related to photosynthesis, is termed photomorphogenesis. Because of the central role that l i g h t plays in the l i f e of plants, understanding the mechanisms by which l i g h t regulates growth and development i s of considerable importance, both from a theoretical standpoint, and also in terms of p r a c t i c a l a p p l i c a t i o n . As an example, the p r a c t i c a l implications of photoperiodism research, a branch of photomorphogenic research, have been of great benefit to greenhouse flower production. To date, much of the research on photomorphogenesis has centred on the i d e n t i f i c a t i o n of the photoreceptors involved, and the elucidation of their mechanism of action. While much progress has been made in understanding the nature of the photoreceptors, their elusive mechanism of action remains the holy g r a i l of photomorphogenic research. Studies on the i n h i b i t i o n of stem elongation by l i g h t have contributed extensively to our understanding of photomorphogenesis. However, although the long term effects of l i g h t on stem elongation have been known since the time of Sachs (125) and studied intensively for several decades, the kinetics of the response to l i g h t remain scantly studied and poorly understood. Only in very recent years has the relevance of short term high resolution growth studies been recognized in photomorphogenic research. In contrast, the importance of kinetics of growth had been recognized in auxin research for 2 some time. Such studies have contributed enormously to understanding the mechanisms by which auxin influences c e l l elongation (110). The purpose of t h i s study was to examine in d e t a i l the kinetics of the photoinhibition of stem elongation in young seedlings. The germination and early growth of the seedling is a c r u c i a l period in plant establishment. The seedling must reach the surface and begin photosynthesizing before i t s energy reserves are exhausted. Thus, dicot seedlings germinated and grown in darkness divert much of their stored reserves towards extension growth, and have elongated stems and poorly developed leaves. Exposure to l i g h t has profound effects upon seedling development. Stem elongation i s inhibited, the plumule hook opens, and leaf expansion and chlorophyll production i s promoted (125). Thus, the effects of l i g h t on seedling development are of physiological and ecological importance. However, more germane to t h i s study i s the fact that dark-grown (etiolated) seedlings are free of screening pigments, and thus are ideal for photomorphogenic studies. In addition, hypocotyl elongation can be continuously monitored using high resolution growth measurement techniques. Much of the early work on the photocontrol of stem elongation was done using e t i o l a t e d seedlings. In recent years, however, more attention has been given to the role that l i g h t plays in regulating stem elongation in light-grown (de-etiolated) plants. Nevertheless, our understanding of the e t i o l a t e d system remains incomplete. Therefore, this thesis 3 w i l l deal with the kinetics of hypocotyl elongation and i t s photocontrol in e t i o l a t e d seedlings. The f i r s t step in this study involved the design and construction of a high resolution growth measuring apparatus, that could provide an instantaneous measure of the rate of stem elongation, under highly controlled, and e a s i l y manipulated environmental conditions. Preliminary studies with t h i s system were concerned with resolving the kinetics of hypocotyl elongation in darkness, as a prelude to studies on the photocontrol of stem elongation. The bulk of t h i s thesis deals with the kinetics of the growth responses to brief and prolonged i r r a d i a t i o n s with monochromatic l i g h t , and the implications such responses have in our understanding of the so-called high irradiance and low energy responses of plant photomorphogenesis. 4 I I . INTRODUCTION 2.1 Light And Plant Development The growth and development,of plants i s - intimately linked to their radiation environment. Plants have evolved photoreceptor systems that permit them to u t i l i z e l i g h t 1 in two d i s t i n c t ways: as a source of energy, and as a means of obtaining information about their environment. Energy transducing photoreceptors such as chlorophyll and photosynthetic accessory pigments convert l i g h t energy into forms of chemical potential energy, such as ATP and NAD(P)H. Other energy transducing photoreceptors such as protochlorophyll and photoreactivating enzyme use l i g h t to drive one step chemical reactions (117). Plants also possess at least two s p e c i f i c s i g n a l -transducing photoreceptors that allow them to monitor and regulate their development in response to changes in their l i g h t environment. These are phytochrome, a photoreversible chromoprotein that absorbs primarily in the red and far red wavebands, and a UV-blue l i g h t absorbing photoreceptor (BAP) sometimes referred to as cryptochrome (117). The identity of the BAP i s not known with certainty but considerable evidence indicates i t is a flavoprotein (117). The regulation of plant 1For ease of expression, the term l i g h t w i l l be used in this thesis as a synonym for electromagnetic radiation encompassing the u l t r a v i o l e t and infared portions of the spectrum near the v i s i b l e portion, as well as the v i s i b l e wavelengths. 5 g r o w t h a nd d e v e l o p m e n t by t h e s e a nd p e r h a p s o t h e r u n d i s c o v e r e d s i g n a l - t r a n s d u c i n g p h o t o r e c e p t o r s i s t e r m e d p h o t o m o r p h o g e n e s i s . I n a n g i o s p e r m s , t h e number a n d d i v e r s i t y o f p h o t o m o r p h o g e n e t i c r e s p o n s e s known t o be m e d i a t e d by p h y t o c h r o m e i s s u r p r i s i n g l y l a r g e , a n d s e v e r a l h u n d r e d p h y t o c h r o m e - m e d i a t e d r e s p o n s e s h a v e been r e p o r t e d ( 1 5 5 ) . T h e s e i n c l u d e e f f e c t s a t t h e m o l e c u l a r l e v e l , s u c h a s t h e r e g u l a t i o n o f enzyme a c t i v i t y ( 1 4 4 ) , a n d g r o s s m o r p h o l o g i c a l e f f e c t s , s u c h a s t h e p h o t o p e r i o d i c i n d u c t i o n o f f l o w e r i n g ( 1 6 8 ) . A few o t h e r m a j o r p r o c e s s e s w h i c h a r e a t l e a s t p a r t i a l l y r e g u l a t e d by p h y t o c h r o m e i n c l u d e s e e d g e r m i n a t i o n ( 1 5 2 ) , p l u m u l a r hook o p e n i n g ( 7 5 ) , stem, e l o n g a t i o n ( 2 7 , 5 6 , 9 7 , 1 0 0 , 1 0 2 ) , a n d f l a v o n o i d p r o d u c t i o n ( 1 5 0 , 1 5 6 , 1 5 7 ) . I n c o n t r a s t , t h e r e a r e r e l a t i v e l y few p h o t o r e s p o n s e s i n a n g i o s p e r m s i n w h i c h t h e e f f e c t o f b l u e l i g h t c a n be u n e g u i v o c a b l y a t t r i b u t e d t o t h e a c t i o n o f a s p e c i f i c BAP. T h e s e i n c l u d e : p h o t o t r o p i s m , t h e b e n d i n g o f p l a n t o r g a n s t o w a r d s o r away f r o m l i g h t ( 2 6 ) ; t h e p h o t o i n h i b i t i o n o f s t e m e l o n g a t i o n i n d i c o t s ( 1 6 0 ) ; l i g h t i n d u c e d l e a f u n r o l l i n g i n r i c e ( 7 6 ) ; a n t h o c y a n i n a nd b e t a c y a n i n p r o d u c t i o n i n s e v e r a l s p e c i e s ( 9 8 ) ; and c h a n g e s i n t h e b i o e l e c t r i c p o t e n t i a l o f bean h o o k s ( 4 4 ) . I n many o t h e r s y s t e m s t h e e f f e c t s o f b l u e l i g h t a r e p r o b a b l y m e d i a t e d by p h y t o c h r o m e , w h i c h a l s o a b s o r b s i n t h e U V - b l u e waveband. 6 2.2 The Phytochrome System Phytochrome primarily exists in two interconvertible forms: Pr, a red absorbing form with an absorbance maximum near 660 nm and Pfr, a far red absorbing form with an absorbance maximum near 730 nm. Pfr i s thought to be the b i o l o g i c a l l y active form of phytochrome, while Pr is considered to be inactive . Absorption of l i g h t by either Pfr or Pr results in photoconversion to the other form. Photoconversion i s a f i r s t order process and involves the formation of several intermediates in both directions (80,113). The mechanism of photoisomerization is not yet f u l l y understood, however the structure of the chromophore and biochemical properties of the protein moiety have been characterized, and recently reviewed (113,131). The photochemical reactions of the phytochrome system are shown in a si m p l i f i e d form as follows: k1 Pr v s P f r k2 The rate constants, k1 and k2, are a function of the quantum yield s for phototransformation of Pr and Pfr, the extinction c o e f f i c i e n t s of Pr and Pfr and the photon spectral d i s t r i b u t i o n (54) . Because Pr and Pfr have broad absorption spectra that overlap throughout the UV-visible spectrum, photoconversion i s never complete in either d i r e c t i o n (16). During i r r a d i a t i o n , phytochrome 'cycles' between i t s two forms and eventually comes 7 t o a dynamic e q u i l i b r i u m ( p h o t o e q u i l i b r i u m ) d e s i g n a t e d as * The p h o t o e q u i l i b r i u m r e p r e s e n t s t h e p r o p o r t i o n o f p h y t o c h r o m e i n t h e P f r form, i . e . * = P f r / P t o t . The e f f e c t s o f l i g h t on p h y t o c h r o m e p h o t o c o n v e r s i o n s c a n be t o t a l l y d e s c r i b e d by two p a r a m e t e r s , t h e * = k l / ( k 1 + k 2 ) and t h e p h o t o c o n v e r s i o n ( c y c l i n g ) r a t e =(k1+k2) ( 1 3 0 ) . L i g h t t r e a t m e n t s a r e i d e n t i c a l w i t h r e s p e c t t o t h e p h y t o c h r o m e s y s t e m i f * and t h e c y c l i n g r a t e a r e t h e same ( 1 3 0 ) . In o t h e r words p h y t o c h r o m e can o n l y d e t e c t c h a n g e s i n t h e l i g h t e n v i r o n m e n t v i a c h a n g e s i n * a n d / o r c h a n g e s i n t h e c y c l i n g r a t e . The c y c l i n g r a t e i s a f u n c t i o n o f b o t h f l u e n c e r a t e 1 and w a v e l e n g t h . The r a t e o f a p p r o a c h t o * i s f l u e n c e r a t e - d e p e n d e n t , however t h e P f r r a t i o a t p h o t o e q u i l i b r i u m i s d e p e n d e n t o n l y on t h e w a v e l e n g t h o f t h e i n c i d e n t l i g h t ( 1 5 , 1 1 4 ) . F o r non s a t u r a t i n g l i g h t d o s e s , i . e . i r r a d i a t i o n s t e r m i n a t e d b e f o r e t h e p h o t o e q u i l i b r i u m i s e s t a b l i s h e d , t h e P f r / P t o t r a t i o i s a f u n c t i o n o f d o s e and o b e y s t h e Bunsen-Roscoe r e c i p r o c i t y law ( 9 0 ) . The r e c i p r o c i t y law (law o f p h o t o c h e m i c a l e q u i v a l e n c e ) s t a t e s t h a t t h e p h o t o c h e m i c a l e f f e c t s o f l i g h t a r e c o n s t a n t i f t h e p r o d u c t o f i r r a d i a n c e and e x p o s u r e t i m e a r e c o n s t a n t . Thus, below s a t u r a t i o n , l o n g i r r a d i a t i o n s a t low f l u e n c e r a t e s p r o d u c e t h e same P f r / P t o t r a t i o as s h o r t i r r a d i a t i o n s a t h i g h f l u e n c e r a t e s i f f l u e n c e 1 T o comply w i t h r e c e n t r ecommendations (Photochem. and P h o t o b i o l . 25:237-238) I use t h e t e r m f l u e n c e r a t e and f l u e n c e i n s t e a d o f t h e more ambiguous terms i r r a d i a n c e and d o s e . F l u e n c e r a t e and f l u e n c e may be e x p r e s s e d e i t h e r i n t e r m s o f a b s o l u t e e n e r g y ( u n i t s = Wm"2 and Jirr 2 r e s p e c t i v e l y ) o r i n quantum t e r m s ( u n i t s = mol m " 2 s e c _ 1 and mol n r 2 r e s p e c t i v e l y ) . 8 rate x time i s constant. It must be emphasized that the Pfr/Ptot r a t i o at * is a c h a r a c t e r i s t i c of the light-pigment interaction only. In the presence of competing nonphotochemical reactions the Pfr/Ptot r a t i o established under a given l i g h t source may not be i d e n t i c a l to * . This point w i l l be discussed later in more d e t a i l . The photoequilibrium measured in p u r i f i e d phytochrome in solution or in vivo at OC varies s l i g h t l y among d i f f e r e n t studies , but i s about 0.35 to 0.45 in the blue, 0.70 to 0.80 in the red, and 0.2 to <0.001 in the far red (2,16,54). Typical values for the * under broadband l i g h t sources are 0.59-0.62 for midday sunlight, 0.55 for incandescent l i g h t , and 0.76 for fluorescent l i g h t (56). The e f f e c t s of dichromatic i r r a d i a t i o n s should also be mentioned. A powerful technique for the study of photomorphogenetic responses involves the use of simultaneous i r r a d i a t i o n s with two (or sometimes more) wavelengths of l i g h t (45,90). By varying the photon fluence rate r a t i o of the two wavelengths, v i r t u a l l y any Pfr/Ptot r a t i o located between the photoequi1ibria of the two wavelengths can be established. The phytochrome c y c l i n g rate is also affected during dichromatic i r r a d i a t i o n s . The properties of the phytochrome system in vivo are t r a d i t i o n a l l y summarized in a scheme similar to Figure 1 (e.g. 118,130). In addition to the two f i r s t order l i g h t reactions t h i s scheme includes the de novo synthesis of phytochrome and 9 hv s y n t h e s i s r e v e r s 1 on • • b i o l o g i c a l a c t i o n d e s t r u c t i on F i g u r e 1. Summary of the t r a n s f o r m a t i o n s of the phytochrome system ijn v i v o . 10 two nonphotochemical dark reactions. These are a reversion of Pfr to Pr and an i r r e v e r s i b l e destruction of Pfr with the loss of spectrophotometrically detectable phytochrome. However, as w i l l become apparent, this scheme i s probably too s i m p l i s t i c to explain the observed kinetics of the phytochrome system in vivo. It should also be noted that i f Pfr is the b i o l o g i c a l l y active form of phytochrome, then a l l reactions that affect the concentration of Pfr ( [Pfr] ) are p o t e n t i a l l y of physiological s i g n i f i c a n c e . Because even small amounts of chlorophyll interfere with the spectrophotometric analysis of phytochrome,the phytochrome system in vivo has been studied most extensively in chlorophyll free tissues such as cauliflower curd and e t i o l a t e d seedlings (14,130). Recently, however, the use of the herbicide Sandoz 9689 (norflurazon) has made i t possible to study the phytochrome system in light-grown but e s s e n t i a l l y chlorophyll-free seedlings (37,59,61,62,63). Norflurazon i n h i b i t s carotenoid synthesis leaving chlorophyll open to photodestruction under l i g h t of s u f f i c i e n t i n t e n s i t y . The herbicide has no eff e c t on the phytochrome system and has no eff e c t on many (23,37,59) but not a l l phytochrome-mediated photoresponses (23). In addition, a recently developed radioimmunoassay (RIA) procedure has permitted the measurement of Ptot levels in green seedlings (57,58). Unfortunately the assay cannot di s t i n g u i s h between Pfr and Pr. Dark reversion occurs in most dicots but has not been observed in the monocots nor in the dicot order Centrospermae 11 (130). Dark reversion is f i r s t order and rapid at 25C with a t l / 2 of about 6 min in Sinapis (93,141). Dark reversion i s t y p i c a l l y complete within an hour (e.g. 30 min in Sinapis ), however only a small portion of the Pfr molecules present are reverted (113,137,141). Recent studies on the temperature and fluence rate dependence of the Pfr/Ptot r a t i o indicate the existence of a rapid Pfr to Pr thermal reversion that occurs in the l i g h t but stops after a short time in darkness (51,60). It was found that in e t i o l a t e d Sinapis at 0C the * was rapidly established even at r e l a t i v e l y low fluence rates . At 25C a constant Pfr/Ptot r a t i o was established after a transient time of 1 to 2 hours, however, the Pfr/Ptot r a t i o was not equal to the photoequilibrium. For example, the Pfr/Ptot r a t i o under continuous red l i g h t was about 0.2 at 0.017 Wirr2.and 0.4 at 0.034 Wirr2. Continuous i r a d i a t i o n established a Pfr/Ptot r a t i o equal to * only at fluence rates >0.4 Wirr2 in the red and >25 Wm"2 in the blue (131). The fluence rate dependency of Pfr/Ptot was explained by assuming the existence of a fast thermal Pfr to Pr transformation (t1/2 about 3 min at 25C) that competes with the photoconversion reactions for Pfr. The fluence rate dependency occurs at lower fluence rates in the blue because the quantum effectiveness for photoconversion i s much lower in the blue than in the red (114). Thus, the Pfr/Ptot r a t i o maintained in vivo i s a function not only of the wavelength and fluence rate of the l i g h t source but also the temperature. This quasi-stationary state of Pfr/Ptot is usually termed a photostationary state to d i f f e r e n t i a t e i t 12 from the *. Pfr destruction or decay is defined as "the disappearance of spectrophotometrically detectable Pfr without the concomitant appearance of equimolar quantities of Pr" (118). Pfr destruction occurs in both monocots and dicots but i s absent from a number of tissues such as cauliflower curd (14,90). Destruction occurs during continuous i r r a d i a t i o n and in darkness after a brief l i g h t treatment.- In dicots destruction i s f i r s t order with respect to Pfr. Under continuous i r r a d i a t i o n the rate of destruction is proportional to the Pfr/Ptot r a t i o maintained (97,134).. In monocots, destruction i s e s s e n t i a l l y zero order as destruction i s saturated at very low Pfr/Ptot ratios (133). At high fluence rates the rate of Pfr destruction declines probably because a large proportion of Ptot i s maintained in the form of intermediates unavailable for destruct ion ( 78) . Values for the t1/2 of destruction vary with the species and age of the seedling. In e t i o l a t e d Sinapis the t l / 2 of destruction was reported to be 45 min, 48 hours after sowing and 25 min, 72 hours after sowing (94,97). Unlike dark reversion, Pfr destruction proceeds to completion in darkness removing v i r t u a l l y a l l unreverted Pfr molecules (118). To account for this discrepancy, a fast intervening dark reaction between Pfr formation by l i g h t and Pfr destruction was postulated (118,130,137). Both destruction and reversion are temperature dependent. At 25C, destruction is the predominant mechanism by which Pfr molecules are removed; 13 however, at increasing temperatures reversion increases at the expense of destruction (137). A number of studies indicate that Pfr destruction i s actually biphasic in both e t i o l a t e d and light-grown monocots and dicots (13,50,,59,130). In e t i o l a t e d seedlings,rapid f i r s t order destruction removed most but not a l l of the detectable Pfr after a saturating red l i g h t pulse (13,50,130,131). Destruction of the remaining Pfr was much slower. In light-grown herbicide treated seedlings with low Ptot, slow destruction was predominant (50,59). • These results were taken to indicate the existence of two separate pools of phytochrome , i . e . 'stable' phytochrome with slow destruction and ' l a b i l e ' phytochrome with fast destruction (13,130,131). The alternative hypothesis, that there i s one pool of phytochrome with fast destruction above a threshold l e v e l of Pfr, was not supported by these experiments. The existence of two pools of phytochrome, i . e . 'active' phytochrome and 'bulk'. phytochrome had been postulated previously to account for the so-called phytochrome paradoxes (53). However, i t should also be noted that there is a considerable body of evidence from physiological experiments that suggests that there is a single pool of phytochrome with l i t t l e or no Pfr destruction below a threshold concentration of Pfr (6,99). Several recent studies ' suggest that although Pfr destruction i s the main pathway for Pfr removal in both monocots and dicots, Pr i s also subject to destruction after i t has 14 cycled through Pfr, i . e . after a red-far red i r r a d i a t i o n sequence (59,62,130,158). In corn and Amaranthus, Pr was subject to destruction only i f the molecule had remained in the Pfr form for several seconds (59,130). Pr destruction removed only a small proportion of the molecules that had cycled through Pfr, and destruction stopped after 1 to 2 hours of darkness. However, destruction resumed after a second R-FR i r r a d i a t i o n sequence. These results were explained by assuming a competition between Pr destruction and a relaxation reaction that made Pr unavailable for destruction (59,130,131). Schafer (130) and Jabben (59) have presented similar schemes which are consistent with the kinetics of phytochrome in  vivo (Fig.2). The scheme includes separate 'reversion' and 'destruction' pools of phytochrome separated by a rapid dark reaction (78). It takes into account the fact that Pr destruction only occurs after i t has cycled through Pfr, and assumes that Pr destruction competes with a relaxation reaction (79). The scheme does not take into account the biphasic k i n e t i c s of Pfr destruction, nor ' l i g h t activated' Pfr dark reversion. It i s interesting to note that studies on in vivo phytochrome p e l l e t a b i l i t y have led to a similar dynamic scheme (87, 113,116,119,120,121). The mechanism of phytochrome destruction i s not understood, however immunocytochemical studies indicate that a proteolytic degradation of the molecule i s involved, at least for Pfr (112,115). It has been suggested that the red l i g h t induced i n t r a c e l l u l a r r e d i s t r i b u t i o n of phytochrome from a diffuse 15 hv s y n t h e s i s •• P r ^ P f r hv P r P r d e s t r u c t i on P f r d e s t r u c t i on F i g u r e 2. M o d i f i e d scheme o f p h y t o c h r o m e t r a n s f o r m a t i o n s i n v i v o . 16 d i s t r i b u t i o n throughout the c e l l into discrete regions might be linked to phytochrome destruction (164). Thus, the destruction process might be s p e c i f i c for phytochrome associated with some s p e c i f i c subcellular component, rather than for Pfr or Pr (113). Spectrophotometric and RIA studies have shown that Ptot increases during the early growth of e t i o l a t e d seedlings and after a l i g h t to dark t r a n s i t i o n in both e t i o l a t e d and l i g h t -grown seedlings (58,62,99,118). This increase i s considered to be the result of the de novo synthesis of phytochrome as Pr. A D^ O l a b e l l i n g technique has been used to show that this is true in e t i o l a t e d Cucurbita seedlings (121,122). Phytochrome synthesis i s zero order with respect to Ptot (118). In e t i o l a t e d Sinapis as well as other species the rate of Ptot accumulation in darkness i s unaffected by previous l i g h t treatments (99,118,130). Therefore, i t has been generally assumed that the rate of phytochrome synthesis i s constant and unaffected by l i g h t treatments, except perhaps i n d i r e c t l y , e.g. via the a v a i l a b i l i t y of photosynthetic products (130,118). However, Gorton and Briggs (from 131) recently showed that the rate of Ptot accumulation in corn after a l i g h t to dark t r a n s i t i o n appeared to depend on the Pfr/Ptot r a t i o at the beginning of the dark period. In addition, Gottmann and Schafer (39) presented indirect evidence from in v i t r o studies on phytochrome translation in Avena , that indicates that the capacity for phytochrome synthesis is greater in dark-grown than in light-grown seedlings. Therefore, the general assumption that phytochrome synthesis is constant and unaffected by l i g h t 1 7 may have to be re-evaluated. The size of the phytochrome pool i s believed to be regulated by an equilibrium between a more or less constant rate of phytochrome synthesis and a variable rate of phytochrome destruction (118). During continuous i r r a d i a t i o n , phytochrome destruction reduces Ptot in e t i o l a t e d seedlings. After a transient period in which Ptot declines, a new steady state l e v e l of Ptot i s established (135). It has been shown that the steady state l e v e l of Ptot i s inversely related to the Pfr/Ptot r a t i o maintained by the l i g h t source in both e t i o l a t e d and light-grown herbicide treated seedlings (65,128,136,154). At the steady state i t is believed that phytochrome synthesis i s balanced by phytochrome destruction, however the turnover of phytochrome" molecules has never been measured under these conditions. The steady state of the phytochrome system i s approached very slowly and i s characterized by the fact that a l l measurable parameters of the phytochrome system, i . e . [ P f r ] , [Pr], [Ptot], and Pfr/Ptot are constant. A very important c h a r a c t e r i s t i c of the steady state i s that although Pfr/Ptot, [Ptot] and [Pr] are a function of both wavelength and fluence rate, [Pfr] i s completely independent of both (65,128,136,154). This i s because [Ptot] is inversely related to the Pfr/Ptot r a t i o established by the l i g h t source, while [Pfr] i s d i r e c t l y related In summary, spectrophotometric and RIA studies have indicated that the phytochrome systems of e t i o l a t e d and l i g h t -18 grown seedlings are q u a l i t a t i v e l y the same , however,a number of quantitative differences are apparent (130,131). The phytochrome system of e t i o l a t e d seedlings i s characterized by rapid synthesis and rapid destruction. However, a small stable pool of phytochrome (1-3% Ptot) may also e x i s t . The phytochrome system of light-grown seedlings is characterized by slow destruction and probably by slow synthesis. However, afte r a l i g h t to dark t r a n s i t i o n i t appears that l a b i l e phytochrome i s synthesized and accumulates (58). Ptot levels are much lower in light-grown seedlings, e.g. light-grown pea seedlings contain about 2-8% of the phytochrome found in e t i o l a t e d seedlings of the same age (148). 