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Fine structural changes of Tribonema minus Hazen during desiccation Nakahara, Emmy 1972

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FINE STRUCTURAL CHANGES OF TRIBONEMA MINUS HAZEN DURING DESICCATION by EMMY NAKAHARA B.Sc, University of Toronto, 1 9 7 0 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept th i s thesis as conforming to the required standard The University of B r i t i s h Columbia August, 1 9 7 2 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Botany  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date August 18, 1972 ABSTRACT i i . Pine s t r u c t u r a l changes i n vegetative c e l l s of Tribonema  minus during a i r - d r y i n g were examined. The nucleus became contracted into a dense sphere and chromatin condensation occurred. There was reduction i n width of the c e l l wall, p e r i -nuclear and perlchondrial spaces. Increased width and staining of the plasma membrane and inner mitochondrial membrane was observed. Concomitantly, Increased homogeneity of the mitochondrial and chloroplast matrices occurred. Mitochondrial and chloroplast genophores became indistinguishable from t h e i r matrix. Increased water loss was characterized by the fusion of the three-thylakoid bands within the chloroplasts. L i p i d was synthesized i n the chloroplast and transported through the chloroplast endoplasmic reticulum to accumulating areas continuous with i t . The product appeared to be the means by which the c e l l stored water and metabolites during desiccation. i i i . TABLE. OP CONTENTS page LIST OF TABLES i v LIST OF FIGURES'. v ACKNOWLEDGMENTS v i i INTRODUCTION 1 LITERATURE REVIEW 3 METHODS AND MATERIALS 7 RESULTS AND OBSERVATIONS The ultrastructure of a vegetative c e l l 11 Changes during desiccation 16 DISCUSSION 19 REFERENCES 31 PLATES AND EXPLANATIONS bo APPENDIX 51 i v . LIST OP TABLES page Table 1.. Weight l o s s during d e s i c c a t i o n 9 V , LIST OF FIGURES page Figure 1 . Outline of procedure f o r sampling desiccation stages, 8 Plate I Figure Figure Vegetative c e l l of Trlbonema minus, 2 . Longitudinal view 3 . Transverse view kl Plate II Figure k. Figure 5 . Figure 6 . Figure 7 . Nucleus 1 0 0 $ RH c e l l 60% RH c e l l 50% RH c e l l . 50% RH c e l l k2 Plate III Figure 8 . Figure 9 . Figure 1 0 . Figure 1 1 . Nucleus 30% RH c e l l 20% RH c e l l 10% RH c e l l 0% RH c e l l ^ 3 Plate IV Figure 1 2 , Figure 1 3 . Figure lk, Figure 1 5 , Mitochondria, 60% RH c e l l 60% RH c e l l 20% RH c e l l 20% RH c e l l kk Plate V Figure 1 6 , Figure 1 7 . Figure 1 8 , Figure 1 9 , Chloroplasts, 1 0 0 $ RH c e l l 100% RH c e l l 60% RH c e l l 60% RH c e l l P l a t e VI F i g u r e 2 0 . F i g u r e 21 . F i g u r e 2 2 . F i g u r e 2 3 . C h l o r o p l a s t s , 50% RH c e l l 50% RH c e l l k0% RH c e l l 30% RH c e l l P l a t e V I I F i g u r e 24. F i g u r e 25 . F i g u r e 26. F i g u r e 27. C h l o r o p l a s t s , 20% RH c e l l 20% RH c e l l 10$ RH c e l l 0% RH c e l l P l a t e V I I I F i g u r e 28. F i g u r e 29. F i g u r e 30 . G o l g i apparatus, 60% RH c e l l 20% RH c e l l 20$ RH c e l l P l a t e IX Vacuole , F i g u r e 31 . 20$ RH c e l l F i g u r e 32 . 20% RH c e l l P l a t e X F i g u r e 3 3 . F i g u r e 3k. F i g u r e 35 . C e n t r l o l e s and microtubules 100$ RH c e l l 60$ RH c e l l " RH c e l l ACKNOWLEDGMENTS To Dr. Thana Bisalputra f o r his guidance, encouragement and enthusiasm. To Dr. J.R. Stein and Dr. I.E.P. Taylor f o r t h e i r assistance i n preparation of this t h e s i s . To Dr. D.J. Hibberd* f o r i d e n t i f y i n g the species examined. To my family and friends for t h e i r interest and concern. •Curator of Freshwater Algae The Culture Centre of Algae and Protozoa Cambridge, England. INTRODUCTION Algae have been reported to occur i n a e r i a l , freshwater, marine and s o i l habitats susceptible to desiccation ( 3 , 7 , 1 1 . 1 2 , 2 0 , 2 1 , 3 1 , 3 2 , 3 3 . 3 8 , 3 9 , 4 2 , 5 7 . 6 7 , 9 1 . 1 0 4 ) . Resistance to water loss appears to vary with the phyclological state of the organism and i t s immediate stress condition ( 3 2 , 4 1 , 4 3 , 5 5 , 7 3 , 7 4 ) . Special a l g a l c e l l s such as akinetes, aplanospores, cycts and hypnospores have been observed to be produced to escape and/or endure temporary adverse conditions ( 3 1 , 6 7 , 9 1 ) . Some species appeared to r e t a i n t h e i r vegetative morphology although modifications such as thickened walls, f a t t y p e l l i c l e or c u t i c l e , mucilage and l i p i d storage granules occurred ( 1 3 , 3 1 . 3 2 , 4 3 , 9 1 ) . The effects of complex i n t e r r e l a t e d reactions on b i o l o g i c a l systems at t r i b u t e d to desiccation have not been sharply distinguished from those produced wholly or i n part by another such as growth, aging or n u t r i t i o n a l depletion ( 3 2 , ^ - 3 , 5 5 , 7 3 , 7 4 , 9 1 ) . A comprehensive review of the Xanthophyta was presented by Massalski ( 7 1 ) and the fine structure of several Xanthophycean vegetative c e l l s and zoospores have been examined ( 1 7 , 1 8 , 2 2 , 2 3 , 3 5 . 3 6 , 4 7 . 4 8 , 5 6 , 6 3 , 7 0 , 7 1 , 7 2 , 7 6 ) . Petersen ( 9 1 ) reported that Trlbonema bombyclnum (Ag.) Derb. et S o l . occurred i n shallow bodies of freshwater known to undergo extreme fluctuation"of water stress. Because cysts were not found i n his investigation, vegetative stages were believed to be r e s i s t a n t to drought. Hawlltschka (52) reported akinetes and aplanospores were formed 2. by the same species. Thus, a question arose. How do vegetative c e l l s of Tribonema become modified f o r "desiccation resistance"? In this t h e s i s , adaptations to water stress are reviewed and the f i n e s t r u c t u r a l changes which occur during desiccation of vegetative c e l l s of Tribonema minus Hazen are described. The small size of th i s species allowed the examination of several c e l l s i n one section making i t possible to standardize the age of the s t a i n solutions and the staining time f o r the stages examined. The information may provide the bases f o r chemical, physiological and histochemical studies on the effects of water loss from c e l l s and i t s re l a t i o n s h i p to species resistance. LITERATURE REVIEW WATER Investigations of water as the medium from which l i f e began and i n which i t continues have prompted extensive discussions and speculations (6 ,1^,15 ,2^) . Presently, the diverse properties and functions of l i q u i d water are explained by a " f l i c k e r i n g c l u s t e r " model which describes water exi s t i n g i n a continual f l u x between single and groups of hydrogen bonded water molecules (80). Water i s considered to play three major roles within a c e l l : as a metabolite, a medium and an i n t e g r a l s t r u c t u r a l unit ( 6 ) . As a solvent, water Is responsible f o r the d i s t r i b u t i o n of nutrients within a system and also provides the necessary aqueous medium f o r enzyme a c t i v i t y and metabolic processed such as protein synthesis (6,1*0. R e l a t i v e l y free nonhydrogen bonded molecules flowing through c e l l walls and protoplasmic:membranes can act as substrates i n enzyme catalyzed reactions such as photosynthesis. Moreover, the small size of the water molecules enables them to f i l l small i n t e r s t i c e s i n macromolecules-(1*0 . Advanced studies tn the past few years have considered Szent-Gy8rgyi fs concept of "structured water" and i t s ef f e c t on macromolecules ( '6 ,37 ,53.61) . Hazelwood, Nichols and Chamberlain (53) suggest that water i n protein macromolecules exists i n two»dlstinct phases - the major phase consisting of water molecules which have l o s t considerable motional freedom r e l a t i v e to free water and the minor phase containing water molecules which have less motional freedom than i n the major phase, but more than i n s o l i d s . 4 Evidence supports the occurrence of ordered "bound" water of which the degree of p o l a r i z a t i o n and deformation varies upon s p e c i f i c binding interactions at the interfaces of macromolecules (14,81,82). Within a confined space, organized macromolecules are believed to assemble with bound enzymes (103) to form interconnected membranes which segregate into discreet functional units defined by i t s p a r t i c u l a r environment (114). Thus, c e l l u l a r water i n plant c e l l s exists i n heterogenous aqueous regions! within membrane structures; as one or more layers of "bound" water of macromolecular surfaces; between c l o s e l y paired systems of membranes e.g. perinuclear, perichondrial spaces, between associated thylakoids and"the l n t e r c i s t e m a e of endoplasmic reticulum and Golgi apparatus; enclosed within organelles; and l n the hyaloplasm between various organelles. DESICCATIONS EFFECTS Desiccation.affects the continuum of water existing-within-a l i v i n g c e l l and a l t e r a t i o n of the physical and/or chemical property of the solvent would induce changes to occur i n the cytoplasm (66,107). In p a r t i c u l a r , water stress has been reported to cause a decrease i n free energy and to lower water po t e n t i a l specifying the need f o r a more highly ordered form of water (14). Crafts (14) summarized the effects of desiccation on water structure as follows: 1..increased molecular structuring bringing about thickening of multilayers at interfaces, 2. Increased f r a c t i o n a l resistance to movement re s u l t i n g from increased v i s c o s i t y (immobilization),and 5. 3.- strengthening of menisci and other surface f i l m as a re s u l t of increased surface tension. At macromolecular surfaces, where water normally._occurs,, i t has been proposed, that there is_ a. tendency f o r .molecules of water to be held tenaciously to the evaporation surface forming layers of ordered water (1*0. Furthermore, as water i s l o s t from hydrophobic l i p o p r o t e i n and phospholipid regions, i t was suggest-ed that there may be an accentuated squeezing out of water by the i n t e r a c t i o n of hydrocarbon t a i l s becoming at least p a r t l y con-fluent (15). Modifications such as these are believed to r e s t r i c t the movement of water and water-soluble solutes. Physiological examinations reveal that during water loss i n higher plants there i s an increase i n ribonucleic acids and free amino acids suggesting hydrolysis occurs (1,100,113). Water stress causes i n h i b i t i o n of photosynthesis (115)» c e l l u l o s e synthesis (85), phosphorylation (53.88), protein synthesis (58, 92,93) and g l y c o l y s i s (100). Thus, desiccation favours the a c t i v i t y of a p a r t i c u l a r enzyme or group of enzymes (5*0 which i n turn a f f e c t the properties of the protoplasm. Increases i n concentration of sugar i n the cytoplasm during desiccation of fungi (111) and higher plants (89) were noted. Also, i n o s i t o l , a polyhydroxycyclic saturated hydrocarbon, has been reported to be an e f f e c t i v e protective substance during water loss from b a c t e r i a l c e l l s (112). Parker (89) pointed out that six-carbon sugars are chemically s i m i l a r to i n o s i t o l having numerous hydroxy groups and often occur i n saturated rings. This was correlated to the starchysugar s h i f t s i n plants exposed 6 to drought or cold. In both cases, hydroxy groups were believed to replace bound water within and on-protein macromolecules by hydrogen-bonding thus protecting drought r e s i s t a n t enzymes. MATERIALS AND METHODS 7. Tribonema minus Hazen was obtained from the Culture C o l l e c t i o n of Algae, Indiana University (Collection Number 639. 1 0 6 ) . Nonaxenic cultures were maintained i n 250 ml Erlenmeyer fl a s k s containing 200 ml of B r i s t o l ' s modified l i q u i d medium (see appendix) f o r one week. At the end of t h i s period, the cultures were i n t h e i r exponential growth phase.. A f t e r vigorous a g i t a t i o n of a culture f l a s k , 5 ml aliquots of the culture were removed with s t e r i l e 5 ml glass pipettes (see P i g . l ) . One ml samples were transferred to p e t r i plates containing ca 9 ml s o l i d 1 .0$ (w/v) agar of nutrient medium. To insure an even d i s t r i b u t i o n of c e l l s , only 3 ml of the culture were pipetted unto the plates. Seven plates were prepared l n this manner. A control plate of agar medium was prepared. The plates were then l e f t uncovered, exposed to the a i r , i n a 20+2°C chamber Illuminated with fluorescent l i g h t on a l6:"B~ l i g h t period The extent of desiccation was defined as r e l a t i v e hydration (RH; see Table 1 ) . Vegetative c e l l s i n l i q u i d culture (100# RH) were of a hydration state s i m i l a r to those growing i n shallow freshwater ponds. Cel l s which had been a i r - d r i e d f o r 36 hours (0% RH) were considered to be i n a dehydrated state s i m i l a r to those c e l l s which have been exposed to drought. U l t r a s t r u c t u r a l comparisons of 100, 6 0 , 5 0 , 40, 30 , 2 0 , 10 and 0% RK'stages were ca r r i e d out and the changes examined. 8 Nonaxenlc culture of Tribonema minus Hazen was grown 1 week to exponential growth phase. Culture vessel agitated, 5 ml of T. minus were pipetted out. 1 ml samples were transferred unto 9 ml of 1 .0$ (w/v) nutrient agar plates. 