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Physiological and biochemical studies of Laurencia spectabilis and its symbiont Janczewskia gardneri… Court, Gary James 1978

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PHYSIOLOGICAL AND BIOCHEMICAL STUDIES OF LAURENCIA SPECTABILIS AND ITS SYMBIONT JANCZEWSKIA GARDNERI (CERAMIALES, RHODOPHYCEAE) by GARY JAMES COURT B . S c , C a l i f o r n i a P o l y t e c h n i c State U n i v e r s i t y , S.L.O., 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1978 © Gary James Court In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly a v a i l a b l e for reference and study. I fur ther agree that permission for extensive copying of th is thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion o f th is thes is fo r f i n a n c i a l gain s h a l l not be allowed without my wri t ten permission. Depa rtment The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 6 Research Supervisor: Dr. I a i n E. P. Tay l o r ABSTRACT This t h e s i s i s a two par t study on the biochemistry and physiology of the red algae Janczewskia g a r d n e r i S e t c h e l l & Guernsey and La u r e n c i a  s p e c t a b i l i s P o s t e l s & Ruprecht (Ceramiales, Rhodophyceae). The f i r s t p a r t c o n s i s t s of photosynthesis, t r a n s l o c a t o r y , m i c r o s c o p i c , and c u l t u r a l s t u d i e s of the symbiotic a s s o c i a t i o n between the supposed p a r a s i t e J_. gardneri and i t s host L_. s p e c t a b i l i s . Mature p l a n t s of J_. gardneri were pigmented, contained t y p i c a l red a l g a l c h l o r o p l a s t s when viewed by e l e c t r o n microscopy, and were capable of photosynthesis. Both J_. gardneri and L_. s p e c t a b i l i s 14 14 incorpor a t e d C - l a b e l from NaH CO^ i n t o s i m i l a r compounds, i n c l u d i n g sugars ( f l o r i d o s i d e , i s o f l o r i d o s i d e , g a l a c t o s e , glucose) and amino acid s ( a l a n i n e , 14 a s p a r t i c a c i d , glutamic a c i d , g l y c i n e , s e r i n e ) . T r a n s l o c a t i o n of C-labeled photosynthetic products between mature J_. gardneri and i t s host d i d not occur. Released spores of J_. gardneri were pigmented, contained mitochondria, p r o p l a s t i d s and f l o r i d e a n s t a r c h r e s e r v e s , and germinated i n the absence of the host; however, the germlings died w i t h i n two weeks. This research suggested that mature i n d i v i d u a l s of J_. gardneri were o b l i g a t e epiphytes. The second p a r t of t h i s t h e s i s r e p o r t s on the i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of a proteoglycan from L_. s p e c t a b i l i s . The proteoglycan was i s o l a t e d by e x t r a c t i o n i n a d i l u t e buffer-NaCl s o l u t i o n followed by g e l and i o n exchange chromatography, us i n g c e l l u l o s e acetate s t r i p e l e c t r o -p h o r e s i s f o r monitoring p u r i f i c a t i o n . The non-sulfated proteoglycan contained 92% carbohydrate and 8% p r o t e i n . Galactose (85%) was the major n e u t r a l sugar detected. Uronic a c i d s , glucose, x y l o s e and a t r a c e of arabinose were a l s o present. A small q u a n t i t y of hydroxyproline was present i n the molecule. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . i i i LIST OF FIGURES . v l LIST OF TABLES v i i i ACKNOWLEDGEMENTS i x FRONTISPIECE,. . • x i PART I : STUDIES OF JANCZEWSKIA GARDNERI AND ITS SYMBIOTIC RELATIONSHIP WITH LAURENCIA SPECTABILIS . . . . . . . 1 INTRODUCTION . . 2 C h a r a c t e r i s t i c s Of Red A l g a l Symbiotic R e l a t i o n s h i p s . . 2 Janczewskia gardneri 4 Other Red A l g a l Symbioses 5 Obj e c t i v e s For This Study 10 MATERIALS AND METHODS 11 Chemicals And Solvents 11 Seawater 12 C o l l e c t i o n S i t e s And Procedures 12 L i g h t Microscopy 15 E l e c t r o n Microscopy 15 Photosynthesis And T r a n s l o c a t i o n Experiments 17 A. P r e p a r a t i o n of P l a n t M a t e r i a l 17 B. Ra d i o a c t i v e L a b e l . . . . . 17 C. Photosynthesis . 17 D. T r a n s l o c a t i o n 18 i v TABLE OF. CONTENTS (cont.) Page E. Measurement of Sample R a d i o a c t i v i t y 18 F. E x t r a c t i o n and F r a c t i o n a t i o n of R a d i o a c t i v e Compounds . . . 20 G. I d e n t i f i c a t i o n of Labeled Compounds 21 1. Paper chromatography 21 2. T h i n - l a y e r chromatography 22 3. High voltage paper e l e c t r o p h o r e s i s 22 4. G a s - l i q u i d chromatography (GLC) 22 5. Mass spectrometry and m e l t i n g p o i n t determinations . . 24 6. Amino a c i d a n a l y s i s 24 7. Computer analyses 24 Free Sugars And Amino Acids 25 Cultu r e Of Spores 25 OBSERVATIONS AND RESULTS . . 27 Host L i f e H i s t o r y Stages Selected By Symbiont 27 L i g h t M i c r o s c o p i c Examination Of The Attachment Region 27 Pigmentation And C h l o r o p l a s t s 27 Photosynthesis Experiments 30 A. Uptake of Radi o a c t i v e L a b e l 30 B. I n c o r p o r a t i o n of Radi o a c t i v e L a b e l 36 T r a n s l o c a t i o n Experiments . 57 Free Sugars And Amino Acids Of L i f e H i s t o r y Stages 66 Cultu r e Of Spores 69 DISCUSSION 72 BIBLIOGRAPHY 80 V TABLE OF CONTENTS (cont.) Page PART I I : ISOLATION AND PARTIAL CHARACTERIZATION OF A PROTEOGLYCAN FROM LAURENCIA SPECTABILIS 85 INTRODUCTION . . . . 86 L e c t i n s . . 87 3-Lectins 88 Objectives For This Study 90 MATERIALS AND METHODS 92 Chemicals And Solvents 92 P l a n t M a t e r i a l 93 P r e p a r a t i o n Of P l a n t M a t e r i a l 93 Gel And Ion Exchange Chromatography . 94 C e l l u l o s e Acetate S t r i p E l e c t r o p h o r e s i s 95 8-Lectin D e t e c t i o n 95 A n a l y t i c a l Procedures . 96 General a n a l y s i s 96 N e u t r a l sugar a n a l y s i s 96 Amino a c i d and amino sugar analyses 97 RESULTS . .„ 98 P u r i f i c a t i o n Of A Proteoglycan 98 '$-Lectin Determination Of The Proteoglycan 98 Chemical A n a l y s i s Of The Proteoglycan 105 DISCUSSION 113 BIBLIOGRAPHY 120 v i LIST OF FIGURES Page FIGURE 1 . C o l l e c t i n g s i t e s i n B r i t i s h Columbia (A), Oregon (B), and C a l i f o r n i a (C) . . . . . . . . . . . . . . . 13 2 Attachment region between gardneri and L. s p e c t a b i l i s 28 3 C h l o r o p l a s t s from a v e g e t a t i v e c e l l of J_. gardneri 31 4 E l e c t r o n micrographs of J_. gardneri tetraspores (A) and carpospores (BJ C) before r e l e a s e from the parent p l a n t s 33 5 Radioautograms of "^C-labeled f r a c t i o n s of L. s p e c t a b i l i s (A, B) and J_. g a r d neri (C, D) 38 6 Radioautograms of "^C-labeled " f r a c t i o n a t e d - n e u t r a l " f r a c t i o n s of L_. s p e c t a b i l i s (A) and J_. gardneri (B) 42 7 G a s - l i q u i d chromatogram of a l d i t o l acetate d e r i v a t i v e s of some standard sugars 44 8 G a s - l i q u i d chromatograms of a l d i t o l acetate d e r i v a t i v e s of f r e e sugars from J_. gardneri (A) and L. s p e c t a b i l i s (B) 46 9 G a s - l i q u i d chromatograms of a l d i t o l acetate d e r i v a t i v e s of hydrolyzed and reduced f r e e sugars of J_. gardneri (A) and L_. s p e c t a b i l i s (B) . . 49 10 D i a g n o s t i c p o r t i o n s of mass spec t r a of f l o r i d o s i d e acetate (A) and i s o f l o r i d o s i d e acetate (B) 51 11 T h e o r e t i c a l fragmentation schemes f o r f l o r i d o s i d e acetate (A) and i s o f l o r i d o s i d e acetate (B) during mass spectrometry 53 12 D i a g n o s t i c p o r t i o n s of mass spec t r a of suspected f l o r i d o s i d e / i s o f l o r -i d o s i d e GLGkpeaks from L_. s p e c t a b i l i s (A) and J_. gardneri (B) . . . . 55 13 Radioautogram of the c a t i o n i c f r a c t i o n s of J_. gardneri and L. s p e c t a b i l i s a f t e r s e paration u s i n g paper e l e c t r o p h o r e s i s 58 14 R a d i o a c t i v i t y of L^ . s p e c t a b i l i s and attached J_. gardneri a f t e r 30 min 14 uptake of NaH CO^ fo l l o w e d by t r a n s l o c a t i o n p e r i o d s 62 15 Carpospores of L. s p e c t a b i l i s (A) and J_. gardneri (B) i n c u l t u r e . . . 70 v i i LIST OF FIGURES (cont.) Page FIGURE 16 Sepharose 4B g e l chromatography of L. s p e c t a b i l i s 100% saturate d (NH^SO^ supernatant. . . . . . . . . . . 99 17 C e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s of L_. s p e c t a b i l i s proteo-glycan a f t e r re-chromatography on Sepharose 4B. 101 18 S a l t - g r a d i e n t e l u t i o n of L_. s p e c t a b i l i s proteoglycan from DEAE-Sephadex A-50 and c e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s of f r a c t i o n s . 103 19 G a s - l i q u i d chromatogram of a l d i t o l acetate d e r i v a t i v e s of sugars from p u r i f i e d L_. s p e c t a b i l i s proteoglycan : 107 20 Chromatograms of (A) amino sugars and b a s i c amino acid s and (B) a c i d i c and n e u t r a l amino acid s of p u r i f i e d L_. s p e c t a b i l i s proteoglycan. . . 109 v i i i I Summary of c o n d i t i o n s f o r the t r a n s l o c a t i o n experiments a t 10 C . . 19 LIST OF TABLES Page TABLE °14 I I I Uptake of C - l a b e l by L. s p e c t a b i l i s and J_. g a r d n e r i from seawater c o n t a i n i n g NaH 1 4C0 3 . . . . . . . . . . 35 14 I I I D i s t r i b u t i o n of a s s i m i l a t e d C - l a b e l i n e x t r a c t s and residues from L_. s p e c t a b i l i s and J_. gardneri 37 IV ^Q±U v a l u e s f ° E some standard sugars separated by one dimensional descending paper chromatography 40 14 V Re s u l t s of C-pulse-chase t r a n s l o c a t i o n experiments w i t h L_. s p e c t a b i l i s and J_. gardneri 60 VI R a t i o s of J_. gardneri/L. s p e c t a b i l i s "^C-pulse-chase t r a n s l o c a t i o n values 64 VI I R a d i o a c t i v i t y of "chase" seawater from the "^C-pulse-chase t r a n s l o -c a t i o n experiments #2 and 3 65 V I I I Composition of recovered f r e e amino a c i d s of L. s p e c t a b i l i s . . . . 67 IX Composition of recovered f r e e amino a c i d s of J_. gardneri 68 X Composition of p u r i f i e d proteoglycan from Laurencia s p e c t a b i l i s . . 106 XI N e u t r a l sugar composition of p u r i f i e d Laurencia s p e c t a b i l i s proteoglycan 106 X I I Composition of amino acid s and amino sugars recovered from h y d r o l -yzates of ix. spec tab 1-lis proteoglycan . '. °. I I . I l l i x ACKNOWLEDGEMENTS This research p r o j e c t was supported by grants to Dr. I . E. P. Taylor from the N a t i o n a l Research. C o u n c i l of Canada and The U n i v e r s i t y of B r i t i s h Columbia. I g r a c e f u l l y acknowledge the U n i v e r s i t y Graduate Fellowship support which I r e c e i v e d from The U n i v e r s i t y of B r i t i s h Columbia. I a l s o wish to acknowledge the g i f t s of f l o r i d o s i d e and i s o f l o r i d o s i d e sugars from Dr. J . S. C r a i g i e ( A t l a n t i c Regional Laboratory, H a l i f a x , N. S.) and of n e u t r a l sugar a l d i t o l s and t h e i r acetates from Dr. J . N. C. Whyte ( F i s h e r i e s and Marine S e r v i c e , Vancouver, B. C ) . Dr. G.SG. S. Dutton, UBC Department of Chemistry, made a v a i l a b l e equipment i n h i s l a b o r a t o r y f o r s e v e r a l of the g a s - l i q u i d and i o n exchange chromatography analyses, w i t h the advice and p r a c t i c a l a s s i s t a n c e of h i s graduate students, e s p e c i a l l y (Dr.) K e i t h Mackie, g r e a t l y appreciated. T e c h n i c a l s t a f f of the UBC Department of Chemistry k i n d l y performed the mass spectrometry analyses. Dr. P. E. Reid and Mr. C h a r l i e Ramey, UBC Department of Pathology, provided a s s i s t a n c e and advice on c e l l u l o s e acetate s t r i p e l e c t r o -phoresis techniques. The o f f e r of Dr. Robin L. Anderson to perform the g - l e c t i n a g g l u t i n a t i o n t e s t s was g r e a t l y appreciated i n a time of need. I wish to acknowledge the a s s i s t a n c e of my committee members, Drs. R. E. De Wreede, R. F. Scagel, J . R. S t e i n , I , E. P. Taylor and J . N. C. Whyte, e s p e c i a l l y f o r t h e i r prompt e d i t i n g of the t h e s i s d r a f t . I wish to give s p e c i a l mention of a p p r e c i a t i o n to Dr. Whyte f o r h i s many long conversations w i t h me on the problems of a n a l y s i s and i d e n t i f i c a t i o n of carbohydrate components of the red algae. I a l s o wish to note t h a t the l a t e Dr. EV":Bruce Tregunna, an o r i g i n a l committee member, provided important suggestions f o r the design of the photosynthesis and t r a n s l o c a t i o n s t u d i e s . X ACKNOWLEDGEMENTS (cont.) I a l s o want to acknowledge the advice, encouragement, and f r i e n d s h i p provided by my v a r i o u s lab colleagues during the research and w r i t i n g of t h i s t h e s i s , i n c l u d i n g s p e c i a l thanks to Drs. I l l i m a r A l t o s a a r , Don Cameron and Adrianne Ross, and e s p e c i a l l y to Mr. Don Ma n s f i e l d whose p r a c t i c a l a s s i s t a n c e and " b u l l " sessions helped i n times of need. I extend my a p p r e c i a t i o n to Mr. Mel Davies and Mr. Ken J e f f e r i e s f o r t h e i r prompt t e c h n i c a l a s s i s t a n c e , to Dr. Lynda Goff f o r our conversations on red a l g a l p a r a s i t i s m , and to other members of the Department who helped i n l a r g e and small ways, w i t h s p e c i a l mention to Mr. Mike Higham, Dr. L u i s O l i v e i r a and Mr. L a s z l o Veto f o r a s s i s t a n c e w i t h e l e c t r o n microscopy, and to Mike Hawkes and C h r i s Tanner who were always there to lend a hand, provide ideas and give encouragement. Most of a l l I wish to thank Dr. I a i n T a y l o r who had an e n t h u s i a s t i c i n t e r e s t i n my research and made the completion of t h i s t h e s i s p o s s i b l e . He always had suggestions f o r surmounting the various methodological problems encountered, and the patience to bear my fl o u n d e r i n g s , e s p e c i a l l y during the w r i t i n g of t h i s t h e s i s . I consider myself extremely fortunate to have had him as my research s u p e r v i s o r . I look forward to h i s c o n t i n u i n g f r i e n d s h i p and w i l l always appreciate h i s i n t r o d u c i n g me to the great i n s t i t u t i o n of Rugby Union F o o t b a l l , a r e c r e a t i o n which helped me to maintain my s a n i t y during my graduate s t u d i e s . F i n a l l y , I want to thank my parents and brother who were always encouraging and supporting me towards the completion of t h i s t h e s i s , an accomplishment which belongs very much to them too. JANCZEWSKIA GARDNERI (ARROW) UPON LAURENCIA SPECTABILIS 1 P A R T I STUDIES OF JANCZEWSKIA GARDNERI AND ITS SYMBIOTIC RELATIONSHIP WITH LAURENCIA SPECTABILIS 2 INTRODUCTION Red algae involved i n symbiotic r e l a t i o n s h i p s have been the subjects of recent research (e.g., H a r l i n 1971b; Kugrens 1971; Evans, Callow and Callow 1973; Goff 1975; H a r l i n and Craigie 1975; Turner and Evans 1977). These associations are symbiotic on the basis of De Bary's o r i g i n a l d e f i n i t i o n that symbiosis i s the l i v i n g together of d i s s i m i l a r organisms i n a constant, intimate association (Henry 1966). This broad d e f i n i t i o n encompasses many kinds of associations including epiphytism, endophytism, and parasitism. The partners of the red algae i n these symbioses include b a c t e r i a (Bland and Brock 1973), fungi (Kazama and F u l l e r 1970; Kohlmeyer 1975), sea grasses (Harlin 1971b; McRoy and Goering 1974), green algae (Wilson 1977), brown algae (Markham 1969; Rawlence and Taylor 1970; Hawkes 1977), and other red algae (Kugrens 1971; Evans, et a l . 1973; Goff 1975). The symbiosis between the red algae Janczewskia gardneri S e t c h e l l & Guernsey and Laurencia s p e c t a b i l i s Postels & Ruprecht, both members of the order Ceramiales, was the subject of t h i s research. This r e l a t i o n s h i p has been known since J_. gardneri was f i r s t reported (Set c h e l l 1914) as a parasite of L. s p e c t a b i l i s . Chapman and Chapman (1973) considered species of the genus Janczewskia to be eit h e r hemi- or h o l o p a r a s i t i c , but the exact nature of t h i s r e l a t i o n s h i p was uncertain. C h a r a c t e r i s t i c s of Red A l g a l Symbiotic Relationships The symbiotic r e l a t i o n s h i p s of the algae, e s p e c i a l l y the Rhodophyta, exhibit a broad spectrum of association from epiphytes to obligate 3 p a r a s i t e s ( S e t c h e l l 1918; F r i t s c h 1935, 1945; Scott 1969; Joubert and Rijkenberg 1971; Evans, et a l . 1973; Goff 1975; H a r l i n and C r a i g i e 1975). A number of hypotheses to e x p l a i n the e v o l u t i o n of the v a r i o u s a s s o c i a t i o n s between red algae have been presented ( S e t c h e l l 1918; Sturch 1926; Feldmann and Feldmann 1958; Fan 1961). S e t c h e l l (1918) suggested that mutated - . . . spores of an al g a may germinate on i t s parent p l a n t and i n i t i a t e p a r a s i t i s m . On the other hand, Sturch (1926) and Fan (1961) suggested that i n most instances of red a l g a l p a r a s i t i s m the p a r a s i t e s o r i g i n a t e d from epiphytes which had sunk r a m i f i c a t i o n s i n t o t h e i r hosts to gain b e t t e r attachment. L a t e r these attachment organs may have become adapted to absorb n o u r i s h -ment from the host. I f the food m a t e r i a l was s u i t a b l e , the ' i n t r u d e r ' may have become i r r e v e r s i b l y dependent by l o s s of i t s photosynthetic system and/or become reduced i n s i z e . I f changes occurred w i t h the t r a n s i t i o n from epiphyte to p a r a s i t e , they expected to and d i d f i n d a progression from independence to interdependence between the algae which had entered i n t o the symbiotic r e l a t i o n s h i p s . In other d i s c u s s i o n , S e t c h e l l (1918) i d e n t i f i e d three c r i t e r i a which may be i n d i c a t i v e of p a r a s i t i s m i n the red algae: p e n e t r a t i o n , r e d u c t i o n of t h a l l u s s i z e , and l o s s of c o l o r . He considered these c r i t e r i a to apply to epiphytes, endophytes and p a r a s i t e s , and concluded that as morpholog-i c a l f e atures they provided only p r e l i m i n a r y i n d i c a t i o n s of a p o s s i b l e p a r a s i t i c r e l a t i o n s h i p . He recognized the need to demonstrate metabolite t r a n s f e r from the host to the p a r a s i t e and a dependence of the p a r a s i t e upon the host to e s t a b l i s h the exi s t e n c e of a p a r a s i t i c r e l a t i o n s h i p . S e t c h e l l a l s o pointed out that many of the p a r a s i t i c red algae are found 4 on c l o s e l y r e l a t e d hosts with host and parasite u s u a l l y i n the same family. In the approximately 40 genera of suspected red a l g a l parasites, about 90% belong to the same family or order as t h e i r host (Feldmann and Feldmann 1958; Dawson 1966). Parasites which exhibit such close systematic r e l a -tionships with t h e i r hosts were c a l l e d adelphoparasites, whereas those lacking a close r e l a t i o n s h i p were termed a l l o p a r a s i t e s (Feldmann and Feldmann 1958). Janczewskia gardneri J^. gardneri was f i r s t described as occurring "on Laurencia p i n n a t i f i d a , p a r t i c u l a r l y that broad, coarse form c a l l e d L. s p e c t a b i l i s by Postels and Ruprecht" (Set c h e l l 1914). The range of J. gardneri extends along the eastern P a c i f i c coast from Vancouver Island, B r i t i s h Columbia to Baja C a l i f o r n i a (Abbott and Hollenberg 1976) . L,. s p e c t a b i l i s i s the predominant host species throughout the range of J_. gardneri except for rare reports of JL. splendens as the host species from c e n t r a l C a l i f o r n i a and Baja C a l i f o r n i a . J_. gardneri i s an adelphoparasite on the basis of the termi-..nology of Feldmann and Feldmann (1958), since i t and i t s hosts are c l a s s -i f i e d i n the subdivision Laurencieae of the family Rhodomelaceae i n the Ceramiales (Hommersand 1963). The t h a l l u s of J_. gardneri i s reduced and t u b e r c l e - l i k e , forming pinkish cushions of i r r e g u l a r s i z e (see F r o n t i s p i e c e ) . S e t c h e l l (1914) reported that from the t h a l l u s "slender hyphal branches" penetrated to the host's elongated c e n t r a l c e l l s and that no connections between the two plants were present. On the basis of these morphological and 5 anatomical characters J_. g a r d n e r i was regarded as p a r a s i t i c by S e t c h e l l (1914), F r i t s c h (1945), Chapman and Chapman (1969), Smith (1969), and Abbott and Hollenberg (1976). Kugrens (1971) examined the u l t r a s t r u c t u r e of v e g e t a t i v e and reproductive s t r u c t u r e s of ten genera of p a r a s i t i c red algae, i n c l u d i n g J_. g a r d n e r i . He reported that J_. gardneri possessed " t y p i c a l red a l g a l c h l o r o p l a s t s w i t h respect to t h y l a k o i d a l s t r u c t u r e and presence of phycobilisomes" (He detected c h l o r o p h y l l a. and r- p h y c o e r y t h r i n , but not r-phycocyanin.). Due to i n t e r m i n g l i n g of t i s s u e s , Kugrens d i d not i d e n t i f y any p i t connections between J_. gardneri and L,. s p e c t a b i l i s , nor could he see the d i s t i n c t r h i z o i d a l f i l a m e n t s mentioned i n S e t c h e l l ' s 14 o r i g i n a l d e s c r i p t i o n . Kugrens a l s o t r i e d to determine i f C-labeled photosynthetic products of the host were t r a n s l o c a t e d to the p a r a s i t e or v i c e v e r s a , but he obtained no co n c l u s i v e answers. Nevertheless, he d i d ob t a i n evidence that ^ J . g ardneri was capable of p h o t o s y n t h e t i c a l l y a s s i m i -l a t i n g the l a b e l , although at a much lower r a t e than i t s host. He germinated t e t r a s p o r e s of ^J. g a r d n e ri i n c u l t u r e , but they died w i t h i n two weeks even i n the presence of host p l a n t s . H i s study d i d not r e s o l v e the exact nature of the a s s o c i a t i o n between ^J. g a r d n e r i and L_. s p e c t a b i l i s . Other Red A l g a l Symbioses Two red a l g a l epiphytes have been the subjects of recent research. Smithora naiadum i s an o b l i g a t e epiphyte found normally on the sea grasses P h y l l o s p a d i x s c o u l e r i or Zostera marina. Radioactive t r a c e r s t u d i e s 32 14 ( H a r l i n 1971a, b) w i t h P and C showed t r a n s l o c a t i o n of those elements to proceed i n both d i r e c t i o n s between host and epiphyte. L a t e r work by 6 McRoy and Goering (1974) a l s o i n d i c a t e d a d i r e c t t r a n s f e r of carbon and n i t r o g e n from Z. marina to i t s l e a f epiphytes, i n c l u d i n g S_. naiadum. . A l -though t h i s exchange occurred, f i e l d s t u d i e s by H a r l i n (1971b,.c, 1973a) usin g an a r t i f i c i a l s u b s t r a t e ( f i b r o u s polypropylene s t r i p s ) r e s u l t e d i n the epiphytes a t t a c h i n g and growing i n t o i r r e g u l a r l y - s h a p e d p l a n t s . H a r l i n 1973a) suggested that " t h i s supposedly o b l i g a t e epiphyte does not r e q u i r e a chemical f a c t o r from i t s normal host pl a n t f o r (1) s u b s t r a t e s e l e c t i o n and adhesion, (2) growth of b a s a l cushions and young blades or (3) matur-a t i o n of t h i c k cushions", and " t h a t the primary r o l e of the host i s to provide a p h y s i c a l environment f o r an o p p o r t u n i s t i c s p e c i e s , w h i l e i t s e c o n d a r i l y may provide biochemical f a c t o r s f o r normal development of an epiphyte and subsequent completion of i t s l i f e h i s t o r y " . B b l y s i p h o n i a lanosa occurs upon the brown a l g a Ascophyllum nodosum or o c c a s i o n a l l y upon species of Fucus (Rawlence and Taylor 1970). P_. lanosa d i f f e r s from S_. naiadum i n that i t has h o s t - p e n e t r a t i n g r h i z o i d s . Subsequent s t u d i e s supplied evidence that the p e n e t r a t i o n was due more to chemical than mechanical means (Rawlence and Taylor 1972; Rawlence 1972). Degeneration and almost complete disappearance of host t i s s u e occurred i n advance of and surrounding the r h i z o i d . U l t r a s t r u c t u r a l changes i n d i c a t e d increased c e l l a c t i v i t y f o r P_. lanosa, but no t e s t s f o r enzymes were per-formed. The p h y s i o l o g i c a l aspect of p o s s i b l e metabolite t r a n s f e r between 14 P_. lanosa and A. nodosum was examined w i t h C-labeled glutamic a c i d by 32 86 99 C i t h a r e l (1972a, b) and w i t h i n o r g a n i c r a d i o i s o t o p e s ( P, Rb, Mo, 24 Na) by Penot (1974). These s t u d i e s showed that there was some t r a n s f e r of r a d i o a c t i v e substances from the brown a l g a to the red a l g a . L a t e r 7 s t u d i e s ( H a r l i n and C r a i g i e 1975; Turner and Evans 1977) determined that 14 P. lanosa could a s s i m i l a t e C-bicarbonate p h o t o s y n t h e t i c a l l y and that i t 14 d i d not appear to r e c e i v e nor to depend upon C-labeled products of p h o t o a s s i m i l a t i o n from A. nodosum. H a r l i n and C r a i g i e (1975) suggested that the a s s o c i a t i o n of the two algae was based on the need f o r growth f a c t o r s from the host f o r germination or c e l l d i v i s i o n i n an e a r l y stage of the l i f e h i s t o r y of the epiphyte or on surface c h a r a c t e r i s t i c s of the host c r i t i c a l f o r the attachment of P_. lanosa spores. Turner and Evans (1977) suggested that carbon metabolism need not be the s o l e b a s i s f o r such a r e l a t i o n s h i p between any two algae and that some aspect of epiphyte spore settlement and attachment may be of greater importance. Rawlence (1972) a l s o suggested that "any n u t r i t i o n dependence on the host i s i n c i d e n t a l to attachment". The red a l g a Gonimophyllum s k o t t s b e r g i i , an adelphoparasite, has a s m a l l , l e a f y , p i n k i s h t h a l l u s and i s a s s o c i a t e d w i t h the red alga Botryoglossum rup r e c h t i a n a ( H a r l i n 1971b). In Kugrens (1971) u l t r a -s t r u c t u r a l study of ten genera of ' p a r a s i t i c ' red algae, he reported that G. s k o t t s b e r g i i had ' t y p i c a l ' red a l g a l c h l o r o p l a s t s and could photo-14 synthesize. H a r l i n (1971b, 1973b) a l s o found that i t a s s i m i l a t e d C-l a b e l p h o t o s y n t h e t i c a l l y . In a d d i t i o n , she reported that the ' p a r a s i t e ' t r a n s l o c a t e d the a s s i m i l a t e d "^C-labeled m a t e r i a l s to i t s 'host' as w e l l as r e c e i v i n g t r a n s l o c a t e s from i t s host. Evans, Callow and Callow (1973) examined "the p a r a s i t i c , c h l o r o -p l a s f r f r e e , red a l g a Holmsella pachyderma", an a l l o p a r a s i t e , and i t s red a l g a l host G r a c i l a r i a verrucosa. Their s t u d i e s i n d i c a t e d that metabolite 8 movement occurred from host to p a r a s i t e only as the carbohydrate f l o r i d o -s i d e , the main photosynthetic product of G. verrucosa,. was t r a n s l o c a t e d 14 to the p a r a s i t e . A s i g n i f i c a n t amount of the detected C - l a b e l i n H. pachyderma was found i n mannitol, a compound which was not i d e n t i f i e d i n the host, so H. pachyderma was capable of m e t a b o l i z i n g substances t r a n s -l o c a t e d from i t s host. Since H. pachyderma was shown to be c h l o r o p l a s t -f r e e by e l e c t r o n microscopy and t h e r e f o r e h e t e r o t r o p h i c , the t r a n s f e r of the f l o r i d o s i d e e s t a b l i s h e d that H. pachyderma was indeed p a r a s i t i c on G. verrucosa. I t was suggested that the movement of the carbohydrate was most l i k e l y through the endophytic f i l a m e n t s which a r i s e from the base of the p a r a s i t e and penetrate between the c e l l s of the host. The l a r g e number of mitochondria present i n the endophytic f i l a m e n t c e l l s was sug-gested to be important i n p r o v i d i n g energy f o r the movement of substances between host and p a r a s i t e . Goff (1975) i n v e s t i g a t e d morphological and p h y s i o l o g i c a l aspects of another red a l g a l a l l o p a r a s i t e , H a r v e y e l l a m i r a b i l i s , w i t h i t s red a l g a l hosts Odonthalia f l o c c o s a (the primary host i n the northeast P a c i f i c ) , (). washingtoniensis, and Rhodomela l a r i x . F i e l d and l a b o r a t o r y s t u d i e s of the development and reproduction of H. m i r a b i l i s showed that the completion of i t s reproductive l i f e h i s t o r y was dependent on the presence of a s u i t a b l e host f o r germination and that the development and repro-duction were a f f e c t e d by seasonal changes i n seawater temperature and photoperiod. H. m i r a b i l i s was found to be p h y s i o l o g i c a l l y dependent upon the host 0. f l o c c o s a s i n c e the p a r a s i t e lacked 'normal' red a l g a l c h l o r o -14 p l a s t s and d i d not p h o t o a s s i m i l a t e C-bicarbonate, but i t d i d r e c e i v e 9 C-labeled photoassimilated products from (J. f l o c c o s a . L i g h t microscopic radioautography showed the primary flow of l a b e l to occur through the r h i z o i d a l c e l l s of the p a r a s i t e . A secondary source of l a b e l was from i s o l a t e d host c e l l s dispersed i n the H. m i r a b i l i s p ustule. Carbohydrates (which could not be s p e c i f i c a l l y i d e n t i f i e d ) were apparently the main r a d i o a c t i v e substances t r a n s l o c a t e d from host to p a r a s i t e , although the presence of p r o t e i n s and l i p i d s as w e l l as carbohydrates was a s s o c i a t e d w i t h a v a c u o l e / v e s i c l e t r a n s p o r t system detected i n the r h i z o i d a l c e l l s of H. m i r a b i l i s . From t h i s survey of work on red a l g a l a s s o c i a t i o n s , i t can be seen that the r e l a t i o n s h i p s between the attached organism and i t s host are o f t e n q u i t e complex. There i s evidence f o r a strong degree of host s p e c i f i c i t y which may be r e l a t e d to p h y s i c a l attachment or a biochemical f a c t o r s u p p l i e d by the host. Any of these r e l a t i o n s h i p s has to i n v o l v e the attachment and germination of a spore, which may i n v o l v e the produc-t i o n of enzymes f o r degrading the host's c e l l s . A d d i t i o n a l s ubstrate s p e c i f i c i t y can i n v o l v e the a v a i l a b i l i t y of ' s u i t a b l e ' metabolites of the host f o r the symbiont. The a b i l i t y of a p a r a s i t e to o b t a i n metabolites and to convert them i n t o other u t i l i z a b l e compounds has been e s t a b l i s h e d f o r two a l l o p a r a s i t e s , Holmsella pachyderma (Evans, ;et a l . 1973) and H a r v e y e l l a m i r a b i l i s (Goff 1975). In a d d i t i o n , evidence from both s t u d i e s suggests that the endophytic attachment organs of such algae can be s p e c i a l i z e d f o r a c t i v e l y t r a n s p o r t i n g t r a n s l o c a t e s from t h e i r hosts. 10 Objectives For This Study I decided to i n v e s t i g a t e a red a l g a l symbiotic r e l a t i o n s h i p i n v o l v i n g an adelphoparasite because i t was of i n t e r e s t to increase the l i m i t e d i n f o r m a t i o n about t h i s l a r g e group of red a l g a l p a r a s i t e s . I chose the supposedly p a r a s i t i c Janczewskia g a r d n e r i because of i t s s p e c i f i c assoc-i a t i o n w i t h the red al g a Laurencia s p e c t a b i l i s , i t s unclear p h y s i o l o g i c a l r e l a t i o n s h i p w i t h i t s host, and.'.its abundance almost year-round i n the l o c a l f l o r a . The general purpose of t h i s study was to determine the exact r e l a -t i o n s h i p of an adelphoparasite ( J . gardneri) with: i t s host (_L. s p e c t a b i l i s ) , i n c l u d i n g the extent of independence and i n t e r a c t i o n of the p a r a s i t e w i t h i t s host, and the p o s s i b l e nature of s u b s t r a t e s e l e c t i o n by the p a r a s i t e . The s p e c i f i c o b j e c t i v e s were: (1) to determine photosynthetic r a t e s f o r the host and p a r a s i t e when separate and when a s s o c i a t e d . (2) to i d e n t i f y the photosynthetic products of host and p a r a s i t e . (3) to determine i f t r a n s l o c a t i o n between host and p a r a s i t e can occur i n e i t h e r d i r e c t i o n , and i f so, what substances are t r a n s l o c a t e d . (4) to determine i f there i s a p r e f e r e n t i a l s e l e c t i o n of a host l i f e h i s t o r y stage f o r i n v a s i o n by the p a r a s i t e . (5) to determine i f the reproductive spores of the p a r a s i t e have the ca p a c i t y (e.g., c h l o r o p l a s t s , s t a r c h reserves, mitochondria) to grow independently from the host. (6) to t r y to grow the p a r a s i t e from spores i n c u l t u r e . 11 MATERIALS AND METHODS CHEMICALS AND SOLVENTS Chemicals and s o l v e n t s , reagent grade ACS or b e t t e r , were obtained from the s u p p l i e r s as i n d i c a t e d : g l y c e r o l ( A l l i e d Chemical Canada, L t d . , P o i n t e C l a i r e , Quebec); a c e t i c anhydride, u r a n y l acetate (J.T. Baker Chemical Co., P h i l l i p s b u r g , New J e r s e y ) ; Beckman Amino A c i d C a l i b r a t i o n Mixture Type 1 (Beckman Instruments, I n c., Spinco D i v i s i o n , Palo A l t o , C a l i f o r n i a ) ; D-galac-tose, D-galactosamine HC1, p y r i d i n e AnalaR ACS (used f o r GLC p r e p a r a t i o n s ) , sodium borohydride ( B r i t i s h Drug Houses L t d . , Poole, England); a l l amino acids (Calbiochem, Los Angeles, C a l i f o r n i a ) ; c a p r y l i e C a c i d , t r i f l u o r o a c e t i c a c i d (Eastman Kodak Co., Rochester, New York); lead c i t r a t e , most reagents f o r Spurr's r e s i n ( E l e c t r o n Microscopy Sciences, F o r t Washington, Pennsylvania); germanium d i o x i d e , D-mannose, D-mannitol, " S c i n t i - V e r s e " s c i n t i l l a t i o n c o c k t a i l ( F i s h e r S c i e n t i f i c Co., F a i r Lawn , New J e r s e y ) ; sodium glucuronate (Koch-Light L a b o r a t o r i e s L t d . , Colnbrook, England); L-fucose, D-galacturonic a c i d (mono-hydr a t e ) , D-glucose, D-xylose ( N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio); methyl c e l l o s o l v e , n i n h y d r i n ( P i e r c e Chemical Co., Rockford, I l l i n o i s ) ; g lutaraldehyde, OsO^ ( P o l y s c i e n c e s , Inc., Warrington, Pennsylvania); D-glucos-amine HC1, m y o - i n o s i t o l (Sigma Chemical Co., St. L o u i s , M i s s o u r i ) . F l o r i d o s i d e and i s o f l o r i d o s i d e were provided by Dr. J.S. C r a i g i e , A t l a n t i c Regional Laboratory, H a l i f a x , Nova S c o t i a . D - g a l a c t i t o l , D - g l u c i t o l , D-mannitol and t h e i r a c e t a t e s , f . L - f u c i t o l pentaacetate, and D - x y l i t o l pentaacetate were provided by Dr. J.N.C. Whyte, F i s h e r i e s and Marine S e r v i c e , Vancouver, B r i t i s h Columbia. A l l other chemicals and s o l v e n t s were obtained l o c a l l y and were of reagent grade ACS q u a l i t y or b e t t e r . 12 SEAWATER Seawater from the c o l l e c t i o n s i t e was used to transport fresh plant material to the laboratory. A l l other seawater used i n t h i s study was obtained from Botany Beach near Port Renfrew, B r i t i s h Columbia and before use was membrane-filtered (0.45 ym membrane; M i l l i p o r e Corp., Bedford, Massachusetts). COLLECTION SITES AND PROCEDURES Laurencia s p e c t a b i l i s with and without attached Janczewskia gardneri was obtained from four locations on Vancouver Island, B r i t i s h Columbia (see Fig . IA) between December 1973 and 1976. The main c o l l e c t i o n area was Botany Beach (the north end) approximately 1 km SE of San Juan Point near Port Renfrew, B.C. The other three s i t e s were Cable Beach on Barkley Sound near Bamfield, B.C.; Whiffen Spit at the harbor entrance, Sooke, B.C.; and the harbor entrance breakwater, V i c t o r i a , B.C. Ad d i t i o n a l c o l -l e c t i o n s were made at le a s t once between November 1972 and December 1976 at Saturnina Island, B.C. (Fig. IA), two s i t e s i n Oregon (Fig. IB), and three s i t e s i n C a l i f o r n i a (Fig. IC). Herbarium specimens of _L. s p e c t a b i l i s and J_. gardneri were examined at The University of B r i t i s h Columbia, Vancouver, B.C.; Univ e r s i t y of C a l i f o r n i a , Berkeley, CA.; and C a l i f o r n i a Polytechnic State University, San Luis Obispo, CA. These specimens had been obtained along the e n t i r e known range of ^ J. gardneri on the eastern P a c i f i c coast from Vancouver Island, B r i t i s h Columbia to Baja C a l i f o r n i a . Freshly c o l l e c t e d plants were placed i n p l a s t i c bags containing sea-water and kept on ic e during transport to the laboratory. They were 13 F i g . 1. C o l l e c t i n g s i t e s i n B r i t i s h Columbia (A), Oregon (B), and C a l i f o r n i a (C). (A) S i t e s i n B r i t i s h Columbia were (1) Cable Beach, (2) Botany Beach, (3) Whiffen S p i t , (4) V i c t o r i a breakwater, and (5) Saturnina I s l a n d , one of the F l a t Top I s l a n d s o f f the eastern t i p of G a b r i o l a I s l a n d . (B) S i t e s i n Oregon were (6) Yaquina Head, north of Newport, and (7) South Cove, Cape Arago, south of Coos Bay. (C) S i t e s i n C a l i f o r n i a were (8) Cambria, (9) Cayucos, and (10) Coal O i l P o i n t , n o r t h of the U n i v e r s i t y of C a l i f o r n i a , Santa Barbara campus. FIGURE 1 15 maintained i n open containers with aeration i n a 10°C growth chamber and 3 2 provided with a 12 hr photoperiod (5.6 x 10 ergs/cm /sec l i g h t ; YSI-Kettering Model 65 radiometer). Specimens were preserved by f i x a t i o n with either 30% ethanol or Karpechenko's s o l u t i o n (Papenfuss 1946). The material was dehydrated i n a graded ethanol ser i e s and stored i n 70% ethanol. LIGHT MICROSCOPY Observations were made p r i m a r i l y on fresh material. Reproductive structures were usually examined from squashed plant preparations. The attachment region between the two plants was studied i n ti s s u e sections cut with a freezing microtome and were examined unstained or stained with t o l u i d i n e blue. ELECTRON MICROSCOPY Fresh plant material for electron microscopy was fi x e d on i c e i n the f i e l d or i n the laboratory. J_. gardneri or pieces of L. s p e c t a b i l i s with attached J_. gardneri were fixed for 1 hr i n either (a) 50% glutaraldehyde: 0.07 M sodium phosphate buffer (pH 7.2): seawater (1:4:4 v/v) (adapted from McBride and Cole 1969) or (b) 5% glutaraldehyde i n 0.1 M sodium phosphate buffer (pH 7.0). Both methods of f i x a t i o n were followed by post-f i x a t i o n for 2 hr at 5°C i n a so l u t i o n containing equal volumes of 2% (w/v) Qsp^ and the phosphate buffer used for the f i x a t i o n . The material was washed i n the buffer, dehydrated i n a graded ethanol s e r i e s , and embedded i n Spurr's r e s i n (Spurr 1969). Sections were .cut using glass knives on a Reichert OM U3 ultramicrotome and were p o s t - s t a i n e d f o r 30 min w i t h saturated u r a n y l acetate followed by 8 min w i t h lead c i t r a t e (Reynolds 1963). Sections were viewed i n a Zeiss EM 9S trans m i s s i o n e l e c t r o n microscope. 