2.3 Photoresponses Based on the c h a r a c t e r i s t i c s of the i r r a d i a t i o n required to induce a photoresponse, i t i s possible to distinguish at least three d i f f e r e n t types of photomorphogenetic responses. These are the phototropic responses, the low energy phytochrome-mediated responses, and the high irradiance responses. The phototropic responses involve d i r e c t i o n a l growth in response to asymmetric illumination, i . e . the bending of stems or leaves towards or away from l i g h t . Phototropism has been recently reviewed (26) and w i l l not be dealt with here. The low energy reponses, also termed induction-reversion (152) or s t a t i c mode (74) responses are inductive type responses that can be saturated by a single brief pulse of red l i g h t (R) 19 a n d r e v e r s e d i f t h e R p u l s e i s f o l l o w e d i m m e d i a t e l y by a f a r r e d l i g h t p u l s e ( F R ) . The low e n e r g y r e s p o n s e s a r e u b i q u i t o u s i n h i g h e r p l a n t s . A few m a j o r p r o c e s s e s i n f l u e n c e d by t h e low e n e r g y p h y t o c h r o m e s y s t e m i n c l u d e s e e d g e r m i n a t i o n ( 1 5 2 , 1 6 5 ) , s t e m e l o n g a t i o n ( 9 7 , 1 5 2 ) , f l a v o n o i d p r o d u c t i o n ( 9 7 , 1 5 0 ) a nd t h e p h o t o p e r i o d i c c o n t r o l o f f l o w e r i n g ( 1 6 8 ) . The ' H i g h I r r a d i a n c e R e s p o n s e s ' (HIR) a r e s t e a d y - s t a t e r e s p o n s e s t h a t r e q u i r e p r o l o n g e d i r r a d i a t i o n s a t r e l a t i v e l y h i g h f l u e n c e r a t e s t o e l i c i t a maximum r e s p o n s e ( 9 0 ) . The HIR h a s been i m p l i c a t e d i n a w i d e r a n g e o f d e v e l o p m e n t a l phenomena i n h i g h e r p l a n t s i n c l u d i n g , t h e i n h i b i t i o n o f h y p o c o t y l e l o n g a t i o n ( 1 4 5 ) , t h e i n d u c t i o n o f f l a v o n o i d s y n t h e s i s ( 2 8 , 2 9 , 1 5 0 ) a nd t h e i n h i b i t i o n o f s e e d g e r m i n a t i o n ( 1 5 2 ) . The f i n a l e x p r e s s i o n o f many p h o t o r e s p o n s e s i s i n f l u e n c e d by b o t h t h e low e n e r g y p h y t o c h r o m e s y s t e m a n d t h e HIR ( 9 0 ) . 2.3.1 Low E n e r g y R e s p o n s e s A c t i o n s p e c t r a f o r l o w e n e r g y r e s p o n s e s h a v e p e a k s o f a c t i v i t y n e a r 660 nm f o r t h e r e d e f f e c t , n e a r 730 nm f o r t h e f a r r e d e f f e c t , c l e a r l y p o i n t i n g t o t h e i n v o l v e m e n t o f p h y t o c h r o m e ( 1 1 8 ) . R-FR r e v e r s i b i l i t y a n d t h e i n d u c t i o n o f t h e r e s p o n s e by a b r i e f p u l s e ( i . e . < 5 min ) a r e t h e c l a s s i c a l l y a c c e p t e d c r i t e r i a f o r e s t a b l i s h i n g t h e i n v o l v e m e n t o f p h y t o c h r o m e i n a p h o t o r e s p o n s e ( 9 0 , 1 1 8 ) . An i m p o r t a n t c h a r a c t e r i s t i c o f i n d u c t i o n - r e v e r s i o n r e s p o n s e s i s t h a t t h e y a r e s a t u r a t e d a t r e l a t i v e l y l ow f l u e n c e s 20 o f r e d l i g h t , i . e . < 2 X 1 0 ~ 3 J r r r 2 ( 9 0 ) . B e l o w s a t u r a t i o n t h e m a g n i t u d e o f t h e r e s p o n s e i s a f u n c t i o n o f t h e f l u e n c e , and w i t h i n l i m i t s o b e y s t h e r e c i p r o c i t y l a w ( 9 0 , 1 1 8 ) . V a l i d i t y o f t h e r e c i p r o c i t y l a w i s u s u a l l y i n t e r p r e t e d a s m e a n i n g t h a t a s i n g l e p h o t o c h e m i c a l r e a c t i o n i s r a t e l i m i t i n g ( 9 0 ) . Q u a n t i t a t i v e l y , t h i s i n d i c a t e s t h a t t h e r e s p o n s e i s a f u n c t i o n o f t h e d e g r e e o f p h o t o c o n v e r s i o n , i . e . t h e r e s p o n s e i s p r o p o r t i o n a l t o [ P f r ] o r P f r / P t o t . B e c a u s e o f t h e s e a p p a r e n t c o r r e l a t i o n s b e t w e e n [ P f r ] and t h e m a g n i t u d e o f some p h o t o r e s p o n s e s , P f r i s c o n s i d e r e d t o be t h e a c t i v e f o r m o f p h y t o c h r o m e ( 7 7 ) . The m e c h a n i s m by w h i c h P f r b r i n g s a b o u t a b i o l o g i c a l r e s p o n s e i s unknown, b u t by l o g i c a l n e c e s s i t y t h e f i r s t s t e p o r ' p r i m a r y r e a c t i o n ' o f p h y t o c h r o m e must i n v o l v e t h e i n t e r a c t i o n of p h y t o c h r o m e w i t h some unknown r e a c t i o n p a r t n e r , u s u a l l y s y m b o l i z e d a s 'X' ( 1 1 8 ) . The f o r m a t i o n o f P f r - X i n i t i a t e s a s e r i e s o f unknown e v e n t s t h a t u l t i m a t e l y l e a d s t o a b i o l o g i c a l r e s p o n s e . The e l u c i d a t i o n o f P f r - X and e v e n t s s u b s e q u e n t t o i t i s t h e p r i m a r y a i m o f most p h o t o m o r p h o g e n e t i c r e s e a r c h . The l a g - p h a s e (97) o r l a t e n t p e r i o d b e t w e e n t h e s t a r t o f t h e i r r a d i a t i o n and t h e f i r s t e x p r e s s i o n o f t h e r e s p o n s e i s h i g h l y v a r i a b l e . Newmann and B r i g g s (107) d e t e c t e d a c h a n g e i n b i o e l e c t r i c p o t e n t i a l i n e t i o l a t e d Avena c o l e o p t i l e s w i t h i n 15 s e c o n d s a f t e r t h e s t a r t o f i r r a d i a t i o n . The l a g - p h a s e f o r a n t h o c y a n i n s y n t h e s i s i n e t i o l a t e d S i n a p i s i s a b o u t 3 h o u r s (97) w h i l e l i g h t - p r o m o t e d l e t t u c e s e e d g e r m i n a t i o n may o c c u r many h o u r s o r e v e n d a y s a f t e r i r r a d i a t i o n ( 7 6 ) . 21 Expression of the photoresponse can often be prevented i f the Pfr formed by R i s immediately removed by FR. However, as the length of an intervening dark period between R and FR i r r a d i a t i o n s is increased, a progressive loss of p h o t o r e v e r s i b i l i t y occurs, u n t i l expression of the response cannot be prevented by FR ( i . e . Pfr removal). Pfr i s then said to have 'potentiated' the response (152). This escape from FR r e v e r s i b i l i t y can occur within minutes, as in the case of the synergism between phytochrome and g i b b e r e l l i n in lettuce seed germination (4), or gradually over the course of many hours as in the normal phytochrome promoted germination of lettuce (82). The time course of escape i s thought to represent the time course of signal transduction from phytochrome to the responding system (138). Escape from FR r e v e r s i b i l i t y does not appear to occur in a number of photoresponses such as the i n h i b i t i o n of hypocotyl elongation (97) and lipoxygenase synthesis (99) in e t i o l a t e d Sinapis. In these cases the continuous presence of Pfr i s apparently required to sustain the photoresponse. An important aspect of photoresponses of this type i s that the response w i l l be affected by the removal of Pfr in darkness by Pfr destruction and reversion. The dose response curves of several low energy responses (e.g. i n h i b i t i o n of mesocotyl elongation in Avena ) show two-steps, separated by l i g h t doses several orders of magnitude apart (6,92). The f i r s t step termed the very low fluence-reponse (VLFR) i s induced and becomes saturated at extremely low 22 fluences ( i . e . < 0.1 Jrrr 2) . The second step termed the low fluence-response (LFR) occurs at fluences more commonly associated with low energy phytochrome-mediated responses (6). Operation of the low energy phytochrome system can account for both the VLFR and the LFR (6,99). Mandoli and Briggs (92) calculated that in e t i o l a t e d Avena the VLFR had a threshold of about 0.01% Pfr and became saturated at 0.4% Pfr, while the LFR was activated at about 2% Pfr and became saturated at 87% Pfr. Thus, the VLFR operates at Pfr levels that are near or below the l i m i t s of d e t e c t a b i l i t y by normal spectrophotometry. Blaauw-Jansen (6) has suggested that two-step fluence response curves are c h a r a c t e r i s t i c of most low energy phytochrome responses, but are usually not observed because handling seedlings under the dim green safelights used in most experinvents i s s u f f i c i e n t to saturate the VLFR. S i m i l a r l y , FR r e v e r s i b i l i t y for the VLFR cannot usually be shown because most FR sources produce s u f f i c i e n t Pfr to saturate the response (6,92) . The mechanism underlying two-step fluence response curves is unknown. Blaau-Jansen (6) proposed that the low energy phytochrome responses are induced at extremely low Pfr leve l s resulting in the f i r s t r i s e of the response curve. Above a threshold l e v e l of Pfr, a Pfr-destroying enzyme i s activated, which e f f e c t i v e l y prevents further increases in Pfr l e v e l s . At increasing fluences, Pfr destruction becomes saturated and the Pfr l e v e l r i s e s resulting in the LFR. . Drumm and Mohr (28) observed a two-step [Pfr] versus 23 r e s p o n s e c u r v e f o r a n t h o c y a n i n s y n t h e s i s i n S i n a p i s , and i n t e r p r e t e d t h e i r f i n d i n g s i n t e r m s o f P f r ' s i n t e r a c t i o n w i t h i t s ' r e a c t i o n m a t r i x ' . They s u g g e s t e d t h a t a t low c o n c e n t r a t i o n s P f r was h i g h l y e f f e c t i v e i n i n d u c i n g a r e s p o n s e , b u t a t h i g h e r l e v e l s a c o n f o r m a t i o n a l c h a n g e i n t h e m a t r i x r e d u c e d t h e e f f e c t i v e n e s s o f a g i v e n amount o f P f r ( 2 8 , 1 5 5 ) . A number o f o t h e r e x p l a n a t i o n s a r e a l s o p o s s i b l e . F o r e x a m p l e , e a c h s t e p i n t h e r e s p o n s e c u r v e c o u l d r e p r e s e n t t h e r e s p o n s e o f a s e p a r a t e a n d d i s t i n c t p o o l o f p h y t o c h r o m e . P h o t o r e s p o n s e s i n w h i c h t h e m a g n i t u d e o f t h e r e s p o n s e a p p e a r t o be i n some way p r o p o r t i o n a l t o [ P f r ] h a v e been t e r m e d ' g r a d e d ' r e s p o n s e s ( 7 7 ) . I n a d d i t i o n t o t h e s e g r a d e d r e s p o n s e s t o P f r , Mohr and h i s c o w o r k e r s (99) have c o n v i n c i n g l y d e m o n s t r a t e d t h a t t h e low e n e r g y p h y t o c h r o m e s y s t e m c o n t r o l s l i p o x y g e n a s e (LOG) s y n t h e s i s i n t h e c o t y l e d o n s o f e t i o l a t e d S i n a p i s v i a a ' t h r e s h o l d ' ( i . e . a l l - o r - n o n e ) r e s p o n s e . The t h r e s h o l d l e v e l o f P f r c o n t r o l l i n g LOG s y n t h e s i s was d e t e r m i n e d i n t h e f o l l o w i n g manner. The l e n g t h o f t h e i n h i b i t o r y p e r i o d f o l l o w i n g a b r i e f R i r r a d i a t i o n (* = 0.8) was d e t e r m i n e d . By a s s u m i n g r a p i d f i r s t o r d e r P f r d e s t r u c t i o n ( t l / 2 = 45 m i n ) a n d t h a t LOG s y n t h e s i s r e s u m e s i m m e d i a t e l y a f t e r P f r f a l l s b e l o w a t h r e s h o l d , t h e t h r e s h o l d c o n c e n t r a t i o n o f P f r c o u l d be c a l c u l a t e d . LOG s y n t h e s i s was r a p i d l y a n d t o t a l l y s u p r e s s e d when a t h r e s h o l d l e v e l o f P f r ( 1 . 2 5 % b a s e d on [ P t o t ] a t t i m e z e r o = l 0 0 % ) was e x c e e d e d . When [ P f r ] was r e d u c e d b e l o w t h e t h r e s h o l d l e v e l by i r r a d i a t i o n , o r a l l o w e d t o d e c a y b e l o w t h e t h r e s h o l d by P f r 24 destruction, LOG synthesis was immediately resumed at a maximum rate. By reducing Ptot with prolonged R i r r a d i a t i o n , i t was shown that the threshold was based on an absolute concentration of Pfr, rather than the Pfr/Ptot r a t i o . Further experiments demonstrated that Pfr destruction stopped when Pfr f e l l below the threshold l e v e l . It is interesting to note that LOG synthesis, which occurred in the cotyledons, was shown to be controlled by phytochrome located in the hypocotyl hook. A rapid inter-organ signal transfer was postulated to account for t h i s observation. Similar rapid inter-organ correlations have been noted for phytochrome-mediated responses in other systems (8,24,25). A similar approach was also used to show that the growth rate of e t i o l a t e d Sinapis hypocotyls in darkness was controlled by a phytochrome-mediated threshold response (97,.145). In t h i s case the threshold was found to be 0.03% Pfr (based on Ptot at time zero=100%). One incongruous point that was not addressed by the authors should also be noted. LOG synthesis and hypocotyl elongation were both found to be controlled by phytochrome located in the hypocotyl hook. Physiological experiments had indicated that in the phytochrome pool c o n t r o l l i n g LOG synthesis, Pfr destruction did not occur when the Pfr l e v e l f e l l below 1.25%. Yet inexplicably, in studies on hypocotyl elongation, i t was assumed that rapid f i r s t order destruction continued in darkness u n t i l the threshold for hypocotyl elongation was reached ( i . e . 0.03% P f r ) . 25 Threshold phenomena are generally considered to be the result of a sharp change in membrane properties ( 9 9 ) .The general model for threshold phenomena holds that "the subunits of a matrix ('membrane') which c a r r i e s the receptor s i t e s for some ligand perform a t r a n s i t i o n with a high degree of cooperativity after the l e v e l of the external ligand has reached the threshold l e v e l " ( 9 9 ) . The s p e c i f i c model for the control of LOG synthesis elaborated by Mohr and Oelze-Karow ( 9 9 ) has several assumptions. These are: any Pfr formed is rapidly bound to the receptor 'X' and X i s not l i m i t i n g ; the cooperative t r a n s i t i o n Pfr-X^Pfr-X' occurs at 1.25% Pfr and i s rapid and Pfr destruction occurs only from Pfr-X'. This model predicts that: below 1.25% Pfr only Pfr-X is present;Pfr destruction does not occur; and LOG synthesis i s unaffected. Above 1.25% Pfr, the model predicts that: only Pfr-X' i s present; LOG synthesis is inhibited; and Pfr i s subject to destruction. Thus, according to t h i s scheme the 'primary reaction' of Pfr in suppressing LOG synthesis involves a reversible threshold reaction, i . e . Pfr + X5=^ Pfr-X^± Pfr-X'. Presumably, i f hypocotyl elongation i s also controlled by a threshold mechanism, then a cooperative t r a n s i t i o n in the 'reaction matrix' must also occur at 0 . 0 3 % Pfr. Mohr and his coworkers ( 3 3 , 9 7 , 9 9 ) as well as others (81) have argued that diverse phytochrome-mediated responses such as LOG synthesis and anthocyanin synthesis (e.g. a graded response) cannot be the attributed to the same primary reaction 26 o f p h y t o c h r o m e . They c o n c l u d e t h a t a m u l t i p l i c i t y o f p r i m a r y r e a c t i o n s must be p o s t u l a t e d t o a c c o u n t f o r t h e many and d i v e r s e p h y t o c h r o m e - m e d i a t e d r e s p o n s e s w h i c h a r e known t o o c c u r ( 3 3 , 9 7 , 9 9 ) . I n c o n t r a s t , S m i t h ( 1 5 3 , 1 5 5 ) c o n t e n d s t h a t t h e i d e a o f a s i n g l e p r i m a r y r e a c t i o n o f P f r n e e d n o t be r e j e c t e d on t h a t b a s i s . R e g a r d l e s s o f w h i c h i n t e r p r e t a t i o n i s c o r r e c t , i t i s o b v i o u s t h a t t h e r e i s no s i n g l e s t a n d a r d r e l a t i o n s h i p b e t w e e n P f r a nd t h e m a g n i t u d e o f low- e n e r g y p h y t o c h r o m e - m e d i a t e d r e s p o n s e s . The c o r r e l a t i o n b e t w e e n s p e c t r o p h o t o m e t r i c a l l y d e t e c t a b l e P f r a nd t h e m a g n i t u d e o f t h e r e s p o n s e h a s been shown t o be r e m a r k a b l y good f o r t h e c o n t r o l o f LOG s y n t h e s i s and a few o t h e r p h o t o r e s p o n s e s , e.g. t h e i n d u c t i o n o f a n t h o c y a n i n s y n t h e s i s i n e t i o l a t e d S i n a p i s ( 2 8 , 1 5 7 ) . However f o r a c o n s i d e r a b l e number o f c a s e s , w h i c h h a v e been t e r m e d p h y t o c h r o m e ' p a r a d o x e s ' , t h e r e d o e s n o t seem t o be any r e a s o n a b l e c o r r e l a t i o n b e t w e e n d e t e c t a b l e P f r a n d t h e m a g n i t u d e o f t h e p h o t o r e s p o n s e ( 5 3 ) . F o r e x a m p l e , i n t h e 'Zea p a r a d o x ' , t h e p h o t o t r o p i c s e n s i t i v i t y o f m a i z e c o l e o p t i l e s i s a f f e c t e d by p r e v i o u s e x p o s u r e t o r e d l i g h t ( 1 1 , 5 3 ) . The e f f e c t i s s a t u r a t e d by v e r y low f l u e n c e s , a t an u n d e t e c t a b l y low l e v e l o f P f r , a p o i n t w h i c h c a n p r o b a b l y be e x p l a i n e d i n t e r m s o f t h e VLFR. However, t h e p a r a d o x a r i s e s f r o m t h e f a c t t h a t t h e r e s p o n s e c a n be r e v e r s e d by FR, a t r e a t m e n t w h i c h p r o d u c e s a m e a s u r a b l e l e v e l o f P f r ! To e x p l a i n t h i s p a r a d o x two d i s t i n c t p o o l s o f p h y t o c h r o m e w i t h d i f f e r e n t p h o t o c h e m i c a l r a t e c o n s t a n t s were p o s t u l a t e d , i . e . a 27 spectrophotometrically detectable but inactive 'bulk' pool and a small 'active' but undetectable pool (11,53). Another class of paradox, of which the 'Pisum paradox' is a c l a s s i c example, could only be explained by assuming the existence of a bulk phytochrome pool with rapid destruction and an active pool with slow destruction (54). It is interesting to note that 'stable' and ' l a b i l e ' pools of phytochrome have once again been postulated, but in this, case to explain the biphasic kinetics of Pfr destruction (13). However, the concept of bulk or l a b i l e phytochrome meaning inactive phytochrome must be rejected, because i t has c l e a r l y been demonstrated that l a b i l e phytochrome plays an important regulatory role in several phytochrome-mediated photoresponses (e.g. LOG synthesis)(99). Instead, i t seems reasonable to assume that both l a b i l e and stable phytochrome pools can have separate regulatory functions (13). Indeed, the suggestion that there may be several or many populations of Pfr, each with d i f f e r e n t properties and each regulating d i f f e r e n t responses cannot be discounted (77,155). 2.3.2 The High Irradiance Responses The most distinguishing c h a r a c t e r i s t i c of high irradiance responses (HIR) is their strong irradiance (fluence rate) dependency. For example, at progressively higher fluence rates of continuous FR, the rate of anthocyanin synthesis in e t i o l a t e d Sinapis increases, while the rate of hypocotyl elongation declines (97). 28 B e c a u s e HIR r e s p o n s e s r e q u i r e p r o l o n g e d i r r a d i a t i o n s t o i n d u c e a nd s u s t a i n a r e s p o n s e , R-FR r e v e r s i b i l i t y i n t h e n o r m a l s e n s e d o e s n o t a p p l y (118) - I n a d d i t i o n , u n d e r most c o n d i t i o n s HIR r e s p o n s e s do n o t show r e c i p r o c i t y , i . e . l o n g low f l u e n c e r a t e i r r a d i a t i o n s a r e more e f f e c t i v e t h a n b r i e f h i g h f l u e n c e r a t e i r r a d i a t i o n s o f t h e same t o t a l f l u e n c e ( 9 0 ) . F a i l u r e o f r e c i p r o c i t y i m p l i e s t h a t a n o n - p h o t o c h e m i c a l p r o c e s s i s r a t e l i m i t i n g , o r t h a t t h e r e s p o n s e i s d e p e n d e n t on an i n t e r a c t i o n b e t w e e n two o r more p h o t o c h e m i c a l s y s t e m s ( 9 0 ) . T h e r e h a v e been numerous s t u d i e s on t h e s p e c t r a l s e n s i t i v i t y o f t h e HIR ( r e v i e w e d i n 9 0 ) . HIR a c t i o n s p e c t r a f o r d a r k - g r o w n p l a n t s u s u a l l y show one o r more p e a k s o f a c t i v i t y i n t h e B, a n d a peak o f a c t i v i t y i n t h e FR, o f t e n c e n t r e d a t a b o u t 720 nm ( e g . 3 , 7 3 , 5 5 , 4 6 , 1 4 9 ) . Many a c t i o n s p e c t r a a l s o show a s h o u l d e r ( 1 2 7 ) o r a peak ( 3 , 5 5 , 7 2 ) o f a c t i v i t y i n t h e R, b u t - i n a number of c a s e s , a c t i v i t y i n t h e R was low o r a b s e n t ( 3 1 , 5 5 ) . The r a t i o o f e f f e c t i v e n e s s o f B/R/FR v a r i e s g r e a t l y among d i f f e r e n t p h o t o r e s p o n s e s a n d d i f f e r e n t s t u d i e s ( 9 0 ) . P r e t r e a t m e n t s w i t h R o r w h i t e l i g h t f r e q u e n t l y h ave been shown t o r e d u c e t h e e f f e c t i v e n e s s o f s u b s e q u e n t FR i r r a d i a t i o n s ( 7 , 3 , 2 9 , 5 5 , 7 3 , 1 6 2 ) , w h i l e h a v i n g l i t t l e o r no e f f e c t on t h e r e s p o n s i v e n e s s t o B o r R i r r a d i a t i o n s ( 7 , 7 3 , 1 6 2 ) . I n r e c e n t l y p u b l i s h e d a c t i o n s p e c t r a f o r t h e i n h i b i t i o n o f h y p o c o t y l e l o n g a t i o n i n S i n a p i s, i t was shown t h a t d u r i n g d e - e t o l i a t i o n , a c t i v i t y i n b o t h t h e B a n d FR wavebands was d i m i n i s h e d , a n d t h a t i n l i g h t - g r o w n s e e d l i n g s o n l y t h e R peak r e m a i n e d ( 3 , 5 5 ) . M a n c i n e l l i (90) h a s p o i n t e d o u t t h a t t h e s p e c t r a l 29 s e n s i t i v i t y of the HIR can be affected by a wide variety of factors including the experimental procedures used, the temperature, the n u t r i t i o n a l status of the plant and i t s age. Thus, great caution must be exercised in interpreting HIR action spectra. One of the most detailed and widely c i t e d (eg. 127) HIR action spectra was prepared by Hartmann (46) for the i n h i b i t i o n of hypocotyl elongation in e t i o l a t e d lettuce. It is characterized by a t r i p l e peaked structure in the B and a narrow peak in the FR, but a c t i v i t y in the R i s conspicuously absent. It i s now believed that the lack of a c t i v i t y in the R observed by Hartmann and others (31) was spurious, and probably related to equal but opposite responses to R occurring in di f f e r e n t parts of the hypocotyl (41,72,74). Hartmann's action spectrum greatly influenced the course of photomorphogenetic research and led to the concept that the maximum a c t i v i t y of the HIR occurs under conditions that result in the presence of Pfr, even at low concentrations for a long period of time. However, th i s concept ignores HIR responses that demonstrate considerable a c t i v i t y in the R wavebands or assumes that the effects of R are confined to simple inductive responses (152). Through the use of dichromatic i r r a d i a t i o n s Hartmann (45,46) demonstrated that the i n h i b i t i o n of hypocotyl elongation in lettuce by FR was in some way related to phytochrome. The peak of the FR response occurred at 717 nm, which established a phytochrome photoequilibrium of about 0.03. Dichromatic 30 i r r a d i a t i o n s with 658 and 760 nm l i g h t , wavelengths that i n d i v i d u a l l y had no i n h i b i t o r y e f f e c t s , were strongly i n h i b i t o r y when a Pfr/Ptot r a t i o of about 0.03 was established. In addition, the inhibitory effect of 717 nm l i g h t was n u l l i f i e d when the Pfr/Ptot r a t i o was shifted by simultaneous i r r a d i a t i o n with 658 or 760 nm l i g h t . Correlations between Ptot and the effectiveness of the FRHIR also indicate the involvement of phytochrome in the FRHIR. Because l i g h t pretreatments are known to reduce Ptot via Pfr destruction, there have been numerous attempts to relate the effectiveness of the FRHIR to Ptot. A s t r i c t or p a r t i a l c o r r e l a t i o n between the effectiveness of the FRHIR and Ptot has been found in many (3,29,55,73) but not a l l cases (40,162). Correlations between Ptot and the effectiveness of FR i r r a d i a t i o n s indicate that the photoequilibrium (or photostationary state) cannot be the only c o n t r o l l i n g factor in the FRHIR (despite Hartmann's experiments) since the photoequilibrium remains unchanged as Ptot l e v e l s decline (29). Suggestions that photosynthesis, in p a r t i c u l a r photosystem I, contributes to the FRHIR (142,143) have been thoroughly discredited (29,90,91). Phytochrome also appears to be the photoreceptor responsible for the RHIR (90). Dichromatic i r r a d i a t i o n s have demonstrated the involvement of photochrome in the i n h i b i t i o n of hypocotyl elongation by continuous R in Sinapis (172). Beggs et a_l. (3) reduced chlorophyll levels in light-grown Sinapis with the herbicide Norflurazon and showed that chlorophyll and 31 photosynthesis in general was not d i r e c t l y involved in the response to R. While i t i s now generally agreed that phytochrome i s the photoreceptor responsible for the response to continuous R and FR i r r a d i a t i o n s , the manner by which phytochrome brings about, these responses remains a matter of great controversy. Models to explain the HIR must take into account the fact that HIR responses require prolonged or repeated i r r a d i a t i o n s , and are fluence rate-dependent even after the phytochrome photoequilibrium has been established (90). In addition i t i s known that under prolonged i r r a d i a t i o n s the phytochrome system reaches a steady state in which the concentration of Pfr becomes independent of both wavelength and fluence rate (64,128,154,156). Thus, i t i s d i f f i c u l t to ascribe a c o n t r o l l i n g role to the l e v e l of Pfr. To overcome these d i f f i c u l t i e s most models assume the existence of two separate effectors for the HIR and low energy inductive responses i . e . two forms of Pfr (45,97) or two d i f f e r e n t associations of Pfr with i t s receptor (89,129,151). Other models assume that some unspecified product of phytochrome cyclin g i s the second ef f e c t o r , in addition to Pfr (64,66,74). Nevertheless, in a review of HIR responses and models Mancinelli (90) concluded that no single model was adequate to explain a l l the features of HIR phenomena. The two models that perhaps continue to receive the greatest attention are Schafer's open phytochrome-receptor model (128,129) and the m u l t i p l i c a t i v e model postulated by Johnson and Tasker (66). 