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The effect of iron and light co-limitation on the oceanic diatom : Pseudo-nitzshia granii El-Sabaawi, Rana 2002

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THE EFFECT OF IRON AND LIGHT CO-LIMITATION ON THE OCEANIC DIATOM, PSEUDO-NITZSCHIA  GRANII  By RANA EL-SAB AAWI B.Sc, The University of Western Ontario, 1999 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERJOF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  April 2002 © Rana W. El-Sabaawi, 2002  UBC  Special Collections - Thesis Authorisation Form  Page 1 of 1  I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the head o f my department o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  http://www.library.ubcxa/spcoll/mesaum.htinl  4/11/02  Abstract  Light and iron (Fe) Co-limitation occur in temperate, Fe-limited regions in seasons where days are short and the mixed layer is deeper than the euophotic zone. In thefirstpart of this study, the effect of light and Fe co-limitation on Pseudo-nitzschia granii isolated from the NE Subarctic Pacific was investigated. This was one of thefirststudies to investigate the effect of Fe and light on the physiology of oceanic, pennate diatoms in artificial seawater. Felimited P. granii grew at slower rates than Fe-replete cells, and the former showed signs of chlorosis and silica deficiency. P. granii was efficient at utilizing light, especially low light. Light and Fe were found to affect different aspects of photosynthesis. Whereas Fe affected the quantum yield of photosynthesis, light affected photochemical efficiency. Despite low growth rates, P. granii was able to survive and grow under Fe-limitation. This was possibly because Fe limitation increased the activity of non-photochemical quenching mechanisms, allowing the cells protection against photodamage. Overall, growth rates were directly related to the rate of linear electron transport through the cells. In the second part of this thesis, interaction of light and temperature on the physiology of P. granii was investigated. Studies on the combined effects of light and temperature, especially in temperate phytoplankton, are rare. Both light and temperature were found to influence growth rates. Temperature dependency decreased with decreasing light. Cellular chlorophyll displayed a modal response to temperature and light. Photosynthetic yield and efficiency were generally depressed at low and high temperatures. Unlike in green algae and some cyanobacteria, low-temperature acclimation in P. granii was not similar to high light acclimation.  Table of Contents Abstract  ii  Table o f Contents  iii  List o f Tables  v  List of Figures  vi  Acknowledgements  ix  Chapter 1: General Introduction A B r i e f H i s t o r y o f O c e a n Station P ( O S P )  2  The Role of Fe in Cellular Physiology  3  L i g h t and P h o t o a c c l i m a t i o n  4  T h e E c o l o g y and P h y s i o l o g y o f D i a t o m s  5  Pseudo-nitzschia  7  The Genus Summary  9  Chapter 2: Pulse Amplitude Modulated (PAM) Chlorophyll Fluorescence C h l o r o p h y l l F l u o r e s c e n c e and T h e K a u t s k y E f f e c t  10  The Development o f P A M fluorometry  14  T e c h n i c a l concerns r e g a r d i n g P A M fluorescence  18  T h e e x c i t a t i o n b e a m and determination o f F  19  0  T e r m i n o l o g y and N o m e n c l a t u r e  20  T h e p h y s i o l o g i c a l status o f the c e l l  20  S p e c i f i c concerns f o r p h y t o p l a n k t o n samples  21  Summary  22  Thesis Objectives  23  Chapter 3: Effect of Light and Fe on The Physiology oi Pseudo-nitzschia granii isolated from the NE Subarctic Pacific Introduction  24  M a t e r i a l s and M e t h o d s  27  D i a t o m Isolation  27  Culture Conditions  27  C h l o r o p h y l l ( C h i cell" ), n o n - m o d u l a t e d c h l o r o p h y l l fluorescence 1  ( F S U ) per unit c h l o r o p h y l l ( F S U C h i  _ 1  )  28  Photosynthetic Efficiency U s i n g D C M U ( F / F D C M U )  29  Biogenic Silica (BSi)  29  V  M  Particulate O r g a n i c C a r b o n and Particulate O r g a n i c N i t r o g e n (POCand PON)  .....29  M o d u l a t e d c h l o r o p h y l l fluorescence  29  Statistical analysis  30  Results  32  D i a t o m Isolation  32  G