17 PHOTOSYNTHESIS AND TRANSLOCATION EXPERIMENTS A. P r e p a r a t i o n of P l a n t M a t e r i a l . Fresh p l a n t m a t e r i a l stored over-n i g h t i n the 10°C growth chamber was used f o r the experiments. P l a n t s f o r photosynthesis experiments #1, 2 and 3 were c o l l e c t e d at Botany Beach on 18 August 1974, 24 February 1975, and 11 J u l y 1976. P l a n t s f o r t r a n s -l o c a t i o n experiments #1 and 3 were from Botany Beach on 24 February 1975 and 11 J u l y 1976, and f o r experiment #2 p l a n t s were from Whiffen S p i t on 31 March 1975. ^J. g a r d n e ri and pieces of L. s p e c t a b i l i s w i t h or without attached J^. gardneri were s e l e c t e d and cleaned of macroscopic epiphytes at l e a s t 14 hr 14 before any uptake of C - l a b e l was allowed. The clean m a t e r i a l was put i n p e t r i p l a t e s (100 x 15 mm) c o n t a i n i n g seawater. The p l a n t s were maintained at ca. 10°C and were kept i n the dark f o r at l e a s t 8 hr followed by a minimal pretreatment of 6 hr l i g h t - or. dark. The pretreatment condi-t i o n ( l i g h t or dark) i n the photosynthesis experiments was the same c o n d i t i o n under which the p l a n t s were allowed to take up the r a d i o a c t i v e l a b e l . The pretreatment c o n d i t i o n s i n t r a n s l o c a t i o n experiments #1 and 2 were the same c o n d i t i o n s as during the experimental t r a n s l o c a t i o n p e r i o d , but i n experiment #3 a l l p l a n t s were pr e t r e a t e d w i t h l i g h t . 14 B. Ra d i o a c t i v e L a b e l . C-sodium bicarbonate (sp. act. 56-60 mCi/mM) was used f o r the experiments. Seawater was a c i d i f i e d to pH 2.0 w i t h HC1 and s t i r r e d to remove CO^. The pH was readjusted to pH 7.9 w i t h NaOH. The r a d i o i s o t o p e was then added to give a f i n a l c o n c e n t r a t i o n of 2 y C i NaH 1 4C0 3/ml. C. Photosynthesis. The algae used i n the three photosynthesis 18 experiments were allowed to take up the l a b e l for between 10 sec and 30 min 5 2 i n the l i g h t (2.6-2.8 x 10 ergs/cm /sec) or dark from 25 ml of radio-a c t i v e seawater i n 100 x 15 mm p e t r i plates. In the three experiments there were three r e p l i c a t e s for each l i g h t and dark.(control) treatment used. A f t e r the uptake period, the plant material was removed immediately, rinsed thoroughly i n seawater, k i l l e d by submersion i n l i q u i d a n < i stored at ca. -70°C. D. Translocation. Table I summarizes conditions of l i g h t and dark pretreatments, l a b e l i n g periods, and l i g h t i n t e n s i t i e s used i n the trans-l o c a t i o n experiments. In the three translocation experiments, pieces of L. s p e c t a b i l i s with J_. gardneri were allowed to assimilate the l a b e l i n the l i g h t and then were removed from the radioactive seawater (25 ml) and rinsed thoroughly with seawater. Three samples were taken, k i l l e d by sub-mersion i n l i q u i d ^ and stored frozen (ca. -70°C), while the other r a d i o a c t i v e plant material was placed i n p e t r i plates containing 25 ml non-radioactive 'chase' seawater. These plants were allowed to trans-locate i n the l i g h t or dark for periods of 2 to 12 hr. Three samples were removed at the end of each translocation period, k i l l e d by submer-sion i n l i q u i d and stored frozen. The chase seawater from transloca-t i o n experiments #2 and 3 was kept for s c i n t i l l a t i o n counting. E. Measurement of Sample Ra d i o a c t i v i t y . Frozen radioactive plant material was thawed and any attached J_. gardneri plants were dissected from t h e i r host material. The plants were blotted, weighed, and digested i n s c i n t i l l a t i o n v i a l s using p e r c h l o r i c acid and hydrogen peroxide (Lobban 1974). The s c i n t i l l a t i o n counting " c o c k t a i l " , 10 ml of " S c i n t i -Verse", was added and mixed thoroughly with the digested material. The TABLE I SUMMARY OE CONDITIONS FOR THE TRANSLOCATION EXPERIMENTS AT 10°C Plan t Pretreatment L a b e l i n g L a b e l i n g Chase Experiment M a t e r i a l Condit^ ons P e r i o d L i g h t Intensity Conditions (ergs/cm /sec) (min) (ergs/cm /sec) (ergs/cm /sec) #1 Botany Beach L i g h t 60 1.4 x 1 0 5 L i g h t 24 February 1975 8 x 1 0 3 8 x 1 0 3 #2a Whiffen S p i t 31 March 1975 #2b whiffen S p i t 31 March 1975 L i g h t 8 x 10' Dark 30 30 8.0 x 10" 8.0 x 10~ L i g h t 8 x 10~ Dark 3 #3a Botany Beach L i g h t 30 8.0 x 10 L i g h t 11 J u l y 1976 8 x 10 3 8 x 1 0 3 #3b Botany Beach L i g h t 30 8.0 x 1 0 3 Dark 11 J u l y 1976 8 x 10 3 20 samples were counted using a Nuclear Chicago Unilux I I - A s c i n t i l l a t i o n counter and counts were correct e d using the c h a n n e l - r a t i o method (Wang and W i l l i s 1965). Residual m a t e r i a l s were a l s o t r e a t e d i n t h i s manner. R a d i o a c t i v i t y i n other samples was determined a f t e r a d d i t i o n of a s u i t a b l e p o r t i o n of the s o l u t i o n to 10 ml of " S c i n t i - V e r s e " . R a d i o a c t i v i t y of paper chromatograms was measured by p l a c i n g p o r t i o n s of the paper i n Bray's f l u i d (Bray 1960) f o r counting. F. E x t r a c t i o n and F r a c t i o n a t i o n of R a d i o a c t i v e Compounds. P l a n t m a t e r i a l s from photosynthesis experiments #1 and 2 were e x t r a c t e d separ-a t e l y and examined f o r r a d i o a c t i v e compounds. ^J. gardneri p l a n t s and I J . s p e c t a b i l i s p o r t i o n s which had been incubated s e p a r a t e l y i n the r a d i o -a c t i v e seawater were used. These algae were exposed to the r a d i o a c t i v e l a b e l i n the l i g h t or dark f o r 30 min. Each p l a n t sample (0.1-0.9'gm wet wt) was e x t r a c t e d f o r 15 min w i t h 100 ml b o i l i n g 80% ethanol, f o r 10 min w i t h 100 ml b o i l i n g 50% ethanol, and again f o r 10 min w i t h 100 ml of b o i l i n g 80% ethanol. The combined e x t r a c t s were d r i e d by r o t a r y evaporation at 40°C and then d i s s o l v e d i n d e i o n i z e d water. The e x t r a c t e d residues were a i r - d r i e d and l a t e r prepared f o r s c i n t i l l a t i o n counting. Chloroform was added to each aqueous e x t r a c t to p a r t i t i o n l i p i d s and pigments. The chloroform-phase was evaporated and counted. The volume of the water-phase was reduced to l e s s than 5 ml by r o t a r y evaporation at 40°C, then desalted and f r a c t i o n a t e d u s i n g a column of Rexyn 101(H) (3 x 25 cm; F i s h e r S c i e n t i f i c Co.) and next a column of D u o l i t e A-4 (3.5 x 16 cm; Diamond A l k a l i Co., Redwood C i t y , CA). The n e u t r a l f r a c t i o n , which c o n s i s t e d of sugars, was c o l l e c t e d a f t e r 21 passage of the e x t r a c t through both columns. Amino acid s were e l u t e d from the Rexyn 101(H) c a t i o n exchange r e s i n w i t h 2 N NH^OH, while organic a c i d s were e l u t e d from the D u o l i t e A-4 anion exchange r e s i n w i t h 0.2 N formic a c i d . The volumes of these three f r a c t i o n s were reduced by r o t a r y evaporation and samples were taken f o r s c i n t i l l a t i o n counting. The i d e n t i f i c a t i o n of sugars i n the n e u t r a l f r a c t i o n was f a c i l i t a t e d by f u r t h e r f r a c t i o n a t i o n using a column of a c i d - f r e e charcoal (2 x 4.7 cm). The samples were a p p l i e d i n l e s s than 5 ml of d e i o n i z e d water and e l u t e d i n water (100 ml) followed by a step-wise gradient (100 ml each) of 5, 10, 20, and 35% ethanol. These 11 f r a c t i o n a t e d - n e u t r a l " f r a c t i o n s were concentrated s e p a r a t e l y by r o t a r y evaporation f o r paper chromatography. G. I d e n t i f i c a t i o n of Labeled Compounds. 1. Paper chromatography. The n e u t r a l , " f r a c t i o n a t e d - n e u t r a l " , c a t i o n i c and a n i o n i c f r a c t i o n s from each p l a n t e x t r a c t , and standard sugar mixtures were analyzed by one-dimensional paper chromatography on Whatman No. 1 paper using two solvent systems (Whyte and Southcott 1970): (a) e t h y l a c e t a t e : p y r i d i n e ( 1 0 : 4 : 3 v/v) and (b) e t h y l a c e t a t e r a c e t i c a c i d : formic a c i d r ^ O (18:3:1:4 v / v ) . Dried chromatograms were covered w i t h t h i n p l a s t i c - w r a p (e.g., "Handi-Wrap", Dow Chemical Co.) before r a d i o -autography, otherwise the e n t i r e chromatogram became s e n s i t i v e to the reagents used to detect sugars. The l a b e l e d compounds were detected by exposure to Kodak Blue Brand BB 14 medical X-ray f i l m . Sugars were l o c a t e d on the chromatograms by m o d i f i c a t i o n s (J.N.C. Whyte, K. Mackie, pers. coram'.) of the s i l v e r n i t r a t e procedure of Trevelyan, P r o c t e r and H a r r i s o n (1950). Dried chromatograms were dipped i n the s i l v e r n i t r a t e 22 reagent, d r i e d , and dipped i n 0.5 n NaOH i n 95% ethanol. Spot development was enhanced a f t e r the NaOH-ethanol dip by steaming the chromatograms f o r a few seconds, being c a r e f u l not to overheat. The steamed chromatograms were placed i n 5% Na^S^O^ s o l u t i o n (25 gm sodium t h i o s u l f a t e , 25 gm sodium ace t a t e , 0.5 ml g l a c i a l a c e t i c a c i d , plus 500 ml ^O) f o r 10-15 min, then washed w i t h tap water before d r y i n g . 2. T h i n - l a y e r chromatography. Organic a c i d f r a c t i o n s were separated on A v i c e l (American Vicose Co., Marcus Hook, PA.) t h i n - l a y e r s (ca. 0.65 mm) which were developed i n one or two d i r e c t i o n s using the same solvent system of n-amyl a l c o h o l : f o r m i c a c i d (97%):^© (20:20:1 v / v ) . Labeled compounds were detected by radioautography as described e a r l i e r . A c i d i c compounds were v i s u a l i z e d by spraying the t h i n - l a y e r w i t h a s o l u -t i o n of bromothymol blue (0.04 gm i n 100 ml of 95% e t h a n o l , adjusted w i t h NaOH to pH 10.0). 3. High voltage paper e l e c t r o p h o r e s i s . N e u t r a l , c a t i o n i c and a n i o n i c f r a c t i o n s , and mixtures of reference amino a c i d s were separat-ed by paper e l e c t r o p h o r e s i s using a Michl-type l i q u i d cooled apparatus ( M i c h l 1951, 1959). The samples were a p p l i e d to Whatman 3MM chromatography paper and e l e c t r o p h o r e s i s was performed f o r 40 min at 3 kV (ca. 52.6 V/cm of paper length) i n a c e t i c a c i d : f o r m i c acidiH^O (8:2:90 v/v, pH 2.0). Labeled compounds were detected by radioautography and amino a c i d s were l o c a t e d on the electrophoregram by dipping i n a cadmium a c e t a t e - n i n h y d r i n s o l u t i o n (Heilmann, B a r r o l l i e r and Watzke 1957). 4. G a s - l i q u i d chromatography (GLC). Non-radioactive n e u t r a l f r a c t i o n s were obtained from f r e s h p l a n t m a t e r i a l s kept i n c o n d i t i o n s s i m i l a r to those of the experimental p l a n t s . A l d i t o l acetate d e r i v a t i v e s 23 of the unknown and standard sugars were prepared as described by Albersheim, e_t a l . (1967). Samples to be analyzed by mass spectrometry were d e r i v a -t i z e d by the method of Bjorndal, Lindberg and Svensson (1967). Samples were hydrolyzed for 1 hr at 110°C i n 2 N t r i f l u o r o a c e t i c acid and dried i n a stream of a i r before d e r i v a t i z a t i o n . Varian Aerograph Model 1740 and Hewlett Packard Model 5710A dual column gas chromatographs were used for the analyses. The gas flow rates i n both machines were 25 ml/min for and H^, and 250 ml/min for a i r . Two a n a l y t i c a l systems were used. (1) Stainless s t e e l columns (.6' x 1/8" o.d.) were packed with 5% (w/w) S i l a r 10C ( A l l t e c h Associates, A r l i n g t o n Heights, IL.) on 100/120 mesh Gas Chrom Q (Applied Sciences Lab. Inc., State College, PA.). Samples were injected at 120°C followed by a programmed increase i n temperature from 120° to 260°C at 2° or 4°/min. (2) Copper columns (6' x 1/8" o.d.) were packed with 3% (w/w) ECNSS-M on 100/120 mesh Gas Chrom Q (prepared by Applied Sciences Lab. Inc.). Samples were i n j e c t e d and held at 120°C for 10 min before an increase i n temperature from 120° to 185°C was programmed at 2°/min. The samples used i n both a n a l y t i c a l systems were in j e c t e d i n var^ ious solvents: a c e t i c anhydride, acetone, chloroform, ethanol, and e t h y l acetate. Samples for mass spectrometry were obtained using a pre-parative column (6' x 1/4" o.d. s t a i n l e s s steel) packed with 5% (w/w) S i l a r 10C on 100/120 mesh Gas Chrom Q i n a F & M Model 720 gas chromato-graph which was run isothermally at 250°C. Samples were c o l l e c t e d i n glass c a p i l l a r y tubes at the machine ex i t port. 24 5. Mass spectrometry and mel t i n g p o i n t determinations. The p u r i t y of samples c o l l e c t e d from the p r e p a r a t i v e S i l a r IOC column was checked by a n a l y s i s of a p o r t i o n using the a n a l y t i c a l S i l a r IOC columns. Mass spectrometry was performed i n a Varian/MAT CH4-B mass spectrometer using the d i r e c t - i n s e r t i o n technique. Probe temperatures were 100° and 120°C, and the source temperature was ca. 150°C. The e l e c t r o n energy was 70 eV. Mass spectra were obtained f o r the unknown component(s) of the m a t e r i a l r e s o l v e d as a s i n g l e peak by GLC and suspected to be f l o r i d o s i d e ( 2 -0-a-D-galactopyranosylglycerol), i s o f l o r i d o s i d e (1-0-a-D-galactopyrano-s y l g l y c e r o l ) , or both. Reference samples of these two compounds were a l s o analyzed. M e l t i n g p o i n t s of a l l samples were determined, except f o r a c e t y l a t e d i s o f l o r i d o s i d e which was not obtained i n s o l i d form; a c e t y l a t e d f l o r i d o s i d e has a value of 101°C (Putman and Hassid 1954) . 6. Amino a c i d a n a l y s i s . Non-radioactive c a t i o n i c f r a c t i o n s were obtained from f r e s h p l a n t m a t e r i a l stored i n the 10°C growth chamber. The e t h a n o l - s o l u b l e f r e e amino acids were analyzed by the method of Spackman, S t e i n and Moore (1958) using a Beckman Amino A c i d Analyzer Model 120C. Basic amino acid s were separated on a 15.5 x 0.9 cm column. .The a c i d i c and n e u t r a l amino acids were separated on a 58 x 0.9 cm column (Cameron 1972). The amino sugars glucosamine and galactosamine were re s o l v e d from each other and from other amino acids i n both of these systems. 7. Computer analyses. Data from the photosynthesis and t r a n s l o c a t i o n experiments were analyzed w i t h the a i d of two computer pro-grams used at the I n s t i t u t e of Animal Resource Ecology Data Centre, The U n i v e r s i t y of B r i t i s h Columbia. Program SASTT was used to c a l c u l a t e the 25 means, variances and standard d e v i a t i o n s of two sets of numbers, and to t e s t f o r s i g n i f i c a n t d i f f e r e n c e s (a=0.05) between the two s e t s u s i n g a T-test. This program was used to analyze the photosynthesis data. Program ANVAR was used to analyze the data from the t r a n s l o c a t i o n e x p e r i -ments. The program performed a one-way a n a l y s i s of v a r i a n c e (a=0.05) and an o p t i o n was used to perform a Schefe's Test f o r m u l t i p l e comparison among the l e v e l s of the one-way a n a l y s i s of variance. FREE SUGARS AND AMINO ACIDS Fresh p l a n t m a t e r i a l of J^. g a r d n e r i and L. s p e c t a b i l i s c o l l e c t e d at Botany Beach (16 A p r i l and 14 May 1976), and L_. s p e c t a b i l i s from Cable Beach (13 May 1976) was sorted according to d e t e c t a b l e l i f e h i s t o r y stages (male or female gametophyte, or tetrasporophyte). The p l a n t s were e x t r a c t -ed f o r 30 min i n b o i l i n g 80% ethanol. The e x t r a c t s of e t h a n o l - s o l u b l e f r e e sugars and amino acid s were prepared and analyzed by GLC and amino a c i d analyzer as described e a r l i e r , except that the e x t r a c t s were not f r a c t i o n a t e d by i o n exchange chromatography. CULTURE OF SPORES M a t e r i a l c o l l e c t e d at the V i c t o r i a breakwater C28 June and 12 J u l y 1976) and at Botany Beach (11 J u l y 1976) was used. J_. gardneri p l a n t s and p o r t i o n s of L_. s p e c t a b i l i s , having e i t h e r carposporangia or t e t r a -sporangia, were cleaned and placed s e p a r a t e l y on g l a s s cover s l i p s i n p e t r i p l a t e s (60 x 15 mm) c o n t a i n i n g 10 ml of c u l t u r e medium [1 1. membrane-filtered seawater, 20 ml enrichment s o l u t i o n (ES from Table 2-5, 26 McLachlan 1973), 5 ml germanium d i o x i d e (250 mg/1. d i s t i l l e d w a t e r ) ] . The p l a n t s were maintained i n a growth chamber (10°C) w i t h a 12 hr photo-3 2 pe r i o d ; the l i g h t was 3.4 x 10 ergs/cm /sec. Spores were u s u a l l y r e l e a s e d w i t h i n 24 h r , and the source p l a n t m a t e r i a l was removed. Medium was changed every two to three days, and some spores were t r a n s f e r r e d on gla s s cover s l i p s or by c a p i l l a r y p i p e t t e s to other p e t r i p l a t e s . C u l t u r e c o n d i t i o n s were a l s o v a r i e d i n an attempt to improve growth by a g i t a t i n g the c u l t u r e s on a shaker t a b l e , or adding pieces of _L. s p e c t a b i l i s to c u l t u r e s of J_. gardneri spores, or both. 27 OBSERVATIONS AND RESULTS HOST LIFE HISTORY STAGES SELECTED BY SYMBIONT Observations on fresh, preserved, and herbarium materials revealed that there was no p r e f e r e n t i a l s e l e c t i o n of host l i f e h i s t o r y stages by the symbiont. Absence of p r e f e r e n t i a l s e l e c t i o n was confirmed during exam-in a t i o n of fourteen c o l l e c t i o n s , which consisted of over 500 plants of each species, from Botany Beach over a two year period (July 1974-76). The l i f e h i s t o r y stages of the symbiotic partners i n any given c o l l e c t i o n was apparently random; indeed, two or more l i f e h i s t o r y stages of the symbiont were commonly observed on one host plant. LIGHT MICROSCOPIC EXAMINATION OF THE ATTACHMENT REGION Examination of sectioned material showed that J_. gardneri had f i l a -ments which penetrated between c e l l s of host ti s s u e (Fig. 2), but there were no v i s i b l e p i t connections between the c e l l s of the two plants. The region i n which the tissues of the two plants intermingle was l i m i t e d . Neither the larger host c e l l s nor the smaller symbiont c e l l s were spread throughout the other organism (Fig. 2). Thus photosynthesis and trans-l o c a t i o n experiments could be undertaken with minimal r i s k of interference from "contaminating" c e l l s . PIGMENTATION AND CHLOROPLASTS Chloroplasts of J_. gardneri and L_. s p e c t a b i l i s were observed by l i g h t microscopy i n sections cut with a freezing microtome. Both whole plants . 2. Attachment region between J_. gardneri and L. s p e c t a b i l i s . The l i g h t micrographs are of unstained s e c t i o n s , cut w i t h a f r e e z i n g microtome, of fl;igardneri (JG) attached to L. s p e c t a b i l i s (LS) . Dotted l i n e s i n d i c a t e the boundaries of the attachment region between the two p l a n t s . Arrows i n both micrographs p o i n t to slender f i l a m e n t s of J_. g a r d n e r i c e l l s p e n e t r a t i n g between the l a r g e host c e l l s . (A) Attached J_. gardneri p l a n t i s male, spermatangial branches v i s i b l e i n upper right-hand corner. (B) Attached J_. gardneri p l a n t i s v e g e t a t i v e . - " 29 30 ( F r o n t i s p i e c e ) and s e c t i o n s of J . gardneri had a red d i s h hue f a i n t e r than the red c o l o r of L_. s p e c t a b i l i s p l a n t s and sectioned m a t e r i a l . The carpo-spores and te t r a s p o r e s of both p l a n t s were pigmented, but c h l o r o p l a s t s were not d i s c e r n i b l e by l i g h t microscopy due to other spore contents. E l e c t r o n microscopic i n v e s t i g a t i o n confirmed that J_. g a r d n e r i had c h l o r o p l a s t s ( F i g . 3). Some c h l o r o p l a s t s had t h y l a k o i d s arranged stack-l i k e , w i t h an outer e n c i r c l i n g t h y l a k o i d . Another common arrangement had only e n c i r c l i n g t h y l a k o i d s which appeared i n c r o s s - s e c t i o n as c o n c e n t r i c r i n g s . Observation of J_. gardneri carpospores and t e t r a s p o r e s before t h e i r r e l e a s e from parent p l a n t s ( F i g . 