7 experimental plates and a control plate (no alga) uncovered; at 20 C Illuminated with fluorescent l i g h t ( l 6 : S ) . Samples (0 .25 cm2) were f l o a t e d on cza the surface of 200 ml l i q u i d / a * medium and placed / \ at 20 C; l 6 : S . > Samples of each stage were taken at a l l r e l a t i v e hydration stages (RH). Samples prepared f o r electron microscope study (Table 1 ) . Figure 1. Outline of procedure f o r sampling desiccation stages. Table 1 . Weight Loss During Desiccation ( i n g): Weight loss from the system of the Inoculum, agar and p e t r i plate was measured. Relative hydration was defined as exposed weight x 1 Q Q ^ T h e l n l t l a l w e i s r h t was the t o t a l weight of 1 .0 ml T. minus i n i t i a l weight — l i q u i d culture, agar and p e t r i plate. The exposed weight was the measured weight a f t e r each exposure (sample) time. A l l weight measurements were made of the plates with t h e i r respective covers which were kept beside them i n the growth chamber during desiccation. Stage {%RH) 100 60 50 40 30 20 10 Control Plate Plate + agar I n i t i a l weight Exposed weight Sample time (hr) Relative hydration 0 100 17 . 6 2 4 2 5 - 9 3 0 2 6 . 9 7 2 2 3.214 28 . 8 0 5 9 . 8 0 1 7 . 0 8 6 25.845 2 6 . 8 7 1 2 1 . 9 1 4 3 0 . 8 3 4 9 . 3 4 17.594 26.408 2 7 . 6 1 6 21.566 31.42 39.63 1 6 . 8 1 3 2 5 . 5 7 5 2 6.644 1 9 . 5 7 1 3 2 . 2 5 2 8 . 0 5 1 7 . 6 2 3 26 . 4 1 5 2 7 . 4 3 6 19.488 3 2 . 3 3 1 9 . 0 1 1 6 . 8 1 4 2 5 . 7 6 8 2 6 . 8 3 7 1 7 . 7 3 4 33.42 9 . 1 8 1 7 . 5 9 6 2 6 . 2 6 2 2 7 . 3 6 0 17 .711 3 6 . 0 0 1 .18 1 7 . 2 5 9 2 6 . 3 4 7 : *26 ;347 1 7 . 3 6 0 3 6 . 0 0 * no culture was added. 10 Samples (0.25 cm^) of each desiccation stage were fl o a t e d on the surface of 200 ml of l i q u i d medium i n 250 ml f l a s k s . V i s u a l evidence of resumed growth was observed i n each case. At the same time, samples f o r u l t r a t h i n sectioning i n preparation f o r electron microscopy were f i x e d f o r 1 hr i n 2.5^ (v/v) glutaraldehyde i n 0.1M sodium cacodylate buffer (pH 6.8) at room temperature. During water l o s s , sucrose was added to the buffer (up to 5-0% (w/v) sucrose f o r 0% RH c e l l s ) to reduce osmotic shock. After three 10 min rinses in buffer, the material was post-fixed with 1 .0$ (w/v) OsOty i n cacodylate buffer f o r 1 hr. Afte r washing thoroughly with d i s t i l l e d water, the material was dehydrated i n a graded ethanol series then i n f i l t r a t e d and embedded i n Spurr*s medium ( 105) . Following polymerization i n a vacuum oven at 65 C f o r 11 hr, sections were cut with a du Pont diamond knife on a Reichert Omu 3 ultramicrotome. The sections were post stained with saturated uranyl acetate i n 70% (w/v) methanol and lead c i t r a t e ( 9 5 ) . C e l l s adjacent to the a p i c a l c e l l s were examined primarily. Six c e l l s of each stage were measured. Observations were made with a Zeiss EM 9A electron microscope. RESULTS AND OBSERVATIONS 11. THE ULTRASTRUCTURE OF A VEGETATIVE CELL The unbranched filaments of Tribonema minus were composed of c y l i n d r i c a l c e l l s (2-3 pm wide x 5-7 pm long) i n which the H-pieces of the wall were conjoined u n t i l t h e i r d i s a r t i c u l a t i o n during growth and reproduction. Surrounding a central nucleus, the cytoplasm contained two to four peripheral chloroplasts, endoplasmic reticulum, a single Golgi apparatus, mitochondria and vacuoles (Fig.2). The morphology of the vegetative c e l l s of various states of hydration s i m i l a r to the 100$ RH c e l l s are indicated. CELL WALL AND PLASMA MEMBRANE: Cell s of 100$ RH had tapering outer cup-like c e l l walls (OW) 2300 +300 A wide which overlapped the shorter inner walls (IW) produced a f t e r nuclear d i v i s i o n (Figs.2 , 3 ) . Wall formation appeared to involve the accumulation of wall material within the c e l l wall membrane (CM) ca 100A wide ex t e r i o r to, but separated by a liol space from the 100A wide plasma membrane ( F i g . 3 ) . NUCLEUS: Nuclei were t y p i c a l of those found i n eukaryotic c e l l s (28). S l i g h t l y elongated interphase n u c l e i (N) of c e l l s between 100 and 20$ RHwere 1.5-2.0 um x 2.0-2.5 jxm (Figs.2,*4-9). The outer nuclear membrane (ON), 70A wide, was often associated with ribosomes (R) and extended into the cytoplasm as rough endoplasmic reticulum (RER, Figs. 5 , 6 , 8 ) . Vesiculation of the outer nuclear membrane occurred along the flattened faces of the i n the v i c i n i t y of the forming face of the Golgi (G, Figs. 4,5,8, 12. 28-30). In comparison, the inner nuclear membrane (IN), ca 70A wide, was associated with chromatin and was separated from the outer nuclear membrane by a perinuclear space (PN) 200 - 2*K)A wide (Figs.4 - 9 ) . Nuclear pore complexes (NP) 400A wide interrupted the continuity of the nuclear membrane (Fig.9). The c i r c u l a r pore o appeared surrounded by an annulus 100A wide which encircled a medium dense pore area 200A i n diameter. Nucleoli (NU) were densely stained areas associated with chromatin (Figs.2,5 ,7 - 9 )• V i s i b l e in oblique sections, the n u c l e o l i extended over an area ca 700A across consisting of a pars granulosa (PG), pars f i b r o s a (PF), pars chromosoma (PC) and a central "vacuole" (Nv) devoid of f i b r i l s (Fig.7). MITOCHONDRIA: Mitochondria maintained a d e f i n i t e s p a c i a l r e l a t i o n s h i p , c l o s e l y associated to the chloroplasts, the Golgi apparatus and the nucleus. The double membrane bound mito-chondria (M) of 100 to 20$ RH c e l l s were polymorphic. They appear-ed as large spherical organelles 2 x 3 pa (Fig.2) and elongate or disc-shaped (Figs.12,14,15,28,29,3D. The outer mitochondrial membrane (OM) was ca 50A wide and separated by an 80A perichondrial space (PCS) from the wider, ca 6 0 A , intensely stained inner membrane (IM). The l a t e r invagin-ated to form the tubular c r i s t a e (CR, Figs.12-15). CHLOROPLASTS: Peripheral chloroplasts (CH) were bound by a double membrane envelope. Ce l l s of 100 to 20$ RH had outer (OC) and inner (IC) chloroplast membranes, 70I wide, separated from 13 each other by an i r r e g u l a r space ca 90A wide (Pig.19).