r o w t h rates and b i o c h e m i c a l parameters  32  Chlorophyll  fluorescence  kinetics  34  Discussion  50  G r o w t h rates  50  C h l o r o p h y l l and c h l o r o p h y l l  fluorescence  Particulate o r g a n i c c a r b o n a n d n i t r o g e n  52 54  Biogenic silica  55  Photosynthetic efficiency  56  PAM  fluorescence  results  58  Quantum yield o f P S 2  58  P h o t o c h e m i c a l q u e n c h i n g , r e d o x poise and non-photochemical quenching  59  Conclusions  61  Chapter 4: Effect of Temperature and Irradiance on the Physiology of Pseudo-nitzschia granii Introduction  63  Materials and Methods  66  Results G r o w t h rate and photosynthetic p i g m e n t s  67 67  Chlorophyll  67  fluorescence  Discussion G r o w t h rates and C e l l u l a r C h l o r o p h y l l  75 75  C h l o r o p h y l l Fluorescence A r e t h e r m o a c c l i m a t i o n and p h o t o a c c l i u m a t i o n s i m i l a r  77  processes i n P. graniil  79  Conclusions  81  Future Research  82  Bibliography  83  Appendix 1  94  Appendix 2  95  iv  List of Tables Table 3.1: E q u a t i o n s o f photosynthetic parameters calculated f r o m c h l o r o p h y l l fluorescence kinetics. F  v  is v a r i a b l e fluorescence, F , is m a x i m a l fluorescence, F is b a s a l fluorescence, F  is steady state fluorescence, F '  M  M  0  is variable fluorescence d u r i n g steady state, F' is basal  fluorescence f o l l o w i n g steady state, Q  0  A  is the p h a e o p h y t i n A , I is incident light intensity a n d  D C M U , is 3-(3,4- dichlorophenyl)-1,1 d i m e t h y l urea  31  s  List of Figures  Fig 2.1: A s c h e m a t i c o f the K a u t s k y effect i l l u s t r a t i n g the k i n e t i c s o f f l u o r e s c e n c e o b s e r v e d w h e n plants are transferred f r o m darkness to h i g h light. F is basal fluorescence, P is the 0  fluorescence peak after the sample is r e m o v e d f r o m the dark into actinic light, F M is m a x i m a l fluorescence, F is v a r i a b l e fluorescence and F i s steady-state fluorescence v  s  13  Fig 2.2. T h e fluorescence trace generated u s i n g P u l s e A m p l i t u d e M o d u l a t e d f l u o r e s c e n c e i n dark-adpated  Pseudo-nitzschia granii. F is basal fluorescence after dark-adaptation, F M is 0  m a x i m u m fluorescence, Fs is steady-state fluorescence under a c t i n i c c o n d i t i o n s , F ' M i s m a x i m u m fluorescence under steady-state c o n d i t i o n s , F ' is basal fluorescence p r o c e e d i n g 0  steady-state  15  Figure 3.1: A ) S c a n n i n g electron m i c r o g r a p h o f Pseudo-nitzschia granii after one m o n t h o f i s o l a t i o n . A r r o w points to d e f o r m a t i o n i n shape. B ) U n d e f o r m e d c e l l after three m o n t h s o f i s o l a t i o n (bar = 1 0 p m ; Photos were taken b y B r i a n D. B i l l , U n i v e r s i t y o f W a s h i n g t o n ) , a n d C ) c l o s e - u p o f surface features (bar =1 p m ; P h o t o g r a p h t a k e n b y A d r i a n M a r c h e t t i , University of British Columbia)  36  Figure 3.2: L i g h t m i c r o g r a p h s o f P. granii bar = 10 p m )  37  Figure 3.3: G r o w t h rate (p.) v s . irradiance for P. granii g r o w n under Fe-replete c o n d i t i o n s ( b l a c k circles) and Fe-deplete c o n d i t i o n s (white circles) ( A ) and, ratio o f Fe-deplete to F e replete g r o w t h rates v s . irradiance ( B ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e  38  Figure 3.4: C h i cell" v s . irradiance ( A ) , and C c h l v s . irradiance ( B ) f o r P. granii g r o w n 1  under Fe-replete c o n d i t i o n s ( b l a c k circles) and Fe-deplete c o n d i t i o n s ( w h i t e c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n invisible  39  Figure 3.5: F S U c h l v s . irradiance f o r P. granii g r o w n under Fe-replete c o n d i t i o n s ( c l o s e d c i r c l e s ) a n d Fe-deplete c o n d i t i o n s (open c i r c l e s )  40  Figure 3.6: C : N m o l a r ratios v s . irradiance ( A ) , and ( B ) C cell" v s . irradiance and ( C ) N cell" v s . irradiance f o r P. granii g r o w n under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete 1  c o n d i t i o n s (white circles)  41  Figure 3.7: B S i : N m o l a r ratios v s . irradiance (a), and B