4) showed many p r o p l a s t i d s as w e l l as f l o r i d e a n starch, mitochondria, and dictyosomes present. PHOTOSYNTHESIS EXPERIMENTS A. Uptake of Radioactive Label. The r e s u l t s of photosynthesis e x p e r i -ment #1 are shown i n Table I I . S i m i l a r r e s u l t s were obtained i n e x p e r i -ments #2 and 3. Both J_. gardneri and L,. s p e c t a b i l i s took up the r a d i o -14 a c t i v e l a b e l from the seawater c o n t a i n i n g NaH 00^. This uptake by J_. gardneri occurred whether or not i t was attached to the host. P l a n t s kept i n the dark took up l i t t l e of the l a b e l and from t h i s I conclude that the net i n c o r p o r a t i o n by both p l a n t s during the l i g h t treatment i s a l i g h t -dependent phenomena. The q u a n t i t y of l a b e l taken up by each a l g a (whether separate or attached to one another) d i d not i n most instances d i f f e r s i g n i f i c a n t l y a t the 5% l e v e l i n the three experiments. One exception was i n experiment #1 (Table I I ) , when the i n c o r p o r a t i o n by "separate" (non-attached) L. s p e c t a b i l i s i n the l i g h t f o r 30 sec d i f f e r e d s i g n i f i c a n t l y from the uptake of "attached" p l a n t s . The other occurrence was i n e x p e r i -31 F i g . 3. C h l o r o p l a s t s from a v e g e t a t i v e c e l l of J_. g a r d n e r i . The arrow p o i n t s to a region where two c h l o r o p l a s t s (p) are d i v i d i n g , a l s o present are s e v e r a l mitochondria (m) and a nucleus ( n ) . (Plant m a t e r i a l was f i x e d f o r e l e c t r o n microscopy using method b_ i n the M a t e r i a l s and Methods.). 33 F i g . 4. E l e c t r o n micrographs of J_. gardneri tetraspores (A) and carpospores (B, C) before r e l e a s e from the parent p l a n t s . P l a n t m a t e r i a l was f i x e d using method a i n the M a t e r i a l s and Methods. (A) Two te t r a s p o r e s are present and are d i v i d e d by a t h i c k c e l l w a l l ' • (cw). Both tetraspores c o n t a i n numerous p r o p l a s t i d s (p) which c o n t a i n a s i n g l e e n c i r c l i n g t h y l a k o i d . Mitochondria (m) and f l o r i d e a n s t a r c h (s) are evident as are dictyosomes ( d ) . (B) This carpospore has numerous p r o p l a s t i d s (p) present p e r i p h e r a l l y and c e n t r a l l y as w e l l as f l o r i d e a n s t a r c h ( s ) . (C) D i v i d i n g p r o p l a s t i d s (p) are i n d i c a t e d by the arrow i n t h i s s e c t i o n from a carpospore. A l s o present are f l o r i d e a n s t a r c h ( s ) , mitochon-d r i a (m), and a dictyosome (d) and ass o c i a t e d v e s i c l e s . 34 35 TABLE I I UPTAKE OF 1 4C-LABEL BY L. SPECTABILIS AND J . GARDNERI FROM SEAWATER CONTAINING 14 NaH C0 3 J_. g a r d n e r i and pieces of L_. s p e c t a b i l i s w i t h and without attached J_. gardneri 14 were incubated i n seawater c o n t a i n i n g NaH CO^ f o r the times shown under the con d i t i o n s of l i g h t or dark. The k i l l e d m a t e r i a l was prepared f o r s c i n t i l l a -t i o n counting and the " ^ C - l a b e l uptake was measured (see M a t e r i a l s and Methods). The r e s u l t s , c o r r e c t e d f o r background, are f o r a s i n g l e experiment (#1) and are averages of three samples from each treatment + the standard e r r o r of the mean. S i g n i f i c a n t d i f f e r e n c e s at the 5% l e v e l were e s t a b l i s h e d u s i n g a T-test (SASTT computer program). SEPARATE DURING UPTAKE ATTACHED DURING UPTAKE L_. s p e c t a b i l i s J_. gardneri L_. s p e c t a b i l i s J_. g a r d n e r i LIGHT (dpm/mg wet wt) (dpm/mg wet wt) (dpm/mg wet wt) (dpm/mg wet wt) 10 sec 162 + 5 5 64 + 17 160 + 18 110 + 29 30 sec *195 + 3 126 + 14 *174 + 6 1 1 2 + 2 5 min 1490 + 122 484 + 16 1094 + 214 464 + 183 30 min 3822 +503 1584 + 403 3057 + 232 1123 + 80 DARK 30 sec 15 + 3 6 + 2 16 + 2 8 + 2 30 min, 6 0 + 13 111+ 21 8 4 + 9 106+ 25 * — S i g n i f i c a n t l y d i f f e r e n t from one another at the 5% l e v e l . 36 merit #3, when the means of separate and attached C-uptake (62 and 156 dpm/mg wet wt, respectively) by J_. gardneri i n the l i g h t for 30 sec were s i g n i f i c a n t l y d i f f e r e n t . B. Incorporation of Radioactive Label. A l l f r a c t i o n s prepared from the two algae i n photosynthesis experiments #1 and 2 were rad i o a c t i v e (Table I I I ) . The quantities incorporated varied between experiments and thus the r e s u l t s serve only to show which groups of compounds became labeled. Most of the l a b e l was present i n the neutral and c a t i o n i c f r a c -tions with very l i t t l e l a b e l i n the l i p i d f r a c t i o n s . No attempt was made to e s t a b l i s h the cause of the quantitative differences between the r e s u l t s of these two experiments. V a r i a t i o n i n season, l i g h t i n t e n s i t i e s , extrac-t i o n procedures, or severe color quenching during s c i n t i l l a t i o n counting seemed most l i k e l y causes. Figure 5 shows the radioautograms of the neutral, c a t i o n i c , and anionic f r a c t i o n s of L_. s p e c t a b i l i s and J_. gardneri a f t e r separation 14 by paper chromatography. The areas of C - a c t i v i t y for the neutral f r a c -tions coincided with areas on the paper chromatograms where sugars were detected. Results from s c i n t i l l a t i o n counting of paper s t r i p s cut from chromatograms of the neutral f r a c t i o n s of L. s p e c t a b i l i s (Fig. 5 A,B) and 14 J_. gardneri (Fig. 5 C,D) indicated that 67-70% of the C - a c t i v i t y was located i n areas corresponding to standards of galactose, glucose, f l o r i d -oside, i s o f l o r i d o s i d e , and mannitol (Table IV), but the a c t i v i t y could not be s p e c i f i c a l l y associated with i n d i v i d u a l sugars due to lack of res o l u -t i o n . The solvent ji chromatograms of the anionic f r a c t i o n s from both algae showed f a i n t "sugar-positive" areas corresponding to galacturoriic TABLE I I I DISTRIBUTION OF ASSIMILATED 1 4C-LABEL IN EXTRACTS AND RESIDUES FROM L. SPECTABILIS- AND. J . GARDNERI 14 14 The algae were exposed to the C- l a b e l i n seawater c o n t a i n i n g NaH CO^ f o r 30 min i n the l i g h t or dark. The m a t e r i a l was extr a c t e d i n ethanol and f r a c t i o n s were prepared (see M a t e r i a l s and Methods). The r e s u l t s , corrected f o r background, are from photosynthesis experiments #1 and 2. L I G H T * D A R K L. s p e c t a b i l i s J . gardneri L. s p e c t a b i l i s J_. gardneri N e u t r a l f r a c t i o n Exp. #1 Exp. #2 31.8% 17.8% Exp. #1 Exp. #2 36.2% 32.1% Exp. #1 Exp. #2 11.6% 7V.9% Exp. #1 Exp. #2 27.7% 3.8% C a t i o n i c f r a c t i o n 31.8 64.5 36.8 52.9 43.8 87.1 59.4 91.0 An i o n i c f r a c t i o n 10.9 8.4 20.2 6.2 16.0 2.7 8.5 2.2 L i p i d f r a c t i o n 0.4 <0.1 0.3 1.2 1.9 <0.1 0.3 <0.1 Residue 25.1 8.9 6.4 7.7 26.6 2.3 4.1 3.0 * - L i g h t i n t e n s i t y : Exp. #1 - 2.6 x 10 5 / 2 / ergs/cm /sec , Exp. #2 - 2.8 x 10^ ergs/ cm /sec ** - P l a n t s from Botany Beach: Exp. #1 - 18 August 1974, Exp. #2 - 24 February 1975 . 5. Radioautograms of """^C-labeled f r a c t i o n s of 1. s p e c t a b i l i s (A,B) and J_. gardneri (C,D) . 14 14 The p l a n t s were exposed to C - l a b e l i n seawater c o n t a i n i n g NaH CO^ f o r 30 min i n the l i g h t before e x t r a c t i o n w i t h ethanol and f r a c t i o n a t i o n on i o n exchange r e s i n s . N e u t r a l , . c a t i o n i c , and a n i o n i c f r a c t i o n s of the two algae were separated by one-dimensional paper chromatography using solvent a. f o r #5A and C, and s o l v e n t _b f o r #5B and D. (See M a t e r i a l s and Methods.),. The r e s u l t s are from photosynthesis experiment #1. S c i n t i l l a t i o n counting of paper s t r i p s cut from the chromatograms used f o r each radioautogram showed 67-70% of the " ^ C - a c t i v i t y to be l o c a t e d i n Zones A, B, and C corresponding to s e v e r a l ^ r e f e r e n c e sugars: mannitol, glucose, f l o r i d o s i d e , i s o f l o r i d o s i d e , and galactose (Table.IV). Zones A and B correspond to r a d i o a c t i v e spots #1 and 2, r e s p e c t i v e l y . RQ- u^ values f o r the r a d i o a c t i v e spots of, the radioautograms ar e : F i g . 5A — #1= 1.03, #2= 0.90, and #3= 0.14 B — #1= 1.17 and #2= 1.02 C — #1= 0.98, #2= 0.88, and #3= 0.13 D — #1= 1.22 and #2= 1.05 ' 40 TABLE IV R VALUES FOR SOME STANDARD SUGARS SEPARATED BY ONE DIMENSIONAL DESCENDING GLiU PAPER CHROMATOGRAPHY. Sample's'were a p p l i e d to Whatman No. 1 chromatography paper and developed i n e i t h e r solvent a,: e t h y l acetate - p y r i d i n e - ^ 0 (10r:4:3 v / v ) , or solvent b: e t h y l acetate - a c e t i c a c i d - formic a c i d - ^ 0 (18:3:1:4 v / v ) . The l o c a t i o n of the sugars a f t e r chromatography was determined by an a l k a l i n e s i l v e r n i t r a t e procedure (see M a t e r i a l s and Methods). The r e s u l t s are the average of values determined from a t l e a s t two d i f f e r e n t chromatograms. RGLU VALUES Solvent a Solvent b Mannose 1.12 1.15 Mannitol 1.03 1.25 Glucose 1.00 1.00 F l o r i d o s i d e 0.92 1.20 Galactose 0.85 0.94 I s o f l o r i s o s i d e 0.82 0.93 Sucrose r, 0.77 0.36 Trehalose 0.53 0.38 Glucuronic a c i d 0.16 1.01 Gala c t u r o n i c a c i d 0.14 0.92 41 a c i d and g l u c u r o n i c a c i d ; both were substances (uronic acids) u n l i k e l y to adsorb to the anion exchange r e s i n . In both algae, the r a d i o a c t i v e spot #3 ( F i g . 5 A,C) was detected a f t e r development i n solvent a_, but was absent i n the solvent b_ radioautograms ( F i g . 5 B,D). This spot d i d not 14 correspond to any of the standard sugars examined. C - a c t i v i t y i n chromatograms of the c a t i o n i c f r a c t i o n s of both algae d i d not correspond to any of the standard sugars, nor were any " s u g a r - p o s i t i v e " areas detected f o r these f r a c t i o n s a f t e r chromatography. The n e u t r a l f r a c t i o n s were f u r t h e r f r a c t i o n a t e d by adsorption onto a charcoal column followed by e l u t i o n w i t h a step-wise ethanol gradient. When these " f r a c t i o n a t e d - n e u t r a l " f r a c t i o n s from both algae were f u r t h e r examined by paper chromatography and radioautography, the l a b e l e d sugars i n each f r a c t i o n were resolv e d more s a t i s f a c t o r i l y w i t h solvent a_ ( F i g . 6), than w i t h solvent b_. However, the radioautograms revealed that the a d d i t i o n a l f r a c t i o n a t i o n procedure d i d not provide complete separation of l a b e l e d compounds. The major l a b e l e d compounds shown i n F i g . 6 appeared to be the same (galactose, glucose, f l o r i d o s i d e , i s o f l o r i d o s i d e , and mannitol) as shown i n F i g . 5. Since the sugar standards were not w e l l r e s o l v e d by paper chromatography (Table IV) i n the s o l v e n t s used, GLC was used f o r f u r t h e r separation and a n a l y s i s of n o n - r a d i o a c t i v e e x t r a c t s from both algae. Separation of reference sugars by GLC i s shown i n F i g . 7. Mannitol was not detected i n e i t h e r alga by GLC analyses ( F i g . 8). Galactose and glucose were present i n t r a c e amounts. The major GLC peak (#6) appeared to be e i t h e r f l o r i d o s i d e , i s o f l o r i d o s i d e , or both. Co-chromatography of auth e n t i c f l o r i d o s i d e or i s o f l o r i d o s i d e w i t h samples from both algae a l s o . 6. Radioautograms of 1 4 C - l a b e l e d " f r a c t i o n a t e d - n e u t r a l " f r a c t i o n s of L. s p e c t a b i l i s (A) and J . gardneri (B). 14 14 The p l a n t s were exposed to C - l a b e l i n seawater c o n t a i n i n g NaH CO^ f o r 30 min i n the l i g h t before e x t r a c t i o n w i t h e t h a n o l , f r a c t i o n a t i o n on i o n exchange r e s i n s , and f u r t h e r f r a c t i o n a t i o n of the n e u t r a l f r a c t i o n by a d s o r p t i o n on a c h a r c o a l column followed by e l u t i o n w i t h H^O and then 14 a step-wise ethanol gradient (5, 10, 20, and 35%). The samples pl u s C-l a b e l e d glucose were separated by one-dimensional paper chromatography using solvent a. or solvent b_. The r e s u l t s showiiiareaafter^chromatography i n s o l v e n t system a_ of p l a n t m a t e r i a l from photosynthesis experiment #2. The r a d i o a c t i v e spots appear to correspond (corroborated using solvent system b) to the reference sugars mannitol, glucose, f l o r i d o s i d e , i s o f l o r -i d o s i d e , and galactose (Table I V ) , but l a c k of r e s o l u t i o n d i d not permit a s s o c i a t i o n of each r a d i o a c t i v e spot w i t h an i n d i v i d u a l sugar. The values and probable corresponding sugars f o r the r a d i o a c t i v e spots a r e : (A) L. s p e c t a b i l i s — #1= 0.98 (glucose/mannitol), #2= 0.84 ( i s o f l o r -i d p s i d e / g a l a c t o s e ) , #3= 0.89 ( f l o r i d o s i d e ) , and #4= 0.90 ( f l o r -i d o s i d e ) . (B) J . gardneri — #1= 0.97 ( f l o r i d o s i d e / g l u c o s e / m a n n i t o l ) , #2= 0.81 ( i s o f l o r i d o s i d e / g a l a c t o s e ) , #3= 1.00 (glucose/mannitol), and #4=.0.89 ( f l o r i d o s i d e ) . 44 F i g . 7. G a s - l i q u i d chromatogram of a l d i t o l acetate d e r i v a t i v e s of some standard sugars. The sample of sugars was reduced and, a f t e r a d d i t i o n of the i n t e r n a l standard m y o - i n o s i t o l , a c e t y l a t e d w i t h a c e t i c anhydride (see M a t e r i a l s and Methods). The sample i n e t h y l acetate was i n j e c t e d i n t o a Hewlett-Packard Model 5710A gas chromatograph w i t h 5% S i l a r IOC columns and temperature programmed from 120°-260°C at 4°/min. 45 FIGURE 7 STANDARD SUGARS LU CO o o_ CO LU o (— u LU I-UJ Q i f : r.V. "| I'll 11T- nn r nrrmr TTTTT iTT^Tp-rT' • "V. i r r r r T H T t t TfT ITT! : : : : ; i j i '.; i; iii! III! ilin j •|! k e y • . : : lit! I'll m ii'i • ' ' i l l I;:: • !ir. : iiiiii ..-. •; : - : ' -1: ; • i' : • t ii i' li!! j !;!!!! • : : !!!! pi! TH- -l i : ; : .- J .1.: 1 '. • 1 i ! ; .i: 1 r • !:h i i i: i I I - i: i". • !!!! Trrr \-\\ • =!!h . • 1 J •; i ' r ; I i .. i • 1 " • 11 . ;i. • ! ! Ij: • -i •t ' •Al ill: ! • . t t • ; ; . ! I I I i * • .iii -i -: '"•}'• c lit' : It- '. ' 1' • ! i !-| !• ; : ; , | : •f! • \ r i ! j ! " ' ! : ; . ! • • i ' i i i l l : ^ •r 1 • — - • L„„ ,. , _ " i i : ; , . : ! • 2)1 7" - i l l i 1" . - ! — • ( . . . i *. i: . ;_iLi. - — " " , i • 1, ii;i j : : ! - - ' -;- "i :. i L ._. •i jl ! • 1 1 ; i l . i --- --• — . __ __h_ • ! I. : I* : ; V -; -• •• i 1 : v i i i -' © i • ! • • — ii : : l . ' ! . . _ _ "• - -it : 1 • : ! : ;ir. :: i • ; t; ' • ' j ~ — -! : : . •.Ij::;. 1 ' , 1 ' , ; ; ! - : — ~— • i-' ' • 1 . : : : : . |:-:ir; !:l! '•pi " :! ; i.'v •JL. - J . !il:|:i!i4;i;:iiii!|^|rr:i !iii|ii|!i!!ii ' II i:<" •I!-0 10 20 T I M E 30 (MIN) 40 50 Standard Sugars Corresponding To The Peaks Are: 1. g l y c e r o l 5. galactose 2. fucose 6. glucose 3. xylose 7. myo-inositol 4. mannose 8. f l o r i d o s i d e / i s o f l o r i d o s i d e , which are not resolved . 8. G a s - l i q u i d chromatograms of a l d i t o l acetate d e r i v a t i v e s of f r e e sugars from J_. gardneri (A) and L. s p e c t a b i l i s (B). P l a n t s were e x t r a c t e d w i t h ethanol and a p o r t i o n of the e x t r a c t was -d r i e d , reduced and, a f t e r a d d i t i o n of the i n t e r n a l standard m y o - i n o s i t o l , a c e t y l a t e d w i t h a c e t i c anhydride (see M a t e r i a l s and Methods). The samples i n e t h y l acetate were i n j e c t e d i n t o a Var i a n Aerograph Model 1740 gas chromatograph w i t h 5% S i l a r 10C columns and temperature pro-grammed from 120°-260°C at 4°/min. FIGURE 8 47 A J , GARDNERI - T E T R A S P O R I C FROM BOTANY BEACH ^-===^-•-»: :^t= i T i i r T r T ' . iMt i i t t i tmi iMtt t i t -| ipMi[ i t i '| i t i i| : i i i| ; i i : iM! i i i int f nirTiMU!tMt;iit}'H}iii UJ •z. o D_ CO Ui DC OC O H O U l Peaks I d e n t i f i e d 1. unknown #1 2. unknown #2 3. g a l a c t o s e 4. glucose 5. m y o - i n o s i t o l 6. f l o r i d o s i d e / i s o f l o r i d o s i d e MAY 1 9 7 6 10 20 30 T I M E ( M I N ) 40 B L . S P E C T A B I L I S - FEMALE FROM BOTANY B E A C H , MAY 1 9 7 6 . i •. 11 > •: • n 1 • 11 IM 1111111' 1111II • I) • • 11' •M|i':-I -"'I' . 30 ( M I N ) 48 indicated that e i t h e r sugar, or both, may be present. The r e s u l t s (Fig. 9) of analysis of neutral f r a c t i o n s a f t e r hydrolysis supported the hypothesis that the substance(s) was one or more g a l a c t o s y l - g l y c e r o l compounds. There was a decrease i n peak #6, an increase i n the galactose peak, and the appearance of a g l y c e r o l peak. Mass spectrometry was used to examine f l o r i d o s i d e , i s o f l o r i d o -side, and the unknown compound(s) composing GLC peak #6 (Figs. 8, 9). The mass spectra of the two sugar standards showed that high mass peaks for the compounds were at m/e 446 f o r f l o r i d o s i d e , and m/e 433 and 446 f o r i s o f l o r i d o s i d e , with the peak at m/e 433 being d i a g n o s t i c a l l y s i g n i f i c a n t (Fig. 10; see Fig. 11 for fragmentation scheme). Mass spectrometry analysis (Fig. 12) of the unknown substance(s) of peak #6 from both L_. s p e c t a b i l i s and J_. gardneri showed peaks at m/e 433 and 446, with the former being diagnostic f or i s o f l o r i d o s i d e . However, a comparison of the r a t i o of peak i n t e n s i t i e s at m/e_ 433 and 446 i n the known (Fig. 10) and unknown (Fig. 12) spectra revealed an enhancement of the peak at m/e 446 i n the unknown spectra and i l l u s t r a t e d the probable presence of f l o r i d o s i d e i n the samples analyzed. I conclude that both compounds were i n fact present i n both algae. The melting point of the c r y s t a l l i n e samples before mass spectrom-etry was between 98° and 100°C, which i s i n agreement with the l i t e r a t u r e value of 101°C for acetylated f l o r i d o s i d e (Putman and Hassid 1954), whereas the acetylated i s o f l o r i d o s i d e made i n t h i s study was a syrupy, non-crystal-l i n e substance. This further supports the conclusion that the unknown material was a mixture of f l o r i d o s i d e and i s o f l o r i d o s i d e . 49 F i g . 9. G a s - l i q u i d chromatograms of a l d i t o l acetate d e r i v a t i v e s of hydrolyzed and reduced f r e e sugars of J . gardneri (A) and L. s p e c t a b i l i s (B). Drie d e t h a n o l i c e x t r a c t s c o n t a i n i n g s o l u b l e f r e e sugars from each a l g a were hydrolyzed, reduced and, a f t e r the a d d i t i o n of the i n t e r n a l standard m y o - i n o s i t o l , a c e t y l a t e d w i t h a c e t i c anhydride (see M a t e r i a l s and Methods). The samples i n e t h y l acetate were i n j e c t e d i n a Hewlett-Packard Model 5710A gas chromatograph w i t h 5% S i l a r IOC columns. Column temperature was h e l d f o r 8 min at 120°C and then progammed from 120°-260°C at 4°/min. FIGURE 9 50 . J . G A R D N E R I B m I LU CO 2: o ci-ty} LU cc DC o u LU f -LU O 1 i n 30 ( M I N ) L. S P E C T A B I L I S m I'!; ~!KiiiH!:ili:::hi:jl!:li?!:lJI:!!ll!|: Peaks I d e n t i f i e d 1. g l y c e r o l 2. x y l o s e 3. g a l a c t o s e 4. glucose 5. rnyjo-inositol 6. f l o r i d o s i d e / ::l:::t ;:i::s: •::L;: . 1:: r: : i : : : • ;: i : i 30 ( M I N ) 51 F i g . 10. D i a g n o s t i c p o r t i o n s of the mass spec t r a of f l o r i d o s i d e acetate (A) and i s o f l o r i d o s i d e acetate (B). Pure f l o r i d o s i d e and i s o f l o r i d o s i d e (obtained from Dr. J . S. C r a i g i e ) was a c e t y l a t e d (see M a t e r i a l s and Methods) before mass spectrometry, which was performed i n a Varian/MAT CH4-B mass spectrometer us i n g the d i r e c t - p r o b e i n s e r t i o n technique. (A) P o r t i o n of the mass spectrum of reference f l o r i d o s i d e acetate showing the high mass peaks. The probe temperature was 100°C f o r t h i s a n a l y s i s . (B) P o r t i o n of the mass spectrum of reference i s o f l o r i d o s i d e acetate showing the high mass peaks, i n c l u d i n g i t s d i a g n o s t i c peak at m/e 433. The probe temperature was 120°C f o r t h i s a n a l y s i s . FIGURE 10 A F L O R I D O S I D E A C E T A T E i -i—t CO / m/e 446 It. .1 , ,1. ,i I. .1 . .1, _J JL 0 330 370 410 m/e 450 490 B I S O F L O R I D O S I D E A C E T A T E GO LU IK m/e 433, \ i i " — r JLi ,^m/e 446 I T " ,1, 0 330 370 410 m/e 450 490 53 F i g . 