- Examin-a t i o n of the cross (Pigs.17,2 6 ,2?) and l o n g i t u d i n a l : s e c t i o n s (Pigs.16,18 - 2 5 ) r e v e a l e d i n t e r n a l membrane systems comprised of three c l o s e l y a s s o c i a t e d t h y l a k o l d s . C h l o r o p l a s t bands were ca 820A wide and Included three t h y l a k o l d s each 245A wide (80A wide membrane surrounding an 85A wide l o c u l u s ) which were sepa r a t e d by a space ca 40JL The p e r i p h e r a l bands (PL) and each of the u s u a l l y f i v e c e n t r a l bands (CL) appeared t o be separated by a 580A stroma space (Pigs.21,22,24). The c h l o r o p l a s t stroma (S) took up s t a i n more h e a v i l y than the c y t o p l a s m i c matrix i n c e l l s of 100 to 20$ RH. D i s t i n c t l i p i d d r o p l e t s (L) were found i n the stroma between the t h y l a k o l d e bands ( F i g s . l 6 - l 8 , 2 0 , 2 4 , 2 6 ) . E l e c t r o n t r a n s p a r e n t regions of the c h l o r o p l a s t stroma c o n t a i n i n g c h l o r o p l a s t genophores (CG) were c l e a r l y v i s i b l e ( Figs. 1 6 ,20 - 2 3 ) . Extensions of the outer n u c l e a r membrane e n c i r c l e d the c h l o r o p l a s t forming the c h l o r o p l a s t endoplasmic r e t i c u l u m (9; CER, P i g . 9 ) . The CER was c l o s e l y adpressed to or continuous with the c h l o r o p l a s t membrane (Figs.18 , 1 9 ), extending from the c h l o r o p l a s t and a s s o c i a t e d w i t h polyribosomes forming RER ( F i g s . 4 , 2 3 ) , or connected to a system of l i p i d storage areas (LS, F i g s . 18,24 - 2 7 ) . ENDOPLASMIC'RETICULUM:AND RIB0S0MES: Rlbosomes occ u r r e d f r e e l y i n the cytoplasm of 100 to 0% RH c e l l s (Figs. 5 , 6,8-11)., In a d d i t i o n , polyribosomes (PR, Fig.6 ) were a t t a c h e d t o membranes of the RER (Figs.10,11 , 2 3 ) or t o tubules extending from the o u t e r 14. n u c l e a r membrane ( F i g s . 6 , 8 ) . On the oth e r hand, a s s o c i a t i o n of ribosomes on v e s i c l e s a l o n g the f l a t t e n e d n u c l e a r f a c e formed the i n i t i a l phase of dictyosome for m a t i o n (Figs.8,28, 2 9 ). In o b l i q u e planes of s e c t i o n i n g , ER appeared as sheets of c i s t e r n a e ( F i g . 2 3 ) . GOLGI APPARATUS: A mentioned p r e v i o u s l y , v e s i c u l a t l o n (NV) of the n u c l e a r membrane of 100 to 20$ RH c e l l s o c c u r r e d a t the proxi m a l f a c e of the G o l g i apparatus (Px). T h i s a c t i v i t y appeared as the primary f a c t o r i n m a i n t a i n i n g the G o l g i apparatus which was 1 pm i n diameter (G, F i g s . 2 , 4 , 5 , 8 , 2 8 - 3 0 ) . However, v e s i c l e s . f o r m e d from the extended p o r t i o n s of the n u c l e a r membrane were added t o the G o l g i c i s t e r n a e ( F i g . 2 9 ) . Seven G o l g i c i s t e r n a e were present i n 100 to 30$ R H " c e l l s ( F i g s . 4 , 8 , 2 8 ) . D i f f e r e n t i a t i o n of the G o l g i c i s t e r n a e ( Figs.28 - 3 0 ) i n v o l v e d 1. the enlargement of i n t e r c i s t e r n a l space along the circumference o f the c e n t r a l G o l g i p l a t e s from 150 to 300A, 2 . the l o s s o f ribosomes towards the d i s t a l f a c e ( D l ) , 3 . i n c r e a s e d s t a i n a b i l i t y of the c i s t e r n a l membrane towards the mature f a c e , and 4 . v e s i c u l a t l o n along the mature f a c e producing s i n g l e membrane bound (80 - 100A t h i c k ) G o l g i v e s i c l e s (GV) and coated G o l g i v e s i c l e s (CGV). Some v e s i c l e s produced by the G o l g i apparatus j o i n e d to form p l a t e s of storage and/or t r a n s p o r t systems (*,,Fig.28). 15 VACUOLES: Vacuoles (V) of c e l l s of 100$ RH were ca 1 . 3 urn l n diameter and c e n t r a l l y l o c a t e d ( P i g . 1 7 ) . Bound by a s i n g l e membrane (80A wide), the vacuoles appeared t o c o n t a i n v e s i c l e s and l i p i d g l o b u l e s . CENTRIOLES AND MICROTUBULES: A p a i r of c e n t r i o l e s (CN) p e r p e n d i c u l a r to each other o c c u r r e d near the nucleus where the w a l l p i e c e s overlapped i n 100 and 60$ RH c e l l s ( P i g s . 3 3 , 3 4 ) . C e n t r i o l e s (2000l i n diameter) c o n s i s t e d of a r i n g of nine t r i p l e t s of microtubules (200A i n diameter) arranged l i k e a cartwheel (Pig. 3 3 ) . The proximal ends of the c e n t r i o l e s showed dense condensations p r o j e c t i n g from the A-tubules toward the centre t o form a s e r i e s of n f e e t " meeting c e n t r a l l y i n a dense mass (44). The i n t e r c o n n e c t i o n of tubules resembling a 9-polnted s t a r d e s c r i b e d a t the d i s t a l ends of the b a s a l bodies (71) were not seen. Surrounding each c e n t r i o l e was a r e l a t i v e l y c l e a r a r e a c a l l e d the centrosphere (CS) which c o n t a i n e d few ribosomes and f i b r o u s i n c l u s i o n s (45 , P i g . 3 3 ) . M i c r o t u b u l e s (MT) 200A i n diameter were of v a r i o u s lengths appearing p a r a l l e l to or r a d i a t i n g from the c e n t r i o l e a r e a . In c e l l s o f 100 to 20$ RH, microtubules were found between G o l g i v e s i c l e s and near the c e l l s u r f a c e ( P i g s . 8 , 9 , 3 4 , 3 5 ) . 16. CHANGES DURING: DESICCATION CELL WALL AND PLASMA MEMBRANE: During desiccation, the c e l l wall decreased i n width from less than 2000A of 100$ RH c e l l s to 1600 +100A i n 0$ RH c e l l s (Fig.2 7 ) . The inner and outer c e l l walls of 100$ RH c e l l s did not take up st a i n (Fig.3 ) . "but those walls of 0$ RH c e l l s appeared stained i n multilayers as seen by alter n a t i n g dark and opaque layers (Figs.11 , 2 7 ) . o The width of the plasma membrane increased from 100A i n 100$ RH c e l l s (Fig.3) to 160A i n 10$ RH c e l l s (Fig.26), and to ca 200A i n 0$ RH c e l l s (Fig.2 7 ) . Increased s t a l n a b l l i t y of the membrane occurred d i r e c t l y with increased water l o s s . NUCLEUS: Nuclei of 10 and 0$ RH c e l l s appeared c i r c u l a r with a diameter of 1.5 pm (Figs.1 0 ,11) as compared to elongate i n c e l l s of > 2 0 $ RH. The perinuclear space decreased from ca 200A i n 100$ RH c e l l s to ca 150A wide i n 10 and 0$ RH c e l l s (Figs. 1 0 ,11). Cell s of 60 to 20$ RH were observed to have undergone chrom-a t i n condensation (CC) with increased staining along the inner nuclear membrane (Figs.5-9). The nucleolar regions of 10 and 0$ RH c e l l s were unrecognizable and the chromatin during the l a t t e r stages of desiccation was highly condensed and deeply stained (Figs.10,11). In addition, water loss was accompanied by i n -creased density of the nucleoplasm. The nuclear pores maintained t h e i r t y p i c a l structure (40) throughout desiccation; The pore region of c e l l s of 0$ RH appeared occupied by a fibrous component (Fig.11). 17 MITOCHONDRIA: Water l o s s caused a decrease i n the p e r i -c h o n d r i a ! space from 80A i n 100 and 60$ RH c e l l s (Pigs.3 , 1 3 ) to 50A i n 20$ RH c e l l s ( F i g . 