32 Schafer's scheme i s based on two hypothetical associations of Pfr with i t s receptor "X". Schafer proposed that Pfr-X i s produced tran s i e n t l y upon i r r a d i a t i o n but rapidly decays to a more stable form Pfr-X'. It was calculated that the concentration of Pfr-X would be fluence rate-dependent in the B and FR, with a peak of concentration at about 720 nm. An important aspect of this model i s that Pfr-X would be independent of fluence rate in the R. The steady state level of Pfr-X' would be independent of both fluence rate and wavelength. Thus, according to this scheme Pfr-X would be responsible for the HIR and Pfr-X' would regulate inductive responses. The two separate effectors are assumed to operate in an additive manner. The model proposed by Johnson and Tasker (66) was based on the observation that HIR action spectra resemble the action spectra for the comparative rates of phytochrome c y c l i n g . They proposed that the HIR is controlled by an additive or m u l t i p l i c a t i v e interaction between Pfr and some unspecified product of the P r ^ P f r photoconversion. Evidence was later presented that strongly supported a m u l t i p l i c a t i v e model (64). A m u l t i p l i c a t i v e model implies two d i f f e r e n t effectors, both of which are required to bring about a response (64). Thus, according to t h i s scheme the response under inductive conditions would be controlled primarily by the l e v e l of Pfr. Under prolonged i r r a d i a t i o n s the response would become increasingly dependent on the rate of phytochrome c y c l i n g . It was suggested that phytochrome cycling could drive the transport, or activation of some c r i t i c a l metabolite (64). Smith (152) had 33 previously proposed that phytochrome photoconversions might mediate the transport of some c r i t i c a l metabolite across a membrane. At present there is no evidence for a membrane pump driven by phytochrome c y c l i n g . However, Whitelam and Johnson (171), and Johnson and Whitelam (67), presented evidence from studies on nit r a t e reductase a c t i v i t y in Sinapis that demonstrated a temporal separation of two d i s t i n c t components of phytochrome action, i . e . Pfr and a fluence rate-dependent effect that could only be exhibited in the presence of Pfr. An important difference between these two models relates to their predictions for the effects of prolonged R i r r a d i a t i o n s . Johnson and Tasker's model (66) predicts a strong fluence rate dependency in the R and assumes that responses to both R and FR represent true HIR responses. One of the p r i n c i p a l predictions of Schafer's model is a lack of fluence rate dependency in the R and that, in contrast to the FRHIR, the response to R represents a simple inductive response. Most HIR responses are characterized by a strong fluence rate dependency in the B and FR wavebands (90). However, there have been contradictory reports on the e f f e c t s of prolonged R i r r a d i a t i o n s . In a number of studies l i t t l e or no fluence rate dependency was observed (eg. 169) but in others a strong fluence rate dependency was demonstrated (3,72,171). A number of recent reports have concluded that the apparent lack of fluence rate dependency during the red can be attributed to the screening effects of chlorophyll formed during prolonged R i r r a d i a t i o n s (55,64,67,71,171). In Norflurazon treated 34 seedlings, RHIR responses show a strong fluence rate dependence. (55,65,71,171). Heim and Schafer (51) and Jabben et a l . (60) showed that because of competing nonphotochemical reactions the Pfr/Ptot r a t i o in e t i o l a t e d Sinapis hypocotyls at 25C was strongly fluence rate-dependent. Therefore, they concluded that the fluence rate dependence of the so c a l l e d R HIR could be attributed to the fluence rate dependency of the Pfr/Ptot r a t i o (51,54). Evidence to d i s t i n g u i s h between the actions of R and FR l i g h t was provided by comparative studies on the effects of pulsed and continuous irradations. Heim and Schafer (51) and Schafer et a l . (132) showed that for the promotion of anthocyanin synthesis and the i n h i b i t i o n of hypocotyl elongation in e t i o l a t e d Sinapis , the e f f e c t s of continuous R i r r a d i a t i o n s could be largely substituted for by hourly 5 minute l i g h t pulses. In contrast, hourly l i g h t pulses could not substitute for the e f f e c t s of continuous FR. Therefore, i t was concluded (132) that the response to continuous R represents a 'multiple induction' response. Hartmann (45,46) developed a model which attempted to explain the effects of both the B and FRHIR so l e l y in terms of phytochrome. A number of studies have shown that for some HIR responses (eg. induction of anthocyanin synthesis in Sinapis ) the effects of B can be attributed exclusively to the action of phytochrome (47,98,172). However, for many other HIR responses, the e f f e c t s of B can only be explained in terms of a separate BAP (98,117,160). For example, several l i n e s of evidence, 35 including differences in the ki n e t i c s of the response to R and B, indicate that in a large number of species, the inhi b i t o r y e f f e c t s of B on hypocotyl elongation are mediated by a s p e c i f i c but as yet unidentified BAP (1,7,8,19,21,34,95,161). The main candidates for the BAP are the f l a v i n s and the carotenoids (117). The arguments for and against each have been reviewed many times (10,113,139,160). It i s now generally agreed that one or more flavoproteins i s the most l i k e l y candidate, although the evidence i s not yet conclusive. Most of the information pertaining to the identity of the BAP has been obtained from studies on fungi and lower plants (10,113). However, studies on phototropism in higher plants also strongly suggest a flavoprotein photoreceptor (140,166). There have not been s p e c i f i c attempts to identi f y the BAP responsible for the B HIR. It i s not known whether separate or i d e n t i c a l BAP's mediate phototropism and the B HIR in higher plants. 2.4 High Resolution Measurement Of Plant Growth Techniques for the continuous measurement of stem elongation have been used since Sachs (124) described his autographic auxanometer in 1882. In a recent review, Penny and Penny (110) described many of these early techniques as well as more recent methods used for the high resolution measurement of stem elongation. It is worth noting that some of the early techniques were very sensitive. For example, the 'cresograph' described by Bose (117) in 1927 could probably resolve 36 elongation to within 1 urn. Since their f i r s t introduction by Meijer (95) in 1968, linear displacement transducers have become the primary method of measuring elongation in stem segments, or intact seedlings (110). Linear displacement tranducers permit the continuous measurement of stem length, with a resolution of less than 0.1 urn (e.g. 111). In 1972 Addink and Meijer (1) described the use of a linear displacement transducer in combination with an electronic d i f f e r e n t i a t o r , that permitted growth rate to be plotted d i r e c t l y . However, this technique has not been widely used, and in most studies of intact seedlings, the growth rate has been calculated from the slope of the stem length: time relat i o n s h i p (e.g. 34,86,101,103). 37 III . MATERIALS AND METHODS 3.1 Plant Materials Seeds of white mustard ( Sinapis alba L. Lot WES-475 and Lot 8-CM-416-1859),radish ( Raphanus sativus L. cv. Scarlet Globe), cucumber ( Cucumis sativus L. cv. National P i c k l i n g ) , sunflower ( Helianthus annuus L. Sunbred Brand 265) and oat ( Avena sativa L.) were obtained from Buckerfield's Ltd. Vancouver B.C. Seeds were sown between two moistened s t r i p s (25x4 cm) of Whatman No 4 f i l t e r paper. The paper s t r i p s were r o l l e d into loose cylinders and placed standing upright in 100 ml beakers containing about 1 cm of d i s t i l l e d water. As the seeds germinated and grew their hypocotyls or c o l e o p t i l e s extended above the f i l t e r paper while their roots were securely held in place between the layers of paper. This approach permitted the seedlings to be manipulated with minimal disturbance to their root system. A l l seeds were germinated and grown in darkness in a l i g h t tight box (24x24x13 cm) at 25±1C. When the seedlings reached a height of 1-2 cm (60-72 hours after sowing at 25C ), the paper s t r i p s were cut into sections, each section holding one seedling. Seedlings with straight hypocotyls and tight hypocotyl hooks were selected for growth measurement experiments. 38 3.2 Growth Measurement The rate of seedling elongation was measured with two s p e c i a l l y constructed growth measuring systems, each consisting of a linear displacement transducer (Trans-Tek 241-000,core No.C04-005) coupled to a custom made signal d i f f e r e n t i a t o r (modified from Sargent 126). The p r i n c i p l e of operation was as follows. Movement of a thin metal rod, the transducer core, within the c y l i n d r i c a l transducer c o i l , generated a voltage change in the transducer output proportional to the displacement. By l i n k i n g the elongating seedling to the transducer core, a continuous high resolution record of seedling height was obtained. The rate of elongation could be calculated from the height versus time re l a t i o n s h i p . However, the transducer signal ( i . e . voltage) was e l e c t r o n i c a l l y d i f f e r e n t i a t e d , to obtain d i r e c t l y dv/dt, a signal proportional to the instantaneous rate of elongation. For growth measurements, a seedling was enclosed within a glass cuvette. The cuvette and growth measuring apparatus (Fig. 3) were mounted on heavy bases cushioned by foam pads to help iso l a t e the system from room vibrations. The bases consisted of a concrete pad in one system and a heavy steel plate in the second system. Each growth measuring apparatus and cuvette were enclosed within a l i g h t tight box. The box also also helped to iso l a t e the system from rapid fluctuations in room temperature. The transducer c o i l was mounted on a mechanical stage which in turn was attached to the s l i d e of a microscope body. The 39 MECHANICAL^ STAGE MICROSCOPE BODY o CONCRETE BLOCK FOAM RAD • PULLEY COUNTERWEIGHT •TRANSDUCER COIL -TRANSDUCER CORE -GLASS ROD •CUVETTE r-1 0 cm Figure 3 . Growth measuring apparatus. 40 c o a r s e a n d f i n e a d j u s t m e n t g e a r s o f t h e m i c r o s c o p e a l l o w e d v e r t i c a l p o s i t i o n i n g o f t h e t r a n s d u c e r c o i l , w h i l e t h e m e c h a n i c a l s t a g e p e r m i t t e d h o r i z o n t a l p o s i t i o n a d j u s t m e n t s i n two d i m e n s i o n s . The t r a n s d u c e r c o i l was l i n k e d t o t h e s e e d l i n g by a m e t a l hook on t h e end of a t h i n g l a s s r o d . The hook was e i t h e r s l i p p e d u n d e r t h e h y p o c o t y l hook o f t h e s e e d l i n g , o r l i n k e d t o a c o l l a r t h a t c o u l d be f i t t e d a b o u t t h e s t r a i g h t p o r t i o n o f t h e h y p o c o t y l ( F i g . 4 ) . The c o l l a r c o n s i s t e d o f a r u b b e r r i n g t h a t g r i p p e d t h e h y p o c o t y l a n d a w i r e l o o p t h a t p r o v i d e d a p o i n t o f a t t a c h m e n t f o r t h e c o r e a s s e m b l y . The c o r e a s s e m b l y ( t r a n s d u c e r c o r e , g l a s s r o d , and m e t a l h o ok) were c o u n t e r b a l a n c e d o v e r a p u l l e y w i t h s m a l l w e i g h t s . The t e n s i o n p l a c e d on t h e s e e d l i n g c o u l d be v a r i e d b u t was 200 mg e x c e p t where o t h e r w i s e i n d i c a t e d . The t r a n s d u c e r c o i l was p o w e r e d by an A n a t e k M o d e l 50-1S r e g u l a t e d DC power s u p p l y . By v a r y i n g t h e e x c i t a t i o n v o l t a g e o f t h e t r a n s d u c e r c o i l , t h e t r a n s d u c e r o u t p u t was c a l i b r a t e d so t h a t a 1 mm d i s p l a c e m e n t c o r r e s p o n d e d t o a 5V c h a n g e i n t r a n s d u c e r o u t p u t . A m i c r o m e t e r s c a l e on t h e f i n e a d j u s t m e n t g e a r o f t h e m i c r o s c o p e s l i d e was u s e d f o r c a l i b r a t i o n . The t r a n s d u c e r o u t p u t was i n i t i a l l y r e c o r d e d on a s t r i p c h a r t b u t i n l a t e r e x p e r i m e n t s was r e c o r d e d a t a t 1 o r 10 m i n u t e i n t e r v a l s by a d i g i t a l v o l t m e t e r / d a t a l o g g e r . The d i f f e r e n t i a t e d s i g n a l was r e c o r d e d on a s t r i p c h a r t ( F i g . 5 ) . By c o m p a r i n g t h e g r o w t h r a t e s c a l c u l a t e d f r o m t h e d i g i t i z e d t r a n s d u c e r o u t p u t w i t h t h e d i f f e r e n t i a t o r o u t p u t i t was d e t e r m i n e d t h a t a 1 mmh"1 g r o w t h r a t e c o r r e s p o n d e d t o a 41 GLASS ROD-COLLAR-WATER INLET RUBBER STOPPER I rui GAS OUTLET -WATER OUTLET HOOK -SEEDLING -FILTER PAPER METAL CLIP GAS INLET BASE 2 c m Figure 4. Cuvette, seedling and c o l l a r assembly. 42 DIGITAL DATA LOGGER TRANSDUCER OUTPUT (voltage) CHART RECORDER mm time DIFFERENTIATOR dt mm-h" time Figure 5. Methods of recording transducer output. 43 d i f f e r e n t i a t e d s i g n a l of 42 mv. Under n o r m a l e x p e r i m e n t a l c o n d i t i o n s t h e t r a n s d u c e r o u t p u t was s t a b l e a t t h e 1 mv r a n g e , p r o v i d i n g a u s e a b l e r e s o l u t i o n of a b o u t 0.2 urn. The d i f f e r e n t i a t o r o u t p u t was f i l t e r e d w i t h a low p a s s f i l t e r (RC c i r c u i t , 22 s e c . t i m e c o n s t a n t ) t o remove h i g h f r e q u e n c y n o i s e . Under n o r m a l e x p e r i m e n t a l c o n d i t i o n s t h e r e s o l u t i o n of t h e d i f f e r e n t i a t e d s i g n a l was a p p r o x i m a t e l y 6 umh" 1 . The r e l a t i o n s h i p between t r a n s d u c e r o u t p u t and c o r e d i s p l a c e m e n t was l i n e a r f o r 20V, p e r m i t t i n g 8 mm of s e e d l i n g g r o w t h w i t h o u t manual i n t e r v e n t i o n . The c o a r s e a d j u s t m e n t g e a r of t h e m i c r o s c o p e s l i d e was u s e d t o r e p o s i t i o n t h e t r a n s d u c e r c o i l when s e e d l i n g e l o n g a t i o n r a i s e d t h e c o r e beyond t h e t r a n s d u c e r ' s l i n e a r r a n g e . 3.3 C u v e t t e And S e e d l i n g E n v i r o n m e n t F o r most g r o w t h measurement e x p e r i m e n t s , t h e s e e d l i n g was e n c l o s e d w i t h i n a c y l i n d r i c a l d o u b l e - w a l l e d g l a s s c u v e t t e ( F i g . 4 ) . The s e e d l i n g was h e l d i n p l a c e by a m e t a l c l i p w h i c h f i r m l y g r i p p e d t h e f i l t e r p a p e r s u r r o u n d i n g t h e r o o t s o f t h e s e e d l i n g . The m e t a l c l i p was mounted i n t o a r u b b e r s t o p p e r t h a t was u s e d t o s e a l t h e base of t h e c u v e t t e . The c u v e t t e i t s e l f was mounted on a movable base t h a t p e r m i t t e d t h e s e e d l i n g t o be p r e c i s e l y a l i g n e d w i t h t h e t r a n s d u c e r c o r e . The r o o t s of t h e s e e d l i n g were b a t h e d i n 10 mM M E S - T r i s b u f f e r , pH 6.25. T e m p e r a t u r e c o n t r o l was p r o v i d e d by a water 44 bath which c i r c u l a t e d water through the cuvette jacket. The temperature within the cuvette was measured with a copper/constantan thermocouple connected to an Omega 2176A d i g i t a l thermometer. A gas stream was introduced into the cuvette via a syringe needle pushed through the rubber stopper. The gas stream was bubbled through the buffer and vented through the top of the cuvette. Before entering the cuvette the gas stream was humidified and temperature conditioned by passage through a water column immersed in the temperature controlled water bath. In most experiments medical grade a i r containing 330 ul l " 1 carbon dioxide was flushed through the cuvette at 50 or 100 mlmin" 1. Flow rates were measured with Matheson 601 Flowmeters. 3.4 Technical Problems A number of technical problems associated with the operation of the growth measuring system deserve comment. In the early stages of t h i s study, the growth measuring system was located in a temperature controlled growth room. However, i t eventually became apparent that the normal cycli n g of temperatures within the growth room was a f f e c t i n g the transducer output via expansion and contraction of the growth measuring system. In addition , switching of the solenoids in the growth room cooling system affected transducer output by e l e c t r i c a l induction. To overcome these, problems, and to isol a t e the system from rapid fluctuations in room temperature, 45 the growth measuring system was moved to a laboratory darkroom, and enclosed within a small wooden chamber. None of the results reported in th i s thesis were obtained from the early studies in the growth room. The s t a b i l i t y of the growth measuring system was p e r i o d i c a l l y checked by doing control experiments in which a small metal hook was used in place of the seedling. Under these conditions, perfectly f l a t displacement and growth rate curves were always obtained (Fig.6). A temperature s h i f t caused an apparent change in 'length' of wire due to expansion or contraction of some component of the system. The temperature within the cuvette had usually s t a b i l i z e d within 30 minutes following a 5°C temperature s h i f t . The temperature s h i f t had only a small transient effect on the d i f f e r e n t i a t o r output at the s e n s i t i v i t y used in most experiments. In preliminary experiments, f r i c t i o n between the transducer core and c o i l led to s t i c k i n g of the core and step-like increments in height. A teflon based lubricant (Crown 6075 Dry Film Lubricant) sprayed on the core and inside the c o i l helped a l l e v i a t e t h i s problem. Nevertheless, in some experiments 'noise' due to s t i c k i n g of the transducer core was apparent. In previous studies involving the use of linear displacement transducers to measure the growth of intact seedlings, the seedling has usually been linked to the transducer core by a fine thread t i e d about the hypocotyl. However, this technique was found to be impractical for my study because the seedling was enclosed within a small cuvette. In 46 25C -30C 30C -25C J 1 1 1 1 1 I I I I I L 30 60 90 120 TIME (<Mn) Figure 6. System s t a b i l i t y and response to temperature s h i f t s . A small metal hook was used in place of the seedling. A l l other conditions were i d e n t i c a l to those used during measurements of seedling growth. See Figure 8 for standard experimental conditions. 47 addition , the length of the thread could be affected by changes in humidity. In contrast, the core assembly used here was not affected by changes in humidity, and the metal hook could be e a s i l y slipped under the hypocotyl hook with minimal disturbance to the seedling. In addition, the point of attachment could be shifted from the hypocotyl hook to the c o l l a r assembly. This permitted the measurement of the the t o t a l hypocotyl growth rate, or the growth rate of that portion of the hypocotyl below the c o l l a r . However, in the experiments reported here, the transducer core was always linked d i r e c t l y to the hypocotyl hook of the seedling, unless otherwise stated. 3.5 Light Treatments Narrow bandwidth light.was obtained from a 150W xenon arc lamp or a modified 150W s l i d e projector, which had a quartz-iodide bulb, in combination with PTR (PTR Optics, Waltham, Mass.) interference f i l t e r s (half power bandwidth ca. 10 nm). The peaks of transmission of the f i l t e r s and the fluence rates of the standard l i g h t treatments are shown in Table 1. In a number of experiments Carolina B i o l o g i c a l Supply Plexiglas filters,CBS 650 and CBS 750, were used singly or in combination with the interference f i l t e r s . The transmission spectra of the CBS f i l t e r s , measured with Unicam SP 800B spectrophotometer are shown in Fig.7. The fluence rates of the l i g h t sources were measured inside the cuvette with a YSI Kettering Model 65A radiometer and adjusted to the appropriate value with PTR 4 8 T a b l e 1. C h a r a c t e r i s t i c s of s t a n d a r d l i g h t t r e a t m e n t s u s i n g PTR i n t e r f e r e n c e f i l t e r s . Peak W a v e l e n g t h (nm) P h o t o e q u i 1 i br i a 2 ( P f r / P t o t ) F l u e n c e R a t e 3 (Wm-2) 779 ( 7 8 0 ) 1 <.004 9 760 <.004 16 741 (740) .004 13 731 (730) .008 17 720 .021 ' 13 710 . 1 12 698 (700) .37 15 670 .8 16 660 .8 16 600 .8 12 550 - 16 500 - 16 450 - 16 402 (400) - 7 379 - 5 'For c o n v e n i e n c e of e x p r e s s i o n t h e s e l i g h t t r e a t m e n t s were r e f e r e d t o i n t e x t by t h e w a v e l e n g t h e n c l o s e d i n b r a c k e t s . 2The p h o t o e q u i l i b r i a e s t a b l i s h e d by t h e f i l t e r s u s e d i n t h i s s t u d y were not d e t e r m i n e d . The v a l u e s p r e s e n t e d a r e e s t i m a t e s d e r i v e d f r o m d a t a p u b l i s h e d f o r s i m i l a r f i l t e r s ( 5 5 , 6 0 , 9 9 ) . 3 F l u e n c e r a t e s of l i g h t t r e a t m e n t s were measured w i t h i n t h e c u v e t t e and v a r i e d by a p p r o x i m a t e l y ±10% a c r o s s t h e l i g h t f i e l d . 49 450 500 550 600 650 700 750 800 WAVELENGTH (nm) Figure 7. Transmission spectra of CBS650 red and 730 far red f i l t e r s and dark yellow green Roscolux NO91 acetate f i l t e r . Transmission spectra were obtained with a Unicam SP 800B spectrophotometer. 50 neutral density f i l t e r s . The seedlings were always irradiated from the side. A s t r i p of aluminum f o i l placed behind the seedling outside of the cuvette acted as a mirror and helped provide more uniform illumination. For dichromatic i r r a d i a t i o n s the seedling was irr a d i a t e d simultaneously from opposite sides and no mirror was used. A l l manipulations of the seedlings were done under a dim green safelight consisting of cool white fluorescent tubes wrapped in a t r i p l e layer of dark yellow green Roscolux No 91 acetate f i l t e r ( ^  max 520 nm ,<0.25 WITT 2 at 30cm) (transmission spectrum Fig.7). 51 IV. RESULTS AND DISCUSSION 4.1 Growth In Darkness In preliminary experiments, the growth rate of e t i o l a t e d Sinapis seedlings was measured in darkness at 25C. The growth rate varied considerably among seedlings but was usually between 0.5 and 2 mmh"1. When the growth rate measurements were started immediately after mounting the seedling within the cuvette, a d i s t i n c t decline in growth rate could often be detected; however, the growth rate usually increased within an hour. A surprising observation was that under constant environmental conditions the growth rate of most seedlings was not constant but varied rhythmically (Figs.8 and 9). Some Sinapis seedlings exhibited two types of o s c i l l a t i o n s : a short ( t y p i c a l l y 3-20 min at 25C) period o s c i l l a t i o n (SPO) and a long period o s c i l l a t i o n (LPO). The period of the LPO varied considerably among seedlings, ranging from 50 minutes to more than 2 hours. In some cases the SPOs appeared to be superimposed on the LPO, but both types could occur singly (Fig.8A). Under constant environmental conditions these o s c i l l a t i o n s could be sustained for 48 hours (and presumably longer). SPOs were observed in most seedlings. D i s t i n c t , highly regular LPOs of the type shown in Fig.8A (lower trace) occurred infrequently. In a few seedlings extremely large amplitude LPOs occurred (Fig.8B). A curious feature of these large amplitude LPOs was that the growth rate trace sometimes f e l l below zero, i . e . for brief periods of time the seedlings 52 Figure 8. Representative traces showing A) SPOs, and LPOs, and B) large amplitude LPOs in the growth rate traces of three d i f f e r e n t Sinapis seedlings. The growth rate was measured by l i n k i n g the transducer core to the hypocotyl hook. 330ul 1~1 carbon dioxide in a i r was flushed through the cuvette at 50 or 100ml min" 1. Tension on the seedling was 200mg. Cuvette temperature was 25C. These conditions are referred to as standard experimental conditions. 53 A 1.5 1.0 TIME (min) Figure 9. Short period o s c i l l a t i o n s in the growth rate of Sinapis . A) and B) are traces from two d i f f e r e n t seedings. Growth measurements commenced immediately after mounting the seedling within the cuvette. Standard experimental conditions. 54 a p p e a r e d t o b e s h r i n k i n g ! P r e l i m i n a r y s t u d i e s h a d i n d i c a t e d t h a t v a r i a t i o n s i n t r a n s d u c e r o u t p u t c o u l d b e c a u s e d b y a v a r i e t y o f s y s t e m r e l a t e d p r o b l e m s . T h e r e f o r e , t h e q u e s t i o n a r o s e , w e r e t h e s e o s c i l l a t i o n s b i o l o g i c a l i n o r i g i n , i . e . g e n e r a t e d b y t h e s e e d l i n g , o r w e r e t h e y a r t e f a c t s r e l a t e d i n s o m e w a y t o t h e g r o w t h m e a s u r i n g s y s t e m ? A s p r e v i o u s l y d i s c u s s e d , c o n t r o l e x p e r i m e n t s u s i n g t h e c o m p l e t e g r o w t h s y s t e m a l w a y s y i e l d e d p e r f e c t l y f l a t g r o w t h r a t e t r a c e s u n d e r c o n s t a n t e n v i r o n m e n t a l c o n d i t i o n s ( F i g . 6 ) . T h e r e f o r e , t h e o s c i l l a t i o n s w e r e n o t r e l a t e d t o r h y t h m i c f l u c t u a t i o n s i n t e m p e r a t u r e , l i n e v o l t a g e o r o t h e r s y s t e m e r r o r s o f t h a t t y p e . T h i s c o n c l u s i o n i s s u p p o r t e d b y t h e f a c t t h a t o s c i l l a t i o n s i n t h e g r o w t h r a t e s o f t w o p l a n t s m e a s u r e d s i m u l t a n e o u s l y ( i . e . i n t h e t w o g r o w t h m e a s u r i n g s y s t e m s ) w e r e t o t a l l y i n d e p e n d e n t . T h u s , t h e o s c i l l a t i o n s w e r e c l e a r l y g e n e r a t e d i n s ome m a n n e r b y t h e s e e d l i n g s t h e m s e l v e s . I n m a n y s e e d l i n g s t h e S P O s a p p e a r e d t o b e v e r y n e a r l y s i n u s o i d a l i n f o r m . T h e p e r i o d , a m p l i t u d e a n d s h a p e o f t h e s e o s c i l l a t i o n s v a r i e d b o t h w i t h i n a n d a m o n g s e e d l i n g s . T h e p e r i o d o f t h e o s c i l l a t i o n w a s d e t e r m i n e d b y c o u n t i n g t h e n u m b e r o f p e a k s o c c u r r i n g i n a g i v e n t i m e . I n i n d i v i d u a l s e e d l i n g s t h e p e r i o d w a s n o r m a l l y q u i t e s t a b l e ( e . g . F i g s . 8 a n d 9 ) . H o w e v e r , a s p o n t a n e o u s c h a n g e i n p e r i o d s o m e t i m e s o c c u r r e d , o f t e n i n v o l v i n g a d o u b l i n g o r h a l v i n g o f t h e p e r i o d ( F i g . l O A , l o w e r t r a c e ) . I n a f e w c a s e s a m o r e c o m p l e x c h a n g e i n t h e s h a p e a n d p e r i o d o f t h e o s c i l l a t i o n o c c u r r e d ( F i g . l O A , u p p e r t r a c e ) . T h e r e w a s n o a p p a r e n t r e l a t i o n s h i p b e t w e e n t h e p e r i o d a n d 55 § 2h to o-_l I I l_ 1 ' ' ' • _l I I 1——J I I I— 30 60 90 TIME (min) 120 150 Figure 1 0 . Variations in the period, amplitude and occurrence of SPOs in individual seedlings. Growth measurements commenced immediately after mounting the seedling within the cuvette. A l l traces from d i f f e r e n t seedlings. Standard experimental conditions. 56 F i g u r e 11. E f f e c t s o f i n c r e a s i n g t e n s i o n on t h e g r o w t h r a t e o f S i n a p i s . T e n s i o n on t h e s e e d l i n g was 200mg p r i o r t o a d d i t i o n o f w e i g h t s . S t a n d a r d e x p e r i m e n t a l c o n d i t i o n s e x c e p t f o r t e n s i o n . 57 amplitude of the SPO and the average growth rate. In some seedlings the growth rate appeared to be e r r a t i c , with l i t t l e or no evidence of a rhythmic v a r i a t i o n in rate (Fig.lOB, upper trace). However, even in these seedlings a highly regular o s c i l l a t i o n could a r i s e , seemingly spontaneously, and then disappear (Fig.lOB, lower trace). There did not appear to be any minimum growth rate below which SPOs did not occur. Highly regular SPOs were obtained at growth rates as low as 0.1 mmh"1. The period and amplitude of the SPOs, as well as the long term growth rate, were unaffected by increasing the tension on the seedling in 100 mg increments (Fig.11). Tensions of up to about 1 g had no apparent e f f e c t on the growth rate; however, at higher tensions the hypocotyl hook was slowly pulled open. The period of the SPO was temperature dependent. A 5 C temperature decrease always caused very rapid decline in growth rate, and an increase in the period of the o s c i l l a t i o n (Fig.12A). A 5°C increase in temperature had the opposite effect (Fig.12B). A 10°C decrease in temperature usually caused a large rapid decline in growth rate followed by 2 or 3 large o s c i l l a t i o n s . The SPOs were often damped out, but usually resumed at the lower growth rate after several hours (Fig.13). The temperature c o e f f i c i e n t for the period of the SPO was less than 0.5 because of the negative relationship between period and temperature. The more meaningful rel a t i o n s h i p i s the temperature c o e f f i c i e n t for the change in frequency. This ranged from 2.0-2.5 among individual seedlings in the 25-15C i n t e r v a l . 