11. T h e o r e t i c a l fragmentation schemes f o r f l o r i d o s i d e acetate (A) and i s o f l o r i d o s i d e acetate (B) during mass spectrometry. (A) F l o r i d o s i d e a c etate. The e l i m i n a t i o n of a c e t i c a c i d (60 mass u n i t s ) from the parent molecule (MW 506) provides the high mass peak at m/e_ 446. Cleavage between C^ and C2, or C^ and C^ of the g l y c e r o l moiety i s u n l i k e l y . (B) I s o f l o r i d o s i d e acetate. In a d d i t i o n to the hig h mass peak at m/e 446 due to the e l i m i n a t i o n of a c e t i c a c i d (60 mass u n i t s ) from the parent molecule, cleavage occurs2m6feyreadily between C2 and C^ of the g l y c e r o l moiety which e l i m i n a t e s CI^OAc (73 mass u n i t s ) from the parent molecule (MW 506) to produce the high mass peak at m/e 433. The peak at m/e 433 i s d i a g n o s t i c a l l y s i g n i f i c a n t f o r i s o f l o r i d o s i d e acetate. 54 FIGURE 11 A. AcO CHxOAc OAc H 0—-CH -AcOH OAc (60 mass units) m/e 446 o If ^c= -C-CH, FLORIDOSIDE ACETATE (MW 506) B. AzO -AcOH (60 mass units) m/e 446 OAc ®Ctf OAc o u CHx0Ac Ac" -C-CH3 XHxOA< (73 mass units) ^ m/e 433 ISOFLORIDOSIDE ACETATE (MW 506) 12. D i a g n o s t i c p o r t i o n s of mass spec t r a of suspected f l o r i d o s i d e / i s o -f l o r i d o s i d e GLC peaks from L_. s p e c t a b i l i s (A) and J_. gardneri (B) . Samples from e t h a n o l i c e x t r a c t s of L_. s p e c t a b i l i s and J_. gardneri were d e r i v a t i z e d as a l d i t o l acetates followed by GLC usi n g a p r e p a r a t i v e column of 5% S i l a r IOC (see M a t e r i a l s and Methods). Samples of sus-pected f l o r i d o s i d e / i s o f l o r i d o s i d e peaks were c o l l e c t e d i n g l a s s c a p i l l a r y tubes f o r mass spectrometry, which was performed i n a Varian/MAT CH4-B mass spectrometer using the d i r e c t - p r o b e i n s e r t i o n technique. (A) Spectrum of the unknown GLC peak from L. s p e c t a b i l i s showing the peak at m/e 433 considered d i a g n o s t i c f o r i s o f l o r i d o s i d e acetate. Note the d i f f e r e n c e i n the r a t i o of peak i n t e n s i t i e s of m/e 433 to 446 f o r t h i s spectrum compared w i t h the reference spectrum ( F i g . 10B). The probe temperature was 100°C f o r t h i s a n a l y s i s . (B) Spectrum of the unknown GLC peak from J_. gardneri showing the peak at m/e 433 considered d i a g n o s t i c f o r i s o f l o r i d o s i d e a c etate. Note the d i f f e r e n c e i n the r a t i o of peak i n t e n s i t i e s of m/e 433 to 446 f o r t h i s spectrum compared w i t h the reference spectrum ( F i g . 10B). The probe temperature was 120°C f o r t h i s a n a l y s i s . 56 FIGURE 12 A CO 2: UJ L. S P E C T A B I L I S SAMPLE 0 330 m/e 446 m/e 433 / i—r i — r \ 1 U i 370 410 ni/e ' ' I I ' » 1 450 490 r z3 B J . GARDNERI SAMPLE CO •z. LU m/e 446 V m/e 433 It I 1- 1 \ 1 " 1 " f 1 1 ' ' » ' * 1 1 1 ' ' T r 0 330 370 410 m/e 450 490 57 The conclusions from the paper chromatography, radioautography, g a s - l i q u i d chromatography, and mass spectrometry r e s u l t s are that the major l a b e l e d sugars of both algae were galactose, glucose, i s o f l o r i d o s i d e , and f l o r i d o s i d e . The r e s u l t s a f t e r the t h i n - l a y e r chromatography of the organic a c i d f r a c t i o n s ( a n i o n i c f r a c t i o n s ) of both algae were ambiguous because of the low l e v e l of dete c t a b l e r a d i o a c t i v i t y and the low conc e n t r a t i o n of organic a c i d s . There were f a i n t " a c i d - p o s i t i v e " spots which had m o b i l i t i e s corresponding to c i t r a t e , malate, and g l y c o l a t e . These organic a c i d s have been reported from other red algae (Bean and Hassid 1955; Kremer and Vogl 1975). Figure 13 i s a radioautogram made of the c a t i o n i c f r a c t i o n s of J_. g a r d n e r i and L. s p e c t a b i l i s a f t e r separation by high v o l t a g e paper 14 e l e c t r o p h o r e s i s . The unknown C-labeled amino a c i d s had m o b i l i t i e s cor-responding to those of a l a n i n e , a s p a r t i c a c i d , glutamic a c i d , g l y c i n e , and s e r i n e . The presence of these f i v e amino a c i d s i n both algae was confirmed by analyses of non - r a d i o a c t i v e c a t i o n i c f r a c t i o n s from the algae using an amino a c i d analyzer. TRANSLOCATION EXPERIMENTS The r e s u l t s of the three t r a n s l o c a t i o n experiments are given i n Table V. There was no s i g n i f i c a n t (a=0.05) net movement of the " ^ C - l a b e l i n e i t h e r d i r e c t i o n between the attached algae during the t r a n s l o c a t i o n p e r i o d f o r each experiment. This c o n c l u s i o n holds r e g a r d l e s s of whether the pre-treatment c o n d i t i o n before l a b e l i n g was l i g h t or dark, whether the algae 13. Radioautogram of the c a t i o n i c f r a c t i o n s of J_. gardneri and L. s p e c t a b i l i s a f t e r s e p a r a t i o n u s i n g paper e l e c t r o p h o r e s i s . 14 14 P l a n t s were exposed to the C - l a b e l i n seawater c o n t a i n i n g NaH CO^ f o r 30 min i n the l i g h t (1) or dark (d) before e x t r a c t i o n w i t h ethanol and f r a c t i o n a t i o n u s i n g i o n exchange r e s i n s . The amino acid s i n the c a t i o n i c f r a c t i o n s were re s o l v e d u s i n g h i g h v o l t a g e paper e l e c t r o -phoresis a t £a. 52.6 V/cm of paper length f o r 40 min at pH 2.0 (see M a t e r i a l s and Methods). This radioautogram was from photosynthesis experiment #2. The amino a c i d s detected i n f r a c t i o n s from p l a n t s l a b e l e d i n the l i g h t were g l y c i n e ( g l y ) , a l a n i n e ( a l a ) , s e r i n e ( s e r ) , glutamic a c i d ( g l u ) , and a s p a r t i c a c i d (asp). A s p a r t i c a c i d was the major l a b e l e d amino a c i d finm p l a n t s exposed to the l a b e l i n the dark. 59 TABLE V RESULTS OF 14C-PULSE-CHASE TRANSLOCATION EXPERIMENTS WITH L. SPECTABILIS AND J . GARDNERI 14 Pieces of L. sp e c t a b i l i s with attached J . gardneri were Incubated i n seawater containing NaH CO^, then trans-ferred to non-radioactive "chase" seawater for various translocation periods. A summary of conditions for the experiments i s presented i n Table I. The algae were k i l l e d at the end of each of the translocation periods and were prepared separately for s c i n t i l l a t i o n counting (sea Materials and Methods). These values, corrected for background are an average of 3 samples (except for the 0 hr- values f o r experiment #3 which are based on 6 samples) ± SEM. An analysis of variance, performed by the ANVAR computer program (see Materials and Methods), determined that the values for each alga during the translocation periods for each experiment did not d i f f e r s i g n i f i c a n t l y (a= 0.05). TRANSLOCATION PERIOD EXPERIMENT 0 hr > hr 4 hr 8 hr 12 hr dpm/mg wet wt dpm/mg wet wt dpm/mg wet wt dpm/mg wet wt dpm/mg wet wt #1 J . gardneri 1428 ± 225 1855 + 513 2221 + 158 2348 + 258 L. sp e c t a b i l i s 4393 ± 1658 5460 + 1426 4786 + 753 6930 + 1941 y/2a J . gardneri 2826 + 465 2418 + 444 2178 + 202 2026 + 255 2721 ± 526 L. spectabilis 5259 ± 819 5281 + 864 5714 + 1087 6457 + 479 6585 ± 393 #2b J. gardneri 5094 + 416 6102 + 1835 5441 + 714 4210 ± 367 L. sp e c t a b i l i s 11739 + 2082 12264 + 812 13181 + 1523 13280 + 1501 #3a J_. gardneri 1470 ± 165 1658 + 166 1738 + 156 1813 + 105 1786 ± 79 L. sp e c t a b i l i s 4019 ± 438 6444 + 993 5838 + 666 5181 + 135 5268 ± 173 #3b J . gardneri 1470 ± 165 1651 + 233 1424 + 54 1424 + 215 1406 ± 72 L. s p e c t a b i l i s 4019 ± 438 4815 + 721 4609 + 46 4317 + 83 3127 ± 382 O 61 took up the l a b e l f or 30 or 60 min, or whether the algae were allowed to translocate for a short (2 hr) or long (12 hr) period. Figure 14 shows that the amount of l a b e l i n J_. gardneri a f t e r an i n i t i a l 30 min l a b e l i n g pulse remained steady during a l i g h t or dark trans-l o c a t i o n period. T^ . s p e c t a b i l i s material also remained generally steady a f t e r the i n i t i a l l a b e l i n g pulse.except for some change during the f i r s t 4 hours of the l i g h t t r a n s l o c a t i o n period and the l a s t 4 hours of the dark translocation period. However, Scheffe's Test for Multiple Comparisons indicates no s i g n i f i c a n t d i f f e r e n c e (a=0.10) between the various l i g h t or dark r e s u l t s . The data used to determine the values i n Table V were also expressed as a . r a t i o of J_. gardneri value/L. s p e c t a b i l i s value. These r a t i o s (Table VI) were also compared by the ANVAR computer program. I f the r a t i o of J_. gardneri/L. s p e c t a b i l i s tended toward 1.000, the l a b e l was assumed to be moving into J_. gardneri, while a s h i f t towards 0.000 indicates the l a b e l i s moving into JL. s p e c t a b i l i s . The r e s u l t s showed there was no tendency (a=0.10) for the r a t i o to s h i f t i n e i t h e r d i r e c t i o n during the three ex-14 periments. Thus, C-labeled photosynthetic products did not move between the algae under the experimental conditions used i n t h i s study. The t r a n s l o c a t i o n experiments also established the amount of l a b e l passing from the algae to the "chase" seawater during the t r a n s l o c a t i o n period. The r e s u l t s f or experiments #2 and 3 (Table VII) showed that the amount of l a b e l released to the seawater did not increase during the tr a n s l o c a t i o n period. Since the l a b e l was apparently not continuously released into the chase seawater and only accounted for about 5% or l e s s 62 F i g . 14. R a d i o a c t i v i t y of L. s p e c t a b i l i s and attached J_. gardneri a f t e r 14 30 min uptake of NaH C0^ followed by t r a n s l o c a t i o n p e r i o d s . P l a n t s p r e t r e a t e d w i t h l i g h t were exposed to the " ^ C - l a b e l i n seawater 14 c o n t a i n i n g NaH C0 3 f o r 30 min i n the l i g h t before r i n s i n g and p l a c i n g them i n t o the "chase" seawater (except the p l a n t s sampled at zero t r a n s l o c a t i o n t i m e ) . The p l a n t s were k i l l e d at the end of each t r a n s -l o c a t i o n p e r i o d and prepared f o r s c i n t i l l a t i o n counting (see M a t e r i a l s and Methods). The graph presents data from experiments #3a and 3b which had l i g h t and dark t r a n s l o c a t i o n p e r i o d s , r e s p e c t i v e l y . The means are an average of 3 samples, except the i n i t i a l readings which are based on 6 samples. Bars = SEM 63 FIGURE 14 DPM/MG WET WT 8000 7000 6000 5000 1000 3000 LIGHT DARK L> S P E C T A B I L I S ° Q * i ! . . GARDNERI O — o • 2000 1000 -4 ' • i . . . . i . • i . f 2 1 6 8 10 TRANSLOCATION PERIODS (HR) 64 TABLE VI RATIOS OF J . GARDNERI/L. SPECTABILIS 1 4C-PULSE-CHASE TRANSLOCATION VALUES 14 The C-pulse-chase t r a n s l o c a t i o n data used to c a l c u l a t e these r a t i o s were the same as those used to c a l c u l a t e the r e s u l t s reported i n Table V. Each r a t i o was obtained by d i v i d i n g a J_. gardneri value by i t s corresponding L. s p e c t a b i l i s value and i s the mean of 3 such r a t i o s f o r each treatment, except f o r the 0 hr r a t i o s of experiment #3 which are an average of 6 r a t i o s . An a n a l y s i s of v a r i a n c e , performed by the ANVAR computer program (see M a t e r i a l s and Methods), determined that the r a t i o s d u r ing the t r a n s l o c a t i o n p e r i o d f o r each experiment d i d not d i f f e r s i g n i f i c a n t l y (a= 0.10). RATIOS FOR TRANSLOCATION PERIOD (hr) EXPERIMENT 0 hr 2' h r 4 hr 8 hr 12 hr #1 ( L i g h t ) * 0.379 0.335 0.483 0.465 _ _ _ #2a ( L i g h t ) 0.544 0.454 0.421 0.311 0.424 #2b (Dark) _ _ _ 0.471 0.488 0.422 0.321 #3a (L i g h t ) 0.374 0.261 0.300 0.351 0.340 #3b (Dark) 0.374 0.345 0.309 0.330 0.457 * - Co n d i t i o n of l i g h t or dark during t r a n s l o c a t i o n p e r i o d . 65 TABLE V I I RADIOACTIVITY OF "CHASE" SEAWATER FROM THE 14C-PULSE-CHASE TRANSLOCATION EXPERIMENTS #2 AND 3 14 The amount of C- l a b e l released by the algae i n t o the "chase" seawater during each t r a n s l o c a t i o n p e r i o d of t r a n s l o c a t i o n experiments #2 and 3 was determined by s c i n t i l l a t i o n counting, c o r r e c t e d f o r background, and d i v i d e d by the wet wt of a l g a l m a t e r i a l present (see M a t e r i a l s and Methods). These r e s u l t s are an average of 2 samples taken from the chase seawater. TRANSLOCATION PERIOD EXPERIMENT 2 hr 4 hr 8 hr 12 hr dpm/mg wet wt dpm/mg wet wt dpm/mg wet wt dpm/mg wet wt #2a ( L i g h t ) * 61 (0.8%) 53 (0.7%) 55 (0.7%) 95 (1.0%) #2b (Dark) 328 (2.0 ) 363 (2.0 ) 291 (1.6 ) 368 (2.1 ) #3a (Light) 74 (1.0 ) 87 (1.2 ) 79 (1.1 ) 79 (1.1 ) #3b (Dark) 225 (3.5 ) 327 (5.4 ) 250 (4.4 ) 233 (5.1 ) * - Con d i t i o n of l i g h t or dark during t r a n s l o c a t i o n p e r i o d . * - Percentage of the t o t a l recovered l a b e l present i n chase-seawater. i 66 of the t o t a l C - a c t i v i t y recovered (based on the r e s u l t s of Table V and VII), the l a b e l probably got into the medium from the surface of the algae despite the c a r e f u l r i n s i n g procedure. FREE SUGARS AND AMINO ACIDS OF LIFE HISTORY STAGES The ethanol-soluble free sugar content of material from each l i f e h i s t o r y stage of both algae was determined a f t e r analysis by GLC. The same sugars (galactose, glucose, and f l o r i d o s i d e / i s o f l o r i d o s i d e , which co-chromatograph) and several unknowns were detected a f t e r analysis of extracts from L. s p e c t a b i l i s male and female (Fig. 8B) gametophyte, and tetrasporophyte stages from Botany Beach ( A p r i l , May 1976) and Cable Beach (May 1976). Male gametophyte and tetrasporophyte (Fig. 8A) stages (no female plants were available) of J_. gardneri from Botany Beach ( A p r i l , May 1976) also had these same sugars as well as several unknown substances. The ethanol-soluble free amino acids from each l i f e h i s t o r y stage of the two algae (except for female J_. gardneri) are shown i n Tables VIII and IX. The major free amino acids were detected i n approximately s i m i l a r amounts i n each l i f e h i s t o r y stage examined for each alga from both Botany Beach c o l l e c t i o n s . The same free amino acids were detected i n L_. spectab- i l i s c o l l e c t e d i n May at both Botany Beach and Cable Beach, with most amino acids found i n s i m i l a r quantities except alanine, a s p a r t i c acid, and glutamic acid. 67 TABLE V I I I COMPOSITION OF RECOVERED FREE AMINO ACIDS OF L. SPECTABILIS P l a n t s were c o l l e c t e d at Botany Beach (16 A p r i l , 14 May 1976) and at Cable Beach (13 May 1976). Before e x t r a c t i o n of et h a n o l - s o l u b l e f r e e amino acid s (see M a t e r i a l s and Methods), the p l a n t s were grouped according to l i f e h i s t o r y stages. The r e s u l t s are from one a n a l y s i s . Tr = t r a c e , l e s s than 0.1% of recovered amino a c i d s . TETRASPORIC MALE FEMALE Botany Beach Cable Beach Botany Beach Cable Beach Botany Beach Cable Beach A p r i l May May A p r i l May May A p r i l May May Asp 3.5% A. 8% 13.2% A. 6% 3.7% 23.5% 7.8% 7.A% 13.8% Thr 0.7 0.9 1.6 0.7 1.1 2.9 1.0 1.0 1.9 Ser 2.0 3.3 A.8 2.0 3.0 8.5 3.3 2.8 5.8 Glu 75.1 7A.9 61.A 73.0 76.2 A2.8 73.6 7A.5 59.0 Pro 1.1 1.2 3.0 1.0 1.1 2.A 1.6 1.2 1.7 Gly 0.6 0.7 0.6 0.7 0.6 0.6 0.9 0.8 O.A Ala 13.7 11.2 A.7 14.8 10.2 3.6 7.1 9.0 A.9 Val 0.7 0.5 3.1 0.7 1.0 3.6 1.1 0.8 3.0 Met 0.1 Tr 0.3 0.2 Tr O.A 0.2 Tr Tr Ile- 0.5 0.5 2.1 O.A 0.7 2.9 0.7 0.7 2.A Leu 0.5 0.6 2.8 0.5 0.8 A.l 0.9 0.7 3.2 Tyr 0.5 0.6 1.0 0.5 0.7 1.7 0.7 0.6 1.9 Phe 0.1 O.A 1.1 0.1 0.5 1.8 0.2 0.2 l.A Lys O.A 0.2 0.3 0.3 0.2 0.9 O.A 0.2 0.3 His 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.1 0.1 Arg 0.2 0.1 0.1 0.2 0.2 0.3 0.3 0.1 0.1 TABLE IX COMPOSITION OF RECOVERED FREE AMINO ACIDS OF J . GARDNERI P l a n t s were c o l l e c t e d at Botany Beach (16 A p r i l , 14 May 1976). Before e x t r a c t i o n of e t h a n o l - s o l u b l e f r e e amino a c i d s (see M a t e r i a l s and Methods), the p l a n t s were grouped according to l i f e h i s t o r y stages (no female p l a n t s were c o l l e c t e d ) . The r e s u l t s are from one a n a l y s i s . Tr = t r a c e , l e s s than 0.1% of recovered amino a c i d s . TETRASPORIC MALE Botany Beach Botany Beach A p r i l May A p r i l May Asp 4.4% 4.4% 4.0% 6.7% Thr 2.2 1.6 2.4 2.3 Ser 6.9 4.6 7.8 6.4 Glu 39.5 56.2 46.1 41.1 Pro 2.2 2.3 2.7 1.5 Gly 11.2 1.6 4.5 3.3 A l a 26.8 21.6 24.1 28.2 V a l 1.6 2.0 1.8 2.6 Met 0.3 0.5 0.1 Tr H e 1.6 1.4 1.6 2.1 Leu 1.9 1.6 2.2 2.4 Tyr 0.3 0.6 1.7 1.1 Phe 0.3 0.7 0.7 0.8 Lys 0.3 0.4 0.1 0.1 His 0.3 0.3 0.1 1.0 Arg 0.3 0.1 0.1 0.1 69 CULTURE. OF SPORES M a t e r i a l c o n t a i n i n g carposporangia and t e t r a s p o r a n g i a of both genera when placed i n p e t r i p l a t e s w i t h seawater medium r e a d i l y r eleased spores which adhered to cover s l i p s and bottoms of p e t r i p l a t e s and then became rounded ( F i g . 15). Germination of the red d i s h L_. s p e c t a b i l i s spores was i n c o n s i s t e n t as only t e t r a s p o r e s germinated from p l a n t s c o l l e c t e d at V i c t o r i a (12 J u l y 1976), whereas only carpospores germinated from the m a t e r i a l obtained at Botany Beach (11 J u l y 1976). The L. s p e c t a b i l i s spores which germinated produced a 2-4 c e l l c o l o r l e s s r h i z o i d (sometimes branched). The upper part of the spores would d i v i d e s e v e r a l times to form between 8-60 colo r e d c e l l s . The medium was changed every 2-4 days and the germlings appeared healthy (no l o s s of pigmentation or b a c t e r i a l contamination) f o r 1-2 weeks. The spores of J_. gardneri when released were p i n k i s h i n c o l o r . The only J^. g a r d n e r i spores to germinate were the carpospores ( F i g . 15B) from a Botany Beach c o l l e c t i o n (11 J u l y 1976). The J_. gardneri germlings ( F i g . 15B) d i d not s u r v i v e as long as those of L_. s p e c t a b i l i s . They d i d form a r h i z o i d of one or two c e l l s , but there was no d i v i s i o n of the upper, p i g -mented part of the spores. Most of the germlings l o s t t h e i r c o l o r i n l e s s than 1 week and b a c t e r i a became apparent d e s p i t e changes of medium, a d d i t i o n of pieces of _L. s p e c t a b i l i s , or both. This experience was s i m i l a r to that reported by Kugrens (1971), except that i n h i s study t e t r a s p o r e s , not carpospores, germinated. 70 F i g . 15. Carpospores of L_. s p e c t a b i l i s (A) and J_. gardneri (B) i n c u l t u r e . (A) Elongate carpospores released from female p l a n t s of L. s p e c t a b i l i s ( c o l l e c t e d at V i c t o r i a 28 June 1976) attached by t h e i r " t a i l s " and then became rounded. These d i d not germinate. - • ~A. (B) Elongate carpospores r e l e a s e d from female p l a n t s of J_. gardneri ( c o l l e c t e d at Botany Beach 11 J u l y 1976) attached and became rounded w i t h i n 24 hr of t h e i r r e l e a s e . These germinated about three days a f t e r t h e i r r e l e a s e , w i t h no f u r t h e r development observed. 