1 5 ) . The i n n e r m i t o c h o n d r i a l membrane i n c r e a s e d i n width from ca 60A. i n 60$ RH c e l l s (Pigs.12,13) to 80A a f t e r 20$ RH ( F i g s .14,15). Also, the m i t o c h o n d r i a l matrix (MM) appeared to s t a i n more i n t e n s e l y i n 20$ RH c e l l s than i n 60$ RH c e l l s . The genophore r e g i o n became g r a d u a l l y i n d i s t i n -g u i s h a b l e w i t h i n c r e a s e d d e s i c c a t i o n (MG, Pig. 1 5 ) . CHLOROPLASTS: The extent of a s s o c i a t i o n between the t h y l a -k o lds determined the width of the bands. With Increased water l o s s , there was a t i g h t e r a s s o c i a t i o n of the t h y l a k o l d s . C e l l s of 40$ RH had bands 580A wide ( P i g . 2 2 ) . C e l l s of 10 and 0$ RH had bands ca 500A wide se p a r a t e d by ca 330A stroma space ( F i g s . 2 6 , 2 7 ) . However, the d e f i n i t e s p a c i a l arrangement of the p e r i p h e r a l and c e n t r a l bands was r e t a i n e d . Upon d e s i c c a t i o n , there was a d e f i n i t e l a c k of s t a i n uptake by the t h y l a k o l d s ( F i g . 2 7 ) . With i n c r e a s e d water s t r e s s , the l i p i d d r o p l e t s became l e s s d i s t i n c t i n 10 and 0$ RH c e l l s and the homogenous stroma was very l i g h t l y s t a i n e d ( P i g s . 2 6 , 2 7 ) . The genophore r e g i o n was no l o n g e r d l s t i n g u i h a b l e i n 0$ RH c e l l s ( P i g . 2 7 ) . GOLGI APPARATUS: There was continued v e s i c u l a t i o n of the c i s t e r n a e along the d i s t a l f a c e of the G o l g i apparatus, but the number of c i s t e r n a e was reduced from seven i n 100 to 30$ RH c e l l s to f o u r l n 20$ RH c e l l s ( F i g . 2 9 ) . A G o l g i apparatus was not d e t e c t e d i n c e l l s o f 10 and 0$ RH although v e s i c l e s were present 18 (Pigs.2 6 , 2 7 ). However, the exact o r i g i n of the ve s i c l e s could not "be determined by structure alone. VACUOLES: Vacuoles became reduced i n size from ca 1 .3 pm l n diameter at 100$ RH (Fig.1 7 ) to 0 . 7 pm i n diameter by 20$ RH (F i g . 3 2 ) . No vacuoles were found i n 10 or 0 $ RH c e l l s . CENTRIOLES'AND MICROTUBULES: The presence of centrioles were not detected i n a l l stages because s e r i a l sections wrere not taken. The morphological conihuity of the centrioles of T. minus i s believed to have occurred s i m i l a r to that observed i n animal c e l l s ( 4 5 ) . Microtubules were not seen i n 10 or 0 $ RH c e l l s . 1 9 . DISCUSSION1 CELL WALL AND PLASMA MEMBRANE The H-pleces of the c e l l wall of Tribonema minus appeared to be composed of a homogenous opaque substance. The cup-shaped walls f i t t e d together l i k e "dove-tails" ( 1 0 9 , P i g . 2 ) . Tiffany, l n 1924 ( 1 0 9 ) , found by s o l u b i l i t y , hydrolysis and o p t i c a l tests that the c e l l walls of species of Tribonema contain pectic acid, pectose and c e l l u l o s e . The v a r i a t i o n was believed to be a matter of age, the older filaments containing less pectic compounds and a greater amount of c e l l u l o s e . Haas and H i l l (50) defined pectose, a galacturonic a c i d derivative i n higher plants, as the insoluble precursor of true pectins and pectic a c i d as the demethylated form of pectin. More recent work by N i c o l a i and Preston (83) has proven that c e l l u l o s e , which i s mainly glucose, exists i n c e l l walls of Tribonema sp.. Although c e l l u l o s i c c e l l walls are uncommon i n the Xanthophyceae ( 6 0 ) , c e l l u l o s e has been reported to occur i n some filamentous species ( 6 0 , 6 8 , 8 7 , 9 7 ) . Multilayers of c e l l u l o s e i n T. minus was evidenced by the staining pattern of dark and l i g h t layers i n c e l l s which had been a i r - d r i e d . Similar layered form-ations occurring not i n the wall, but i n the p r o t e i n - r i c h c u t i c l e of two Chlorophyceae - Cladophora rupestrls (L.) Kutz. and Chaetomorpha me lag on 1 urn (Weber et Mohr) Kiitz. - are believed to r e f l e c t d i f f e r e n t chemical compositions of the heavily and l i g h t l y stained areas ( 5 1 ) . It was suggested that the dark 20. staining m i c r o f i b r i l l a r portion was a protein f r a c t i o n and the other, carbohydrate. The c e l l u l o s e l n the c e l l wall of T. minus may be coupled by another f r a c t i o n , possibly protein, which can undergo changes i n s t r u c t u r a l configuration due to water l o s s . Constriction of the c e l l wall may indicate increased structuring of water on the macromolecular surfaces composing the c e l l wall which was followed by the r e s t r i c t i o n of further movement of cytoplasmic water and water-soluble solutes s i m i l a r to that proposed to occur i n higher plants during drought (14). Increase i n width of the plasma membrane from 100A at 100$ RH to ca 200A at 0% RH and the increased s t a i n uptake due to water loss suggest the c o n s t r i c t i o n of membrane subunlts and perhaps the addition of a protective substance to the plasma membrane to l i m i t any further loss of water from the cytoplasm (Figs.3.19,24,26,27). NUCLEUS The condensation of chromatin during desiccation was s i m i l a r to that seen i n spermiogenesis of animal c e l l s during which chromatin f i b e r s shortened and condensed into coarse, ^ i r r e g u l a r dense granules which l a t e r became compacted into a dense homogenous mass ( 6 5 ) . Du Praw (29) has interpreted s i m i l a r chromatin aggregates as various c o i l e d stages of DNA fi b e r s with histones and non-histone proteins. Furthermore, the 21. a c t i v i t y of DNA tr a n s c r i p t i o n has been related to the c o i l i n g I.e. only those portions of uncoiled, naked DNA which appears electron transparent are ac t i v e . I f this analogy i s continued, the interphase nucleus (Pigs.4-9) was a c t i v e l y transcribing between 100 and 20$ RH a f t e r which there was increased DNA c o i l i n g i n 10 and 0$ RH c e l l s (Pigs.10,11) such that t r a n s c r i p t i o n was blocked, RNA production ceased and the remaining RNA i n the nucleus was released. The o v e r a l l effect was r e f l e c t e d i n the decreased perinuclear space and reduced v e s i c u l a t i o n towards Golgi formation.. MITOCHONDRIA The morphology of mitochondria i n 100 to 20$ RH c e l l s was s i m i l a r to that reported i n Xanthophyceae (17,35,56,63,71,72,76) and other eukaryotes (see reviews 28,64,103). However, poly-morphism of mitochondria occurred i n T. minus r e f l e c t i n g changes i n the a c t i v i t y of the mitochondrial and the cytoplasmic DNA as discussed by Du Praw ( 2 9 ) . The inner membrane i s believed to be composed of multi-enzyme systems associated i n globular complexes such that l i p i d s form l i p o p r o t e i n complexes and also patches of l i p i d bilayers extending between aggregates of protein molecules ( 1 0 3 ) . The res u l t s show that desiccation causes an increase i n the width and staining of the