S i c e l l ' v s . irradiance (b) f o r P. granii g r o w n under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete c o n d i t i o n s (white 1  circles). E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the S'y m b o l s w h e n i n v i s i b l e  42  vi  Figure 3.8:  P. granii g r o w n under Fe-replete c o n d i t i o n s  FV/FM(DCMU) VS. irradiance f o r  (black c i r c l e s ) and Fe-deplete c o n d i t i o n s (white c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e  Figure 3.9:  43  L i n e a r regression between F V / F M (DCMU) and F V / F M m e a s u r e d u s i n g the P A M  fluorometer  44  Figure 3.10:  T h e r e l a t i o n s h i p b e t w e e n F V / F M (DCMU) and C h i cell" f o r P. 1  granii g r o w n under  Fe-replete c o n d i t i o n s ( b l a c k circles) and Fe-deplete c o n d i t i o n s (white c i r c l e s ) . S o l i d lines are linear r e g r e s s i o n lines  Figure 3.11:  45  Q u a n t u m photosynthetic e f f i c i e n c y (^PS2) v s . irradiance ( A ) , and relative  electron transport  (J ) ( p m o l e" m" s" ) vs. irradiance (B), f o r P. granii g r o w n under F e 1  2  1  e  replete c o n d i t i o n s ( b l a c k circles) and Fe-deplete c o n d i t i o n s (white c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n invisible  46  Figure 3.12:  L i n e a r regression o f J  e  and p f o r  P. granii g r o w n under Fe-replete c o n d i t i o n s  ( b l a c k c i r c l e s ) and F e - l i m i t e d c o n d i t i o n s (white circles)  Figure 3.13:  47  P h o t o c h e m i c a l e f f i c i e n c y ( P ) v s . irradiance ( A ) , and r e d o x p o i s e ( 1 - P ) v s . q  q  irradiance (B), f o r P. granii g r o w n under Fe-replete c o n d i t i o n s (black c i r c l e s ) a n d Fe-deplete c o n d i t i o n s (white c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e  Figure 3.14:  48  N o n - p h o t o c h e m i c a l q u e n c h i n g ( Q ) vs. irradiance f o r n  P. granii g r o w n under F e -  replete c o n d i t i o n s (open circles) and Fe-deplete c o n d i t i o n s (closed c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n invisible  Figure 4.1:  49 A ) G r o w t h rate (p.) f o r  Pseudo-nitzschia granii g r o w n at f o u r irradiances v s .  temperature. E r r o r bars are standard error (n = 3) and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e . B ) Q i n f o r P. granii v s . irradiance  69  u  1  Figure 4.2:  2 1  C h i cell" for P  granii g r o w n at 150, 100, 50 and 2 0 p m o l photons m" s" v s .  temperature. E r r o r bars are standard error (n = 3) and are s m a l l e r t h a n the s y m b o l w h e n invisible  70  2 1  Figure 4.3:  F V / F M f o r P.  granii g r o w n at 150, 100, 50 and 2 0 p m o l photons m" s" v s .  temperature. E r r o r bars are standard error (n= 3) and are s m a l l e r than the s y m b o l w h e n invisible  Figure 4.4:  71 A)  0PS2, and B) J o f P. granii g r o w n at 150, 100, 50 and 2 0 p m o l photons m"  2  e  s" v s . temperature. E r r o r bars are standard error (n= 3) and are s m a l l e r than the s y m b o l w h e n 1  invisible  72  vii  Figure 4.5: A ) P , and b) 1-P , o f P. granii g r o w n at 150, 100, 50 and 20 p m o l photons m" s z  q  1  q  vs. temperature. E r r o r bars are standard error (n=3) and are s m a l l e r than the s y m b o l w h e n  invisible  73  Figure 4.6: Q for P. granii g r o w n at 150, 100, 50 and 20 p m o l photons m" s" v s . 2  1  n  temperature. E r r o r bars are standard error (n=3) and are smaller than the s y m b o l w h e n invisible  74  viii  Acknowledgements I would like to thank my supervisor, Dr. Paul J. Harrison for giving me a space in his lab and for encouraging me to pursue my ideas with freedom and support. My committee members, Dr. Max Taylor and Dr. Beverly Green were always supportive of my work, especially in the last few months. Veronica Oxtoby and Lebby Balakshin reminded me of deadlines and kept me alert. Thanks to Botany and Earth and Ocean Sciences for being so welcoming. My deepest thanks to all members of the Harrison lab: Tawnya, Philippe, Robert, Michael, Mike, Joe, Heather, Ming, Shannon, Nelson and Adrian for their support technically and emotionally. Maureen Soon generously analyzed my carbon and nitrogen data, and Dr. Norm Huner helped