71 72 DISCUSSION The r e s u l t s show that mature J_. gardneri i s capable of photosynthesis. The alga has a pinkish to reddish hue (see Frontispiece) probably due to the presence of r-phycoerythrin which has been i d e n t i f i e d from J_. gardneri extracts (Kugrens 1971). I t possesses " t y p i c a l " red a l g a l chloroplasts (Fig. 3) also reported by Kugrens (1971) and i t i s capable of l i g h t -14 14 dependent carbon f i x a t i o n from NaH CO^. The rate of C-uptake by the symbiont under l i g h t conditions (Table II) was lower than that of i t s host. The conditions chosen for t h i s study were the same for both organisms and may not have been as close to optimum for J_. gardneri as for L. s p e c t a b i l i s . On the other hand, the symbiont i s much smaller i n s i z e than the host (see Frontispiece) and may have a slower metabolism, and 14 consequently a lower photosynthetic rate. The C-label was detected i n amino acid - , l i p i d - , organic a c i d - , and sugar-containing f r a c t i o n s (Table III) obtained from both algae a f t e r they had been separated and then exposed to the radioactive precursor. The labeled amino acids and sugars were the same i n both algae. These r e s u l t s indicated that J_. gardneri was capable of u t i l i z i n g p h o tosynthetically-fixed carbon for the b i o -synthesis of the various compounds which one would expect to i s o l a t e from a " t y p i c a l " s e l f - s u f f i c i e n t Ceramialian red alga such as L_. s p e c t a b i l i s . 14 The major C-labeled amino acids i d e n t i f i e d (Fig. 13) a f t e r photo-synthesis by both algae were alanine, a s p a r t i c acid, glutamic acid, 14 glycine, and serine. The major C-labeled sugars were f l o r i d o s i d e , usually the most intensive radioactive spot (Fig. 5, 6), i s o f l o r i d o s i d e , 73 galactose, and glucose. These compounds have been i d e n t i f i e d as some of the photosynthetic products of other red algae (Bean and Hassid 1955; B i d w e l l 1958; Majak, C r a i g i e and McLachlan 1966; C r a i g i e , McLachlan and Tocher 1968; Fiege 1973; Kremer and Vogl 1975). Mannitol was not detected by g a s - l i q u i d chromatography i n e i t h e r a l g a , which i s contrary to re p o r t s f o r some other red algae, i n c l u d i n g members of the Ceramiales (Majak, Craigie.and McLachlan 1966; Fiege 1973). This l a c k of mannitol may support the c l a i m of Kremer (1976) that mannitol should not be "regarded as a n a t u r a l metabolite ( a s s i m i l a t e ) of Rhodophyceae"; however, i t s absence i n t h i s study as an a s s i m i l a t e may be due to a seasonal decrease or disappearance not noted because analyses were not conducted throughout a one year p e r i o d . I n t e r e s t i n g l y , Kremer d i d accept the p o s i t i v e i d e n t i -f i c a t i o n of mannitol from the p a r a s i t i c red al g a Holmsella pachyderma (Evans, et a l . 1973), but he suggested that the alga's p a r a s i t i c h a b i t may have caused i t not to " e x h i b i t the normal metabolic s i t u a t i o n " f o r a red a l g a . 14 Some d a r k - a s s i m i l a t i o n of the C - l a b e l occurred f o r both algae, w i t h the m a j o r i t y of the l a b e l detected i n the c a t i o n i c f r a c t i o n s (Table I I I ) . The r a t e f o r t h i s process, u n l i k e that f o r photosynthesis, was s i m i l a r f o r both algae (Table I I ) . For both algae, the only product suf-f i c i e n t l y l a b e l e d to be detected by radioautography was a s p a r t i c a c i d ( F i g . 13). This amino a c i d was a l s o i d e n t i f i e d as the major d a r k - a s s i m i l a -t i o n product f o r the red algae G i g a r t i n a c a n a l i c u l a t a ( J o s h i , e_t a l . 1962) and P o l y s i p h o n i a lanosa ( C r a i g i e 1963). The apparent s i m i l a r i t i e s of the dark a s s i m i l a t i o n process f o r J_. gardneri and L_. s p e c t a b i l i s do not 74 suggest a s p e c i a l r o l e f o r i t i n e i t h e r a l g a . Yet, the amount of l a b e l taken up by J_. gardneri during 30 min i n the dark was about 7-10% of the amount taken up during 30 min i n the l i g h t which suggests that t h i s process i s of some importance to the a l g a f o r a s s i m i l a t i o n of carbon. The pulse-chase t r a n s l o c a t i o n experiments showed no s i g n i f i c a n t move-14 ment of a s s i m i l a t e d C - l a b e l from one a l g a to the other during the t r a n s -l o c a t i o n periods used (Tables V and V I ) . This r e s u l t was obtained even when the algae were kept i n the dark f o r s e v e r a l hours before being exposed to the r a d i o a c t i v e l a b e l and then given time f o r t r a n s l o c a t i o n ( i f any) to occur. T r a n s l o c a t i o n d i d not occur regardless of whether the algae were provided w i t h l i g h t or dark c o n d i t i o n s f o r the t r a n s l o c a t i o n periods. The maximum t r a n s l o c a t i o n p e r i o d of 12 hr used f o r these experiments was s e l e c t e d because i t was longer than the 8 hr period which was used by Evans, e_t a l . (1973) to e s t a b l i s h that t r a n s l o c a t i o n d i d occur from G r a c i l a r i a verrucosa to i t s p a r a s i t e Holmsella pachyderma. The observation ( F i g . 2) of h o s t - p e n e t r a t i n g f i l a m e n t s from J_. gardneri confirms that by S e t c h e l l (1914), but i s contrary to that of Kugrens (1971) who could not d i s t i n g u i s h them. The r e s u l t s of the t r a n s -l o c a t i o n experiments suggested that the only f u n c t i o n of the f i l a m e n t s of mature J_. g a r n d e r i was f o r attachment to the host. This i s u n l i k e the f i l a m e n t s of the non-photosynthetic red a l g a l p a r a s i t e s Holmsella pachy- derma (Evans, et a l . 1973) and H a r v e y e l l a m i r a b i l i s (Goff 1975) which appeared to f u n c t i o n i n absorption of t r a n s l o c a t e s from t h e i r hosts. However, i t i s evident that these t r a n s l o c a t i o n experiments do not r u l e 14 out a small l o c a l i z e d exchange of C-labeled compounds between the symbiont's 75 endophytic f i l a m e n t s and adjacent c e l l s of L_. s p e c t a b i l i s , nor do the r e s u l t s provide any i n s i g h t about the p o s s i b l e exchange of non-labeled substances between the two p l a n t s . 14 Examination of C - l a b e l passing from the algae i n t o the chase sea-water (Table VII) showed that u s u a l l y only 2% or l e s s of the t o t a l l a b e l present leached out of the algae. This d i d not appear to be a continuous phenomenon since the amount detected f o r each t r a n s l o c a t i o n p e r i o d of an experiment remained s i m i l a r , even under dark c o n d i t i o n s when l i g h t was not present to enable r e a s s i m i l a t i o n of the l a b e l . The s i m i l a r i t i e s i n the amounts of "leachate" during the t r a n s l o c a t i o n periods of each e x p e r i -ment suggested that the presence of t h i s l a b e l i n the seawater was not a 14 r e s u l t of l e a c h i n g . Instead, i t may be from unincorporated C - l a b e l which was present i n or on the surfaces of the c e l l w a l l s of the algae a f t e r the.uptake p e r i o d and was not r i n s e d away before p l a c i n g the algae i n the chase seawater. I t was a l s o n o t i c e d that the presence of l i g h t d uring the t r a n s l o c a t i o n periods d i d reduce the amount of l a b e l i n the 14 chase seawater. However, the small amounts of C - l a b e l which d i d appear to be r e a s s i m i l a t e d suggest a minor r o l e f o r t h i s process. This c o n t r a s t s w i t h r e s u l t s from s t u d i e s on the e p i p h y t i c red a l g a Smithbra riaiadum 15 32 which a s s i m i l a t e d leached C-, N-, and P- c o n t a i n i n g compounds from i t s seagrass hosts ( H a r l i n 1971b; McRoy and Goering 1974). E l e c t r o n microscope examination and c u l t u r e of spores both suggested that J_. gardneri had the p o t e n t i a l to be s e l f - s u f f i c i e n t . Both carpospores and tetraspores had a p i n k i s h c o l o r when released from parent p l a n t s i n c u l t u r e , which c o n t r a s t s w i t h the report of c o l o r l e s s spores f o r J_. 76 verrucaeformis (Feldmann.and"Feldmann 1958). Both kinds of spores from J_. gardneri were shown by electron microscopy (Fig. 4) to contain d i c t y o -somes, f l o r i d e a n starch reserves, mitochondria, and proplastids. These observations indicated that these spores had the p o t e n t i a l to germinate and become photosynthetic. Indeed, carpospores obtained from J_. gardneri plants c o l l e c t e d i n July 1976 at Botany Beach germinated i n culture (Fig. 15B). Tetraspores were not germinated i n t h i s study, but Kugrens (1971) was successful i n t h i s endeavour. In both studies the germlings died within two weeks despite changes of culture media and the presence of pieces of host plants. The amount of development seen for the carpospores i n F ig. 15B was s i m i l a r to that obtained by Feldmann and Feldmann (1958) for J_. verrucaeformis 3-6 days a f t e r germination upon glass s l i d e s . The J_. verrucaef ormis spores also deteriorated at t h i s stage and was apparently concomitant with the absence of any more f l o r i d e a n starch reserves i n the spores. Whether a s i m i l a r occurrence. prevented further development of J_. gardneri spores i s not known since the presence of starch reserves was not checked. But, at t h i s stage (Fig. 15B) the J_. gardneri spores were s t i l l i n t a c t , with l i g h t pigmentation and various c e l l contents. Recently Nonamura (pers. comm.), working at the University of C a l i f o r n i a , Berkeley, succeeded i n " i n f e c t i n g " L. s p e c t a b i l i s with J_. gardrieri. He also succeeded i n " i n f e c t i n g " L_. rtipponica from Japan with i t s p a r a s i t i c symbiont J_. morimotoi. He observed that both J_. gardneri and J_. morimotoi germlings had an i n h i b i t o r y e f f e c t on the growth of infected segments (ca. 1-5 cm long) of host material. Although t h i s e f f e c t may be due to a wound response, Nonamura suggested that the plants 77 were d e r i v i n g f o o d s t u f f s from t h e i r hosts because germlings of both p l a n t s change c o l o r from p i n k i s h to w h i t i s h a f t e r a few days growth and continue to increase i n s i z e . A f t e r about three weeks, the p l a n t s become pigmented again. I t would seem from these observations that both J_. gardneri and J_. morimotoi are p a r a s i t i c as germlings. However, the evidence i s circumstan-t i a l , based on morphological f e a t u r e s , s i n c e the necessary p h y s i o l o g i c a l s t u d i e s of photosynthesis and t r a n s l o c a t i o n are l a c k i n g as i s an e l e c t r o n microscopic examination of the " w h i t i s h " p l a n t s f o r c h l o r o p l a s t s . Nonamura's c u l t u r e work (pers. comm.) d i d provide some i n s i g h t i n t o the host s p e c i f i c i t y of a Janczewskia species. He was able to i n f e c t p l a n t s of a Laurencia species which i s not normally a host f o r J_. mormitoi as i t grows i n other parts of Japan outside of the d i s t r i b u t i o n range of the p a r a s i t e . This r e s u l t l i k e the re p o r t of J_. gardneri p a r a s i t i z i n g L. splendens (Abbott and Hollenberg 1976) suggests that species of Janczewskia are not n e c e s s a r i l y r e s t r i c t e d to one host species. Numerous p o t e n t i a l hosts, i n c l u d i n g s e v e r a l species of Laurencia, are found w i t h i n the d i s t r i b u t i o n ranges of both J_. gardneri and J_. morimotoi. I t may be that i n nature the two algae are r e s t r i c t e d to only one or two s p e c i f i c species of Laurencia by other f a c t o r s , f o r example, the type of surrounding s u b s t r a t e (sand vs. r o c k ) , the amount of wave a c t i o n , the temperature or s a l i n i t y of surrounding waters, or the amount of exposure to d e s i c c a t i o n . The f a c t o r s (such as temperature or wave ac t i o n ) could operate i n a gen-e r a l manner by l i m i t i n g only a few p o t e n t i a l hosts to the same l o c a l i t y as the symbionts, or the f a c t o r s (such as d e s i c c a t i o n or substrate) could operate i n a p a r t i c u l a r l o c a l i t y to l i m i t the v e r t i c a l d i s t r i b u t i o n of p o t e n t i a l hosts and symbionts. 78 There was no p r e f e r e n t i a l s e l e c t i o n by J_. gardneri f o r a p a r t i c u l a r l i f e h i s t o r y stage of L_. s p e c t a b i l i s as determined by examination of f r e s h , preserved, and herbarium specimens. There was no d i f f e r e n c e i n the n u t r i e n t pool (Table V I I I ) of f r e e (ethanol-soluble) amino a c i d s and sugars from I J . s p e c t a b i l i s at Botany Beach and at Cable Beach, but the occurrence of J_. g a r d n eri upon ~L. s p e c t a b i l i s was r a r e at Cable Beach [recorded once during t h i s study (May 1976); not recorded p r e v i o u s l y at Cable Beach and v i c i n i t y (Scagel 1973)] and common year-round at Botany Beach. This suggests that some f a c t o r ( s ) other than the host's l i f e h i s t o r y stage or n u t r i e n t pool of f r e e amino acid s and sugars accounted f o r the r a r e occurrence of J_. gardneri upon L_. s p e c t a b i l i s at Cable Beach. The aim of t h i s research was to determine the type of symbiotic r e l a t i o n s h i p which e x i s t s between J_. gardneri and i t s host L_. s p e c t a b i l i s . Most of the i n f o r m a t i o n sought was p h y s i o l o g i c a l , i n c l u d i n g the a b i l i t i e s of both algae to photosynthesize and to t r a n s l o c a t e photosynthetic products to one another. I t was e s t a b l i s h e d that both algae photosynthesize, that 14 C - l a b e l i s a s s i m i l a t e d i n t o s i m i l a r compounds and that the l a b e l e d compounds are not t r a n s l o c a t e d between the algae. These p h y s i o l o g i c a l s t u d i e s i n d i c a t e that mature J_. gardneri i s not a p a r a s i t e , but an o b l i g a t e epiphyte. The c u l t u r e work by Nonamura suggests that the r e l a t i o n s h i p between the two algae i s even more complex, w i t h morphological observations suggesting that the germlings of J_. gardneri are p a r a s i t i c on host m a t e r i a l ; however, the necessary confirmatory p h y s i o l o g i c a l s t u d i e s are l a c k i n g . U n t i l J_. g ardneri i s shown to be a p h y s i o l o g i c a l p a r a s i t e , i t i s probably best to consider i t an o b l i g a t e epiphyte l i k e Smithora naiadum ( H a r l i n 1971b, 1973a). J_. gardneri and S_. naiadum are p h o t o s y n t h e t i c , germinate i n the 79 absence of the host, and appear to need a f a c t o r , probably chemical, from the host to enable normal development and completion of t h e i r l i f e h i s t o r y . The f a c t o r f o r J_. gardneri may not be a host photosynthetic m e t a b o l i t e s i n c e i t s spores are pigmented and co n t a i n p r o p l a s t i d s as w e l l as f l o r i d e a n s t a r c h reserves. The b a s i s f o r the s p e c i f i c i t y of J_. gardneri f o r L. s p e c t a b i l i s i s uncle a r . I f J_. gardneri should r e q u i r e a f a c t o r from i t s host f o r s u c c e s s f u l development from a germling tor-a mature p l a n t , such a f a c t o r ( i f s p e c i f i c to L_. s p e c t a b i l i s ) could e x p l a i n t h i s a s s o c i a t i o n . A l s o , as discussed e a r l i e r , c e r t a i n environmental f a c t o r s may l i m i t only a few p o t e n t i a l hosts to the same l o c a l i t i e s or i n t e r t i d a l areas where J_. gardneri may grow s u c c e s s f u l l y . Another p o s s i b i l i t y i s that J_. gardneri w i l l only develop s u c c e s s f u l l y a f t e r germination i f i t has c e r t a i n s p e c i f i c r e c o g n i t i o n i n t e r a c t i o n s w i t h some surface macromolecule from i t s host; a p o s s i b i l i t y which was examined i n the second p a r t of t h i s t h e s i s . 80 BIBLIOGRAPHY Abbott, I . A. and Hollenberg, G. J . 1976. Marine Algae of C a l i f o r n i a . 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Radiotracer Methodology i n B i o l o g i c a l  Science. P r e n t i c e - H a l l , Inc. Englewood C l i f f s , New Jersey. 382 pp. Whyte, J . N. C. and Southcott, B. A. 1970. An extraction procedure f o r plants: extracts from the red alga Rhodomela l a r i x . Phytochemistry 9:1159-1161. Wilson, L. 1977. Seasonal d i s t r i b u t i o n of red a l g a l epiphytes on Codium  f r a g i l e . J_. Phycol. 13(suppl.) :74. (Abstr.) P A R T I I ISOLATION AND PARTIAL CHARACTERIZATION OF A PROTEOGLYCAN FROM LAURENCIA SPECTABILIS INTRODUCTION A wide range of proteoglycans (major component carbohydrate), as d i s t i n c t from glycoproteins (major component pr o t e i n ) , are known from higher plants and algae. Hydroxyproline-rich arabinogalactan-proteins have been i s o l a t e d from wheat endosperm by extraction i n water (Fincher and Stone 1974), from c e l l walls of Chlamydomonas using the chaotropic agent sodium perchlorate (Catt, H i l l s and Roberts 1976), from various plant c e l l walls by a l k a l i n e extraction (Lamport 1967; M i l l e r , et a l . 1974; Monro, Bailey and Penny 1974), and from numerous higher plant sources a f t e r extraction i n d i l u t e buffer-NaCl solution (Jermyn and Yeow 1975; Anderson, et a l . 1977). Acid-soluble proteoglycans from brown algae have been shown to have fucose, mannose or glucuronic acid as the predominant monosaccharide (Larsen, Haug and Painter 1966; Abdel-Fattah, Hussein and Salem 1973; Abdel-Fattah and Edrees 1977; Medcalf and Larsen 1977) and, i n a proteoglycan from Ascophyllum nodosum (Medcalf and Larsen 1977), to contain a small amount (ca. 0.2%) of hydroxyproline i n the protein moiety. In the red algae, two d i f f e r e n t water-soluble extra-c e l l u l a r proteoglycans have been i s o l a t e d from the culture media of the u n i c e l l u l a r algae Porphyridium cruentum (Jones 1962; Heaney-Kieras and Chapman 1976; Heaney-Kieras, Roden and Chapman 1977) and Rhodella  maculata (Evans, et a l . 1974). Both proteoglycans contained xylose (major sugar), galactose, glucose, uronic acids and s u l f a t e as w e l l as a protein moiety (1.5-16% by weight). Another red a l g a l proteoglycan, a 87 hexose oxidase, was e x t r a c t e d i n aqueous b u f f e r from Chondrus c r i s p u s and contained galactose (major sugar), x y l o s e , and a p r o t e i n moiety (ca. 20% of the molecule) ( S u l l i v a n and Ikawa 1973). Hydroxyproline was not reported i n the p r o t e i n components of these three red a l g a l proteoglycans. The i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of a proteoglycan from the red a l g a Laurencia s p e c t a b i l i s P o s t e l s & Ruprecht (Ceramiales) was the subject of t h i s research. This was an attempt to o b t a i n a macromolecule which might have c e l l r e c o g n i t i o n p r o p e r t i e s s i m i l a r to those f o r the macro-molecular " l e c t i n s " i n higher p l a n t s (Callow 1975; Clarke, Knox and Jermyn 1975; Sharon 1977) and which might a l l o w the red a l g a Janczewskia gardneri S e t c h e l l & Guernsey (Ceramiales), an o b l i g a t e symbiont of _L. s p e c t a b i l i s (Smith 1969; Abbott and Hollenberg 1976), to recognize i t s host p l a n t . L e c t i n s The term " l e c t i n " (from the L a t i n l e g e r e , to choose or p i c k out) was o r i g i n a l l y used by Boyd and Sharpleigh (1954) to r e f e r to substances of p l a n t o r i g i n which a g g l u t i n a t e d red blood c e l l s e x h i b i t i n g blood group s p e c i f i c i t y . L a t e r authors (Liener 1976; Kauss and Bowles 1976; Marx 1977; Sharon 1977) considered l e c t i n s , such as concanavalin A (Con A), r i c i n , wheat germ a g g l u t i n i n and v a r i o u s Phaseolus l e c t i n s , to be a c l a s s of p r o t e i n s or g l y c o p r o t e i n s which had at l e a s t one of two p r o p e r t i e s : a g g l u t i n a t i o n of c e l l s , i n c l u d i n g red blood c e l l s , lymphocytes, f i b r o -b l a s t s , spermatozoa or b a c t e r i a ; or m i t o g e n i c i t y of c e l l s , e s p e c i a l l y of lymphocytes. The word " l e c t i n " was used by Jermyn and Yeow (1975) to describe any macromolecule that was capable of s p e c i f i c non-covalent 88 binding to carbohydrates. I w i l l use th i s broader d e f i n i t i o n . There i s no implication about the chemical nature of the macromolecule, nor about i t s interactions with various c e l l s . The d e f i n i t i o n i s i n agreement with Callow (1975) who also recognized the past problems of the narrow d e f i n i -tion and use of the term l e c t i n . He suggested that " i n the broadest sense, l e c t i n s might simply be defined as those proteins, or glycoproteins, of plant, animal, or b a c t e r i a l o r i g i n , which bind to c e l l surfaces through s p e c i f i c carbohydrate-containing receptor s i t e s " . 