inner mitochondrial membrane from oa 60A i n c e l l s of 60$ RH (Pigs.12,13) to 80A i n c e l l s of 20$ RH (Pigs.14, 15). As water was l o s t , the hydrophobic bonded l i p o p r o t e i n and 2 2 . phospholipid regions appear to have undergone an accentuated water loss and therefore, may have become at least p a r t l y confluent (14). Baxter (2) suggested that the more "soluble' 1 enzymatically active proteins i s synthesized at extra-mitochondrial s i t e s under the genetic control of nuclear DNA. Thus, the outer mitochondrial membrane must act as a d i f f e r e n t i a t i n g membrane regulating the entrance and output of metabolites and products. Furthermore, the perichondrial space would be a temporary storage and perhaps a c t i v e l y transporting area being the intermediate s i t e between two very d i f f e r e n t areas of a c t i v i t y . The decrease i n the perichondrial space from 80A of 60$ RH c e l l s (Fig.13) to 50A of 20$ RH c e l l s (Fig.15) due to water loss implies reduced a c t i v i t y of th i s region. The mitochondrial matrix stained more intensely than the cytoplasmic matrix during desiccation. Electron transparent genophore areas ( 7 8 , 7 9 ) containing the mitochondrial DNA occurred. With desiccation of c e l l s of 100 to 20$ RH, there was increased density of the mitochondrial matrix (Figs . 1 2 - 1 5 ). This increased s t a i n a b i l i t y and homogeneity of the matrix occurred at the same time the genophore became Indistinguishable. 23. CHLOROPLASTS Chloroplasts of T. minus were similar to those described by Massalski and Leedale for T. vulgare Pascher (72). The photosynthetic membranes occurred as peripheral and central bands composed of three-thylakoid lamellae common for Xanthophyceae (17,18,35,56,71.72,76), Chrysophyceae and Dino-phyceae (25, see review 46) and Bacillariophyceae and Phaeo-phyceae (27,34,46). Increased water loss caused a decrease in the width of the bands from 820A at 100$ RH, 580A at 40$ RH to 500A by 0$ RH. The close association of the thylakolds within the bands may have been due to the exclusion of water from the interband region caused by hydrophobic bonding as discussed by Kirk for higher plants (59). The d i f f i c u l t y ln distinguishing the occurrence and frequency of fusion of thylakolds in two filamentous Xanthophytes Is reviewed by Massalski and Leedale (72) and may be c l a r i f i e d by the freeze etching method of examination. The Inability of the chloroplast thylakolds in c e l l of 0% RH to take up stain suggests changes occurred in the molecular configuration of the membrane components. Nevertheless, the peripheral and central spatial relationship of the bands was maintained (Pig.27) as found in a drought resistant moss, Antltrlchla californlca S u l l . (69). This spatial regulation by the substance of the stroma reflects the possibility of a structure similar to water. 24. F a t t y a c i d s and s t e r o l s are some of the p h o t o s y n t h e t i c products of the Xanthophyceae ( 1 3 . 7 5 ) . In T. minus, l i p i d d r o p l e t s were present between l a m e l l a e i n the dense c h l o r o p l a s t m atrix ( F i g s . 17,18,20,24,26).. With i n c r e a s e d water l o s s , the l i p i d d r o p l e t s became l e s s d i s t i n c t due to reduced s t a i n a b i l i t y o f t h i s r e g i o n , e s p e c i a l l y i n the low RH s t a t e s (10,0$). The cause of the l o s s l n s t a i n uptake may have been due to the s a t u r a t i o n of double bonds of the l i p i d storage product (102). Concomitantly, the c h l o r o p l a s t genophore such as d e s c r i b e d by B i s a l p u t r a and B i s a l p u t r a (8) and R i s and P l a u t (96) became u n r e c o g n i z a b l e . Benson and Shibuya (5) propose t h a t l i p i d s of algae are l a r g e l y s u r f a c t a n t ( i . e . having s u r f a c e a c t i v e p r o p e r t i e s ) which f u n c t i o n both as s t r u c t u r a l elements and as p h o t o s y n t h e t i c m e t a b o l i t e s . L i p i d s , l i k e carbohydrates, r e l e a s e hydrogen and oxygen molecules d u r i n g metabolism; thus, the c o n t i n u a t i o n of l i p i d f o r m a t i o n and accumulation i m p l i e s the b i n d i n g of water to new m e t a b o l i t e s and the recombination of such i n t o s y n t h e t i c processes as noted by C r a f t s f o r h i g h e r p l a n t s (14). I t appears t h a t d u r i n g d e s i c c a t i o n , l i p i d s y n t h e s i z i n g enzymes are favoured. To t e s t t h i s theory, one c o u l d study the q u a n t i t a t i v e changes i n the l i p i d s y n t h e s i z i n g enzymes by e x t r a c t i o n and l a b e l l i n g , or examine the r e s u l t of b l o c k i n g l i p i d s y n t h e s i z i n g routes d u r i n g d e s i c c a t i o n . 2 5 . The s i m i l a r i t y i n s t i n i n g property of the stored contents and the chloroplast stroma of T. minus suggest that the chloro-p l a s t i s the production centre. The continuity of cytoplasmic membranes would enhance the flow of information allowing rapid transformations, yet r e t a i n the p a r t i c u l a r c i s t e r n a l Integrity as proposed f o r fungi by Bracker and Grove ( 1 0 ) . THE "VACUOME" SYSTEM A continuous membrane system of endoplasmic reticulum was postulated ( 8 6 ) . De Duve (19) defined s i m i l a r membrane systems as the "vacuome" system encompassing two phases: the Endoplasmic Phase and the Exoplasmic Phase. The func t i o n a l l y synthesizing Endoplasmic Phase included the nuclear membrane, nuclear v e s i c l e s , rough endoplasmic reticulum and the forming face of the Golgi. The second Exoplasmic Phase functioned to transport, store, anabolize and metabolize c e l l products. This l a t t e r phase Involved the smooth endoplasmic reticulum, maturing Golgi face, lysosome, vacuoles and secretory granules. Although the i n t e r r e l a t i o n s h i p i s unclear the continual nature of membrane systems xylthin a c e l l i s evident ( 1 0 , 1 9 , 8 6 ) . A discussion of the interconnection of the s t r u c t u r a l l y d i s t i n c t stages may i l l u c i d a t e the c y t o l o g i c a l changes seen during water s t r e s s . I n i t i a l l y the blebbing and extension of the outer nuclear membrane of T. minus resulted i n the formation of nuclear v e s i c l e s and endoplasmic reticulum, respectively (Figs. 4 - 1 1 ) . Similar 26 relationships were reported i n other Xanthophyceae ( 3 5 . 3 6 ) , Bacillarlophyceae ( 2 6 , 2 7 , 1 0 8 ) , Chrysophyceae (103) and Phaeophyceae ( 9 ) . The association of polyribosomes on the surface of nuclear v e s i c l e s (Fig.2 8 ) and the tubules and plates of endoplasmic reticulum (Figs. 5 - 8 ) suggest protein synthesis occurred as discussed by Schjeide f o r animal c e l l s ( 9 8 ) . Along flattened faces of the nucleus, the nuclear v e s i c l e s fused to form stacks of Golgi cisternae ( 7 7 , F i g s . 