58 a 25C -20C 20C-25C TIME (min) F i g u r e 12. E f f e c t s of 5°C temperature s h i f t s on SPOs in S i n a p i s . A) and B) are t r a c e s from two d i f f e r e n t s e e d l i n g s . Standard experimental c o n d i t i o n s . 59 Figure 13. Effects of a 25 to 15C temperature s h i f t on the growth rate of Sinapis . Standard experimental conditions except for temperature. 60 The possible occurrence of these 'growth' o s c i l l a t i o n s was tested in a number of other dicot seedlings and in one monocot ( i . e . oat). To measure the growth of oat co l e o p t i l e s the transducer core was linked to the c o l e o p t i l e via the c o l l a r assembly, which was placed around the c o l e o p t i l e , just below the t i p . The results of these experiments are summarized in Table 2. Long period o s c i l l a t i o n s were not observed in oat; however, only a limited number of seedlings were tested. Short period o s c i l l a t i o n s , however, did occur (Fig.14A). The growth rate of sunflower was exceptionally stable, with no evidence of SPOs. In one p a r t i c u l a r seedling the growth rate was precisely 0.5 mmh"1 for more than 12 hours. However, in 2 seedlings, large amplitude LPOs arose spontaneously after long periods of very uniform growth rate (e.g.. Fig.15A). LPOs and SPOs were found to occur singly or together in both radish and cucumber (Fig.14). An interesting observation was that in radish the LPOs sometimes appeared to be paired, i.e two v i r t u a l l y i d e n t i c a l o s c i l l a t i o n s would occur, followed by a pair of o s c i l l a t i o n s with a s l i g h t l y d i f f e r e n t pattern (Fig.15B). In most experiments sharp spikes such as those shown in Fig.15B were considered to be noise related to core s t i c k i n g . However, the fact that the fine structure of these peaks could be repeated, c l e a r l y indicates that these p a r t i c u l a r spikes were not random noise. As with Sinapis the growth rate traces sometimes f e l l below zero during LPOs in both radish and sunflower, in some 61 Table 2 . Occurrence of SPOs and LPOs i n dark grown s e e d l i n g s . P l a n t Species LPO SPO Mustard S i n a p i s a l b a L. Lot WES. + + Lot 8-cm + + Sunflower (n=5) H e l i a n t h u s annuus L. + Oat (n=4) Avena s a t i v a L. + Radish Raphanus s a t i v u s L. + + Cucumber Cucumis s a t i v u s L. + + 62 Figure 14. Representative traces showing the growth of A) oat, B) radish and C) cucumber seedlings. The transducer core was linked to the oat c o l e o p t i l e via the c o l l a r assembly. Otherwise, standard experimental conditions. 63 64 TIME (hours) Figure 15. Examples of large amplitude LPOs in the growth rate traces of e t i o l a t e d A) sunflower and B) radish seedlings. Standard experimental conditions. 65 cases by as much as 1 mmh"1 (Fig.15). This observation is i l l o g i c a l and suggests that these large amplitude LPOs do not r e f l e c t a true variation in the rate of hypocotyl elongation. One possible cause of these LPOs i s hypocotyl hook opening and c l o s i n g . Galston et a l . (35) reported 'bobbing' movements in the hook of e t i o l a t e d pea seedlings, with a period of 1-2 hours. However, hypocotyl hook opening and closing was not involved in the LPOs observed here. Large amplitude LPOs were found to occur in mustard, sunflower and radish seedlings even when the seedling was linked to the transducer core via the c o l l a r assembly (e.g. Fig.16). Use of the c o l l a r should eliminate any contribution of hypocotyl hook opening and closing to the 'apparent' growth rate. Instead, the probable o r i g i n of these LPOs involves'another rhythmic growth movement, nutation. In addition to extension growth, young seedlings undergo rhythmic bending movements (nutations) (68). Nutations often occur as e l l i p t i c a l movements around the reference d i r e c t i o n (e.g. the plumbline) so that the t i p of the growing seedling traces a h e l i c a l path in space (circumnutation). Nutation may also occur as a pendulum type movement r e s t r i c t e d to one plane. The period of circumnutation is c h a r a c t e r i s t i c of a species and generally f a l l s into the range of one to several hours. Bending in excess of 10 degrees from the plumbline is not unusual (68,116). The important point is that any change in seedling i n c l i n a t i o n must lead to a change in the 'apparent' height of the seedling due to arcing of the hypocotyl t i p . Thus, bending away from the plumbline decreases 66 Figure 16. Large amplitude LPOs in the growth rate trace of radish measured using the c o l l a r assembly. Standard experimental conditions. 67 t h e a p p a r e n t h e i g h t o f t h e s e e d l i n g , w h i l e b e n d i n g t o w a r d s t h e p l u m b l i n e i n c r e a s e s t h e a p p a r e n t h e i g h t . T h i s e f f e c t c a n be c o n s i d e r a b l e . A 10 d e g r e e bend o v e r a 1 cm h y p o c o t y l segment c a u s e s an a p p a r e n t d e c r e a s e i n h e i g h t o f 152 urn. The s p e c i f i c e f f e c t o f n u t a t i o n on t h e m e a s u r e d ' g r o w t h r a t e ' w o u l d d e p e n d on a number o f f a c t o r s i n c l u d i n g t h e a m p l i t u d e a n d p a t t e r n o f n u t a t i o n , t h e r a t e o f b e n d i n g , a n d t h e g e o m e t r y o f t h e g r o w t h m e a s u r i n g s y s t e m and t h e s e e d l i n g . I n t h e p r e s e n t s t u d y c a r e f u l v i s u a l o b s e r v a t i o n o f s e e d l i n g s w i t h i n t h e c u v e t t e , u s i n g a g r e e n s a f e l i g h t , r e v e a l e d t h a t v i s i b l e r h y t h m i c b e n d i n g movements were o c c u r r i n g w h e n e v e r l a r g e a m p l i t u d e LPOs were o b t a i n e d . A number o f a t t e m p t s were made t o o b t a i n a p h o t o g r a p h i c r e c o r d o f t h e s e b e n d i n g movements, w h i l e s i m u l t a n e o u s l y m e a s u r i n g g r o w t h . U n f o r t u n a t e l y t h e s e a t t e m p t s were n o t s u c c e s s f u l . N e v e r t h e l e s s , I h a v e l i t t l e d o u b t t h a t t h e l a r g e a m p l i t u d e LPOs were a d i r e c t r e s u l t o f c i r c u m n u t a t i o n . I t i s u n c l e a r i f t h e low a m p l i t u d e LPOs, t h a t s o m e t i m e s o c c u r r e d i n r a d i s h , S i n a p i s and c u c u m b e r , were a l s o a r e s u l t o f n u t a t i o n . R h y t h m i c b e n d i n g of t h e h y p o c o t y l c o u l d n o t be d e t e c t e d d u r i n g low a m p l i t u d e L POs, e i t h e r w i t h o r w i t h o u t t h e a i d o f a m i c r o s c o p e . However, i n r a d i s h and i n S i n a p i s l o w a m p l i t u d e LPOs s o m e t i m e s e v o l v e d d i r e c t l y i n t o h i g h a m p l i t u d e LPOs w i t h o u t a c h a n g e i n p e r i o d , w h i c h s t r o n g l y s u g g e s t s t h a t b o t h a r e t h e r e s u l t o f n u t a t i o n . The o r i g i n o f t h e p a i r e d p e a k s t h a t s o m e t i m e s o c c u r r e d ( e . g . F i g . l 5 B ) i s u n c l e a r . One p o s s i b l e e x p l a n a t i o n r e l a t e s t o t h e f a c t t h a t d u r i n g one c y c l e o f n u t a t i o n t h e s e e d l i n g 68 a p p r o a c h e s and moves away f r o m t h e p l u m b l i n e t w i c e . T h e r e f o r e , two o s c i l l a t i o n s i n g r o w t h r a t e s h o u l d o c c u r f o r e a c h c o m p l e t e c y c l e o f n u t a t i o n . T h u s , i f t h e p a t t e r n o f n u t a t i o n c h a n g e d s l i g h t l y b e t w e e n e a c h c o m p l e t e c y c l e , d i f f e r e n t p a i r s o f LPOs c o u l d p o s s i b l y a r i s e . The p o s s i b l e i n f l u e n c e o f c i r c u m n u t a t i o n on h i g h r e s o l u t i o n g r o w t h s t u d i e s i n i n t a c t p l a n t s h a s a p p a r e n t l y n o t been c o n s i d e r e d p r e v i o u s l y . C o s g r o v e ( 1 9 , 2 2 ) f o u n d t h a t w h i l e some e t i o l a t e d c u c umber a n d s u n f l o w e r s e e d l i n g s h a d v e r y s t a b l e g r o w t h r a t e s o t h e r s e x h i b i t e d l a r g e o s c i l l a t i o n s i n r a t e s i m i l a r t o t h e l a r g e a m p l i t u d e LPOs r e p o r t e d h e r e ; h o w e v e r , t h e s e • o s c i l l a t i o n s were c o n s i d e r e d t o r e f l e c t t r u e v a r i a t i o n s i n t h e g r o w t h r a t e . Low a m p l i t u d e o s c i l l a t i o n s w i t h a p e r i o d o f a b o u t one h o u r a r e a l s o a p p a r e n t i n t h e g r o w t h r a t e t r a c e s p u b l i s h e d by A d d i n k a n d M e i j e r (1) a n d Gaba and B l a c k ( 3 4 ) . T h e s e o s c i l l a t i o n s may n o t be r e l a t e d t o n u t a t i o n ; n e v e r t h e l e s s , t h e f a c t t h a t n u t a t i o n i s u b i q u i t o u s i n g r o w i n g s e e d l i n g s s u g g e s t s t h a t c a u t i o n must be u s e d when i n t e r p r e t i n g d a t a f r o m h i g h r e s o l u t i o n g r o w t h s t u d i e s . Do t h e SPOs r e f l e c t a t r u e v a r i a t i o n i n t h e r a t e o f v e r t i c a l e x t e n s i o n , o r a r e t h e y a l s o a r t e f a c t s ? E x p e r i m e n t s i n w h i c h t h e s e e d l i n g was l i n k e d t o t h e t r a n s d u c e r c o r e v i a t h e c o l l a r a s s e m b l y c l e a r l y d e m o n s t r a t e d t h a t h y p o c o t y l hook ' b o b b i n g ' was n o t i n v o l v e d i n t h e g e n e r a t i o n o f SPOs. F i g u r e 17A shows an e x p e r i m e n t i n w h i c h t h e c o l l a r was p l a c e d a b o u t 5 mm b e l o w t h e t i p o f t h e h y p o c o t y l , i n a 20 mm t a l l S i n a p i s s e e d l i n g . As w o u l d be e x p e c t e d , t h e g r o w t h r a t e m e a s u r e d a t t h e 69 E E ~ 0 HYPOCOTYL HOOK •• COLLAR COLLAR •• HYPOCOTYL HOOK J i i i I i_ o HYPOCOTYL HOOK •* COLLAR 30 60 90 120 TIME (min) 150 F i g u r e 17. G r o w t h r a t e m e a s u r e m e n t s o f S i n a p i s made a t t h e h y p o c o t y l hook o r u s i n g t h e c o l l a r a s s e m b l y . The c o l l a r was p l a c e d a p p r o x i m a t e l y A) 5mm b e l o w t h e h y p o c o t y l hook i n a 20mm t a l l s e e d l i n g a n d B) 8mm b e l o w t h e h y p o c o t y l hook i n a 10mm t a l l s e e d l i n g . S t a n d a r d e x p e r i m e n t a l c o n d i t i o n s . 70 c o l l a r was less than that of the entire seedling. In addition, SPOs c l e a r l y occurred, with a period similar to that measured at the hypocotyl hook. However, i t should be noted that in some experiments, the period and shape of the SPOs measured at the hypocotyl hook and the c o l l a r were not the same. In a similar experiment, the c o l l a r was placed about 2 mm above the base of the hypocotyl in a seedling 10 mm t a l l (Fig.17B). The growth rate was very low; however, SPOs were evident. These results suggest that o s c i l l a t i o n s in the rate of stem elongation occur throughout the length of the hypocotyl. However, the results of Heathcote (48) should also be considered. Heathcote (48) discovered small amplitude, high frequency o s c i l l a t i o n s superimposed on the normal e l l i p t i c a l circumnutation patterns of bean seedlings. The period of these 'micronutations' ranged from 12-30 minutes depending on the temperature, with a Q of 2.22 for the 15-25C i n t e r v a l . Micronutations and nutation occurred both singly and together. Micronutation appeared to involve stem displacements of much less than 1 mm. The c h a r a c t e r i s t i c s of these micronutations (e.g. Q J 0 and relationship to nutation) are similar to the SPOs observed here. Therefore, the question a r i s e s , are the SPOs observed here the result of small amplitude micronutations? The fact that SPOs could be detected near the base of the hypocotyl (e.g. Fig.17B) weighs against this p o s s i b i l i t y since the base of the seedling was firmly fixed in position. Nevertheless, i t i s conceivable that bending near the base of the hypocotyl could generate SPOs. It should be noted that the 71 use of the c o l l a r would not prevent the generation of SPOs by micronutation, since s l i g h t bending of the hypocotyl would also pivot the c o l l a r . It can be argued, however, that because SPOs were sometimes superimposed on LPOs (that were caused by nutation), micronutation a p r i o r i cannot be involved in the generation of SPOs. Collar experiments had demonstrated that neither LPOs nor SPOs were related to hypocotyl hook bobbing. Therefore, o s c i l l a t i o n s in the growth rate trace could only be caused by variations in the true growth rate, or by bending towards or away from the plumbline. Bending towards the plumbline adds to the true growth rate, while bending away from the plumbline subtracts from the true growth rate. Thus, nutation or micronutation can only cause o s c i l l a t i o n s about the true growth rate. Since i t i s almost certain that LPOs were the result of nutation ( i . e . the o s c i l l a t i o n s were artefacts and did not represent the true growth rate), SPOs superimposed on the LPOs could not be the result of micronutation. This argument suggests that SPOs represent genuine variations in the rate of stem elongation. Nutations (and presumably micronutation) ari s e because of rhythmically varying but d i f f e r e n t rates of c e l l u l a r elongation on opposite sides of the hypocotyl (68,69). In other words, bending occurs because o s c i l l a t i o n s in the rate of c e l l u l a r elongation are out of phase in a l a t e r a l cross section across the stem. For o s c i l l a t i o n s in the rate of v e r t i c a l extension to occur, the o s c i l l a t i o n s in the rate of c e l l u l a r elongation must be in phase in a l a t e r a l cross section of the stem. Thus, i t i s 72 apparent that at the c e l l u l a r l e v e l there i s no real difference between micronutations and a variation in the rate of v e r t i c a l extension. For either to occur, the rate of c e l l u l a r elongation in an individual c e l l must vary rhythmically. The only difference between the two relates to the c e l l to c e l l coupling of these o s c i l l a t i o n s . Thus, regardless of whether SPOs are the result of micronutation (48) or true variations in the rate of v e r t i c a l extension, their occurrence implies that the rate of c e l l u l a r elongation in young seedlings must vary rhythmically. However, for these o s c i l l a t i o n s to be detected at the whole plant l e v e l , there must be a high degree of synchrony of elongation among large populations of c e l l s within the hypocotyl (or c o l e o p t i l e ) . It i s unclear i f a l l the tissue i s synchronized in i t s growth (e.g. as suggested by Fig.17A) or i f d i f f e r e n t populations of c e l l s are sychronized but d i f f e r in phase or period of their o s c i l l a t i o n s . However, i t does appear that o s c i l l a t i o n s of some type occur throughout the straight portion of the hypocotyl e.g. even in tissues very near the base of the hypocotyl (Fig.17B). Nevertheless, there were numerous cases in which the growth rate of Sinapis seedlings as well as other species, was very stable, with no evidence of o s c i l l a t i o n s , or else was e r r a t i c with irregular variations in rate. In addition, SPOs sometimes spontaneously arose, and then disappeared (e.g. Fig.lOB). The absence of o s c i l l a t i o n s in the growth rate trace could be related to a damping out of the o s c i l l a t i o n in the rate of c e l l u l a r elongation. A l t e r n a t i v e l y , o s c i l l a t i o n s in the rate of 73 c e l l u l a r elongation might be sustained; however, a loss of c e l l to c e l l coupling of these o s c i l l a t i o n s ( i . e . a loss of synchrony) would cause them to 'average out', leading to stable or perhaps sometimes e r r a t i c growth rates. These two p o s s i b i l i t i e s cannot be distinguished on the basis of data presented here. O s c i l l a t i o n s in the growth rate of intact seedlings have been reported previously, but have not been systematically studied (9,19,101,106). Sustained short period o s c i l l a t i o n s are apparent in previously published traces of seedling height (103). Cosgrove (21) showed a growth rate trace of hypocotyl elongation in Sinapi s which also seemed to show rhythmic v a r i a t i o n in growth rate; however, these variations were considered to represent '(random variation) noise'. O s c i l l a t i o n s in the rate of elongation of hypocotyl segments may also occur (e.g. 109). It should also be mentioned that Scott (146) very c l e a r l y demonstrated that the rate of elongation in V i c i a faba roots varied rhythmically with a period of about 15-20 minutes. Thus, there i s evidence for the occurrence of sustained o s c i l l a t i o n s in the growth rate of elongating stems and roots, although, t h i s phenomenon has not been widely recognized. Damping o s c i l l a t i o n s in the growth rate of intact seedlings, after some type of perturbation, are also known to occur (110). In a f i n a l series of experiments on t h i s topic, the possible involvement of the roots and the apex or cotyledons in the generation of SPOs was tested. SPOs were sustained after 74 ROOT EXCISED • ' i • ' ' i i I i 1— 1.0 • B ON COLLAR SEEDLING DECAPITATED _ i i i i ' • 1 1 i i 1 * 1 i 30 60 90 120 150 180 210 TIME (mm) Figure 18. Effects of root excision or decapitation on SPOs in Sinapis . A) The root was excised immediately prior to the start of growth measurements. B) the seedling was decapitated below the cotyledons leaving the hypocotyl hook i n t a c t . Growth measurements made at c o l l a r . Standard experimental conditions. 7 5 t h e e x c i s i o n o f t h e r o o t s , a l t h o u g h t h e g r o w t h r a t e u s u a l l y d e c l i n e d w i t h t i m e ( e . g . F i g . l 8 A ) . T h e e f f e c t s o f d e c a p i t a t i o n ( l e a v i n g t h e h y p o c o t y l h o o k i n t a c t ) o n S P O s w e r e l e s s c l e a r c u t . I n e v e r y c a s e ( n = 7 ) t h e S P O s c o n t i n u e d f o r a n h o u r o r m o r e , a f t e r d e c a p i t a t i o n ; h o w e v e r , t h e g r o w t h r a t e a l w a y s b e c a m e e r r a t i c w i t h t i m e . R e g u l a r S P O s w e r e n e v e r o b t a i n e d m o r e t h a n t w o h o u r s a f t e r e x c i s i o n , a l t h o u g h v e r y r e g u l a r L P O s d i d o c c u r . T h e s e r e s u l t s c l e a r l y i n d i c a t e t h a t t h e r o o t s w e r e n o t r e q u i r e d f o r t h e g e n e r a t i o n o f S P O s , b u t t h e y s u g g e s t t h a t s o m e s i g n a l f r o m t h e a p e x o r c o t y l e d o n s w a s r e q u i r e d t o s u s t a i n t h e S P O s . T h e a b i l i t y o f s u s t a i n e d L P O s t o a r i s e a f t e r d e c a p i t a t i o n o f t h e s e e d l i n g i s c o n s i s t e n t w i t h t h e i r b e i n g t h e r e s u l t o f n u t a t i o n , a s i t i s w e l l k n o w n t h a t n u t a t i o n c a n o c c u r i n d e c a p i t a t e d s e e d l i n g s ( 6 8 ) . T h e o r i g i n a l p u r p o s e o f t h e s e s t u d i e s w a s t o o b t a i n a n i m p r e s s i o n o f h y p o c o t y l g r o w t h i n d a r k n e s s , a s a p r e l i m i n a r y t o s t u d i e s o n t h e e f f e c t s o f l i g h t i n h y p o c o t y l e l o n g a t i o n . H o w e v e r , t h i s w o r k h a s s h o w n t h a t t h e a p p a r e n t e l o n g a t i o n r a t e i n d a r k n e s s i s n o t v e r y s t a b l e . L o n g p e r i o d o s c i l l a t i o n s , w h i c h I c o n c l u d e r e s u l t f r o m n u t a t i o n a l m o v e m e n t s , a n d s h o r t p e r i o d o s c i l l a t i o n s i n r a t e c a n o c c u r i n a n u m b e r o f s p e c i e s . I s u g g e s t t h a t t h e s h o r t p e r i o d o s c i l l a t i o n s r e s u l t f r o m r h y t h m i c v a r i a t i o n s i n t h e r a t e o f c e l l u l a r e x p a n s i o n . T h e s e s t u d i e s h a v e a l s o i n d i c a t e d t h a t c a u t i o n m u s t b e u s e d i n i n t e r p r e t i n g d a t a f r o m h i g h r e s o l u t i o n g r o w t h s t u d i e s , b e c a u s e o f t h e p o s s i b l e c o n f o u n d i n g i n f l u e n c e o f n u t a t i o n . 76 4.2 L i g h t P u l s e E x p e r i m e n t s The e f f e c t s o f b r i e f ( i . e . <30 m i n u t e s ) l i g h t p u l s e s on h y p o c o t y l e l o n g a t i o n were t e s t e d i n a number o f p l a n t s p e c i e s . S e e d l i n g s were a l l o w e d t o e q u i l i b r a t e i n t h e c u v e t t e f o r 1 t o 2 h o u r s p r i o r t o l i g h t t r e a t m e n t s . To a v o i d t h e p o s s i b l e c o n f o u n d i n g e f f e c t s o f n u t a t i o n , s e e d l i n g s w h i c h e x h i b i t e d e x t r e m e l y e r r a t i c g r o w t h r a t e s , o r h i g h a m p l i t u d e l o n g p e r i o d o s c i l l a t i o n s , were d i s c a r d e d . 4.2.1 E f f e c t s Of B l u e L i g h t P u l s e s A r a p i d i n h i b i t i o n o f h y p o c o t y l e l o n g a t i o n by b l u e l i g h t h a s been shown t o o c c u r i n a number o f s p e c i e s ( 1 9 , 2 1 , 3 4 , 9 5 ) . I n a p r e l i m i n a r y s e r i e s o f e x p e r i m e n t s t h e p o s s i b l e o c c u r r e n c e of a r a p i d b l u e l i g h t r e s p o n s e was t e s t e d i n e t i o l a t e d c u c umber and r a d i s h s e e d l i n g s a n d i n S i n a p i s s e e d l i n g s f r o m two d i f f e r e n t s e e d l o t s . R e p r e s e n t a t i v e t r a c e s s h o w i n g t h e e f f e c t s o f b r i e f B450 l i g h t p u l s e s a r e shown i n F i g . 1 9 . The p a t t e r n o f r e s p o n s e t o a B450 l i g h t p u l s e showed some g e n e r a l f e a t u r e s w h i c h were s i m i l a r i n a l l c a s e s . I r r a d i a t i o n w i t h B450 c a u s e d a l a r g e ^ r a p i d d e c l i n e i n g r o w t h r a t e a f t e r a v e r y b r i e f l a g ( i . e . < 2 m i n ) . The g r o w t h r a t e u s u a l l y r e t u r n e d t o t h e o r i g i n a l d a r k l e v e l w i t h i n an h o u r a f t e r a s h o r t ( i . e . 5 min) l i g h t p u l s e . D u r i n g p r o l o n g e d i r r a d i a t i o n s t h e g r o w t h r a t e r e m a i n e d s u p p r e s s e d f o r t h e d u r a t i o n o f t h e i r r a d i a t i o n . A l t h o u g h t h e s e g e n e r a l f e a t u r e s o f t h e r e s p o n s e were 77 Figure 19. E f f e c t s of a 5 minute B450 l i g h t pulse on the growth rate of A) cucumber, B) radish, and C) Sinapis seedlings. Standard experimental conditions. 78 TIME (• in) 79 s i m i l a r , t h e d e t a i l s o f t h e r e s p o n s e t o a B450 l i g h t p u l s e d i f f e r e d somewhat among s p e c i e s . I n c u c u m b e r , g r o w t h was i n h i b i t e d a f t e r a l a g o f l e s s t h a n 30 s e c o n d s . D u r i n g t h e r e c o v e r y p e r i o d f o l l o w i n g t h e l i g h t p u l s e , t h e g r o w t h r a t e u s u a l l y e x h i b i t e d s h o r t p e r i o d o s c i l l a t i o n s ( F i g . l 9 A ) . I n r a d i s h t h e l a g was a b o u t 1.5 m i n u t e s a nd t h e g r o w t h r a t e u s u a l l y u n d e r w e n t one o r two l a r g e o s c i l l a t i o n s b e f o r e s t a b i l i z i n g ( F i g . l 9 B ) . I n s e e d l i n g s f r o m b o t h S i n a p i s s e e d l o t s , t h e t y p i c a l r e s p o n s e t o a B450 p u l s e c o n s i s t e d o f a r a p i d d e c l i n e i n g r o w t h r a t e a f t e r a l a g o f a b o u t 1 m i n u t e , f o l l o w e d by an a b r u p t r e c o v e r y s t a r t i n g a b o u t 10-20 m i n u t e s a f t e r t h e e n d o f t h e i r r a d i a t i o n ( F i g . l 9 C ) . The k i n e t i c s o f t h e r e c o v e r y v a r i e d c o n s i d e r a b l y among s e e d l i n g s . I n some s e e d l i n g s t h e g r o w t h r a t e o v e r s h o t t h e i n i t i a l d a r k v a l u e a n d u n d e r w e n t a few l a r g e o s c i l l a t i o n s b e f o r e s t a b i l i z i n g , w h i l e i n o t h e r s t h e g r o w t h r a t e s i m p l y l e v e l l e d o f f a t a r a t e n e a r o r s l i g h t l y b e l o w t h e o r i g i n a l d a r k v a l u e . I n one e x p e r i m e n t a S i n a p i s s e e d l i n g was d e c a p i t a t e d b e l o w t h e h y p o c o t y l hook, a nd t h e g r o w t h r a t e o f t h e s t r a i g h t p o r t i o n o f t h e h y p o c o t y l was m e a s u r e d by u s i n g t h e c o l l a r a s s e m b l y t o a t t a c h t h e h y p o c o t y l t o t h e t r a n s d u c e r c o r e . A l t h o u g h t h e g r o w t h , r a t e o f t h e e x c i s e d s e e d l i n g was r e l a t i v e l y l o w , t h e t i m i n g a n d g e n e r a l c h a r a c t e r i s t i c s o f t h e r e s p o n s e t o a B450 l i g h t p u l s e were n o t a l t e r e d ( n o t s h o w n ) . T h e s e r e s u l t s c o n f i r m t h e o c c u r r e n c e o f a r a p i d b l u e l i g h t r e s p o n s e i n cucumber ( 1 , 1 9 , 9 5 ) a n d S i n a p i s (21) a n d e x t e n d t h e s e o b s e r v a t i o n s t o r a d i s h . A r a p i d i n h i b i t i o n o f h y p o c o t y l 80 elongation by blue l i g h t , similar to that reported here, was f i r s t demonstrated in e t i o l a t e d cucumber by Meijer (95) and Addink and Meijer (1). A rapid blue l i g h t growth response has also been shown to occur in light-grown cucumber (34), e t i o l a t e d sunflower (19) and a number of other dark-grown seedlings (19). Through the use of shading experiments, Cosgrove (19) showed that the blue l i g h t photoreceptor was located in the growing region of the hypocotyl. The results of the excision experiment reported here confirms t h i s conclusion and demonstrates that neither the cotyledons nor the hypocotyl hook are required for the growth i n h i b i t i o n process. An important c h a r a c t e r i s t i c of the rapid blue l i g h t response reported here, and in other studies (1,34), is that the growth rate recovers rapidly in darkness following the i r r a d i a t i o n . However, Cosgrove (19) demonstrated that in some species, and even in some v a r i e t i e s of cucumber, blue l i g h t pulses also induced a long term suppression of growth. Because of differences in the timing of the photoinhibition caused by blue and red l i g h t , the rapid e f f e c t s of blue are considered to be mediated by a s p e c i f i c BAP (19,36,95). However, i t is assumed that the long term suppression of growth i s mediated by phytochrome (19,20). 