8-Lectins Many of the hydroxyproline-rich arabinogalactan-proteins isolated from higher plant sources have been characterized as B-lectins (Jermyn and Yeow 1975; Anderson, et a l . 1977; Jermyn 1977). This group of " a l l - 8 " l e c t i n s was characterized by Jermyn and Yeow (1975) as having binding s p e c i f i c i t y for 8-D-glycosyl linkages when reacted with Yariv a r t i f i c i a l 8-glycosyl antigens (Yariv, Rapport and Graf 1962). A l l the B-lectins so far described contain both a protein and a carbohydrate moiety. The proportion of protein i n the 8 - l e c t i n preparations ranges from 1-50%; hence, 8-lectins may be referred to as proteoglycans. Typically, prepar-ations of 8-lectins from seeds contain 10-20% protein, and those from leaves, 3-5% protein. The protein i s described as "hydroxyproline-rich" because hydroxyproline i s a major component found by analysis after acid hydrolysis, usually accounting for 10-25% of the protein composition. The carbohydrate portion of 8-lectins contains about 90% galactose (the major sugar) plus arabinose. 89 3 - l e c t i n s have been i s o l a t e d from 91 of 104 higher p l a n t f a m i l i e s , i n c l u d i n g s i x gymnosperm, eight monocotyledon and 78 d i c o t y l e d o n f a m i l i e s (Jermyn and Yeow 1975). These l e c t i n s have been detected from v a r i o u s p l a n t p a r t s , i n c l u d i n g seeds, stems and leaves, and, i n the case of angiosperms, from flower and f r u i t p a r t s (Jermyn and Yeow 1975; Knox, et  a l . 1976; Knox and Clarke 1977). This general d i s t r i b u t i o n of 3 - l e c t i n s among higher p l a n t f a m i l i e s suggests that there may be p h y s i o l o g i c a l r o l e s f o r these macromolecules i n the p l a n t s . There i s h i s t o l o g i c a l evidence that 3 - l e c t i n s are a s s o c i a t e d w i t h the c e l l membrane and are concentrated i n c e l l w a l l s and i n t e r c e l l u l a r spaces of bean cotyledon t i s s u e ( C l a r k e , et a l . 1975). I t was suggested that they "may f u n c t i o n as s e l f - r e c o g n i -t i o n f a c t o r s i n p l a n t c e l l s , determining the r e l a t i o n s h i p s between vege-t a t i v e c e l l s i n t i s s u e s and organs" and t h a t they may " f u l f i l l some r o l e i n c e l l communication". Leaves, p e t i o l e s , and stems of a number of species contained determinants which bound to the r e d d i s h - c o l o r e d Y a r i v a r t i f i c i a l 3 - g l u c o s y l antigen and were g e n e r a l l y a s s o c i a t e d e i t h e r w i t h the c e l l membrane i n t e r f a c e s , i n c l u d i n g phloem t i s s u e of v e i n s and m i d r i b s , and s e c r e t o r y c e l l s , or w i t h lumen m a t e r i a l i n s e c r e t o r y canals (Knox and Clarke 1977). The p i s t i l s and p o l l e n g r a i n s of G l a d i o l u s gandavensis contained 3 - g l u c o s y l determinants a s s o c i a t e d w i t h stigma s u r f a c e s , mucilage i n s t y l e s e c r e t o r y canals (which act as p o l l e n tube guides to the ovary), and p e r i p h e r a l cytoplasm of s t y l e canal c e l l s and p o l l e n g r a i n s (Knox, et a l . 1976; Knox and Clarke 1977). Knox, et a l . (.1976) suggested that the 3 - l e c t i n s may " f u n c t i o n as a n u t r i e n t source and p h y s i c a l support f o r the developing p o l l e n tube" of G. gandavensis "as w e l l as a c t i n g as an 90 adhesive f o r cap t u r i n g p o l l e n grains at the stigma surface". Objectives For This Study I could not f i n d any d e s c r i p t i o n s of l e c t i n s nor any r e p o r t s of attempts to i s o l a t e l e c t i n s from any a l g a . Since 3 - l e c t i n s have proven to be of general occurrence i n higher p l a n t s of a broad taxonomic range, I decided to i n v e s t i g a t e the presence of these or r e l a t e d molecules i n the algae. The a l g a Laurencia s p e c t a b i l i s P o s t e l s & Ruprecht was chosen as the study organism because i t i s the only host along the northeastern P a c i f i c Ocean c o a s t l i n e of the o b l i g a t e symbiont Janczewskia gardneri S e t c h e l l & Guernsey (Smith 1969; Abbott and Hollenberg 1976), and I speculated that some surface macromolecule had a r o l e i n s p e c i f i c r e c o g n i t i o n i n t e r a c t i o n s w i t h might l e a d to establishment of the symbiosis. I f such types of i n t e r a c t i o n s occur, they may be s i m i l a r to those.proposed f o r 3 - l e c t i n s i n higher p l a n t s , and L. s p e c t a b i l i s could be a good source of macro-molecules l i k e the 3 - l e c t i n s . The use of _L. s p e c t a b i l i s as the study organism o f f e r e d other f a v o r a b l e circumstances, i n c l u d i n g year-round a v a i l a b i l i t y of the organism f o r a n a l y s i s , and my f a m i l i a r i t y w i t h sev-e r a l aspects of the physiology and biochemistry of both i t and J_. gardneri. The o b j e c t i v e s j f o n t h i s p r e l i m i n a r y study; were (1) to i s o l a t e proteoglycans from L^ . s p e c t a b i l i s using methods f o r o b t a i n i n g 3 - l e c t i n s . (2) t o determine, u s i n g Y a r i v a r t i f i c i a l antigens, i f any "8-l e c t i n s " were i s o l a t e d . 91 (3) to determine the chemical composition of any @-lectins or other proteoglycans which were i s o l a t e d . (4) to determine m i c r o s c o p i c a l l y the i n s i t u l o c a t i o n s of any $-l e c t i n s i n L. s p e c t a b i l i s as w e l l as the symbiont J_. ga r d n e r i . 92 MATERIALS AND METHODS CHEMICALS AND SOLVENTS Chemicals and s o l v e n t s , reagent grade ACS or b e t t e r , were obtained from the s u p p l i e r s as i n d i c a t e d : a c e t i c anhydride (J.T. Baker Chemical Co., P h i l l i p s b u r g , New J e r s e y ) ; Beckman Amino A c i d C a l i b r a t i o n Mixture Type 1 (Beckman Instruments, Inc., Spinco D i v i s i o n , Palo A l t o , C a l i f o r n i a ) ; b a r b i tone, D-galactose, D-galactosamine HC1, sodium borohydride ( B r i t i s h Drug Houses L t d . , Poole, England); a l l amino a c i d s , jD-nitrophenyl-B-D-glucopyranoside (Calbiochem, Los Angeles, C a l i f o r n i a ) ; c a p r y l i c a c i d , chloramine T, N,N-dimethyl-m-phenylenediamine d i - H C l , N,N-dimethyl-p_-phenylenediamine HC1, m-phenylphenol (m-dihydroxydiphenyl), t r i f l u o r o a c e t i c a c i d (Eastman Kodak Co., Rochester, New Y o r k ) ; a l c i a n blue 8GS, b a r b i t a l sodium, D-mannose, D-mannitol, Phenol Reagent S o l u t i o n ( F o l i n - C i o c a l t e a u Reagent), p h l o r o g l u c i n o l , t o l u i d i n e blue 0 (Fishe r S c i e n t i f i c Co., F a i r Lawn, New J e r s e y ) ; sodium glucuronate (Koch-Light L a b o r a t o r i e s L t d . , Coin-brook, England) ; jp_-dimethylamino benzaldehyde, platinum oxide (Matheson Coleman & B e l l , Norwood, Ohio); D-arabinose, D-galacturonic a c i d (mono-hydr a t e ) , D-glucose, D-xylose ( N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio); methyl c e l l o s o l v e , n i n h y d r i n ( P i e r c e Chemical Co., Rockford, I l l i -n o i s ) ; bovine serum albumin ( c r y s t a l l i z e d l x and l y o p h i l i z e d ) , D-glycosamine HC1, m y o - i n o s i t o l , Trizma Base (Sigma Chemical Co., St. L o u i s , M i s s o u r i ) . D - g a l a c t i t o l , D - g l u c i t o l , D-mannitol and t h e i r a c e t a t e s , D - a r a b i t o l penta-ac e t a t e , and D - x y l i t o l pentaacetate were provided by Dr. J.N.C. Whyte, F i s h -e r i e s and Marine S e r v i c e , Vancouver, B r i t i s h Columbia. A l l other chemicals and s o l v e n t s were obtained l o c a l l y and were of reagent grade ACS q u a l i t y or b e t t e r . 93 PLANT MATERIAL Laurencia s p e c t a b i l i s P o s t e l s & Ruprecht was c o l l e c t e d at Botany Beach near Port Renfrew on Vancouver I s l a n d , B r i t i s h Columbia i n A p r i l and November 1976. The f r e s h m a t e r i a l was kept on i c e i n p l a s t i c bags f i l l e d w i t h seawater during t r a n s p o r t a t i o n back to the l a b o r a t o r y . The m a t e r i a l was cleaned, b l o t t e d dry, and stored at -15°C. PREPARATION OF PLANT MATERIAL P l a n t m a t e r i a l (ca. 300 gm f r e s h wt) was thawed and homogenized i n small p o r t i o n s i n a Waring Blendor i n ca. 800 ml of 10 mM potassium phosphate b u f f e r (pH 7.0) c o n t a i n i n g 1% (w/v) NaCl and 3 mM NaN^ (sodium a z i d e ) . A d d i t i o n a l b u f f e r (500-700 ml) was added and the s l u r r y s t i r r e d f o r 2-6 hr at 5°C then f i l t e r e d through four l a y e r s of cheesecloth. The i n i t i a l e x t r a c t was stored at 5°C and the residue was r e - e x t r a c t e d i n 10 mM potassium phosphate b u f f e r (pH 7.0) c o n t a i n i n g 5% (w/v) NaCl and 3 mM NaN^, and f i l t e r e d . The two e x t r a c t s were combined. (NH^^SO^ was added stepwise to 40, 70, and 100% s a t u r a t i o n at room temperature, and i n s o l u b l e m a t e r i a l was removed a f t e r each a d d i t i o n by c e n t r i f u g a t i o n at 4800 x f o r 15 min. The r e s u l t i n g 100% (NH^^SO^ saturated supernatant was d i a l y z e d against running tap water f o r 48 hr and concentrated, but not d r i e d , by r o t a r y evaporation at 45°C. I t was important that complete d r y i n g of the sample be avoided at t h i s step and at l a t e r i s o l a t i o n steps because the s o l u b i l i t y of substances, i n c l u d i n g the proteoglycan l a t e r i s o l a t e d , i n aqueous s o l u t i o n s was e f f e c t e d . 94 GEL AND ION EXCHANGE CHROMATOGRAPHY Samples (10-20 ml) were a p p l i e d to a column (50 x 3.4 cm) of Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) e q u i l i b r a t e d w i t h 10 mM potassium phosphate b u f f e r (pH 7.0) c o n t a i n i n g 1% (w/v) NaCl and 3 mM NaN^. The column was el u t e d w i t h the same b u f f e r . The eluate was monitored f o r carbohydrate (Dubois, et a l . 1956) and f o r p r o t e i n (Lowry, et a l . 1951; Eggstein and Kreutz 1955). F r a c t i o n s from the second carbohydrate-c o n t a i n i n g peak (which contained the proteoglycan m a t e r i a l ) were pooled, d i a l y z e d against running tap water f o r 24 h r , concentrated (but not dried) by r o t a r y evaporation, and re-chromatographed on Sepharose 4B. The f r a c -t i o n s c o n t a i n i n g carbohydrate and p r o t e i n were pooled, desalted on a column (50 x 2.3 cm) of Sephadex G-25-300 (Sigma Chemical Co., St. L o u i s , MO.), and concentrated (but not dried) by r o t a r y evaporation. Samples (2-5 ml) i n deionize d water were a p p l i e d to a column (25-27 x 1.7 cm) of DEAE-Sephadex A-50 (Pharmacia Fine Chemicals) e q u i l i b r a t e d w i t h 200 mM sodium phosphate b u f f e r (pH 8.0) c o n t a i n i n g 3 mM NaN^- The column was e l u t e d by a l i n e a r s a l t gradient w i t h the flow r a t e maintained between 15-20 ml/hr. The l i n e a r gradient was formed by mixing 300 ml of e q u i l i b r a -t i o n b u f f e r w i t h 300 ml of e q u i l i b r a t i o n b u f f e r c o n t a i n i n g 1.8 M NaCl. The m o l a r i t y of the elu a t e was monitored using a c o n d u c t i v i t y meter (Type COM 2d No. 56606, Radiometer, Copenhagen, Denmark). Column e l u t i o n was stopped when the e l u a t e m o l a r i t y reached 0.5 M. Eluate samples were monitored f o r the presence of carbohydrate. U s u a l l y , only one peak of carbohydrate a c t i v i t y was detected. The homogeneity of the carbohydrate-c o n t a i n i n g f r a c t i o n s was monitored r o u t i n e l y by c e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s . 95 CELLULOSE ACETATE STRIP ELECTROPHORESIS Samples (5-10 y l ) of the carbohydrate-containing f r a c t i o n s from the ion exchange column were applied to 15.2 x 2.5 cm Sephaphore III c e l l u l o s e polyacetate s t r i p s (Gelman Instrument Co., Ann Arbor, MI.). Electrophor-e s i s was performed at 300 V for 40 min at room temperature i n a Gelman electrophoresis chamber (model No. 51170-1) using 60 mM T r i s - b a r b i t a l -sodium b a r b i t a l buffer (pH 8.8). The s t r i p s were cut i n h a l f and were stained with a l c i a n blue (method a. i n Reid, et: a l . 1972) to detect poly-anionic substances.or with high i r o n diamine (Reid, et a l . 1972) to detect h a l f s u l f a t e esters. The f r a c t i o n s which contained one band of s i m i l a r electrophoretic and s t a i n i n g behavior were combined, desalted by passage through Sephadex G-25-300, and concentrated (but not dried) by rotary evaporation to 5-10 ml. Some of t h i s s o l u t i o n was kept f o r t e s t i n g with Yariv a r t i f i c i a l antigen f o r the presence of a 3 - l e c t i n , while the remain-der was l y o p h i l i z e d p r i o r to chemical a n a l y s i s . 3-LECTIN DETECTION Several unsuccessful attempts were made to produce the 3-glucosyl form of Yariv a r t i f i c i a l antigen using the method described by Yariv, et_ a l . (1962) with some attempts under conditions for the synthesis suggested by Professor Bruce A. Stone (pers. comm.). The gel d i f f u s i o n cross-p r e c i p i t a t i o n test for 3 - l e c t i n s with a 3-glucosyl determinant (Jermyn and Yeow 1975) was done by Dr. Robin L. Anderson (.formerly of Professor Stone's Laboratory). An approximate 1% (w/v) so l u t i o n of the L. spectab- i l i s proteoglycan was provided for the sample test s o l u t i o n , and pea c e l l 96 supernatant s o l u t i o n and soybean c u l t u r e medium were used as c o n t r o l s . A 0.1% (w/v) s o l u t i o n of Y a r i v a r t i f i c i a l 8-glucosyl antigen was used f o r the t e s t s . ANALYTICAL PROCEDURES General a n a l y s i s . T o t a l carbohydrate was determined by the phenol-s u l f u r i c a c i d method (Dubois, et a l . 1956) using galactose as standard. T o t a l p r o t e i n was determined by m o d i f i c a t i o n (Eggstein and Kreutz 1955) of the Folin-Lowry procedure (Lowry, et a l . 1951).using bovine serum albumin as standard. T o t a l u r o n i c a c i d s was determined u s i n g the method of Blumenkrantz and Asboe-Hansen (1973) w i t h a 1:1 mixture of g a l a c t u r o n i c and g l u c u r o n i c a c i d s as standard. T o t a l s u l f a t e was determined by the method of Nader and D i e t r i c h (1977). N e u t r a l sugar a n a l y s i s . Samples (1.5-4 mg) were hydrolyzed w i t h 1 ml of 2 N t r i f l u o r o a c e t i c a c i d i n sealed tubes at 110°C f o r 1 hr. The n e u t r a l sugars were analyzed as a l d i t o l acetate d e r i v a t i v e s (Albersheim, et a l . 1967) by g a s - l i q u i d chromatography, using a Varian Aerograph dual column gas chromatograph Model 1740 (Varian Aerograph, Walnut Creek, CA.) equipped w i t h flame i o n i z a t i o n d e t e c t o r s . The flow r a t e s of 1$ and were 25 ml/min and of a i r were 250 ml/min. D e r i v a t i v e samples or standard sugars i n chloroform were i n j e c t e d i n t o s t a i n l e s s s t e e l columns (1.8 m x 3 mm o.d.) of 5% (w/w) S i l a r 10C ( A l l t e c h A s s o c i a t e s , A r l i n g t o n Heights, IL.) on 100/120 mesh Gas Chrom Q (Applied Sciences Lab. Inc., State 97 Co l l e g e , PA.) which were temperature programmed l i n e a r l y from 120°C (at i n j e c t i o n ) to 260°C at 2°C/min. Amino a c i d and amino sugar analyses. Samples (1-4 mg) were hydrolyzed i n vacuo w i t h ca. 0.5 ml of 6 N HC1 at 110°C f o r 20 hr. Hydrolyzates were d r i e d i n vacuo and analyzed by the method of Spackman, S t e i n and Moore (1958) using a Beckman Amino A c i d Analyzer Model 120C Instruments, Inc., Palo A l t o , CA.). The b a s i c amino acid s and the amino sugars were resolve d on a 16 x 0.9 cm column (Cameron and Taylor 1976). The a c i d i c and n e u t r a l amino a c i d s were r e s o l v e d on a 58 x 0.9 cm column. Hydroxyproline was determined a f t e r a c i d h y d r o l y s i s u s i n g the spectro-photometry method of Bergman and Loxley (1970). 98 RESULTS PURIFICATION OF A PROTEOGLYCAN Figure 16 shows the carbohydrate and p r o t e i n p r o f i l e f o r the e l u t i o n of L. s p e c t a b i l i s 100% (NH.)-SO. saturated supernatant from Sepharose 4B. -- — —' 4 l 4 Carbohydrate was l o c a t e d i n three peaks (A, B, and C). P r o t e i n was detected i n peaks B and C, but only peak B contained proteoglycan m a t e r i a l . A f t e r re-chromatography of peak B, the f r a c t i o n s forming the s i n g l e carbohydrate peak were pooled. The m a t e r i a l was shown to co n t a i n at l e a s t s i x components when analyzed by c e l l u l o s e acetate s t r i p e l e c t r o -phoresis ( F i g . 17). A l l the bands (No. 4 and 6) reacted w i t h the high i r o n diamine s t a i n ( s u l f a t e groups present). When t h i s mixture was sep-arated by ion exchange chromatography, a s i n g l e carbohydrate peak was ob-t a i n e d ( F i g . 18). However, e l e c t r o p h o r e t i c examination of the f r a c t i o n s i n t h i s peak revealed that the f r o n t of the peak ( F i g . 18, f r a c t i o n s No. 27-31) contained two a l c i a n blue r e a c t i v e substances. The major one was a proteoglycan (band No. 2). E l e c t r o p h o r e t i c a n a l y s i s of f r a c t i o n s which emerged l a t e r revealed only a s i n g l e component which corresponded to the proteoglycan. Only those f r a c t i o n s shown to be homogeneous by e l e c t r o -phoresis were used f o r B - l e c t i n d e t e c t i o n and f o r chemical a n a l y s i s . The minimum y i e l d of the p u r i f i e d proteoglycan was approximately 32 mg dry wt from 800 gm f r e s h wt of a l g a . B-LECTIN DETERMINATION OF THE PROTEOGLYCAN The L_. s p e c t a b i l i s proteoglycan s o l u t i o n d i d not form c r o s s - p r e c i p i t a -t i o n l i n e s w i t h the Y a r i v a r t i f i c i a l B - g l u c o s y l antigen. Only the c o n t r o l 99 F i g . 16. Sepharose 4B g e l chromatography of L. s p e c t a b i l i s 100% satura t e d (NHj^SO^ supernatant. A 15 ml sample of L. s p e c t a b i l i s e x t r a c t (see M a t e r i a l s and Methods f o r preparation) was subject t o g e l chromatography on a Sepharose 4B column (50 x 3.4 cm). The running b u f f e r was 10 mM potassium phosphate (pH 7.0) c o n t a i n i n g 1% (w/v) NaCl and 3 mM NaN^. Carbohydrate (Dubois, ejt a l . 1956; • — — # ) and p r o t e i n (Lowry, et a l . 1951 as modified by Eggstein and Kreutz 1955; O o) were detected i n the e l u a t e . V = v o i d volume V = t o t a l volume o t 100 FIGURE 16 0 100 160 220 280 340 400 , ELUTION VOLUME (ML) 101 F i g . 17. C e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s of L. s p e c t a b i l i s proteoglycan a f t e r re-chromatography on Sepharose 4B. The proteoglycan a f t e r re-chromatography on Sepharose 4B was subject to e l e c t r o p h o r e s i s , and then s t a i n e d w i t h a l c i a n blue or w i t h high i r o n diamine (see M a t e r i a l s and Methods). This drawing shows the r e s u l t s a f t e r s t a i n i n g of the s t r i p s . Band No. 2 i s the proteoglycan i n v e s t i g a t e d i n t h i s study. FIGURE 17 777/ ® 4 ORIGIN e A L C I A N B L U E (POLYANION GROUPS) HIGH IRON DIAMINE ( S U L F A T E GROUPS 103 F i g . 