4 , 5 ) . The proximal face of the Golgi was s i m i l a r to that of the rough endoplasmic reticulum. Towards the opposite pole, ribosomes were l o s t and membrane transformation occurred ( 4 9 ) . At the margins of the cisternae during maturation and also along the maturing face, secretory v e s i c l e s were produced ( F i g s . 2 8 - 3 1 ) . Morre, Mollenhauer and Bracker (78) proposed that membrane transformation involved the metabolism of membrane l i p i d s and additions of sugars. E a r l i e r , Perry and Kelley (90) stated that new protein for the secretory products and f o r the formation of membranes appeared to be synthesized on the polyribosomes and associated messenger RNA of the BER. Functions, other than the general scheme of concentrating and transporting, were ascribed to Golgi apparati to explain the movement of proteins being secreted through t h i s system of membranes ( 6 2 ) . The add i t i o n a l functions involved the synthesis of polysaccharides and (less established) providing a location f o r protein maturation f o r export. 27 G o l g i v e s i c l e s served to compartmentalize m a t e r i a l s f o r t r a n s p o r t t o the c e l l s u r f a c e (Figs. 2 9 ,31, 3 5 , 3 6 ) as previously-r e p o r t e d f o r h i g h e r p l a n t and animal c e l l s (114). The l i m i t i n g membranes of the v e s i c l e s were m o r p h o l o g i c a l l y s i m i l a r (80-100A t h i c k ) t o the plasma membrane which suggests the c a p a c i t y of the former to fuse with the l a t t e r as i n other p l a n t s ( 4 9 , 9 4,101). During chromatin condensation, the reduced number of G o l g i c i s t e r n a e from seven i n 100$ RH c e l l s to f o u r i n 20$ RH c e l l s i m p l i e s a r e l a t i o n s h i p to the apparent b l o c k i n DNA t r a n s c r i p t i o n and RNA p r o d u c t i o n . Some G o l g i v e s i c l e s may become promary lysosmes which con-t a i n h y d r o l y t i c enzymes capable of f u s i n g with vacuoles (Fig.17) which l o s e water w i t h i n the cytoplasm causing h y d r o l y s i s of i t s contents as i n other organisms ( 3 0,84). T h e r e f o r e , the m u l t i -v e s i c u l a r bodies ( F i g s . 3 1 , 3 2 ) were probably i n t e r m e d i a t e d i g e s t i v e s t a g e s . I t appears that h y d r o l y t i c enzymes are a c t i v e d u r i n g d e s i c c a t i o n s i m i l a r t o that o c c u r r i n g i n h i g h e r p l a n t s (1,100 , 1 1 3 ) . Endoplasmic r e t i c u l u m e x i s t e d i n c l o s e a s s o c i a t i o n with the nucleus and c h l o r o p l a s t as r e p o r t e d f o r the Xanthophyceae ( 3 5 . 3 6 , 4 7 . 5 6 , 7 1 ) . Extensions of the outer n u c l e a r envelope formed RER (Fig. 2 3 ) or l o s t the polyribosomes becoming smooth endoplasmic r e t i c u l u m (SER, 16). Membranes c l o s e l y adpressed to the c h l o r o -p l a s t outer membrane i n T. minus were SER, but ribosomes were a t t a c h e d to t h a t p o r t i o n d i s t a n t from i t forming RER (Fig.14) The RER f u n c t i o n e d to s y n t h e s i z e p r o t e i n and s t o r e i t ; whereas, the SER m o d i f i e d p r e v i o u s l y accumulated p r o t e i n s and s y n t h e s i z e d l i p i d or carbohydrate (114). 28 The chloroplast endoplasmic reticulum (CER) appeared to be a transport system continuous ivith large reservoirs of l i p i d (Figs.18,24 -26). During extreme desiccation (10 and 0% RH), there was further expansion of this storage area providing a p e r i -pheral layer of l i p i d ( F i g s . 2 6 , 2 7 ) . The loss i n water caused shrinkage i n the c e l l and the membranes of the "vacuome" system became cl o s e l y associated, but remained d i s t i n c t (Figs.24-26). CENTRIOLES AND MICROTUBULES. The centrioles of T. minus appeared s i m i l a r to those reported previously for the Xanthophyceae (17,18,35,56,71,72,76). The p a i r of centrioles were observed to l i e t y p i c a l l y at right angles to each other (44,45) near the c e l l surface (Figs.33,35)• Microtubules of T. minus extended r a d i a l l y from the centrioles maintaining a close association with the centrioles i n the cartwheel region. The occurrence of microtubules emanat-ing from centrioles and located among Golgi v e s i c l e s suggests that the microtubules and centrioles function as part of a conducting system as discussed by Tilney (110). During desiccation, microtubules were evident up to 20% RH, but were not detected l n the cytoplasm i n 10 and 0% RH. The loss of microtubules i s s i m i l a r to t h e i r i n a b i l i t y to withstand stress conditions such as hydrostatic pressure and lour temperature (110). 29 SUMMARY Desiccation of the vegetative c e l l s of Trlbonema minus was characterized by the following: 1. Condensation of chromatin. 2. Contraction of the nucleus into a dense sphere. 3. Reduction i n width of the c e l l wall, perinuclear and perichondrlal space. 4. Reduction i n size and apparent fusion of chloroplast three-thylakoid bands. Reduced "lnterband" space. 5. Increased width and staining of plasma membrane and -inner mitochondrial membrane. 6. Increased homogeneity of the mitochondrial and chloro-plast matrices. 7. Mitochondrial and chloroplast genophores became indistinguishable from the matrices. 8. Production of a l i p i d component i n the chloroplast and i t s accumulation l n storage areas continuous with the chloroplast endoplasmic reticulum. During water stress, increased l i p i d sysnthesis appeared to be the means by which T. minus conserved water and produced new metabolites. For further understanding of this process, i t i s necessary to 1. Determine the composition of the l i p i d substance. 2. Examine the effects of various rates of drying by extraction and electron microscopy. 3 0 . 3 . -Compare the structure of a i r - d r i e d c e l l s to those grown axenically i n culture as reported by Hawlitschka (52) and Belcher and Fogg (4) at various osmotic pressures. 4 . Examine c e l l s grown at defined humidities observing the differences l n a) rate of r e s p i r a t i o n , b) rate of photosynthesis, c) d i f f e r e n t i a l enzyme a c t i v i t y , and d) comparison of mitochondrial and chloroplast DNA to nuclear DNA a c t i v i t y . 