81 4.2.2 Effects Of Red Light Pulses The possible action of phytochrome operating under inductive conditions was investigated in the same seedlots by exposing seedlings to a brief red (660 or 670 nm) l i g h t pulse. The photoequilibria established by 660 and 670 nm light' are almost i d e n t i c a l (2) and R660 and R670 l i g h t pulses were used interchangeably throughout this work. The response to a red l i g h t pulse varied considerably among the three species. In cucumber, a red l i g h t pulse had no detectable effect on the growth rate within the f i r s t hour after i r r a d i a t i o n (not shown). In radish, a red l i g h t pulse usually caused a gradual decline in growth rate, beginning 15-45 minutes after the i r r a d i a t i o n . In some seedlings a transient growth promotion occurred prior to the decline in growth rate (Fig.20A). After the decline, the growth rate usually remained inhibited for several hours before undergoing a slow recovery. In Sinapis (Lot WES), a red l i g h t pulse t y p i c a l l y caused a sharp decline in growth rate similar to that produced by blue; however, the lag was consistently longer, ranging from 3-9 minutes (Fig.20B). Another c h a r a c t e r i s t i c feature of the rapid response to red li g h t was that the i n i t i a l decline in growth rate was usually terminated by a transient increase in rate, i . e . the decline had the general shape of a backwards J. The growth rate following a red l i g h t pulse was quite variable, but in most seedlings the rate was inhibited by about 40 to 50%. In some seedlings the growth rate was r e l a t i v e l y stable (e.g. Fig.20B), however in others the growth rate underwent large 82 _i ' • • • • i i i i 30 60 90 120 150 180 210 T I M E (uln) F i g u r e 20. E f f e c t s of a b r i e f R660 l i g h t pulse on the growth r a t e of A) r a d i s h and B) S i n a p i s s e e d l i n g s . A) 5 min p u l s e B) 10 min p u l s e . Standard experimental c o n d i t i o n s . 83 o s c i l l a t i o n s b e f o r e s t a b i l i z i n g ( e . g . F i g . 2 1 A ) . S h o r t p e r i o d a n d / o r l o n g p e r i o d o s c i l l a t i o n s o f t e n r e s u m e d a t t h e new l o w e r g r o w t h r a t e s h o r t l y a f t e r t h e l i g h t p u l s e ( F i g . 2 1 B ) . The g r o w t h r a t e u s u a l l y r e t u r n e d t o t h e o r i g i n a l d a r k l e v e l b e t w e e n 2 a n d 6 h o u r s a f t e r a r e d l i g h t p u l s e , h o w e v e r i n a number o f c a s e s t h e g r o w t h r a t e r e m a i n e d s u p p r e s s e d f o r t h e d u r a t i o n o f t h e e x p e r i m e n t ( e . g . up t o 12 h o u r s ) . T h e r e was no e v i d e n c e o f h y p o c o t y l hook o p e n i n g i n r e s p o n s e t o b r i e f r e d l i g h t p u l s e s . The r e s p o n s e t o a r e d l i g h t p u l s e was l e s s c o n s i s t e n t i n s e e d l i n g s f r o m L o t 8-CM ( d a t a n o t s h o w n ) . A l a r g e r a p i d d e c l i n e i n g r o w t h r a t e o c c u r r e d a f t e r a l a g o f a b o u t .5 m i n u t e s i n a b o u t h a l f t h e s e e d l i n g s t e s t e d . I n t h e o t h e r s , no r e s p o n s e o r o n l y a s l i g h t t r a n s i e n t r e s p o n s e was o b s e r v e d . I t i s c l e a r f r o m t h e r e s u l t s p r e s e n t e d i n F i g u r e s 19, 20 and 21 t h a t t h e e f f e c t s o f b r i e f r e d and b l u e i r r a d i a t i o n s a r e d i s t i n c t i n t h e s p e c i e s t e s t e d h e r e . Even i n S i n a p i s t h e s m a l l b u t c o n s i s t e n t d i f f e r e n c e i n t h e l a g b e f o r e t h e r e s p o n s e t o r e d and b l u e i r r a d i a t i o n s ( i . e . 3-9 min v e r s u s c a . 1 min) s u g g e s t s t h a t t h e r e s p o n s e s a r e m e d i a t e d v i a s e p a r a t e p h o t o r e c e p t o r s . The p r e s e n t r e s u l t s a l s o s u g g e s t t h a t , u n l i k e t h e r e s p o n s e t o b l u e , t h e r e d o e s n o t a p p e a r t o be a s t a n d a r d r e s p o n s e t o b r i e f r e d i r r a d i a t i o n s i n e t i o l a t e d d i c o t s e e d l i n g s . The e f f e c t s o f p r o l o n g e d R o r FR i r r a d i a t i o n s on h y p o c o t y l e l o n g a t i o n h a v e been e x t e n s i v e l y s t u d i e d ( e . g . 3 , 5 4 , 5 5 , 7 3 , 1 6 1 ) , as have t h e e f f e c t s o f b r i e f R o r FR l i g h t p u l s e s g i v e n a s an e n d - o f - d a y o r n i g h t b r e a k t r e a t m e n t s ( e . g . 2 7 , 8 3 , 8 4 , 1 6 7 , 1 7 2 , 1 7 3 ) . However, t h e e f f e c t s o f l i g h t p u l s e s on 84 Figure 21. Effects of 10 minute R670 l i g h t pulse the growth rate of Sinapis . Standard experimental conditions. 8 5 hypocotyl elongation in dark-grown seedlings have received comparatively l i t t l e attention. In the present study, a s i g n i f i c a n t response to a brief red l i g h t pulse was observed in radish and Sinapis but not in cucumber. However, i t should be noted that because of the v a r i a b i l i t y of dark growth rates, a small gradual response or a response occurring after a long lag ( i . e . >1 hour) would not have been detected. In previous high resolution studies of hypocotyl growth in e t i o l a t e d cucumber, no response to brief (<30 min) R or FR i r r a d i a t i o n s was detected (19); however, prolonged i r r a d i a t i o n s were shown to i n h i b i t growth after a lag of 30-45 (95) or 90 minutes (19). In de-etiolated cucumber, continuous i r r a d i a t i o n with red or repeated red l i g h t pulses inhi b i t e d growth after a lag of about 5 hours (34). In perhaps the only report involving e t i o l a t e d radish, Jose (70) indicated that brief exposures to red l i g h t i n h i b i t e d growth s l i g h t l y for a few hours; however no data were presented. There have been contradictory reports on the e f f e c t s of l i g h t pulses on hypocotyl elongation in Sinapis. In an early study, Mohr (96) found that brief R or FR i r r a d i a t i o n s had no s i g n i f i c a n t effect on hypocotyl elongation in e t i o l a t e d Sinapis. A more recent study came to the same conclusion (2). Beggs et a l . (2) found that in dark-grown Sinapi s, 30 minute monochromatic l i g h t treatments (400-750 nm) had no s i g n i f i c a n t e f f e c t on hypocotyl length measured 24 hours l a t e r . However, a 30 minute l i g h t pulse given as an end-of-day treatment in l i g h t -grown seedlings was highly e f f e c t i v e in i n h i b i t i n g elongation. 86 They concluded that a continuous l i g h t pretreatment was a prerequisite for the induction response. In contrast, Schopfer and Oelze-Karow (145,also reported in 97) obtained results quite similar to those presented here. They measured hypocotyl length at frequent intervals (e.g. 1-6 hours) and showed that 5 minute R or FR l i g h t pulses strongly inhibited elongation in Sinapis. The growth rate was suppressed for about 5 hours following i r r a d i a t i o n with FR (* = 0.25)and for about 8 hours after a brief R (* = 0.8) l i g h t pulse. They concluded that the growth rate of e t i o l a t e d Sinapis, in darkness following i r r a d i a t i o n , was controlled by a phytochrome-mediated threshold response. Growth was strongly inhibited above 0.03% Pfr (based on Ptot at time zero = 100%), but resumed at a maximum rate when Pfr destruction reduced Pfr below the threshold l e v e l . It i s unclear i f these c o n f l i c t i n g results r e f l e c t differences in the plant materials used, or i f they are due to differences in experimental procedure. However, i t is l i k e l y that the transient growth responses reported here, and by Schopfer and Oelze-Karow (145) would not have been detected by the low resolution procedures used by Beggs et a l . (2). For example, a red l i g h t pulse that caused a 50% reduction of growth rate for about 5 hours would result in only a 10% i n h i b i t i o n of height measured after 24 hours. Beggs et a_l. (2) noted growth in h i b i t i o n s of about 10% in response to l i g h t pulses, but did not consider them to be s i g n i f i c a n t . In the only previous high resolution study of hypocotyl 8 7 e l o n g a t i o n i n e t i o l a t e d S i n a p i s , C o s g r o v e (21 ) f o u n d t h a t b r i e f o r c o n t i n u o u s R o r FR i r r a d i a t i o n s c a u s e d o n l y a s m a l l g r a d u a l i n h i b i t i o n o f g r o w t h . I n c o m p a r i s o n , a l a r g e r a p i d r e s p o n s e t o r e d l i g h t was u s u a l l y o b s e r v e d i n t h i s s t u d y . The p o s s i b i l i t y t h a t t h i s d i s c r e p a n c y was c a u s e d by a d i f f e r e n c e i n e x p e r i m e n t a l p r o c e d u r e was t e s t e d by c l o s e l y d u p l i c a t i n g t h e p r o c e d u r e s d e s c r i b e d by C o s g r o v e ( 2 1 ) . S e e d l i n g s 2-4 cm i n h e i g h t were s e l e c t e d f o r q r o w t h m e a s u r e m e n t s and m e a s u r e m e n t s were made a t 30C. B r o a d b a n d R was p r o v i d e d by a CBS 650 Red p l a s t i c f i l t e r . D e s p i t e t h e s e m o d i f i c a t i o n s , a r a p i d r e d l i g h t r e s p o n s e was a l w a y s o b t a i n e d . T h e s e r e s u l t s s u g g e s t t h a t d i f f e r e n t S i n a p i s s e e d l o t s c a n d i s p l a y s i g n i f i c a n t l y d i f f e r e n t r e s p o n s e s t o r e d l i g h t . B e c a u s e a r a p i d and c o n s i s t e n t r e s p o n s e t o b o t h r e d a n d b l u e i r r a d i a t i o n s c o u l d be o b t a i n e d i n S i n a p i s ( L o t WES), t h i s s e e d l o t was s e l e c t e d f o r more d e t a i l e d s t u d i e s . 4.2.3 R-FR R e v e r s i b i l i t y P h o t o r e v e r s i b i l i t y s t u d i e s were done t o t e s t t h e i n v o l v e m e n t o f p h y t o c h r o m e i n t h e r a p i d r e s p o n s e t o r e d l i g h t . S e e d l i n g s were e x p o s e d t o a b r i e f R660 o r R670 l i g h t p u l s e f o l l o w e d by a FR740 o r FR760 ('longwave FR') l i g h t p u l s e a f t e r v a r y i n g p e r i o d s o f d a r k n e s s . FR740 o r FR760 l i g h t p u l s e s a l o n e , u s u a l l y h a d l i t t l e o r no e f f e c t on t h e g r o w t h r a t e o f s e e d l i n g s t h a t had n o t r e c e i v e d a r e d l i g h t p u l s e , b u t i n some c a s e s s u c h p u l s e s c a u s e d a s m a l l t r a n s i e n t i n h i b i t i o n o f g r o w t h . T h i s 88 observation w i l l be discussed in section 4.3.4. Far red pulses given 20-60 minutes after a red l i g h t pulse usually caused an abrupt increase in growth rate within 5 to 15 minutes (mean lag = 6.4±0.71 min,n=1l) after the start of the FR i r r a d i a t i o n (Fig.22A). The growth rate often underwent two or three extremely large o s c i l l a t i o n s before s t a b i l i z i n g (Fig.22B). A complete recovery to the i n i t i a l dark rate usually occurred gradually over the course of 1-2 hours after FR. Pulses of FR given up to 3 to 4 hours after a red l i g h t pulse also caused a d i s t i n c t increase in growth rate. However, FR pulses after that time had no promotive effect on elongation. This indicates that Pfr was present and a c t i v e l y i n h i b i t i n g elongation for 3 to 4 hours after a red l i g h t pulse, but was absent or no longer e f f e c t i v e after that time. P h o t o r e v e r s i b i l i t y studies were also done using the c o l l a r assembly. Use of the c o l l a r had no effect on either the rapid red or far red response (Fig.22C). Thus, hypocotyl hook opening and closing were not involved in these rapid growth responses. Decapitating the seedling (the hypocotyl hook was l e f t intact) had no e f f e c t on the timing or general c h a r a c t e r i s t i c s of the response to red l i g h t (not shown). This indicates that the apex or cotyledons were not involved in photoreception or mediation of the response. It was also of interest to determine i f longwave FR i r r a d i a t i o n s given immediately after a brief R pulse could E r r o r s reported in text are standard errors of the mean. 8 9 TIME (min) F i g u r e 22. R e v e r s a l o f r e d l i g h t i n d u c e d i n h i b i t i o n by FR740 l i g h t p u l s e s . G r o w t h m e a s u r e d a t A) and B) t h e h y p o c o t y l hook a n d C) t h e c o l l a r . 5 o r 10 min p u l s e s . S t a n d a r d e x p e r i m e n t a l c o n d i t i o n s . 90 F i g u r e 23. E f f e c t s of v a r y i n g p e r i o d s of darkness between R660 and FR740 l i g h t p u l s e s . A) 1 min R660 l i g h t p u l s e s were fo l l o w e d by FR740 l i g h t p u l s e s a f t e r 1 min and 10 min of darkness. B) 30 sec R660 l i g h t p u l s e s were f o l l o w e d by FR740 l i g h t p u l s e s a f t e r 30 sec and 20 min of darkness. Standard experimental c o n d i t i o n s . 91 prevent the rapid growth response. A transient decline in growth rate was always observed i f the FR i r r a d i a t i o n commenced more than 2 minutes after the start of a red l i g h t pulse (Fig.23A). However, the rapid growth response was almost completely prevented by FR i r r a d i a t i o n commencing within 50 to 60 seconds after the start of a 30 second R pulse (Fig.23B). Because of time constraints associated with changing the interference f i l t e r s , the effects of shorter time periods between i r r a d i a t i o n s were not tested. These experiments c l e a r l y demonstrate that the growth rate of e t i o l a t e d Sinapis i s rapidly and reversibly controlled by the l e v e l of Pfr. The continuous presence of Pfr i s required for the long term suppression of elongation. However, the presence of Pfr for even very short periods is capable of inducing a transient growth i n h i b i t i o n . Thus, the rapid response to red l i g h t i s a low energy phytochrome-mediated response. For convenience of expression t h i s rapid growth response w i l l be referred to as the rapid inductive response. 4.2.4 380-780 nm Light Pulses To compare the kinetics of photoinhibition at d i f f e r e n t wavelengths, seedlings were exposed to brief (10 min) pulses of narrow bandwidth (<10 nm half bandwidth) l i g h t at a number of wavelengths spanning the 380-780 nm waveband. The purpose of the experiment was to determine the wavelengths at which a rapid 'blue l i g h t response' occurred, and to examine the effects of 92 l i g h t p u l s e s t h a t e s t a b l i s h d i f f e r e n t l e v e l s o f P f r . I t s h o u l d be n o t e d t h a t b e c a u s e o f c o m p e t i n g t h e r m a l r e a c t i o n s , l i g h t p u l s e s b e l o w 600 nm p r o b a b l y d i d n o t e s t a b l i s h t h e p h y t o c h r o m e p h o t o e q u i l i b r i a a n d t h u s h ad an i n d e t e r m i n a t e e f f e c t on t h e P f r / P t o t r a t i o ( 5 1 , 6 0 ) . However, l i g h t p u l s e s f r o m t h e 600-780 nm waveband were p r o b a b l y o f s u f f i c i e n t i n t e n s i t y ( f l u e n c e r a t e ) ( s e e T a b l e 1) t o r a p i d l y e s t a b l i s h t h e p h y t o c h r o m e p h o t o e q u i 1 i b r i a . B a s e d on d i f f e r e n c e s i n t h e l e n g t h o f t h e l a t e n t p e r i o d , t h r e e d i s t i n c t t y p e s o f p h o t o r e s p o n s e s c o u l d be d i s t i n g u i s h e d ( T a b l e 3 ) . L i g h t p u l s e s f r o m t h e 380-500 nm waveband c a u s e d a c h a r a c t e r i s t i c b l u e l i g h t r e s p o n s e a f t e r a l a g o f a b o u t 1 m i n u t e , w h i l e l i g h t f r o m t h e 550-710 nm waveband e v o k e d a t y p i c a l r e d l i g h t o r i n d u c t i v e r e s p o n s e a f t e r a l a g o f a b o u t 5 m i n u t e s . The r e s p o n s e t o 720-780 nm l i g h t p u l s e s was n o t c o n s i s t e n t , b u t u s u a l l y i n v o l v e d a s m a l l t r a n s i e n t d e c l i n e i n g r o w t h r a t e a f t e r a 15-20 m i n u t e l a g . The maximum i n h i b i t i o n o f g r o w t h r a t e t h a t o c c u r r e d w i t h i n one h o u r a f t e r a l i g h t p u l s e was d e t e r m i n e d f o r e a c h w a v e l e n g t h . T h e s e d a t a a l s o a p p e a r t o f a l l i n t o t h r e e d i s t i n c t r a n g e s . L i g h t p u l s e s f r o m t h e 380-500 nm waveband i n h i b i t e d g r o w t h by more t h a n 6 5 % , w h i l e t h e i n h i b i t i o n i n d u c e d by 550-710 nm l i g h t p u l s e s r a n g e d f r o m a b o u t 5 4 - 64%. L i g h t p u l s e s f r o m t h e 720-780 nm waveband were c o n s i d e r a b l y l e s s e f f e c t i v e a n d i n many c a s e s h ad no d e t e c t a b l e e f f e c t on e l o n g a t i o n . 93 4.2.5 380-500 nm Waveband Representative traces showing the effects of 380, 400, 450 and 500 nm l i g h t pulses are presented in Fig.24. There were no consistent differences in the k i n e t i c s of the photoresponse at d i f f e r e n t wavelengths. The latent period ranged from about 0.5 to 1.5 minutes among seedlings. An interesting feature of the blue l i g h t response was that growth rate almost • invariably began an abrupt recovery between 15 and 20 minutes after the end of a 10 minute l i g h t pulse. However, as previously noted, the kinetics of the recovery varied considerably among seedlings. In some v a r i e t i e s of cucumber and sunflower, the rapid i n h i b i t i o n of growth by blue l i g h t has been shown to have the general form of an exponential decline to an asymptote (19,21). The exponential character of the rapid blue l i g h t response i s also evident in Sinapis. P l o t t i n g the log of growth rate versus time yi e l d s a straight l i n e confirming the exponential character of the response (not shown). 4.2.6 550-710 nm Waveband A l l wavelengths that were tested in the 550-710 nm waveband induced a large rapid decline in growth rate after a lag of about 5 minutes (Fig.25). The latent period of the photoresponse ranged from 3-9 minutes among seedlings but did not vary greatly at d i f f e r e n t wavelengths (Table 3). Two d i s t i n c t aspects of the photoinhibition were considered: the 'depth' of the photoinhibition ( i . e . percent 94 Table 3. Latent p e r i o d and i n h i b i t o r y e f f e c t s of 10 minute l i g h t p u l s e s i n the 380 to 760 nm waveband. Percent i n h i b i t i o n Latent Wavelength (nm) Number of r e p l i c a t e s p e r i o d (min) r a p i d 1 response maximum2 w i t h i n 60 min 380 4 ca. 1 - 67.814.5" 400 1 ca. 1 - 69.1 450 4 ca. 1 - 79.212.9 500 3 ca. 1 - 75.913.8 550 6 5.11.4 59.115.6 63.614.9 600 4 5.1±.3 57.116.3 57. 116.3 660 19 4.9+.2 56.912.1 57.311.9 670 8 5.0±.3 52.712.3 54.012.9 700 6 5.2+.3 52.415.1 59.7+5.3 710 9 5.11.3 54.613.9 57.812.7 720 7+3NE3 10.611 .4 - 35.013.7 730 9+2NE 14.411 .4 - 39.714.7 740 8 + 2NE 16.51.5 - 20.711 .7 760 2 + 9NE 19.51.5 - 29 780 - + 2NE - - -'Percent i n h i b i t i o n of e l o n g a t i o n r a t e preceding the l i g h t p u l s e , as determined from the f i r s t minimum f o l l o w i n g the p u l s e . 2 P e r c e n t i n h i b i t i o n of e l o n g a t i o n r a t e • preceding the l i g h t p u l s e , as determined from the minimum ra t e o c c u r r i n g w i t h i n 60 minutes f o l l o w i n g the p u l s e . 3NE i n d i c a t e s no e f f e c t on e l o n g a t i o n . " E r r o r s shown are standard e r r o r s of the mean. 95 F i g u r e 24. R e p r e s e n t a t i v e t r a c e s showing the e f f e c t s of 10 minute A) 380, B) 400, C) 450, and D) 500nm l i g h t p u l s e s . Standard experimental c o n d i t i o n s . 96 TIME («1n) Figure 25. Representative traces showing the ef f e c t s of 10 minute A) 550, B) 600, C) 700 and D) 7l0nm l i g h t pulses. Standard experimental conditions. 97 F i g u r e 26. E f f e c t s o f 10 m i n u t e A) 700 and B) 7l0nm l i g h t p u l s e s . S t a n d a r d e x p e r i m e n t a l c o n d i t i o n s . 9 8 i n h i b i t i o n o f t h e p r e i r r a d i a t i o n g r o w t h r a t e ) and t h e d u r a t i o n o f t h e p h o t o i n h i b i t i o n . H o wever, b e c a u s e o f t h e g r e a t v a r i a b i l i t y o f d a r k g r o w t h r a t e s b o t h b e f o r e a n d a f t e r a l i g h t p u l s e , t h e i n h i b i t o r y e f f e c t s o f l i g h t t r e a t m e n t s were a t t i m e s d i f f i c u l t t o a s s e s s . The g r o w t h r a t e f o l l o w i n g a FR710 o r FR700 l i g h t p u l s e u s u a l l y u n d e r w e n t an a b r u p t and s u s t a i n e d r e c o v e r y w i t h i n 1 o r 2 h o u r s a f t e r i r r a d i a t i o n ( F i g . 2 6 , s e e a l s o F i g . 2 5 D a n d 3 6 B ) . The l e n g t h o f t h e i n h i b i t o r y p e r i o d ( A t ) , m e a s u r e d f r o m t h e s t a r t o f t h e i r r a d i a t i o n t o t h e b e g i n n i n g o f an a b r u p t a n d s u s t a i n e d i n c r e a s e i n r a t e , was a b o u t 45 m i n u t e s (mean=44±14,n=6) f o l l o w i n g a FR710 p u l s e , c o m p a r e d t o 130 m i n u t e s (mean=127±12, ,n=5) f o l l o w i n g a FR700 p u l s e . The d u r a t i o n o f t h e p h o t o i n h i b i t i o n f o l l o w i n g 550, 600, 660 a n d 670 nm l i g h t p u l s e s was c l e a r l y l o n g e r t h a n t h a t i n d u c e d by FR710 o r F R700, b u t was d i f f i c u l t t o d e t e r m i n e w i t h p r e c i s i o n . The g r o w t h r a t e u s u a l l y r e t u r n e d t o t h e o r i g i n a l d a r k v a l u e w i t h i n 2-6 h o u r s f o l l o w i n g a l i g h t p u l s e ; h owever i n some c a s e s t h e r e was no a b r u p t i n c r e a s e i n g r o w t h r a t e b u t o n l y a g r a d u a l r e c o v e r y o c c u r r i n g o v e r t h e c o u r s e o f s e v e r a l h o u r s ( e . g . s e e F i g . 2 l A ) . N e v e r t h e l e s s , t h e s e r e s u l t s c l e a r l y s u g g e s t t h a t t h e d u r a t i o n o f t h e p h o t o i n h i b i t i o n i n d a r k n e s s was g o v e r n e d by t h e p h y t o c h r o m e p h o t o e q u i l i b r i u m e s t a b l i s h e d by t h e l i g h t p u l s e . As p r e v i o u s l y n o t e d , t h e g r o w t h r a t e f o l l o w i n g R660 a n d R670 l i g h t p u l s e s s o m e t i m e s d i d n o t r e c o v e r t o t h e o r i g i n a l d a r k l e v e l , e v e n a f t e r v e r y p r o l o n g e d p e r i o d s i n d a r k n e s s . T h i s p r o l o n g e d s u p p r e s s i o n o f g r o w t h r a t e o c c a s i o n a l l y o c c u r r e d a t a l l 99 wavelengths and was not reversed by FR740 l i g h t pulses. This suggests that the apparent long term i n h i b i t i o n of elongation was not d i r e c t l y related to Pfr, but probably r e f l e c t e d a change in the dark (control) growth rate. Several approaches were used to compare the depth of the photoinhibition induced by l i g h t pulses at d i f f e r e n t wavelengths. The magnitude of the rapid growth response ( i . e . the backwards J curve) was measured by comparing the growth rate at the start of the i r r a d i a t i o n to the minimum rate attained at the base of the curve (Table 3). It i s apparent that the magnitude of the response did not d i f f e r s i g n i f i c a n t l y , among wavelengths known to est a b l i s h vastly d i f f e r e n t phytochrome photoeguilibria (e.g. FR710, *=.1; R660, *=.8). S i m i l a r l y , the maximum i n h i b i t i o n of growth rate occurring within one hour after a l i g h t pulse, was not related to the level, of Pfr established by the pulse (Table 2). In addition, when plotted in r e l a t i v e terms (to take into account differences in the i n i t i a l dark rates) the numerically averaged traces of the several experiments suggest that the responses to FR710, FR700 and R660 l i g h t pulses were not fundamentally d i f f e r e n t , at least during the i n i t i a l stages of the photoinhibition (Fig.27). These results indicate that the l e v e l of Pfr established by a l i g h t pulse in the 550-710 nm waveband had l i t t l e or no effect on the depth of the induced photoinhibition. However, the duration of the photoinhibition appeared to be d i r e c t l y related to the l e v e l of Pfr established. 1 0 0 Figure 27. Numerically averaged and normalized curves showing e f f e c t s of 660, 700, and 7l0nm l i g h t pulses. Results are averages from 7, 5, and 9 seedlings for R660, FR700 and FR710 l i g h t pulses respectively. Light pulses were 10 min in duration and commenced at the arrow. Data was normalized to the p r e - i r r a d i a t i o n growth rate. Standard experimental conditions. 101 2-FR720 1-TIME (min) F i g u r e 28. E f f e c t s of 10 minute longwave FR l i g h t p u l s e s . A) and B) are t r a c e s from two d i f f e r e n t s e e d l i n g s . Standard experimental c o n d i t i o n s . 102 4.2.7 720-780 nm Waveband The response to l i g h t pulses from the 720-780 nm waveband (longwave FR) was quite variable. In a few cases FR720 and FR730 l i g h t pulses evoked a rapid inductive response similar to that described for FR710 ( i . e . 3 of 10 seedlings for FR720; 2 of 11 seedlings for FR730). However, in most seedlings, l i g h t pulses from this waveband had either no detectable effect (Fig.28A) or induced a small transient decline in growth rate after a lag of 15-20 minutes (Fig.28B). The d i f f e r e n t inh i b i t o r y e f f e c t s of FR710 (e.g. 5 min lag) and FR720 (e.g. 15 min lag or no effect) l i g h t pulses could be shown to occur in a single seedling. The d i f f e r e n t effects of l i g h t pulses from the 550-710 and the 720-780 nm wavebands strongly support the hypothesis that phytochrome regulates the growth rate of e t i o l a t e d Sinapis in darkness via a threshold response (97,145). For most seedlings, the threshold for the rapid inductive response l i e s between 10% and 2% Pfr, i . e . the levels established by FR710 or FR720 l i g h t pulses. The observation that above the threshold, increasing levels of Pfr had l i t t l e e f f e c t on the depth of the photoinhibition also supports the threshold ( i . e . all-or-none) interpretation. One f i n a l observation supporting a threshold response relates to the fact that the level of Pfr would be continuously declining after a brief l i g h t pulse, due to Pfr destruction and/or reversion. The fact that the growth rate did not respond to t h i s constantly declining level of Pfr, but often underwent an abrupt recovery (e.g. Fig.26A), strongly supports 1 03 t h e c o n c e p t o f a t h r e s h o l d r e s p o n s e , a s o p p o s e d t o a g r a d e d p h y t o c h r o m e r e s p o n s e . The f a c t t h a t FR720 (* = 0.02) and FR730 (* = 0.008) l i g h t p u l s e s c o u l d o c c a s i o n a l l y e v o k e a t y p i c a l r a p i d i n d u c t i o n r e s p o n s e s u g g e s t s t h a t t h e t h r e s h o l d l e v e l o f P f r c a n v a r y among s e e d l i n g s . H o wever, i f t h e t h r e s h o l d i s b a s e d on an a b s o l u t e l e v e l o f P f r ( 9 7 ) ( r a t h e r t h a n P f r / P t o t r a t i o ) , t h e n d i f f e r e n c e s i n t h e r e s p o n s e s t o l i g h t p u l s e s c o u l d s i m p l y r e f l e c t d i f f e r e n c e s i n t h e P t o t l e v e l s among s e e d l i n g s . The t h r e s h o l d l e v e l o f P f r c a n a l s o be r o u g h l y e s t i m a t e d f r o m t h e l e n g t h o f t h e i n h i b i t o r y p e r i o d ( A t ) f o l l o w i n g a l i g h t p u l s e ( F i g . 