me with PAM fluorescence. My mentor Charlie Trick deserved much credit for convincing me of my own strength and capability. I would like to thank my mother and father for their prayers and my sisters for keeping me firmly rooted in myself. This thesis was dedicated to my grandfather, Hussien Al-Yousif whose encouragement of my early career aspirations as a space woman leads me, indirectly, to be an oceanographer! My desses, Kim, Jeana and Jessica are always there for me, and I am forever in their debt. I would especially like to thank Jessica for many an enlightening chat on Kits beach, and for her generosity and wonderful spirit. Thanks to Cheryl for many sanity-preserving mochas on the beach and to Tristan for all the phone calls and postcards of various Ontario rodents. Though this thesis is against everything George McWhirter has thought me, I would like to thank him for his remarkable understanding and constant encouragements.  Chapter 1 General Introduction Aquatic carbon assimilation (primary productivity) accounts for approximately 40% of global carbon fixation (Falkowski and Raven, 1997), a considerable percentage when the implications of carbon dioxide removal in terms of global warming are considered. This increases the need for scientists to understand what controls primary production of marine phytoplankton in the ocean. Traditionally, scientists have investigated how macronutrients, such as nitrate and phosphate, affect marine phytoplankton physiology, with the general consensus that nitrate is the limiting nutrient in most regions of the ocean (Falkowski and Raven, 1997). However, there are many, paradoxical regions in the ocean where the concentration of macronutrients is high, but the photosynthetic biomass (chlorophyll) is low (Hutchins, 1995). Many hypotheses were proposed to explain the limitation of algal growth rates in High nutrient- low chlorophyll (FfNLC) regions. These included active grazing by meso- and microzooplankton, low light levels, strong wind mixing, or unavailability of essential micronutrients (trace metals), most specifically iron (Fe) (Martin et al., 1990). The latter was difficult to discern because of high levels of background contamination and poor trace metal sampling techniques. However, in 1990, trace metal clean shipboard bottle experiments provided evidence to support the role of Fe as a limiting factor of algal growth and biomass in the NE subarctic Pacific (a HNLC region) (Martin et al., 1990). In the decade that followed, further refinement of trace metal clean techniques, numerous shipboard experiments and three large-scale Fe fertilizations unequivocally confirmed Fe as the limiting factor of algal growth in two  1  HNLC regions, namely the Equatorial Pacific (Iron EX1 and 2) and the Southern Ocean (SOIREE) (Boyd et al., 2000; Martin et al, 1994). In these experiments, the addition of Fe stimulated primary productivity and nutrient drawdown, and increased photosynthetic efficiency and phytoplankton biomass, especially for large diatoms (> 20 |am) (Boyd et al., 2000; Martin et al., 1994). It was estimated that over 30% of the world's oceans is Felimited, and one of the most important HNLC, Fe-limited regions was Ocean Station P in the NE subarctic Pacific (Boyd and Harrison, 1999; Harrison, et al. 1999). A brief History Ocean Station P (OSP): Ocean station P (OSP; formerly ocean station Papa), located in the Alaskan gyre (145°W 45°N) was the site of the longest running series of oceanographic data (Harrison et al., 1999). Observation at OSP started in 1950's with the weathership program as implemented by the Canadian government (Boyd and Harrison, 1999; Whitney et al., 1998; Freeland et al., 1997). Initial measurements included temperature, salinity, chlorophyll and nutrients. In 1956, hydrographic casts were conducted on a daily bases and primary productivity as well as zooplankton data were collected. A series of 13 stations were sampled on the way to OSP; they rangedfromcoastal stations (P04) to offshore stations (OSP, also known as P26) (Boyd and Harrison, 1999). This transect, known as line P, provided a valuable opportunity to compare oceanic and offshore regions. The weather ship program was cancelled in the early 1980's (Boyd and Harrison, 1999). Sampling at line P was continued