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PLATES AND EXPLANATIONS LEGEND C = chromatin CC = chromatin condensation CER = chloroplast endoplasmic reticulum CG = chloroplast genophore CGV s= coated Golgi v e s i c l e CH = chloroplast CL = chloroplast central lamellae CR = mitochondrial c r i s t a CS = centrosphere CW = c e l l wall D = dense centre of en t r i o l e Di = d i s t a l face of Golgi apparatus P = feet from A-tubule of centriole G = Golgi apparatus GC = Golgi cisternae GV = Golgi v e s i c l e IC = inner chloroplast membrane IM = inner mitochondrial membrane IN = Inner nuclear membrane IW = inner c e l l wall L = l i p i d LS = l i p i d storage area N = nucleus NP = nuclear pore NU = nucleolus Nv = nucleolar "vacuole" NV = nuclear v e s i c l e M = mitochondrion MG = mitochondrial genophore MM = mitochondrial matrix MT = microtubules OC = outer chloroplast membrane ON = outer nuclear membrane OM = outer mitochondrial membrane OW = outer c e l l wall PC = pars chromosoma PCS = perichondrial space PF = pars f i b r o s a PG = pars granulosa PL = peripheral lamellae PN = perinuclear space PM = plasma membrane PR = polyribosome Px = proximal face of Golgi apparatus RER = rough endoplasmic reticulum S = chloroplast stroma V = vacuole WM = c e l l wall membrane 41. PLATE I Figure 2 . Longitudinal section of a 100$ RH c e l l of Tribonema  minus Hazen. H-pieces of the c e l l wall were conjoined. A central nucleus was surrounded by-cytoplasm containing two peripheral chloroplasts, endoplasmic reticulum, a single Golgi apparatus and mitochondrion, x 1 5 , 0 0 0 Figure 3 . Cross section of the overlapping outer (OW) and inner (IW) walls of a c e l l of 100$ RH. Note the plasma membrane (PM) clos e l y associated to the c e l l wall membrane (WM). x 4 9 , 0 0 0 PLATE II Figure 4. The interphase nucleus (N) of 100$ RH c e l l , x 42 ,000 Figure 5 . The nucleus of 60$ RH c e l l . Attachment of the chromatin (C) to the inner nuclear (IN) membrane was seen, x 36,000 Figure 6. Chromatin condensation along the inner nuclear membrane (IN) l n 50$ RH c e l l , x 30,000 Figure ?. The nucleolus of 50$ RH c e l l , x 32,000 4 3 . PLATE III Figure 8 . The nucleus of 30$ RH c e l l showed further chromatin condensation. Note that Golgi ves i c l e s (GV) from the maturing d i s t a l face (Di) of the Golgi apparatus was interspersed with microtubules (MT). x 2 7 , 0 0 0 Figure 9. Increasing chromatin condensation (CC) occurred i n 20$ RH c e l l . The outer nuclear membrane extended to form the chloroplast endoplasmic reticulum (CER). x 42,000 Figures 10 and 1 1 . Cells of 10 and 0 $ RH, respectively, showed extreme chromatin condensation (CC). Association of l i p i d (L) around the outer nuclear membrane was seen. The c e l l s of 0$ RH had fibrous components i n the pore region, x 3 6 , 0 0 0 x 5 0 , 0 0 0 PLATE" IV Figures 12 and 13. Mitochondria of 60$ RH c e l l s with electron transparent genophores containing DNA (MG). x 48,000 x 100,000 Figures 14 and 15. Increased density of the mitochondrial matrix (MM) i n 20$ RH c e l l s was followed by the genophores becoming unrecognizable, x 80,000 x 60,000 4 5 PLATE' V: Figures 1 6 and 1 7 . Peripheral chloroplasts of 1 0 0 $ RH c e l l s occurred cl o s e l y associated to the plasma membrane. Note that l i p i d droplets (L) were seen i n the stroma (S), i n the space between the two chloroplast membranes (OC and IC), and in membrane bound l i p i d storage areas (LS). x 3 0 , 0 0 0 x 1 9 , 0 0 0 Figure 1 8 . The outer chloroplast membrane of a 6 0 $ RH c e l l was clos e l y associated to the chloroplast endoplasmic reticulum (CER) which appeared to be continuous with the l i p i d storage area, x 3 2 , 0 0 0 Figure 1 9 . Along the length of a chloroplast of 6 0 $ RH c e l l , there was close association of the outer and inner chloroplast membranes, x 5 5 , 0 0 0 46 PLATE" VI Figure 2 0 . Chloroplast of a c e l l of 50$ RH with a l i p i d producing area of the stroma in close proximity to the l i p i d storage area (LS). x 53,000 Figure 21 . Oblique section through a chloroplast of a 50$ RH c e l l showed peripheral (PL) and central (CL) lamellae associating with other bands along i t s length, x 55.000 Figure 22. Chloroplast of a 40$ RH c e l l with c l o s e l y associated or fused thylakolds. x 120,000 Figure 2 3 . Chloroplast endoplasmic reticulum of a c e l l of 30$ RH extended into the cytoplasm and appeared connected to the rough endoplasmic reticulum, x 36,000 47. PLATE" V I I F i g u r e s 24 and 25 . The c h l o r o p l a s t endoplasmic r e t i c u l u m of a 20$ RH c e l l appeared continuous (arrow) with the membrane surrounding the l i p i d storage a r e a , x 24 ,000 x 59,000 F i g u r e 26. L i p i d storage areas of 10$ RH c e l l were c l o s e l y appressed to the plasma membrane, but remained d i s t i n c t from i t . x 15,000 F i g u r e 27 . P e r i p h e r a l and c e n t r a l l a m e l l a e of a i r - d r i e d c e l l s , 0$ RH, were " n e g a t i v e l y " s t a i n e d . Note l a c k of s t a i n uptake, x 30,000 PLATE VIII Figure 28. Golgi apparatus of a 60$ RH c e l l with a large accumulating system (*) at Its d i s t a l face (Di). x 66,500 Figure 2 9 . C e l l of 20$ RH had nuclear v e s i c l e s (NV) which appeared directed towards the Golgi apparatus, x 76,000 Figure 3 0 . C e l l of 20$ RH had Golgi v e s i c l e s blebblng off the mature face of the Golgi apparatus near the c e l l surface, x 60,000 49. PLATE IX Figures 31 and 32. . C e l l s of 20$ RH had small vacuoles (V) containing v e s i c u l a r and membranous inclusions, x 30,000 x 95.000 PLATE X. Figure 3 3 . A pa i r of centrioles i n a 100$ RH c e l l , x 1 0 2 , 0 0 0 Figures 34 and 3 5 . C e l l of 60$ RH with microtubules (MT) radiating from and running p a r a l l e l to the c e n t r i o l e s ( * ) . Note that the microtubules occurred near the surface and between Golgi v e s i c l e s (GV). x 3 6 , 0 0 0 x 4 ? , 0 0 0 APPENDIX 51. M o d i f i e d B r i s t o l ' s Medium Add 10 ml of each of B r i s t o l ' s stock s o l u t i o n s to 9^0 ml of d i s t i l l e d water and 1 ml of Hutner's tr a c e element s o l u t i o n . B r i s t o l ' s Stock S o l u t i o n s (Bold, H.C. 19^9. B u l l Torrey Club 76 :101-108.) Make s i x 400 ml stock s o l u t i o n s . stock s o l u t i o n q u a n t i t y NaN0 3 10 .Og CaCl2 l.Og MgS04.7H20 3 . 0 g K2HPO4 3 . 0 g KH2PO4 7 . 0 g N a C l 2 l.Og Hutner's Trace Element S o l u t i o n (Hutner, S.H.., P r o v a s o l i , L., Schatz, A., and Hawkins, C P . 1950. Proc. Amer. P h i l . Soc. 94:152-170.) Add s a l t s t o 75 ml d i s t i l l e d water. A f t e r each a d d i t i o n , a d j u s t to pH 5»5. B o i l , c o o l s l i g h t l y and adjust t o pH 6.5 w i t h KOH p e l l e t s . D i l u t e to 100 ml. m a t e r i a l q u a n t i t y EDTA 5.00g ZnSOi4..7H20 2.20g H3PO3 l.OOg CaCl2 0.50g MnCl 2.4H20 o.50g PeS04..7H20 o.50g C0CI2.6H2O o.i5g CUSO4.5H2O o.i5g (NH4)6Mo-p024.4H20 0.10g 

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