2 9 ) ( a f t e r M o h r , 9 7 ) . F o r e x a m p l e , a s s u m i n g a A t o f 127 m i n u t e s f o l l o w i n g a FR700 (* = c a . 0.27) p u l s e , a n d r a p i d f i r s t o r d e r d e s t r u c t i o n o f P f r ( t l / 2 = 45 min) ( 9 4 , 9 7 ) , a t h r e s h o l d l e v e l o f 3.6% P f r i s o b t a i n e d ( F i g . 1 1 ) . S i m i l a r l y , a s s u m i n g a At o f 45 m i n u t e s f o l l o w i n g a FR710 p u l s e (* = c a . 0 . 1 ) , a t h r e s h o l d l e v e l o f a b o u t 5.0% P f r i s o b t a i n e d . C o n v e r s e l y , i f a t h r e s h o l d l e v e l o f a b o u t 5.0% i s a c c e p t e d , t h e n a At o f a b o u t 180 m i n u t e s i s c a l c u l a t e d f o r R670 l i g h t p u l s e s , a v a l u e w h i c h seems r e a s o n a b l e ( e . g . s e e F i g . 2 l A ) . T h e s e c a l c u l a t i o n s a r e o b v i o u s l y v e r y c r u d e a n d d e p e n d upon s e v e r a l f a c t o r s t h a t a r e n o t known w i t h p r e c i s i o n , i . e . t h e t 1 / 2 o f P f r d e s t r u c t i o n , a n d t h e P f r / P t o t r a t i o e s t a b l i s h e d by t h e f i l t e r s a n d p r o c e d u r e s u s e d i n t h i s s t u d y . I n a d d i t i o n , P f r r e v e r s i o n a n d t h e b i p h a s i c k i n e t i c s o f P f r d e s t r u c t i o n a r e i g n o r e d . N e v e r t h e l e s s , e v e n t h e s e c r u d e e s t i m a t e s p r o v i d e a u s e f u l b a s i s f o r c o m p a r i s o n . R e g a r d l e s s o f t h e p r e c i s e l e v e l , t h e t h r e s h o l d f o r t h e 1 0 4 100 A t ( h o u r s ) F i g u r e 29. Model showing h y p o t h e t i c a l l o g a r i t h m i c d e c l i n e s i n the P f r / P t o t r a t i o f o l l o w i n g b r i e f 660, 700, 710 and 720nm l i g h t p u l s e s . F i r s t order d e s t r u c t i o n of P f r i s assumed to occur with a t l / 2 of 45 min, while dark r e v e r s i o n i s assumed to be n e g l i g i b l e . B r i e f (10 min) R670, FR710 and FR720 l i g h t p u l s e s are assumed to e s t a b l i s h * of .8, .27, .1 and .021 r e s p e c t i v e l y . 45 min and 127 min r e f e r to the d u r a t i o n of the p h o t o i n h i b i t i o n f o l l o w i n g FR710 and FR700 l i g h t p u l s e s . 105 i n h i b i t i o n of hypocotyl elongation found here appears to be two orders of magnitude above that reported by Schopfer and Oelze-Karow (145) ( i . e . ca. 5% versus 0.03% P f r ) . This discrepancy could conceivably be due to a large (100 fold) difference in Ptot levels between seedlings from the two seedlots. However, there is no reason to reject the p o s s i b i l i t y that phytochrome-mediated threshold responses could occur at vastly d i f f e r e n t Pfr l e v e l s . Indeed, the threshold response for the suppression of LOG synthesis in e t i o l a t e d Sinapis i s known to occur at 1.25% Pfr. Many low energy phytochrome responses show a two-step fluence response curve (6). It i s interesting to note that the threshold for hypocotyl elongation found by Schopfer and Oelze-Karow (145) f a l l s within the levels of Pfr usually associated with the VLFR. In comparison, the threshold for the suppression of LOG synthesis (99,108) and the threshold l e v e l of Pfr found here f a l l within the range of Pfr levels commonly associated with the LFR. Thus, these threshold responses could represent cooperative t r a n s i t i o n s in the Pfr reaction matrix associated with the two d i s t i n c t steps observed in fluence response curves. The small but s i g n i f i c a n t response to l i g h t pulses from 720 to 760 nm waveband suggests that very low Pfr le v e l s ( i . e . below the threshold) can also e l i c i t a growth response in t h i s seedlot. Thus, this small transient response could be a manifestation of the VLFR. A l t e r n a t i v e l y , the response could be related to some aspect of phytochrome c y c l i n g . However, results presented in section 4.3.4 suggest that the response to longwave 106 FR was in some way associated with the l i g h t s - o f f signal at the end of the i r r a d i a t i o n . This finding suggests that the transient response to longwave FR represents an e n t i r e l y d i f f e r e n t type of response than the rapid inductive response. • 4.2.8 Effects Of Broadband Green Light Brief exposures to v i r t u a l l y a l l wavelengths of l i g h t were found to have at least a small effect on hypocotyl elongation. Since a l l seedlings were exposed to a few minutes of low intensity broadband green l i g h t Umax = 520 nm, < 0.25 Wirr2) while being mounted into the transducer apparatus, the possible e f f e c t s of l i g h t from the green 'safelight' were of considerable int e r e s t . At a fluence rate of about 0.25 Wirr2, brief or prolonged exposures to broadband green l i g h t had l i t t l e or no effect on hypocotyl elongation. In a few cases a small transient i n h i b i t i o n of growth rate occurred, similar to that produced by longwave FR. However, a rapid inductive response was never observed. At 10 Wirr2, a 10 minute exposure to broadband green l i g h t sometimes induced a short duration rapid inductive response. The photostationary r a t i o established by green l i g h t i s known to be strongly dependent on the fluence rate at 25C (60). Thus, i t appears that low fluence rates of broadband green l i g h t do not raise the le v e l of Pfr above the threshold required for a rapid inductive response. However, at high fluence rates the threshold can be surpassed. It i s also interesting to note that narrow bandwidth 500 nm 107 l i g h t always induced a rapid blue l i g h t response, yet broadband green U m a x = 520 nm) did not. Action spectra for blue l i g h t responses usually show a sharp cut off at about 500 nm (113). The present results c l e a r l y demonstrate that a very sharp cut off does occur. Thus, i t appears that at least in terms of the blue l i g h t response and the rapid inductive response, the safelight used here does appear to be 'safe'. 4.2.9 Phytochrome And The Response To Blue Light Because phytochrome also absorbs in the UV-blue waveband, the possible role of phytochrome in the response to blue i r r a d i a t i o n s must also be considered. The phytochrome photoequilibria range from about 0.2-0.8 in the 380-500 nm waveband. However, because of competing thermal reactions, blue i r r a d i a t i o n s establish photoequilibria only at fluence rates >25 Wm~2 (60,131). Therefore, the a b i l i t y of a blue l i g h t treatment to cause a phytochrome-mediated response in Sinapis would probably depend on the temperature, the fluence rate and duration of the i r r a d i a t i o n , as well as the threshold l e v e l of Pfr required to induce a response. For example, in the seedlot used by Schopfer and Oelze-Karow (145), the threshold was so low that even very brief low fluence rate i r r a d i a t i o n s could be expected to raise the Pfr/Ptot r a t i o above the threshold l e v e l . Blue l i g h t pulses have been shown to induce a prolonged i n h i b i t i o n of growth in a number of species (19), however the involvement of phytochrome in t h i s response has not been 108 established. In the present study, a prolonged i n h i b i t i o n , consistent with a phytochrome-mediated inductive response, was sometimes observed following a brief UV or blue l i g h t i r r a d i a t i o n (e.g. Fig.24A). The influence of phytochrome in the response to blue i r r a d i a t i o n s was investigated through the use of dichromatic i r r a d i a t i o n s . The results presented in Fig.30A c l e a r l y demonstrated that the growth rate in darkness following a blue l i g h t treatment can be influenced by Pfr formed during the blue i r r a d i a t i o n . A red l i g h t pulse was given during the course of a blue i r r a d i a t i o n to ensure that the Pfr/Ptot r a t i o was above the threshold required for an inductive response. The red l i g h t pulse had no immediately observable effect on elongation; however, the growth rate c l e a r l y did not recover f u l l y after the end of the blue i r r a d i a t i o n . The promotion of growth by a FR760 l i g h t pulse indicates that this i n h i b i t o r y effect was due to the presence of Pfr. In related experiments i t was found that simultaneous i r r a d i a t i o n s with FR740, which should maintain Pfr at a low l e v e l , had * no apparent effect on the timing or general pattern of response to a B450 l i g h t pulse (Fig.30B). Conversely, the timing and general pattern of response to a B450 l i g h t pulse was not greatly affected by a previous exposure to red l i g h t ; an exposure that should have . established a high l e v e l of Pfr (Fig.30C). These and similar experiments suggest that the rapid i n h i b i t o r y e f f e c t s of blue l i g h t , and the rapid recovery following i r r a d i a t i o n s , are dire c t e f f e c t s of the BAP and are 109 independent of the Pfr/Ptot r a t i o . The ef f e c t s of the BAP and phytochrome appear to be independent and additive under some conditions (Fig.30C), although t h i s relationship does not hold when the growth rate i s strongly suppressed by blue l i g h t (Fig.30A). In summary, these studies have c l e a r l y demonstrated that brief l i g h t pulses i n h i b i t hypocotyl elongation in et i o l a t e d Sinapis via a phytochrome-mediated threshold response, and through the action of a separate blue-light-absorbing pigment. The basic observation that brief l i g h t pulses can have a s i g n i f i c a n t e ffect on elongation i s of some importance. As was previously discussed, Beggs et a l . (2) concluded that brief l i g h t pulses had no effect on elongation in e t i o l a t e d Sinapis. It should be noted that this apparently erroneous conclusion is rapidly becoming incorporated into the phytochrome l i t e r a t u r e , and theories on phytochrome action (67,132). The results of t h i s study c l e a r l y suggest that conclusions drawn from long term growth experiments involving end point determinations of height should be viewed with caution. For example, end point determinations of height, made even after a r e l a t i v e l y short period in the present study, would probably have led to the conclusion that a graded response to Pfr was involved! Thus i t is essential to understand the kinetics of the photoresponse when interpreting the action of either phytochrome or the BAP. 110 Figure 30. Effects of simultaneous and/or sequential i r r a d i a t i o n s with B450, R660 and longwave FR. Light pulses were of 5 or 10 min duration. A)-C) are separate experiments with d i f f e r e n t seedlings. Standard experimental conditions. 1 1 1 TIME (""In) 1 12 4.3 Effects Of Prolonged Irradiations And Repeated Light Pulses Hypocotyl elongation in e t i o l a t e d Sinapis i s known to be strongly i n h i b i t e d by continuous i r r a d i a t i o n s with l i g h t from the blue, red and far red wavebands (2). The effects of continuous red and far red i r r a d i a t i o n s are thought to be mediated exclusively through the action of phytochrome (3,2,51,55,132), while the response to blue l i g h t is mediated, at least in part, by the action of a separate BAP (21,55). The spectral c h a r a c t e r i s t i c s and fluence rate dependence of these so-called high irradiance responses have been very extensively studied (2,3,51,54,55,132). However, the ki n e t i c s of the response to continuous i r r a d i a t i o n s i s not well understood, p a r t i c u l a r l y in the red and far red wavebands. In the following section I b r i e f l y report on the response to prolonged ( i . e . >60 min) UV-blue l i g h t i r r a d i a t i o n s . The remainder of t h i s section i s mainly concerned with investigating the ki n e t i c s and significance of the responses to brief and prolonged i r r a d i a t i o n s in the 660-760 nm waveband. 4.3.1 UV-blue Waveband Prolonged i r r a d i a t i o n s ( i . e . >60 min) with B450 reduced the growth rate by up to 95% (e.g. Fig.31A). Continuous i r r a d i a t i o n s with UV380 were somewhat less e f f e c t i v e (e.g. Fig.31B), however, a d i r e c t comparison should not be made because the l i g h t treatments were not adjusted to the same photon fluence rates. One of the most noticeable 1 13 c h a r a c t e r i s t i c s of the response to prolonged UV-blue ir r a d i a t i o n s was the extreme s t a b i l i t y of the growth rate. However, short period o s c i l l a t i o n s usually resumed soon after the end of the i r r a d i a t i o n . The lag between the end of the i r r a d i a t i o n and the start of a d i s t i n c t recovery, decreased as the duration of the l i g h t treatment increased. After a 90 minute B450 i r r a d i a t i o n the lag was about 3 minutes (mean = 3.2±.5 min,n=4). In comparison, the lag after a 10 minute B450 pulse was about 19 minutes (mean = 18.7±.8 min,n=4). The growth rate following prolonged i r r a d i a t i o n s recovered very gradually over the course of several hours. FR r e v e r s i b i l i t y experiments indicated that phytochrome was involved in t h i s long term suppression of growth (Fig.3lB). 4.3.2 The Rapid Blue Light Response And Phototropic Bending An unsuccessful attempt was made to examine the fluence rate dependence of the blue l i g h t response in individual seedlings. Seedlings were exposed to low intensity blue l i g h t and the fluence rate was increased at approximately hourly intervals by a factor of ten. The results of these experiments were inconsistent, and in most cases highly e r r a t i c growth rate traces were obtained. It eventually became apparent, that despite the use of a mirror to provide more uniform illumination, low fluence rates of B450 l i g h t had induced phototropic bending., Therefore, firm conclusions regarding the fluence rate dependence of the rapid blue l i g h t response could 114 ~ 0 o cc <J3 B450 ON UV380 ON B450 OFF FR740 UV380 OFF J 1 I I I I I L. 30 60 90 120 TIME (»1n) 150 F i g u r e 31. E f f e c t s of prolonged A) B450 or B) UV380 i r r a d i a t i o n s , and recovery i n A) darkness or B) a f t e r longwave FR. Standard experimental c o n d i t i o n s . 1 15 not be made. Nevertheless, a number of p o t e n t i a l l y s i g n i f i c a n t observations concerning the occurrence of the rapid blue l i g h t response, and phototropic bending are worth noting. As was previously described, the growth measuring system was extremely sensitive to s l i g h t changes in seedling i n c l i n a t i o n . Therefore, bending could often be detected on the growth rate traces well before i t was v i s i b l e in seedlings within the cuvette. Bending usually caused a rapid but e r r a t i c decline in the growth rate trace, after which the trace become exceptionally noisy. At very low fluence rates (.0016 Wirr 2 ) a rapid blue l i g h t growth response did not occur (Fig.32A). However, phototropic bending occurred after about 45 minutes of continuous i r r a d i a t i o n (as indicated by the rapid decline in the growth rate trace to below zero). At fluence rates ranging from .016-16 Wirr 2 a d i s t i n c t decrease in growth rate always occurred after a lag of about 1 minute. The decline in rate was gradual at .016 Wirr 2 (Fig.32C), but a c h a r a c t e r i s t i c rapid decline always occurred at .16 Wirr 2 (Fig.32B) and at higher fluence rates. Phototropic bending was never observed in response to ir r a d i a t i o n s at 16 W n r 2. The absence of phototropic bending at this fluence rate was also indicated by the s t a b i l i t y of the growth rate traces (e.g. Fig.31A). In contrast, the growth rate trace during i r r a d i a t i o n s at lower fluence rates was extremely variable (e.g. Fig.32B and C). This v a r i a b i l i t y appeared to be due to a combination of both phototropic bending and true variations in growth rate. 116 Figure 32. Effects of continuous B450 i r r a d i a t i o n s at A) .0016, B) .16 and C) .016 Wnr2. In A) phototropic bending became v i s i b l e between 130 and 140 min. Standard experimental conditions. 1 17 Two conclusions arise from these observations. F i r s t of a l l , the rapid blue l i g h t growth response, and the phototropic response appear to be d i s t i n c t phenomena. Secondly, the rapid blue l i g h t growth response occurs only in response to i r r a d i a t i o n s above a minimum fluence rate. This minimum rate f e l l between .0016 and .016 Wm"2 in the present seedlot. It i s interesting to consider t h i s l a t t e r observation in r e l a t i o n to blue l i g h t fluence response curves obtained by low resolution growth studies. Holmes and Schafer (55) presented detailed fluence rate response curves for the i n h i b i t i o n of hypocotyl elongation in e t i o l a t e d Sinapis by UV and blue l i g h t . These curves exhibited a d i s t i n c t break at about I0" 1umol n r 2 sec" 1 ( ca. .026 Wm"2 for 446 nm) . Above lO'^mol m"2 sec" 1 the response was strongly fluence rate-dependent, but below that rate, the response was almost constant at about 10% i n h i b i t i o n . The present findings suggest that the break in the response curve represents the minimum fluence rate at which a rapid blue l i g h t growth response can occur. If that i s the case then, the 10% i n h i b i t i o n induced by very low fluence rates must be due to some other mechanism. Phototropism has been extensively studied in the c o l e o p t i l e s of grasses; however, much less i s known about the phototropic responses of dicot seedlings. In dicots,- true phototropic curvature results from the absorption of blue l i g h t by the hypocotyl i t s e l f (42,160). A c h a r a c t e r i s t i c pattern of response ( i . e . f i r s t p o s i t i v e , f i r s t negative, and second po s i t i v e curvatures) to increasing exposures of u n i l a t e r a l l i g h t 118 occurs in both monocots and dicots. The l i g h t exposures used here probably f a l l into the range of second p o s i t i v e curvatures (26) . It should be mentioned that in unrelated experiments i t was noticed that prolonged or repeated i r r a d i a t i o n s with FR760 also induced phototropic bending (Fig.33). This bending response was apparently due to the leakage of very low leve l s of blue l i g h t through the 760 nm narrowbandwidth f i l t e r . Passing the FR760 l i g h t beam through a CBS 750 far red p l a s t i c f i l t e r (which strongly absorbs blue), had ne g l i g i b l e effect on the fluence rate of the FR760 l i g h t treatment, but prevented phototropic bending. Therefore, in a l l further experiments involving FR760 l i g h t treatments, the combination of narrowbandwidth and broadbandwidth f i l t e r s was always used. It must be emphasized that phototropic bending was never observed at other wavelengths outside the UV-blue waveband. In the absence of a rapid blue l i g h t growth response, the onset of phototropic bending could usually be determined with great precision. Under these conditions bending always began 40-60 minutes after the start of continuous i r r a d i a t i o n (e.g. Fig.32A). Hart and MacDonald (42) noted measurable phototropic curvature after about 90 minutes of continuous blue i r r a d i a t i o n in e t i o l a t e d dicot seedlings. In contrast, bending occurred within 10-15 minutes after the start of a second FR760 l i g h t pulse (Fig.33), yet never occurred in response to a single l i g h t pulse. This suggests that under continuous i r r a d i a t i o n the timing of the onset of bending was limited by nonphotochemical 119 30 60 90 120 150 TIME (min) Figure 33. Induction of phototropic bending by repeated FR760 l i g h t pulses. Standard experimental conditions. 1 20 ( i . e . 'dark') reactions rather than by the absorption of l i g h t . However, the fact that bending occurred only after a second l i g h t pulse, suggests that the absorption of a c r i t i c a l number of photons, or exposure to a minimum duration of i r r a d i a t i o n , was required to i n i t i a t e the response. Another p o s s i b i l i t y i s that phototropism is a two-step process that requires an input of photons during two d i s t i n c t periods. Although i t is obvious that phototropic curvature i s the result of d i f f e r e n t i a l growth on opposite sides of the shoot, the manner in which this d i f f e r e n t i a l growth arises is not well understood. F i r n and Digby (32) l i s t 5 ways in which curvature could arise (e.g. increasing the growth rate of one side, decreasing the growth rate of one side, e t c . ) . In 1919, Blaauw (5) suggested that phototropic curvature was the result of d i f f e r e n t i a l growth i n h i b i t i o n caused by a l i g h t gradient across a u n i l a t e r a l l y i r r a d i a t e d shoot. Because of recent studies demonstrating the existence of a s p e c i f i c BAP involved in the i n h i b i t i o n of hypocotyl elongation, Blaau's model has received renewed attention. There are some s i m i l a r i t i e s between phototropism and the rapid blue l i g h t growth response, e.g. both are due to the absorption of blue l i g h t d i r e c t l y by the hypocotyl. However, the fact that phototropic bending can occur in the complete absence of a rapid blue l i g h t response strongly suggests that bending i s not simply the result of d i f f e r e n t i a l growth i n h i b i t i o n by blue l i g h t . Differences in the timing of the rapid blue l i g h t response and phototropic bending (e.g. 1 min 121 lag versus 40-60 min lag) also support t h i s view. These results strongly suggest that phototropism and the rapid blue l i g h t response are d i s t i n c t phenomena mediated by separate BAPs. Al t e r n a t i v e l y , the bending response and growth i n h i b i t i o n response could represent two separate modes of action of a common BAP. 4.3.3 Phytochrome And The Response To Continuous Irradiations Results presented here and in a previous study (97,145) indicated that under inductive conditions, hypocotyl elongation in e t i o l a t e d Sinapis i s regulated by a phytochrome-mediated threshold response. However, under steady state conditions ( i . e . continuous irr a d i a t i o n s ) the response to red and far red l i g h t is known to be strongly fluence-rate dependent (3,51,54,65,145). It i s generally accepted that in e t i o l a t e d seedlings, the fluence-rate dependence of the response to continuous i r r a d i a t i o n s , at least in the FR waveband, is in some way related to the cycling of phytochrome molecules (64,65,67,73,74). However, the relationship between these so-c a l l e d high irradiance responses and the low energy phytochrome system is not understood. Two current models of phytochrome action are b r i e f l y described to provide a framework for discussion. Schafer (128,129) proposed a model of phytochrome action based on an additive effect of two d i f f e r e n t associations of Pfr with i t s receptor. It was proposed that one form of the Pfr-1 22 receptor complex (Pfr-X) is formed during i r r a d i a t i o n , but decays in a dark reaction to a more stable form (Pfr-X'). This model assumes separate and additive effects of the two Pfr-receptor complexes: Pfr-X responsible for the HIR, while Pfr-X' governs the low energy phytochrome responses. One key aspect of this model i s that i t predicts a strong FRHIR but a low fluence rate dependency in the red waveband (66). Based on comparative studies on the e f f e c t s of continuous i r r a d i a t i o n s and brief l i g h t pulses Schafer et a_l. (132) concluded that the response to continuous red i r r a d i a t i o n s represented a 'multiple inductive response'. The fluence rate dependency of the response to red was attributed to the fluence rate dependency of the phytochrome photostationary state (51). Johnson and Tasker (66) and Johnson (64) recently proposed that phytochrome acts through a m u l t i p l i c a t i v e e f f e c t of Pfr and some product of the phytochrome photoconversion ( c y c l i n g ) . Good evidence for a m u l t i p l i c a t i v e interaction and the existence of a cycling dependent effector of phytochrome action, separate from Pfr has been provided by studies on n i t r a t e reductase a c t i v i t y and anthocyanin synthesis in Sinapis (64,67,171). An important implication of their model is that i t predicts that the so-c a l l e d HIR responses and the low energy phytochrome-mediated responses represent d i f f e r e n t manifestations of a single photoresponse. This model predicts a strong fluence rate dependency in the red wavebands and assumes that the response to both continuous red and far red i r r a d i a t i o n s represent true HIRs (64). 123 The following series of experiments was conducted to examine the kinetics of the response to continuous i r r a d i a t i o n s and to determine i f a d i s t i n c t fluence rate-dependent response ( i . e . HIR), could be distinguished from the phytochrome-mediated threshold response. The basic assumption behind these studies was that i f the phytochrome inductive responses and HIR responses represent d i s t i n c t modes of phytochrome action , then i t might be possible to di s t i n g u i s h these photoresponses on the basis of their k i n e t i c s . 4 . 3 . 4 720-760 nm Waveband In an i n i t i a l series of experiments the spectral dependence of the response to prolonged i r r a d i a t i o n s was investigated Prolonged i r r a d i a t i o n s with l i g h t from the 720-760 nm waveband ( i . e . longwave FR) usually had l i t t l e or no effect on elongation during the course of the i r r a d i a t i o n . A weak but long term i n h i b i t i o n was observed in response to FR720 l i g h t treatments in only one of seven experiments (not shown). The small transient decline in growth rate that sometimes occurred after brief longwave FR l i g h t pulses (e.g. Fig.28B), never occurred during a prolonged i r r a d i a t i o n . However, a d i s t i n c t but transient decrease in growth rate sometimes occurred within 5-10 minutes after the end of a long term i r r a d i a t i o n ( F i g . 