by the Institute of Ocean Sciences (IOS, Sidney, BC) through the world ocean current exchange program (Whitney et al., 1998 and Freeland, et al., 1997). The number of stations increased to 26 and the frequency of sampling decreased to three times a year (spring, summer and winter). Proceeding the world current exchange program, sampling at  2  line P continued through the Canadian chapter of the Joint Global Flux Study (CJGOFS) (Boyd and Harrison, 1999; Harrison et al., 1999). OSP is characterized by a permanent shallow pycnocline (40 m in the summer, and 140 m in the winter because of storm activity) (Whitney et al., 1998 and Freeland et al., 1997). The concentration of macronutrients is high throughout the year, chlorophyll low and, unlike coastal regions, there is no spring bloom of phytoplankton in the spring (Boyd and Harrison, 1999; Harrison et al., 1999). Dominant phytoplankton species are less than 5 p:m and consist of mainly flagellates (prymnesiophytes such as Emiliania huxlyii and Phaeocystis sp.) and cyanobacteria (Synechococcus sp.) (Boyd and Harrison, 1999; Harrison et al., 1999). The absence of larger phytoplankton (specifically diatoms) is due to Fe limitation (Boyd et al., 1996; Boyd et al., 1995a; 1995b; Martin et al., 1990). OSP was the first oceanographic station where trace metal clean techniques were employed in shipboard experiments (Martin et al., 1990). Sources of Fe to OSP in order of significance are dust depositionfromthe Gobi Desert in China, recycling by bacteria and micrograzers, horizontal advection of water from the coast of the Aleutian Islands and vertical mixing (Young et al., 1991). In the July of 2002, OSP will be the site of a large-scale Fe fertilization experiment akin to those conducted in the Equatorial Pacific and the Southern Ocean. The emphasis of the project is to investigate the effect of in situ Fe addition on the flux of biogenic gases (such as carbon dioxide and dimethyl sulfide)fromand to the ocean (Harrison, pers. commun.). The Role of Fe in Cellular Physiology: Though it is the fourth most abundant terrestrial element, Fe is found in trace concentrations in seawater,fromnM (10", in Fe-replete, or coastal areas) to pM (10" , 9  12  3  in Fe-deficient, oceanic areas) (Rue and Bruland, 1997). This d-block element has high redox potential (Fe + e' <-> Fe , E ii = -0.244) within the limits of biological molecules, making 3+  1  +2  ce  it a favorable electron carrier in many metalloproteins and enzymes. Fe is a required micronutrient for all living organisms and it is especially important in photosynthesis, where it is an integral component of a variety of electron carriers, such as heme proteins (cytochromes) (Glover, 1977), or Fe-sulfur proteins such as ferredoxin (Doucette et al., 1996; La Roche et al., 1996; 1995). The function of electron transport in these proteins depends on the redox transition between ferric (Fe ) form and ferrous (Fe ) form. Fe is also involved in the +3  +2  chlorophyll synthesis pathway and Fe limited cells are often chlorotic (Geider and La Roche, 1994). Fe is also required for the structural integrity and function of photosynthetic reaction centers, photosystem 1 and photosystem 2 (PS 1 and PS2, respectively)(Geider and La Roche, 1994). The effect of Fe stress on the physiology of coastal phytoplankton is well studied (Doucette et al., 1996; La Roche et al., 1995; Greene et al., 1992; 1991; Glover, 1977). Phytoplankton follow a combination of two strategies to relieve Fe stress, including the depletion of Fe-dependent photosynthetic electron carriers, or their replacement by functionally equivalent molecules that do not require Fe (Doucette et al., 1996; Glover, 1977). In general, photosynthetic enzymes and components constitute the largest Fe quota in diatoms (Raven et al., 1999). The details of photosynthesis in diatoms, as well as most chlorophyll-c containing organisms are poorly understood compared to terrestrial plants and green algae. Light and Photoacclimation:  4  In addition to Fe limitation, phytoplankton communities in temperate HNLC regions are exposed to low, sub-saturating levels of light, especially in the winter season (Timmermans et al., 2001; Maldonado et al., 1999). Photosynthetic cells acclimate to low light by producing more chlorophyll