18. S a l t - g r a d i e n t e l u t i o n of L_. s p e c t a b i l i s proteoglycan from DEAE-Sephadex A-50 and c e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s of f r a c t i o n s . A proteoglycan sample p u r i f i e d by g e l chromatography was a p p l i e d to the anion exchange column (27 x 1.7 cm) and e l u t e d by a s a l t g r a d i e n t . The carbohydrate content (• •) and the s a l t m o l a r i t y (O O) were d e t e r -mined. E l e c t r o p h o r e s i s was performed on carbohydrate-containing f r a c -t i o n s f o l l o w e d by s t a i n i n g w i t h a l c i a n blue and hig h i r o n diamine. D e t a i l s i n the M a t e r i a l s and Methods. The drawing shows r e s u l t s of the e l e c t r o p h o r e s i s and s t a i n i n g w i t h a l c i a n blue of f r a c t i o n s No. 27-39. No substances reacted w i t h high i r o n diamine. Band No. 2 i s the proteo-glycan i n v e s t i g a t e d i n t h i s study. 104 FIGURE 18 105 s o l u t i o n s of pea c e l l supernatant and soybean c u l t u r e medium formed c r o s s -p r e c i p i t a t i o n l i n e s . Even when the L_. s p e c t a b i l i s proteoglycan s o l u t i o n was concentrated s i x - f o l d , no p r e c i p i t a t i o n l i n e s were formed. The pro-teoglycan d i d not have a 3~glucosyl determinant @-lectin. CHEMICAL ANALYSIS OF THE PROTEOGLYCAN The composition of the apparently homogeneous proteoglycan i s shown i n Table X. The preponderance of carbohydrate over p r o t e i n confirmed the view that the compound i s o l a t e d from I i . s p e c t a b i l i s was a proteoglycan. Uronic a c i d s accounted f o r 8.4% of the t o t a l composition. S u l f a t e was not detected by e i t h e r the assay of Nader and D i e t r i c h (1977) or, a f t e r e l e c t r o p h o r e s i s of the proteoglycan, r e a c t i o n w i t h high i r o n diamine s t a i n (minimum d e t e c t i o n l i m i t of s t a i n ca. 20 yg of s u l f a t e when determined by the Nader and D i e t r i c h assay). The n e u t r a l sugar compositions of two d i f f e r e n t proteoglycan prepara-t i o n s are shown i n Table XI. The proteoglycan-was apparently a galactan w i t h small q u a n t i t i e s of xy l o s e and glucose i n a d d i t i o n to the ur o n i c a c i d s (see F i g . 19). The arabinose detected i n both preparations was present i n only trace q u a n t i t i e s . The amino aci d s and amino sugars recovered (see F i g . 20) from the hydr o l y z a t e s of two d i f f e r e n t preparations of the proteoglycan are given i n Table X I I . The a c i d i c amino aci d s a s p a r t a t e , threonine, s e r i n e , and glutamate accounted f o r approximately 60% of the recovered amino a c i d s . Hydroxyproline and s e v e r a l other amino a c i d s were detected i n small ( l e s s than 1.5%) q u a n t i t i e s , w h i l e n e i t h e r p r e p a r a t i o n contained d e t e c t a b l e 106 TABLE X COMPOSITION OF PURIFIED PROTEOGLYCAN FROM LAURENCIA SPECTABILIS Results are the mean of four analyses (see M a t e r i a l s and Methods) f o r one p r e p a r a t i o n . Carbohydrate 92% Uronic Acids P r o t e i n 8% T o t a l weight 8.4% T o t a l carbohydrate 9.1% Carbohydrate/ ft P r o t e i n R a t i o 12:1 S u l f a t e N.D. * N.D. - not detected; i f present, l e s s than 2.0% of t o t a l weight. TABLE XI NEUTRAL SUGAR COMPOSITION OF PURIFIED LAURENCIA SPECTABILIS PROTEOGLYCAN Res u l t s f o r each p r e p a r a t i o n are the average of two analyses u s i n g gas-l i q u i d chromatography (see M a t e r i a l s and Methods). P r e p a r a t i o n N o . 1 Pr e p a r a t i o n No. 2 Arabinose 1.8% ( 1.5%) 2.3% ( 1.9%) Xylose 6.4 ( 5.4 ) 7.4 ( 6.2 ) Galactose 84.6 (70.7 ) 84.6 (70.7 ) Glucose 7.3 (-6.1 ) 5.7 ( 4.8 ) * Re s u l t s i n ( ) are the proporti o n s of each n e u t r a l sugar by t o t a l weight of the proteoglycan. 107 F i g . 19. G a s - l i q u i d chromatogram of a l d i t o l acetate d e r i v a t i v e s of sugars from p u r i f i e d L_. s p e c t a b i l i s proteoglycan. A sample of p u r i f i e d proteoglycan was hydrolyzed f o r 1 hr i n 2 N t r i f l u o r o a c e t i c a c i d , reduced and, a f t e r a d d i t i o n of the i n t e r n a l standard my_o-inositol, a c e t y l a t e d w i t h a c e t i c anhydride (see M a t e r i a l s and Methods). The sample i n chloroform was i n j e c t e d i n t o a V a r i a n Aerograph gas chromatograph w i t h 5%lSilarG10C columns and temperature programmed from 120O-260°C at 2°/min. 108 FIGURE 19 109 F i g . 20. Chromatograms of (A) amino sugars and b a s i c amino acid s and (B) a c i d i c and n e u t r a l amino a c i d s of p u r i f i e d L_. s p e c t a b i l i s proteoglycan. (A) A sample of p u r i f i e d proteoglycan was hydrolyzed f o r 20 hr i n 6 N HC1 before a n a l y s i s of amino sugars and b a s i c amino acid s using an amino a c i d a n a l y z e r , w i t h a running b u f f e r of pH 5.25 (see M a t e r i a l s and Methods). AGPA (a-amino-g-guanidinopropionic acid) was added to the sample as an i n t e r n a l standard. The detected substances are; (L)X a c i d i c and n e u t r a l amino a c i d s (6) H i s (2) Phe (7) NH 3 (3) GlcN (8) AGPA (4) GalN (9) Arg (5) Lys (B) Sample d e t a i l s f o r the a n a l y s i s of a c i d i c and n e u t r a l amino acid s were as i n (A). The running b u f f e r was pH 3.25 u n t i l peak ( 9 ) , when the b u f f e r was changed to pH 4.30. Norleucine was added to the sample as an i n t e r n a l standard. The detected substances are: (1) Asx (9) b u f f e r change peak (2) Thr (10) Met (3) Ser (11) l i e (4) Glx (12) Leu (5) Pro (13) Nle (6) Gly (14)ATy.r (7) A l a (15) Phe (8) V a l I l l TABLE X I I COMPOSITION OF AMINO ACIDS AND AMINO SUGARS RECOVERED FROM HYDROLYZATES OF L. SPECTABILIS PROTEOGLYCAN Two preparations of p u r i f i e d proteoglycan were hydrolyzed f o r 20 hr i n 6 N HC1 before a n a l y s i s of amino acid s and amino sugars us i n g an amino a c i d analyzer (see M a t e r i a l s and Methods). The r e s u l t s f o r each p r e p a r a t i o n are the average ofrtwo analyses. Prep. No. 1 Prep. No. 2 Prep. No. 1 Prep. No. 2 Asx 23.9% Lys 1.7% 2.7% Thr 9.9 9.5 His 0.6 0.5 Ser 8.8 " 8.0 Arg 0.8 1.0 Glx 15.4 17.7 Pro 11.2 11.7 Gly 5.1 6.5 GlcN 4.9 2.1 A l a 6.6 5.9 GalN 2.3 0.5 Cys 0 0 V a l 5.0 4.3 Met 0.6 0.5 * Hyp 0.3 0.3 H e 0.8 0.7 Leu 0.8 0.7 * Determined s p e c t r o p h o t o m e t r i c a l l y Tyr 0.3 0.3 Phe 0.8 1.2 112 amounts o f • c y s t ( e ) i n e . Glucosamine and galactosamine were present i n both prep a r a t i o n s , although the recovery of both amino sugars was lower i n pr e p a r a t i o n No. 2. 113 DISCUSSION The procedures used to obt a i n the i s o l a t e d proteoglycan from L. s p e c t a b i l i s i n cluded e x t r a c t i o n of the p l a n t m a t e r i a l w i t h a d i l u t e buffer-NaCi s o l u t i o n , followed by p u r i f i c a t i o n steps i n v o l v i n g s o l u b i l i t y i n 100% saturated (NH^^SO^ and g e l chromatography on Sepharose 4B. These procedures were used to obt a i n B - l e c t i n s from higher p l a n t sources (Jermyn and Yeow 1975; Anderson, et a l . 1977). The use of a d i l u t e buffer-NaCl s o l u t i o n f o r e x t r a c t i o n was of importance s i n c e t h i s m i l d c o n d i t i o n , compared w i t h more rig o r o u s a c i d or base e x t r a c t i o n c o n d i t i o n s , was more l i k e l y to leave proteoglycans i n t a c t . An a d d i t i o n a l procedure i n v o l v i n g i o n exchange chromatography w i t h very c a r e f u l gradient e l u t i o n was needed to o b t a i n the p u r i f i e d L_. s p e c t a b i l i s proteoglycan. C e l l u l o s e acetate s t r i p e l e c t r o p h o r e s i s was used to monitor the progress of p u r i f i c a t i o n and to determine the poi n t of emergence of the s i n g l e proteoglycan component from the DEAE-Sephadex A-50 i o n exchange column ( F i g . 18). Cle a r r e s o l u -t i o n of the "contaminant" component from the proteoglycan was obtained only when a "slow" continuous s a l t gradient ( t o t a l v o l 600 ml) formed part of the i o n exchange chromatography procedure. E a r l i e r attempts using a step-wise or a "steep" continuous gradient ( t o t a l v o l . l e s s than 300 ml) di d not provide r e s o l u t i o n of the two substances. Only one major proteoglycan f r a c t i o n (peak B, F i g . 16), c o n t a i n i n g a s i n g l e proteoglycan (band No. 2, F i g . 17), was obtained from L. spectab- i l i s . The other f r a c t i o n c o n t a i n i n g Folin-Lowry p o s i t i v e m a t e r i a l was peak C, which c o n s i s t e d of substances e l u t e d at the t o t a l volume (V t) of 114 the Sepharose 4B column. The "contaminant" component (band No. 1, F i g . 18) which was resolve d from the proteoglycan by i o n exchange chromatography appeared to be a polysaccharide composed mainly of ur o n i c a c i d s , arabinose, and glucosamine, but without any de t e c t a b l e p r o t e i n (according to e l e c t r o -p h o r e t i c , sugar, and amino a c i d analyses of f r a c t i o n s No. 27-31 i n F i g . 18). The four a d d i t i o n a l substances (bands No. 3-6, F i g . 17) detected a f t e r e l e c t r o p h o r e s i s of the proteoglycan f r a c t i o n were not c h a r a c t e r i z e d because they were not recovered a f t e r i o n exchange chromatography, even when the m o l a r i t y of the e l u t i o n b u f f e r was r a i s e d to 2. M. Some methods used to obt a i n 8 - l e c t i n s have proven s u c c e s s f u l f o r i s o -l a t i n g a proteoglycan from a red a l g a . However, the r e s u l t s of the g e l d i f f u s i o n c r o s s - p r e c i p i t a t i o n s t u d i e s of the p u r i f i e d proteoglycan w i t h the Y a r i v a r t i f i c i a l B-glucosyl antigen i n d i c a t e d that the proteoglycan was not a 8 - l e c t i n . The chemical analyses a l s o i n d i c a t e d that the i s o l a t e d proteoglycan was not a 8 - l e c t i n . The 8 - l e c t i n s c h a r a c t e r i z e d to date (Jermyn and Yeow 1975; Anderson, et a l . 1977; Jermyn 1977) have been composed p r i m a r i l y of a carbohydrate component of galactose and arabinose (ca. 4 : 3 . r a t i o of galactose:arabinose) and a smaller p r o t e i n moiety " r i c h " i n hydroxyproline ( u s u a l l y greater than 10% of the amino a c i d composition). The i s o l a t e d proteoglycan from L_. s p e c t a b i l i s had a carbohydrate :protein r a t i o i n the range of those f o r 8 - l e c t i n s and i t contained galactose and arabinose, but the r a t i o of the two sugars (85:2) and the low amount of hydroxypro-l i n e (0.3% of the t o t a l amino a c i d composition) i n d i c a t e d that the molecule d i f f e r e d s u b s t a n t i a l l y from B - l e c t i n s . I t has been noted that hydrox-115 y p r o l i n e plays an important r o l e i n the a c t i v e s i t e of 3 - l e c t i n s (Jermyn 1977; R.L. Anderson, pers. comm.); thus a substance c o n t a i n i n g the low amount of hydroxyproline observed i n the molecule from L. s p e c t a b i l i s was very u n l i k e l y to r e a c t w i t h a Y a r i v a r t i f i c i a l antigen. The composition and r e l a t i v e amounts of sugars i n the carbohydrate p o r t i o n s of the two proteoglycans reported from the red algae Porphyridium cruentum (Jones 1962; Heaney-Kieras and Chapman 1976; Heaney-Kieras, et a l . 1977) and Rhodella  maculata (Evans, et a l . 1974) were s i m i l a r , but only the amount of u r o n i c a c i d s was determined q u a n t i t a t i v e l y f o r both molecules (8.5% by weight f o r j?. cruentum and 12% f o r R. maculata). The q u a n t i t a t i v e n e u t r a l sugar a n a l y s i s (by t o t a l weight) of the molecule from P_. cruentum showed that there was approximately 44% x y l o s e , 38% galactose, and 18% glucose (Heaney-K i e r a s and Chapman 1976; Heaney-Kieras, et a l . 1977). The carbohydrate p o r t i o n of the L_. s p e c t a b i l i s proteoglycan (Tables X, XI) had sugars and u r o n i c a c i d content (8.4%) s i m i l a r t o the other two molecules; however, galactose was by f a r the major n e u t r a l sugar (70.7% by t o t a l weight) and a t r a c e amount of arabinose was present. The hexose oxidase proteoglycan from the red a l g a Chondrus c r i s p u s a l s o contained galactose as the pre-dominant sugar and x y l o s e , but no u r o n i c ac i d s were present ( S u l l i v a n and Ikawa, 1973). The n e u t r a l sugars galactose and glucose, plus t r a c e amounts of arabinose and x y l o s e , were the main c o n s t i t u e n t s of the carbo-hydrate p o r t i o n s of g l y c o p r o t e i n s from the red algae Phyllophora nervosa (Medvedeva and S e l i c h 1968; Medvedeva and Kaganovich 1970; Medvedeva, et a l . 1973) and F u r c e l l a r i a f a s t i g i a t a ( K r a s i l ' n i k o v a and Medvedeva 1975). The main n e u t r a l sugar c o n s t i t u e n t s of Iv. s p e c t a b i l i s c e l l w a l l s were a l s o 116 galactose (ca. 69% of the n e u t r a l sugars), glucose ( 9 % ) , and xylose ( 6 % ) , p l u s mannose (5%) (Court 1972). P r o t e i n accounted f o r 1.5-7% (by weight) of the Porphyridium cruentum proteoglycan (Jones 1962; Heaney-Kieras and Chapman 1976; Heaney-Kieras, et a l . 1977), f o r about 16% of the Rhodella maculata proteoglycan (Evans, et a l . 1974), and f o r about 20% of the Chondrus c r i s p u s proteoglycan ( S u l -l i v a n and Ikawa, 1973). The published amino a c i d compositions of the proteoglycans from P_. cruentum and C. c r i s p u s does not c o n t a i n hydroxy-p r o l i n e , but the authors d i d not report using any of the s p e c i f i c assays a v a i l a b l e to detect t h i s imino a c i d . The L. s p e c t a b i l i s proteoglycan contained 8% (by weight) p r o t e i n and hydroxyproline was present. The presence of hydroxyproline i n red a l g a l p r o t e i n was reported by Lewis and Gonzalves (1962) and Lewis (1973), but not i n the analyses presented by G o t e l l i and C l e l a n d (1968). My r e p o r t of hydroxyproline i n a red a l g a i s based upon r e s u l t s from a more s e n s i t i v e assay (Bergman and Loxley 1970) than used by previous workers as w e l l as upon a n a l y s i s of a p u r i f i e d sub-stance r a t h e r than a whole p l a n t e x t r a c t . E i t h e r or both f a c t o r s could account f o r the disagreement w i t h the r e s u l t s of G o t e l l i and C l e l a n d (1968) whose paper i s o f t e n c i t e d as the most a u t h o r i t a t i v e source on hydroxyproline d i s t r i b u t i o n i n a l g a l p r o t e i n s . The low amount of hydroxyproline detected i n the _L. s p e c t a b i l i s proteoglycan suggests t h a t any r o l e which i t may have i n c e l l w a l l growth or s t r u c t u r e d i f f e r s at l e a s t q u a n t i t a t i v e l y from the r o l e s suggested f o r hydroxyproline i n the h y d r o x y p r o l i n e - r i c h proteoglycans from higher p l a n t s (Lamport 1965, 1970) or green algae (Thompson and Preston 1967; 117 M i l l e r , Lamport and M i l l e r 1972; M i l l e r , et a l . 1974). The linkage between hydroxyproline and arabinose, or galactose i n a green alga ( M i l l e r , et a l . 1972), i s the major protein-carbohydrate linkage i n these proteoglycans. The protein-carbohydrate linkage of Porphyridium cruentum has been i n v e s t i -gated and evidence for an 0-glycosidic linkage of serine or threonine to xylose was obtained (Heaney-Kieras, et a l . 1977). Although these authors claim t h i s report to be the " f i r s t such report i n the plant kingdom" of a "protein-carbohydrate linkage in v o l v i n g serine and threonine", Russian workers (Krasil'nikova and Medvedeva 1975) had e a r l i e r found an 0-glyco-s i d i c bond between serine and glucose i n a glycoprotein from the red alga F u r c e l l a r i a f a s t i g i a t a . In addition, other reports (Medvedeva and S e l i c h 1968; Medvedeva, et a l . 1973) contained evidence for protein-carbohydrate bonds through the 3-carboxyl group of aspartic acid or the hydroxyl group of tyrosine to galactose i n other red a l g a l glycoproteins. Since the L_. s p e c t a b i l i s proteoglycan contains large quantities of a s p a r t i c acid (some of which could be a hydrolysis product from asparagine), serine, threonine, galactose, glucose and xylose, these compounds, rather than hydroxyproline and arabinose, probably form the major protein-carbohydrate linkages of t h i s molecule. S i m i l a r l y i n the brown alga Ascophyllum nodosum, an ascophyllan-like proteoglycan, which contained a small amount of hydroxy-pr o l i n e (and no arabinose), appeared to have the majority of protein-carbohydrate linkages through serine and threonine (Medcalf and Larsen 1977). The presence of s u l f a t e i n red a l g a l g a l a c t a n s has been docu-mented (Pe r c i v a l and McDowell 1967), although some non-sulfated galactans are known, for example, that from the mucilage of Batracho-118 spermum sp. (Turvey and G r i f f i t h s 1973). The a c i d i c proteoglycan from Porphyridium cruentum contained 9-10% (by weight) ester s u l f a t e (Jones 1962; Heaney-Kieras and Chapman 1976; Heaney-Kieras, et a l . 1977), whereas that from Rhodella maculata had 10% s u l f a t e (Evans, et a l . 1974). Sulfate was not detected i n the proteoglycan from Chondrus crispus ( S u l l i v a n and Ikawa 1973) . Sulfate was also absent from the a c i d i c L^ . s p e c t a b i l i s proteoglycan; thus, the uronic acids i n the molecule accounted for i t s reaction with a l c i a n blue s t a i n and i t s behavior during both ion exchange chromatography and c e l l u l o s e acetate s t r i p electrophoresis. It has been established that a proteoglycan can be i s o l a t e d from the red alga L. s p e c t a b i l i s by methods used for obtaining 8-lectins and that the i s o l a t e d proteoglycan i s not a 8-lectin. The chemical composition of the proteoglycan has been determined. These findings accomplish the f i r s t three objectives of t h i s study and, since no B - l e c t i n s were i s o l a t e d , the fourth goal has been obviated. The l o c a t i o n and function of the i s o l a t e d proteoglycan i n the alga remains to be resolved. There are f i v e possible general locations for t h i s proteoglycan i n L. s p e c t a b i l i s : i n an e x t r a c e l l u l a r mucilage (as the two proteoglycans from Porphyridium cruentum and Rhodella macUlata), i n the c e l l w all, i n the c e l l membranes, i n the cytoplasm, or i n any combination of these places. The firm, non-slimey texture of L. s p e c t a b i l i s plants seems to eliminate l o c a t i o n i n e x t r a c e l l u l a r mucilage, whereas the large carbohydrate compo-nent and the water-soluble property of the proteoglycan seems to exclude i t s l o c a t i o n i n c e l l membranes. The proteoglycan may be located i n the cytoplasm, possibly as an enzyme. Since i t s neutral sugar composition i s 119 s i m i l a r to the neutral sugar composition of the c e l l w all, i t also seems reasonable to speculate that some of the molecule i s located i n the c e l l w a l l . I t i s premature to comment on possible r e l a t i o n s h i p s of the pro-teoglycan with the p r o t e i n - r i c h " c u t i c l e " , possibly glycoprotein in. .nature, which has been reported from some red algae (Hanic and Craigie 1969; L i c h t l e 1975; Gerwick and Lang 1977). Possible functions for the proteoglycan include as a s t r u c t u r a l component of the c e l l w a l l , as a non-sulfated galactan "precursor" for synthesis of a s u l f a t e d galactan, as another type of l e c t i n than a 8-l e c t i n , or as an enzyme. The apparently small amount of proteoglycan i s o l a t e d from the alga (minimum of 32 mg dry wt from 800 gm of fresh alga), suggests a non-structural r o l e and a non-involvement as a precursor for su l f a t e d galactans, t y p i c a l components of red a l g a l - ' c e l l walls (Percival and McDowell 1967). I do not know of any reports concerning other l e c t i n types'in the red algae. 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