3 4 ) . It should be recalled that longwave FR l i g h t pulses caused a small transient decline in growth rate s t a r t i n g 5-10 minutes after the end of the i r r a d i a t i o n (Fig.28). These results 124 suggest that the response to brief and prolonged longwave FR i r r a d i a t i o n s was in some way caused by a ' l i g h t s - o f f ' s i g n a l . Unfortunately these inferences were made after the experimental portion of my research was terminated and this hypothesis was not tested further. Cosgrove (19) also noted a similar l i g h t - o f f response in e t i o l a t e d Alaska pea seedlings. Continuous i r r a d i a t i o n s with red l i g h t had no rapid effect on elongation; however, a rapid but transient decline in growth rate occurred when the l i g h t was turned o f f . Naunovic and Neskovic (106) also used a high •o resolution growth measuring technique to study stem elongation in decapitated Alaska pea seedlings. Like Cosgrove (19), they found that a brief red l i g h t pulse caused a rapid but temporary i n h i b i t i o n of growth. Unfortunately, i t cannot be determined from their results i f they were studying a normal phytochrome-mediated response, or a l i g h t s - o f f phenomenon. The underlying mechanism and possible significance of t h i s l i g h t s - o f f phenomenon i s t o t a l l y unknown. 4.3.5 660-710 nm Waveband In contrast to the e f f e c t s of continuous longwave FR, prolonged i r r a d i a t i o n s with l i g h t from the 660-710 nm waveband caused a large highly c h a r a c t e r i s t i c decline in growth rate. The kinetics of the response to prolonged red (Fig.35) and far red (Fig.36) l i g h t treatments were similar and consisted of a sharp decline in growth rate, followed by a transient recovery, 125 F i g u r e 34. E f f e c t s of 1 hour longwave FR i r r a d i a t i o n s . A l l t r a c e s are from d i f f e r e n t s e e d l i n g s . Standard experimental c o n d i t i o n s . 126 Figure 35. Effects of prolonged A) 660 or B) 670nm ir r a d i a t i o n s and B) subsequent FR710 i r r a d i a t i o n . Standard experimental conditions. 127 TIME (hours) Figure 36. Effects of brief or prolonged FR700 and FR710 l i g h t treatments. A) and B) are traces from d i f f e r e n t seedlings. Standard experimental conditions. 128 and gradual decline to a low rate. One of the most notable features of the response was the great s t a b i l i t y of the growth rate during the l i g h t treatments. The d i s t i n c t kinetics of the response to l i g h t pulses and continuous i r r a d i a t i o n s are contrasted in Fig.36 where the e f f e c t s of a brief FR700 l i g h t pulse and the effects of a prolonged FR700 i r r a d i a t i o n are shown for the same seedling. Compared to the effects of brief l i g h t pulses, the response to prolonged i r r a d i a t i o n s was remarkably consistent. A p a r t i c u l a r l y interesting observation was that after prolonged red i r r a d i a t i o n s , FR710 l i g h t treatments promoted elongation! Discussion of this important observation w i l l be delayed u n t i l l a t er in t h i s thesis (page 148). Schopfer and Oelze-Karow (145) also studied the kinetics of the response to continuous i r r a d i a t i o n s in e t i o l a t e d Sinapis However, because of the limited resolution of their technique (seedling height was measured at frequent intervals e.g. hours), the detailed kinetics of the response to continuous i r r a d i a t i o n s could not be determined. However, using similar low resolution procedures, Roth e_t a_l. (123) estimated that continuous FR i r r a d i a t i o n s strongly inhibited growth in e t i o l a t e d Sinapis after a lag of about 15 minutes. In the only previous high resolution study of growth in Sinapis , Cosgrove (21) found that prolonged i r r a d i a t i o n s with broadband R or FR l i g h t caused only a small gradual decline in growth rate. The reason for t h i s disparate result remains unclear. Although the general c h a r a c t e r i s t i c s of the response to red and far red i r r a d i a t i o n s were sim i l a r , these responses d i f f e r e d 129 with respect to the size of the i n i t i a l decline in growth rate. During continuous R660 i r r a d i a t i o n s , the decline ranged from 63-80% (mean = 70.2±2.9%, n=5) while under continuous FR710 ir r a d i a t i o n s the decline was always less than 40% (mean = 34.9%±2.3, n=5). This difference was quite evident in most growth rate traces (e.g. compare Figs.35 and 36). Previous experiments had indicated that the rapid decline in growth rate was an all-or-none-response that occurred whenever Pfr exceeded a threshold l e v e l (Table 3 and Fig.27). However, these l a t t e r results c l e a r l y indicate that the kinetics of the i n i t i a l decline were not constant but varied depending on the nature of the i r r a d i a t i o n . However, l i g h t pulse experiments had indicated that the kinetics of the i n i t a l decline and depth of the photoinhibition were not affected by widely varying levels of Pfr (e.g. Fig.27 and Table 3). This suggests then that some aspect of phytochrome cycli n g was a f f e c t i n g the kinetics of the response to l i g h t during even the very e a r l i e s t stages of the photoresponse. Thus, although i t i s obvious that continuous i r r a d i a t i o n s cause a photoresponse that i s d i s t i n c t from the phytochrome-mediated threshold response, i t cannot be determined from kinetic studies alone whether this response represents the summed eff e c t s of two photosystems, both with very brief lags, or the e f f e c t s of a single ( i . e . m u l t i p l i c a t i v e ) photosystem. The growth rate remained strongly suppressed following prolonged red (not shown) or far red (Fig.36A) i r r a d i a t i o n s and recovered very gradually over a prolonged period in darkness. After 3 hour i r r a d i a t i o n s with FR710 the growth rate did not 130 return to the i n i t i a l dark value even after 16 hours of darkness, however, a gradual increase in growth rate was s t i l l occurring (Fig.37). P h o t o r e v e r s i b i l i t y studies indicated that phytochrome was involved in long term suppression of growth following R660 or FR710 i r r a d i a t i o n s . When a prolonged i r r a d i a t i o n was terminated with a longwave FR l i g h t pulse, an abrupt increase in growth rate always occurred after a 5-15 minute lag (Fig.38). In some cases a f a i r l y rapid recovery to the o r i g i n a l dark growth rate would follow, however, after very long (e.g. >2 hour) R660 or FR710 i r r a d i a t i o n s a much more gradual recovery would sometimes occur (not shown). Discussion of the effects of FR710 shown in Fig.38B s h a l l be deferred. One incongruous point which should be noted i s that the latent period of the response to continuous i r r a d i a t i o n s was on average s l i g h t l y longer than that for 10 minute l i g h t pulses. Since the f i r s t 10 minutes of a continuous i r r a d i a t i o n were i d e n t i c a l in every respect to a 10 minutes l i g h t pulse, this e f f e c t must be related to some change in the measuring system, perhaps a s l i g h t difference in temperature among the two sets of experiments. The results presented here on the effects of prolonged i r r a d i a t i o n s d i f f e r in one minor respect from the findings of Schopfer and Oelze-Karow (145). They found that the duration of the photoinhibition following brief FR l i g h t pulses or prolonged FR i r r a d i a t i o n s was i d e n t i c a l . In both cases the growth rate remained strongly suppressed after the i r r a d i a t i o n but recovered 131 Figure 37. Effects of 3 hour FR710 i r r a d i a t i o n s and recovery in darkness. The instantaneous growth rate of two seedlings was plotted at hourly i n t e r v a l s . Standard experimental conditions. 132 F i g u r e 3 8 . E f f e c t s o f F R 7 4 0 l i g h t p u l s e s f o l l o w i n g p r o l o n g e d A ) 6 6 0 a n d B ) 7 T 0 n m i r r a d i a t i o n s . S t a n d a r d e x p e r i m e n t a l c o n d i t i o n s . 133 rapidly to the control l e v e l after a precisely determined period related to the l e v e l of Pfr established by the l i g h t treatment. In other words, the growth rate following prolonged FR i r r a d i a t i o n s was c l e a r l y controlled by a phytochrome-mediated threshold response. However, i t should be noted that after very long i r r a d i a t i o n s (e.g. 6-18 hours) the growth rate did not recover f u l l y but remained suppressed even after very prolonged periods in darkness. The kinetics of the recovery following prolonged i r r a d i a t i o n s was only b r i e f l y examined in the present study. However, from these limited studies i t appeared that a large abrupt recovery, similar to that observed after FR710 l i g h t pulses, did not occur after prolonged FR710 i r r a d i a t i o n s . In some cases a small but d i s t i n c t increase in growth rate did occur, about 1 hour after the end of a prolonged i r r a d i a t i o n (e.g. Fig.37); however, after t h i s increase the rate continued to recover only very gradually. S i m i l a r l y , the growth rate sometimes recovered very gradually even after longwave FR l i g h t pulses, which should remove v i r t u a l l y a l l Pfr. This suggests that in the seedlot used here, the growth rate following prolonged FR710 i r r a d i a t i o n s was at times limited by factors other than Pfr. Continuous i r r a d i a t i o n s with FR710 and R660 at fluence rates below 12 Wm"2 also evoked the highly c h a r a c t e r i s t i c growth response, but usually appeared to be less i n h i b i t o r y than i r r a d i a t i o n s at the standard fluence rate. At fluence rates less than 1.2 Wm"2, the growth rate was usually quite variable. 134 The results of several experiments were averaged and normalized to take into account s l i g h t differences in control (dark) growth rates (Fig.39). The effects of 10 minute R660 or FR710 l i g h t pulses (from Fig.27) are included for comparison. These data c l e a r l y indicate that the depth of the i n h i b i t i o n caused by continuous R660 and FR710 l i g h t treatments was strongly fluence rate-dependent even during the f i r s t 90 minutes of i r r a d i a t i o n . Increasing fluence rates affect both phytochrome cycli n g and the phytochrome photostationary state (51,60,66). Heim and Schafer (51) attributed the fluence rate dependency of the response to red l i g h t to the fluence rate dependency of the phytochrome photostationary state. Exactly the same argument could be applied to the effects of far red l i g h t . However, i f the threshold hypothesis of Pfr action i s v a l i d , then these arguments are irrelevant in terms of an additive model. If a m u l t i p l i c a t i v e interaction occurs between Pfr and some component of phytochrome cycling then increases in both Pfr and c y c l i n g would be expected to cause a greater i n h i b i t i o n . Holmes e_t a_l. (54) showed that at constant photon fluence rates, vastly d i f f e r e n t phytochrome photoequi1ibria ( i . e . .033-.8) had v i r t u a l l y i d e n t i c a l e f f e c ts on elongation. However, at a given photoequilibrium, the response was strongly fluence rate-dependent. These results suggest that the fluence rate dependency of the response to continuous i r r a d i a t i o n s i s related to some aspect of phytochrome c y c l i n g , and i s not related to the phytochrome photostationary state. It is apparent that in terms of the minimum growth rate 135 Figure 39. Fluence rate dependence of the response to prolonged A) FR710 and B) R660 i r r adiat ions. Results shown are averages of 5 seedlings except 10% FR710 (n=4). I r r a d i a t i o n s commenced at the arrow. 100% FR7lO=12Wirr2 100% R660=i6Wm"2. Data was normalized to the p r e - i r r a d i a t i o n growth rate. Light pulse data from Fig.27. Standard experimental conditions. 136 TIME («m) 1 37 achieved, continuous R660 or FR710 i r r a d i a t i o n s , at 12 Wrrr2 (100%) were both more inhi b i t o r y than a l i g h t pulse of the same wavelength. However, i t i s interesting to note that during the i n i t i a l stages of the photoinhibition, continuous i r r a d i a t i o n s with FR710 were consistently less inhibitory than a FR710 l i g h t pulse. Even after 90 minutes of continuous i r r a d i a t i o n , 10% FR710 had suppressed the growth rate by only 30%. In comparison, a 40-50% reduction in growth rate was usually induced by the phytochrome-mediated inductive response. Thus, i t appears that prolonged i r r a d i a t i o n s with FR710 could be both more inhi b i t o r y or less i n h i b i t o r y than an inductive response, depending on the fluence rate of the i r r a d i a t i o n . Similar results were also obtained by Schopfer and Oelze-Karow (145). They c l e a r l y showed that the rate of elongation in eti o l a t e d Sinapis was logarithmically related to the fluence rate of continuous broadband FR i r r a d i a t i o n s . In their study i t appeared that the growth rate during continuous FR i r r a d i a t i o n s (3.5 Wm"2=l00%) was i d e n t i c a l to that induced by brief R or FR l i g h t pulses. However, i t i s apparent from inspection of their data, that brief R or FR pulses caused a greater i n h i b i t i o n of growth rate than did continuous i r r a d i a t i o n s with 10% or 1% FR. These' results are d i f f i c u l t to explain in terms of an additive e f f e c t between the low energy phytochrome system and the HIR. It would imply that phytochrome -cycling promotes elongation at low fluence rates, but i n h i b i t s elongation at high rates. Thus, these results argue strongly against a s t r i c t l y additive model of phytochrome action, but could be interpreted 138 in terms of interaction between two photosystems or in terms of a m u l t i p l i c a t i v e model. The fluence rate dependence of the response to continuous FR710 could also be demonstrated in individual seedlings by increasing the fluence rate of the i r r a d i a t i o n at various intervals (Fig.40A). The response to increasing fluence rates usually involved a smooth gradual decline to a new steady state rate. The response to increasing fluence rates of R660 was less obvious but in some cases a weak decline in growth rate could be detected (Fig.40B). The involvement of phytochrome in the response to prolonged FR710 i r r a d i a t i o n s was tested by simultaneous i r r a d i a t i o n s with FR710 and FR740 (Fig.41). In repeated experiments, longwave FR given against a constant background of low intensity FR710 always caused a small but d e f i n i t e increase in growth rate after a 5-10 minute lag. When the longwave FR was turned o f f , a d i s t i n c t decrease in growth rate occurred after about a 5-10 minute lag. These results c l e a r l y indicate the involvement of phytochrome in the suppression of growth by long term i r r a d i a t i o n s . Simultaneous i r r a d i a t i o n s with longwave FR should have two main ef f e c t s on the phytochrome system. The phytochrome photostationary state should be decreased (i . e . - the lev e l of Pfr would decline) while the rate of phytochrome cyclin g would be increased. Results presented in Figures 39 and 40 indicated that increasing fluence rates (which increase cycling) lead to a greater reduction in growth rate. Therefore, the effects of simultaneous i r r a d i a t i o n s with longwave FR can 139 Figure 40. Response to increasing fluence rates of A) FR710 and B) R660 i r r a d i a t i o n s . 100% FR710=12Wm-2; 100% R660=16Wnr 2. Standard experimental conditions. 140 TIME (min) Figure 41. Effects of simultaneous i r r a d i a t i o n s with FR710 and FR740. Standard experimental conditions. 141 almost c e r t a i n l y be attributed to a decrease in the l e v e l of Pfr. Thus these results suggest that during continuous i r r a d i a t i o n s with FR710, varying the level of Pfr can have an effect on the rate of elongation. Unfortunately, the phytochrome photostationary state established during these dichromatic i r r a d i a t i o n s is not known. Thus, i t i s unclear i f the response to longwave FR was related to a simple v a r i a t i o n in the l e v e l of Pfr, or more s p e c i f i c a l l y to reducing the Pfr l e v e l below that required for a phytochrome-mediated threshold response. Does the response to red l i g h t represent a true HIR or i s i t a multiple inductive response as concluded by Schafer e_t a l . (132)? Evidence supporting a 'multiple inductive' response was based on the comparative effects of brief l i g h t pulses and continuous i r r a d i a t i o n s on anthocyanin synthesis and hypocotyl elongation in e t i o l a t e d Sinapis . The p r i n c i p a l argument was based on the fact that the effects of continuous red ir r a d i a t i o n s could be mimicked by hourly red l i g h t pulses. This strongly suggests that an inductive response is involved. In contrast, the effects of continuous far red i r r a d i a t i o n s could only be p a r t i a l l y substituted for by hourly far red l i g h t pulses ( i . e . r e c i p r o c i t y f a i l u r e ) . Thus, the basic nature of the photoresponse to the two wavelengths did appear to be d i f f e r e n t . However, the question arises, can these results be explained in terms of the phytochrome threshold reaction, and the kinetics of the photoresponse? To examine the kinetics of the response to repeated l i g h t 142 pulses, seedlings were exposed to 6 min FR710 l i g h t pulses at hourly intervals or 1 minute FR710 l i g h t pulses every 10 minutes. The t o t a l fluence (dose) received during one hour was id e n t i c a l for the two l i g h t regimes and equivalent to a 10% FR710 i r r a d i a t i o n . The results of two separate experiments in which seedlings were exposed to hourly 6 minute l i g h t pulses are shown in Fig.42. As would be expected, the inh i b i t o r y e f fects of brief l i g h t pulses were transient and a complete or p a r t i a l recovery to the o r i g i n a l growth rate occurred within the hour i n t e r v a l . This indicates that the Pfr l e v e l f e l l below the threshold required for the inductive response between l i g h t pulses. After 3 or 4 pulses these FR710 l i g h t treatments ceased to have any large rapid effect on elongation (e.g. Fig . 4253). In some experiments repeated i r r a d i a t i o n s had no long term effect on the growth rate (e.g.Fig.42A), while in others a long term reduction in rate did occur (e.g. Fig . 4233). The results of two separate experiments in which seedlings were exposed to 1 minute l i g h t pulses repeated at 10 minute intervals are shown in Fig.43. It i s apparent that the response produced by such frequent l i g h t pulses was very similar to that obtained by continuous FR710 i r r a d i a t i o n s , much more so than the response to hourly treatments described above. These studies c l e a r l y indicate that repeated FR710 l i g h t pulses can induce a ty p i c a l 'HIR' response. However, to do so, i t appears that the l i g h t pulses must be frequent enough to maintain Pfr above the threshold l e v e l . Clearly, 'continuous' i r r a d i a t i o n s are not required to evoke the 'HIR' response. For comparison, the 143 Figure 42. Effects of hourly, 6 minute FR710 l i g h t pulses. A) and B) are replicate experiments. Standard experimental conditions. Figure 43. Effects of 1 minute 710 l i g h t pul repeated at 10 min i n t e r v a l s . A) and B) are r e p l i c a t e experiments. Standi experimental conditions. 1 45 ef f e c t s of repeated R660 l i g h t pulses are shown in Fig.44. These results and replicate experiments indicated that repeated R660 l i g h t pulses caused a further i n h i b i t i o n of growth, beyond that induced by a single pulse. If the threshold hypothesis for Pfr action i s accepted, t h i s result implies that repeated inputs of phytochrome cycli n g can lead to a further i n h i b i t i o n of growth. Thus, the main difference between the e f f e c t s of hourly R660 and FR710 l i g h t pulses appears to be related to the fact that red l i g h t produces a high enough le v e l of Pfr to enable Pfr to remain above the threshold in the i n t e r v a l between l i g h t pulses. The d i f f e r e n t effectiveness of hourly red and far red l i g h t pulses can be explained e n t i r e l y in terms of a phytochrome threshold response, and the l e v e l of Pfr produced by the pulse. Therefore, these studies do not support the .contention (132) that continuous red and far red i r r a d i a t i o n s act through di f f e r e n t photosystems. However, they do suggest that the phytochrome threshold response plays a key role in the response to intermittent i r r a d i a t i o n s , and thus by association with the response to continuous i r r a d i a t i o n s . One of the most important observations that arise from th i s thesis concerns the spectral dependence of the response to continuous i r r a d i a t i o n s . Light from the 720-760 nm waveband had no detectable effect on hypocotyl elongation (excluding the l i g h t s - o f f response), although appreciable phytochrome cycli n g occurs at those wavelengths (54,66). A fluence rate-dependent response ( i . e . HIR) could only be detected at wavelengths that 146 induced a phytochrome-mediated threshold response. In other words i t appeared that phytochrome cycling could only have an effect on elongation i f a minimum le v e l of Pfr was present. These results suggest that a phytochrome threshold reaction i s prerequisite to, or an integral component of the HIR. The involvement of a phytochrome threshold reaction in the so-called HIR responses implies that a s t r i c t l y additive model of phytochrome action is untenable. Some type of sequential interaction between the low energy phytochrome system ( i . e . Pfr) and the fluence rate-dependent component i s obligatory. As previously described, i t was recently proposed that phytochrome acts through a direct mutiplicative interaction between Pfr and some component of phytochrome c y c l i n g . However, Johnson (65) later found that in de-etiolated Sinapis, increasing phytochrome cyclin g at Pfr levels ( i . e . *<.5) that had l i t t l e or no effect on elongation, could not bring about a further reduction of growth. This r e s u l t , which i s analogous to that reported here, cannot be explained in terms of a d i r e c t m u l t i p l i c a t i v e interaction between the Pfr and the second e f f e c t o r . Therefore, Johnson (65) rejected the o r i g i n a l hypothesis and concluded that the interaction could only occur 'at a subsequent step between Pfr and the response'. However, a direct interaction between Pfr and the second effector need not be rejected i f a phytochrome threshold reaction is assumed to be a prerequisite for the interaction. Indeed, Wall and Johnson (170) later showed that in their seedlot a phytochrome threshold reaction occurred at a photoequilibrium of about 0.5 in de-etiolated 147 Sinapis. The possible relationship between th i s threshold reaction and the HIR was not commented upon. Further evidence for the involvement of a phytochrome threshold reaction in the HIR was obtained in experiments in which the l e v e l of Ptot in the seedlings was reduced. Red l i g h t pretreatments cause a reduction in Ptot through phytochrome destruction. Under continuous red i r r a d i a t i o n s Ptot declines u n t i l a new steady state l e v e l of Ptot i s established (135). The precise e f f e c t s of red l i g h t pretreatments on Ptot le v e l s in this study cannot be determined; however, i t i s known that a large (e.g. ca. 85%) reduction in Ptot occurs in Sinapis during 2 hours of continuous red i r r a d i a t i o n (93,97). Phytochrome-mediated threshold responses have been shown to be governed by an absolute l e v e l of Pfr, rather than the photoequilibrium (97,99,145). In the present study the phytochrome threshold response was found to occur at a Pfr l e v e l of about 5%. The Ptot l e v e l at the time of the i r r a d i a t i o n w i l l be considered to be equal to 100%. The photoequilibrium established by FR710 i s about 0.1 (10% P f r ) . After a 50% reduction in Ptot, FR710 i r r a d i a t i o n s would e s t a b l i s h a Pfr l e v e l of 5% (based on Ptot at time zero = 100). Therefore, i f the phytochrome-mediated threshold response i s based on an absolute l e v e l of Pfr, reducing Ptot by more than 50% should eliminate the rapid inductive response at FR710. Si m i l a r l y , i f the phytochrome threshold response i s an obligatory component of the HIR, then the response to continuous FR710 should also be l o s t . 1 48 In repeated experiments i t was found that FR710 ir r a d i a t i o n s always promoted elongation a f t e r red l i g h t pretreatments of about 1 hour or longer (Fig.35B and Fig.45). The growth rate underwent an abrupt recovery after a 5-10 minute lag, suggesting that the FR710 i r r a d i a t i o n had reduced the l e v e l of Pfr below that required for the threshold response. Irradiations with B450 remained strongly i n h i b i t o r y even after the i n h i b i t o r y effects of FR710 were t o t a l l y l o s t (Fig.44). In related experiments, seedlings were given a red l i g h t pretreatment and then Pfr was removed by a FR740 l i g h t pulse. The growth rate was allowed to recover and then the e f f e c t s of FR710 i r r a d i a t i o n s were tested (e.g. Fig.38B). These experiments c l e a r l y showed that both the rapid inductive response (at FR710), and the response to continuous FR710 ir r a d i a t i o n s were l o s t . In a single experiment i t was found that the inhibitory effects of FR710 were lost after a 2 hour red l i g h t pretreatment, however, FR700 remained strongly i n h i b i t o r y . This finding of course requires further v e r i f i c a t i o n , but i t implies that Ptot had declined s u f f i c i e n t l y to eliminate the response to FR710, but remained high enough to allow i r r a d i a t i o n s with FR700 (*=.37) to raise Pfr above the threshold. In similar experiments i t was found that a R660 li g h t pulse remained strongly i n h i b i t o r y (not shown). These studies strongly support the hypothesis that a phytochrome threshold reaction is an integral component of the FRHIR. Pretreatments with red or white l i g h t have been shown to reduce the effectiveness of the FRHIR in a number of 149 TIME (min) Figure 44. Effects of hourly, 6 min R660 l i g h t pulses. Standard experimental conditions. 150 photoresponses, including the i n h i b i t i o n of hypocotyl elongation in e t i o l a t e d Sinapis (3,7,29,55,73,162). HIR action spectra for the i n h i b i t i o n of hypocotyl elongation in Sinapis show a more or less synchronous loss of both the B and FRHIR peaks following red l i g h t pretreatments (3,55). In light-grown Sinapis the response to both B and FR are t o t a l l y l o s t . The fact that B450 remains strongly in h i b i t o r y even when the inhib i t o r y e f fects of FR710 are lost (Fig.45) suggests that the loss of the FRHIR and BHIR are not d i r e c t l y coupled. Because l i g h t pretreatments are known to decrease Ptot there have been a number of attempts to relate the loss of the FRHIR to a decline in Ptot. A cor r e l a t i o n between the magnitude of the FRHIR and spectrophometrically determined phytochrome has been found in some cases (3,29,55,73) but not in others (40,162). Jose and Vince-Prue (73) found a p a r t i a l c o r r e l a t i o n between Ptot and the loss of the FRHIR for the i n h i b i t i o n of hypocotyl elongation in e t i o l a t e d radish. However, they concluded that the decline in Ptot and loss of the FRHIR were independent components of the ov e r a l l de-etiolation process. This p o s s i b i l i t y is not ruled out by the present r e s u l t s ; however, the fact that the FRHIR could be lost so rapidly (Fig.26) argues against this concept. Most previous studies in the loss of the FRHIR have involved testing the effectiveness of very prolonged (e.g. 24 hour) FR i r r a d i a t i o n s . An analogous experiment (to those reported here) done by Schopfer and Oelze-Karow (145) i s also very relevant. They found that transferring seedlings to continuous FR l i g h t after 151 2 -/•—•* f—t 1 JZ -E E R660 ON FR710 ON B450 ON RATE 1 J I 1 3: t— —S> O i s \ 0 1 • 1 1 1 1 t R660 OFF 1 1 1 1 1 1 TFR710 OFF -• 1 1 1 1 30 60 90 120 150 TIME (min) Figure 45. Eff e c t s of continuous FR710 or B450 ir r a d i a t i o n s following a 1 hour R660 pretreatment. Standard experimental conditions. 1 52 two hours of continuous red l i g h t pretreatments did not promote elongation. They concluded that reducing Ptot by approximately 85% had no effect on the FRHIR. However, a c r u c i a l point i s that the threshold for phytochrome action in their seedlot was about 0.03% Pfr (based on Ptot at time zero = 100%). Therefore, according to the hypothesis proposed here, an 85% reduction in Ptot would not be expected to eliminate the FRHIR. Thus, the fact that a 2 hour red l i g h t pretreatment had no eff e c t in their study, strongly suggests that the loss of the response to FR710 observed here i s s p e c i f i c a l l y related to a decline in Ptot, rather than some other factor associated with d e - e t i o l a t i o n . If a phytochrome threshold reaction is a prerequisite to the so-called HIR responses, then the le v e l of Pfr at which the threshold reaction occurs is of great importance. In the seedlot used by Schopfer and Oelze-Karow (145) the threshold for phytochrome action occurred at an extremely low l e v e l of Pfr i. e . .03% Pfr (*=.0003). According to Mohr (97) i t i s impossible to establish a photostationary state below .03% Pfr. Therefore, in their seedlot, the operation of the HIR could not be limited by the phytochrome threshold' reaction since a l l wavelengths of l i g h t would raise Pfr above the threshold l e v e l . Thus, i f a very low phytochrome threshold occurs, the spectral dependence of the HIR must be limited by other factors, perhaps the r e l a t i v e rate of phytochrome c y c l i n g as proposed by Johnson and Tasker (66). Si m i l a r l y , i t i s unlikely that the threshold reaction would play a key role in the loss of the FRHIR (as i t does here), i f the threshold occurs at a very low l e v e l of Pfr. 153 Under these conditions the loss of the FRHIR must be related to some other factor, perhaps a simple c o r r e l a t i o n between Ptot and the a c t i v i t y of the HIR (3,29,55). 1 54 V. GENERAL DISCUSSION This thesis was concerned primarily with the kinetics of the photoinhibition of hypocotyl elongation in e t i o l a t e d Sinapis However, this research has also provided new perspectives on the dynamics of hypocotyl elongation, and the relationship between the rapid blue l i g h t growth response and phototropism. These studies have also contributed to the understanding of the relati o n s h i p between the low energy phytochrome system and the so-called HIR. A comment on the growth measurement apparatus developed for this study is in order. Linear displacement transducers have been widely used to ^obtain continuous high resolution measurements of stem elongation in intact plants (e.g. 1,18,19,21,22,34,85,86,95,101,103,111,163). A common feature of most of these reports was that the seedling environment was not well controlled, or else could not be routinely manipulated. In the present study the seedling was enclosed within a glass cuvette which provided precise control over temperature, but permitted the seedling to be irr a d i a t e d from the side. In addition, the gaseous composition of the seedling environment could be rapidly modified by adjusting the composition and flow of gases through the cuvette. Thus, the system was ideal for investigations on the ef f e c t s and interactions of environmental factors such as l i g h t , temperature, and atmospheric constituents on stem elongation. Because of my focus on photoresponses, many of the p o s s i b i l i t i e s for research afforded by thi s system were not f u l l y u t i l i z e d during the studies reported within this 155 thesis. One of the more interesting observations a r i s i n g from th i s research concerns the occurrence of short period o s c i l l a t i o n s in the growth rate traces of many seedlings. While th i s observation i s not without precedent (e.g. 9,19,101,106), clear evidence for sustained high frequency o s c i l l a t i o n s in the rate of stem elongation has not been previously presented. As stated e a r l i e r , regardless of whether these o s c i l l a t i o n s are artefacts related to micronutation (48), or are the result of genuine variations in the rate of v e r t i c a l extension, their occurrence implies that the rate of c e l l u l a r elongation. in ,young seedlings must vary p e r i o d i c a l l y . The occurrence of SPOs in the growth rate traces of a monocot, e t i o l a t e d and de-etiolated dicots (101) as well as in the roots of a dicot (146) suggests that such o s c i l l a t i o n s might somehow be basic to the mechanism of c e l l u l a r expansion. O s c i l l a t i o n s in the rate of c e l l u l a r expansion could be due to variations in the forces driving and/or r e s t r i c t i n g c e l l enlargement. The Q J 0 of the frequency of the SPOs (2-2.5 in Sinapis ) suggests that they are under metabolic c o n t r o l . The following example gives one possible mechanism by which o s c i l l a t i o n s in the rate of c e l l elongation could a r i s e . Cleland (17) proposed that c e l l wall extension occurs by str a i n hardened p l a s t i c deformation. In other words, c e l l elongation occurs in discrete steps, with intervening periods where no increase in length occurs. Thus, a synchronization of such a process among c e l l s would lead to o s c i l l a t i o n s in the 1 56 rate of elongation in a tissue. Heathcote and Ashton (49) proposed that the alternation between such 'expansive' and 'consolidative' phases of c e l l growth was controlled by an o s c i l l a t o r situated within each c e l l , and that the phasing of individual c e l l u l a r o s c i l l a t o r s was controlled by information received from adjacent c e l l s . This model was developed to explain circumnutation, but could also be applied to the SPOs observed here. It should be noted, however, that the occurrence of o s c i l l a t i o n s in the rate of c e l l u l a r expansion does not necessarily imply the existence of individual c e l l u l a r o s c i l l a t o r s (68). For example, o s c i l l a t i o n s in the rate of c e l l elongation could be the result of a p e r i o d i c i t y in the transport of auxin or other growth substances that a f f e c t c e l l elongation (147). Johnsson (68) has recently reviewed the mechanisms by which o s c i l l a t i o n s in the rate of c e l l u l a r elongation could a r i s e . Sweeney (159) provides a general review of rhythmic phenomena in plants. However, because th i s study was concerned primarily with the photocontrol of elongation, a thorough discussion of o s c i l l a t o r y processes i s beyond the scope of t h i s thesis. The experiments presented here on the growth rate of e t i o l a t e d seedlings in darkness were intended to provide a basis for further studies on the effects of l i g h t on stem elongation. However, these studies have c l e a r l y demonstrated that, at least in intact seedlings, stem elongation is more dynamic than was previously believed. The v a r i a b i l i t y of the dark growth rate of most seedlings 157 caused some d i f f i c u l t y in carrying out the photomorphogenic studies. Extensive studies on the effects of l i g h t on stem elongation in radish would have been d i f f i c u l t because large amplitude LPOs ( i . e . nutation) occurred in v i r t u a l l y every seedling (n>20). In some respects i t might have been preferable to work with a species such as sunflower which exhibited extremely stable growth rates; however, nutation (and i t s confounding effects on the growth rate trace) also appeared to be common in t h i s species. Because of the widespread occurrence of nutation in young growing seedlings and i t s large effect on growth rate measurements, i t i s surprising that the possible influence of nutation on high resolution growth measurements has not been previously considered. The response t o ' l i g h t treatments in Sinapis proved to be rapid, large, and highly c h a r a c t e r i s t i c . Thus, despite the v a r i a b i l i t y of i t s dark growth rate, the response to l i g h t treatments could be e a s i l y detected, although the duration and depth of the photoinhibition was often d i f f i c u l t to assess, p a r t i c u l a r l y in cases where the photoinhibition appeared to be prolonged. Based on differences in the latent periods, kinetics and spectral dependence of the reponses to l i g h t observed here,a t o t a l of fi v e d i s t i n c t types of photoresponses could be distinguished. These are: the rapid i n h i b i t i o n of growth by blue l i g h t , phototropism, the phytochrome threshold response, the fluence rate-dependent response (HIR) in the 660-710 nm waveband, and a 'li g h t s o f f ' response in the 720-760 nm waveband. 158 At the outset of t h i s study, one of the main goals was to compare the kinetics of the response to blue and red l i g h t in e t i o l a t e d Sinapis. Low resolution growth studies had suggested that there was no evidence for the involvement of a separate BAP for the i n h i b i t i o n of hypocotyl elongation in Sinapis by blue l i g h t (3,172). However, on the basis of differences in the fluence rate response curves of red and blue l i g h t , Holmes and Schafer (55) concluded that the e f f e c t s of blue l i g h t could not be accounted for by phytochrome alone. This controversy was recently resolved by Cosgrove (21) who showed that blue l i g h t caused a rapid i n h i b i t i o n of growth in Sinapis, while red or far red i r r a d i a t i o n s had only a small gradual effect on elongation. In the present study, both red and blue l i g h t treatments caused a large rapid i n h i b i t i o n of growth. However, the consistent difference in the latent periods, of the two photoresponses, as well as their d i f f e r e n t kinetics ( i . e . rapid recovery after blue), strongly suggests that the responses to blue and red l i g h t are mediated by separate photoreceptors. However, the effects of blue l i g h t were not mediated exclusively through the action of the BAP, as the prolonged i n h i b i t i o n following the blue l i g h t treatments was apparently related to phytochrome. This p o s s i b i l i t y had been mentioned in the l i t e r a t u r e (19) but had not previously been tested. In Sinapis the influence of c e l l d i v i s i o n on hypocotyl elongation i s negligible and hypocotyl lengthening i s due almost exclusively to c e l l elongation (97). Thus, the inhibitory e f f e c t s of l i g h t on hypocotyl growth must be caused by an 159 i n h i b i t i o n of c e l l elongation. Cosgrove (18,20) concluded that blue l i g h t i n h i b i t e d growth by a l t e r i n g the y i e l d i n g properties of c e l l walls. It seems unlikely that t h i s effect i s mediated via a growth hormone because of the very brief latent period involved in the response to blue ( i . e . 1 minute or l e s s ) . The shortest latent periods that have been reported for the e f f e c t s of auxins or g i b b e r e l l i n s on stem elongation are of the order of 5-10 minutes (106,110). The latent period for the response to red l i g h t reported here was about 5 minutes, which does not rule out the p o s s i b l i l i t y that the effects of phytochrome are mediated via a growth hormone. One point that should be noted is that differences in the latent periods and kinetics of the responses mediated by the BAP and phytochrome does not necessarily imply that they affect c e l l elongation through d i f f e r e n t mechanisms. These differences could be related e n t i r e l y to differences in signal transduction between the photoreceptors and the responding system. A p a r t i c u l a r l y s i g n i f i c a n t finding of t h i s study concerns the r e l a t i o n s h i p between phototropism and the rapid growth response to blue l i g h t . It was shown here, perhaps for the f i r s t time, that phototropism and growth i n h i b i t i o n by blue l i g h t are d i s t i n c t phenomena, at least in e t i o l a t e d Sinapis. This was shown by the fact that continuous i r r a d i a t i o n s with very low fluence rates of blue l i g h t did not cause the c h a r a c t e r i s t i c rapid i n h i b i t i o n of growth, but did induce phototropic bending after a long (40-60 min) lag. This conclusion i s further supported by studies which indicate that 160 the i n h i b i t o r y e f fects of blue l i g h t on stem elongation are lost or greatly reduced in de-etiolated Sinapis (3,100,172), although blue l i g h t does induce phototropic bending in such seedlings (43). It i s unclear i f the d i s t i n c t i o n between phototropism and the blue l i g h t growth response also holds for monocots. In c o l e o p t i l e s , the action spectra and dose response relationship for phototropism and growth i n h i b i t i o n by blue l i g h t are similar (30). It should be mentioned that Cosgrove (19) also recently concluded that the blue l i g h t i n h i b i t i o n of growth and phototropism were d i s t i n c t ; however, th i s conclusion was based on a comparison of the c h a r a c t e r i s t i c s of phototropism derived from c o l e o p t i l e studies and the blue l i g h t i n h i b i t i o n of growth in dicots. I suggest that high resolution growth measurement techniques might be usefully adapted for studies on phototropism. Phototropism research has been dominated by a single experimental approach, i . e . the measurement of the degree of curvature induced by l i g h t treatments. In most studies, the effects of these l i g h t treatments on extension growth i s unknown. The use of high resolution growth measurement techniques, perhaps in combination with time lapse photographic techniques already in use (e.g. 42), would allow the in t e r r e l a t i o n s h i p s between growth and the kinetics of bending to be studied in d e t a i l . The results of l i g h t pulse and p h o t o r e v e r s i b i l i t y studies demonstrated that hypocotyl elongation in e t i o l a t e d Sinapis can 161 be rapidly and reversibly controlled by a phytochrome threshold reaction. This confirms the o r i g i n a l hypothesis of Schopfer and \ Oelze-Karow (145) which was based on low resolution kinetic studies of hypocotyl elongation. The phytochrome threshold reaction occurred at about 5% Pfr in the present study. In previous studies the thresholds for the control of LOG synthesis and hypocotyl elongation in et i o l a t e d Sinapis were found to occur at 1.25 and .03% Pfr respectively (99,145). Clearly, phytochrome threshold responses can occur at widely varying le v e l s of Pfr. However, i t should be noted that these values were obtained from studies using d i f f e r e n t seedlots. It i s pertinent to ask whether d i f f e r e n t phytochrome threshold reactions control d i f f e r e n t photoresponses, or whether there i s a single threshold reaction in a given seedlot. In other words, are LOG synthesis and hypocotyl elongation controlled by the same or d i f f e r e n t phytochrome threshold reactions? Studies on th i s question could provide insight into the issue of whether phytochrome has one or many primary reactions. Does the phytochrome threshold reaction disappear during de-etiolation or does i t also play a role in the photoresponses of light-grown plants? There i s very recent evidence which suggests that under continuous i r r a d i a t i o n , hypocotyl elongation in light-grown Sinapis can be influenced by a phytochrome threshold reaction (170). Thus, phytochrome threshold responses may be more widespread and of greater importance than was previously believed. 162 Nevertheless, i t is unwise to generalize on the occurrence of phytochrome-mediated threshold responses, even within Sinapis. In the present study a large rapid response to red l i g h t pulses was consistently obtained in one seedlot, but not in the other. S i m i l a r l y , broadband red l i g h t treatments caused only a small gradual i n h i b i t i o n of growth in the seedlot tested by Cosgrove (21). It i s not known i f these differences in the responses to l i g h t are due to fundamental differences in the phytochrome system and transduction chain or to differences in the responding system. The responses to red and far red i r r a d i a t i o n s shown here are among the most rapid phytochrome-mediated responses yet reported. It i s perhaps s i g n i f i c a n t that the latent periods of these photoresponses ( i . e . about 5-6 min for i n h i b i t i o n by R and reversal by FR) are similar to the latent period for the eff e c t s of supplementary FR on internode elongation in more mature green plants. A number of studies have shown that a l t e r i n g the * by adding supplementary FR to background white l i g h t (lowers * ) , causes a rapid increase in the stem extension rate of light-grown seedlings (86,88,101,103). Supplementary FR causes an increase in the growth rate of young light-grown Sinapis alba seedlings after a lag of 10-15 minutes (101) . The rapid response to supplementary FR occurred only when the internode i t s e l f was irr a d i a t e d . The rapid response to R and FR l i g h t pulses reported here were caused by l i g h t perceived d i r e c t l y by the hypocotyl. Thus, the response to supplementary FR in green plants, and the phytochrome threshold response of 163 e t i o l a t e d Sinapis are similar with respect to the location of the photoreceptor, and timing of the response. This p a r a l l e l i s m between the two photoresponses suggests that the fundamental mechanism by which l i g h t regulates stem elongation may be similar at these two d i f f e r e n t stages of stem elongation. However, there is an important difference between the two types of photoresponses. Studies of internode elongation in light-grown plants indicate that there is an inverse linear r e l a t i o n s h i p between the stem extension rate and the photoequilibria (101,102,103,104,105). Smith (154) contends that t h i s response is not governed by the l e v e l of Pfr, but i s controlled in some unspecified manner by the r a t i o of Pfr to Ptot. This i s contrary to the current view that Pfr i s the only active form of phytochrome, and that photoresponses are governed by the absolute amount of Pfr present (29,65). In the present study the response to both brief l i g h t pulses and continuous i r r a d i a t i o n s was c l e a r l y governed by the l e v e l of Pfr and not the Pfr/Ptot r a t i o . This was shown by the fact that red l i g h t pretreatments, which would reduce Ptot but would not af f e c t the Pfr/Ptot r a t i o established by subsequent i r r a d i a t i o n s , completely eliminated the effectiveness of FR710 i r r a d i a t i o n s . Thus, these experiments strongly support the concept of Pfr being the only active form of phytochrome. Studies on the kinetics of the responses to prolonged and repeated i r r a d i a t i o n s have provided several new perspectives on the r e l a t i o n s h i p between the low energy phytochrome system and the HIR. Prolonged i r r a d i a t i o n s with either R or FR (FR700 or 164 FR710) caused a rapid fluence rate-dependent response that was d i s t i n c t from the ef f e c t s of brief l i g h t pulses. Frequent FR710 li g h t pulses could mimic the kinetics of the response to continuous FR710 i r r a d i a t i o n s , however, hourly FR710 l i g h t pulses of the same t o t a l fluence could not. Mancinelli and Rabino (90) suggested that the re c i p r o c i t y f a i l u r e of the HIR could be explained on the basis of the l e v e l of Pfr maintained by d i f f e r e n t l i g h t regimes, i . e . l i g h t regimes that maintain some Pfr throughout the treatment period would be expected to be more ef f e c t i v e than l i g h t regimes that allow Pfr levels to decline. The results on the comparative e f f e c t s of hourly and more frequent FR710 l i g h t pulses support th i s interpretation, and suggest that a phytochrome threshold reaction can play a co n t r o l l i n g role in the response to intermittent i r r a d i a t i o n s . If t h i s i s true then i t i s not necessary to postulate the action of two separate photosystems to account for differences in the comparative effects of hourly and continuous R and FR ir r a d i a t i o n s (132). Two additional observations also suggested that the response to continuous i r r a d i a t i o n s was in some way dependent on the phytochrome threshold reaction. F i r s t , the spectral dependence of the response to continuous i r r a d i a t i o n s and brief l i g h t pulses was i d e n t i c a l in the 660-760 nm waveband. Secondly, red l i g h t pretreatments completely eliminated the inhibitory e f fects of FR710 i r r a d i a t i o n s i . e . the rapid inductive response at FR710 and the response to continuous FR710 ir r a d i a t i o n s were simultaneously l o s t . In other words, i t 165 appeared that phytochrome cycli n g had no ef f e c t on elongation in the absence of a phytochrome threshold response. This conclusion i s not consistent with current models of phytochrome action. Figure 46 diagrams a scheme that can account for the involvement of a phytochrome threshold reaction in the response to continuous i r r a d i a t i o n s ( i . e . the HIR). It i s presented primarily to provide a basis for further experimentation; however, i t appears to be consistent with the observations of thi s thesis and much of what i s known about the HIR in Sinapis. The scheme is derived from the threshold model of phytochrome action as elaborated by Mohr and Oelze-Karow (M16) and the m u l t i p l i c a t i v e model of phytochrome action proposed by Johnson and Tasker (66) and Johnson (64). The scheme assumes that Pfr i s rapidly bound to the receptor 'X'; the cooperative t r a n s i t i o n P f r - X ^ P f r - X ' occurs at 5% Pfr (based on [Ptot] at time zero=l00%); Y i s some product or result of phytochrome c y c l i n g ; Y can only act in the presence of Pfr-X'. Thus, the basic assumption of this proposal i s that the product of phytochrome cycli n g can have no effect on elongation at Pfr lev e l s below 5% (in th i s p a r t i c u l a r seedlot). The nature Of the interaction between the phytochrome threshold reaction and Y cannot be predicted on the basis of results presented here; however, a direct m u l t i p l i c a t i v e effect between Pfr ( i . e . Pfr-X') and the product of phytochrome cycling i s not ruled out, as was previously believed (65). Another p o s s i b i l i t y is that 166 Y Pr % Pfr + X % P f r - X P f r - x ' b i o l o g i c a l act ion 5 * Pfr Figure 46. Hypothetical scheme of phytochrome action. 167 the threshold reaction might simply act as a trigger (on-off) switch in a sequential interaction between the two effectors of phytochrome action. The scheme presented here was based on studies of hypocotyl elongation in e t i o l a t e d Sinapis. The general a p p l i c a b i l i t y of the scheme for other HIR responses in Sinapis remains to be tested. However, as previously discussed there i s also evidence to support the involvement of a phytochrome threshold reaction in the e f fects of continuous i r r a d i a t i o n s on hypocotyl elongation in de-etiolated Sinapis (170). In addition, Johnson (65) found that in light-grown Sinapis, "where there i s l i t t l e or no Pfr-promoted i n h i b i t i o n of extension, i t i s not possible to induce such i n h i b i t i o n by increasing the fluence rate". This finding i s consistent with the scheme outlined in Fig.46 and suggests that a phytochrome threshold reaction may be involved in the response to continuous i r r a d i a t i o n s in both e t i o l a t e d and light-grown Sinapis. The high irradiance responses of plant photomorphogenesis have been the subject of intensive study and debate for more than 25 years. However, l i t t l e real progress has been made in understanding the nature of processes involved in the HIR. The results of t h i s study have perhaps provided a valuable new insight on the role that phytochrome plays in the HIR. 1 68 VI. CONCLUSIONS 1. Under constant environmental conditions the growth rate traces of Sinapis, cucumber, radish and oat seedlings often exhibited sustained highly regular o s c i l l a t i o n s with a period of less than 30 minutes. It was concluded that a high frequency o s c i l l a t i o n in the rate of c e l l u l a r elongation can occur in elongating hypocotyls and c o l e o p t i l e s . 2. Brief i r r a d i a t i o n s with red or blue l i g h t both caused a large rapid i n h i b i t i o n of growth in e t i o l a t e d Sinapis Differences in the latent periods and kinetics of these responses indicated that the effects of blue l i g h t were mediated by a BAP d i s t i n c t from phytochrome. 3. Differences in the fluence rate dependence and latent periods of phototropism, and the rapid i n h i b i t i o n of growth mediated by the BAP, suggest that these two photoresponses are d i s t i n c t phenomena. 4. Pho t o r e v e r s i b i l i t y studies, measurements of the depth and duration of the photoinhibition, and the spectral dependence of the response to brief l i g h t pulses indicated that hypocotyl elongation in et i o l a t e d Sinapis was rapidly and reversibly controlled -by a phytochrome threshold reaction. 5. There was i d e n t i c a l spectral dependence of the responses to brief and continuous i r r a d i a t i o n s in the 660-760 nm waveband. It was concluded that the response to continuous i r r a d i a t i o n s was in some way dependent on the phytochrome threshold reaction. This conclusion was further supported by studies showing that the i n h i b i t o r y effects of 710 nm i r r a d i a t i o n s were t o t a l l y lost 169 following red l i g h t pretreatments. 6. 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