and more photosynthetic components to maximize light absorption (Sunda and Huntsman, 1997). Low light acclimation increases Fe requirements considerably, and if Fe is limiting in the environment, the cells will be under a double stress. In the spring and summer, Fe addition (shipboard experiments) at OSP results in a bloom of large diatoms (Boyd et al., 1995a). This bloom is not as pronounced in the winter unless irradiance is also increased during the Fe addition (Maldonado et al., 1999). During winter at OSP, light levels are low because days are short, cloud cover is high and the pycnocline is often deeper than the euphotic zone due to storm-induced mixing (Maldonado et al., 1999). Photoacclimation involves changes in pigment concentration, cellular composition, cellular volumes and carbon fixation within the genetic limitation of the organism (Aiming et al., 2000). It is thought to be a relatively fast process occurring within the span of one generation or less depending on the organism's growth rate (Anning et al., 2000). There are two strategies for photoacclimation in marine algae: altering the number of photosynthetic units available without affecting the absorption cross-section of the photosynthetic unit, or altering the cross-section by changing the composition of the photosynthetic unit (Greene et al., 1992). The Ecology and Physiology of Diatoms: Diatoms are ubiquitous in freshwater and marine environments. Unique to them is a siliceous, decorative frustule that resembles petri dishes. In the coastal environment, diatoms (Bacillariophyceae) account for 20% of new production, and on the west coast of Canada,  5  fast growing diatoms (such as Skeletonema costatum) dominate the spring bloom (Taylor and Haigh, 1996). Diatoms are grazed upon by mesozooplankton (such as the calanoid copepod Neocalanusplumchrus), which are then grazed upon by pelagicfish(Mackas et al., 1998). Therefore, the dynamics of diatom blooms on the west coast relate indirectly to fluctuations in fish populations (Mackas et al., 1998). In the open ocean, diatoms are small and less abundant than phytoflagellates and cyanobacteria (Boyd and Harrison, 1999). Unlike flagellates and cyanobacteria, diatoms have the ability to sink and are an important source of carbonfluxin the open ocean, and are also important in the geochemical cycle of silica. Diatoms are divided into two groups, centric and pennate, depending on the symmetry of their silica frustules, radial and bilateral respectively. Each can exist as single cells or colonies. Centric diatoms are non-motile and planktonic, whereas pennate diatoms are predominantly motile (through raphes) and benthic. Several genera of pennate diatoms (such as Pseudo-nitzschia) exist planktonically in the coastal and offshore environments (Haigh and Taylor, 1996). Both pennate and centric diatoms reproduce asexually until they reach a certain size at which gamete production is triggered and sexual reproduction between different strains commences (Hiltz et al, 2000). The gametes of centric diatoms are morphologically different; certain cells become large sessile gametes (eggs) while others produce small, flagellated cells (sperm) in a process known as oogamy. In pennate diatoms, gametes are indistinguishable; both are small, and the overall process is known as isogamy (Davidovich and Bates, 1998). Sexual reproduction is triggered in the laboratory by nitrate depletion or by varying the length and intensity of the light cycle (Hiltz et al, 2000). Pennate diatoms are  6  also thought to require a substrate on which to mate, as well as the presence of a different clonefromthe same species (Davidovich and Bates, 1998). Diatoms have chlorophyll a, cl, c2 , fucoxanthin as well as two xanthophyll pigments, didinoxanthin and diatoxanthin, that are restricted to them (Olaizola and Yamamoto, 1994). Diatoms are easier to grow in the laboratory than other phytoplankton. Therefore, a wealth of information about their physiology exists in the scientific literature. Most of these studies have focused on so called "weed" species that are easy to grow in the laboratory but are not true representatives of all species. In addition, most studies so far have focused on coastal or estuarine species of diatoms, which have been shown to be physiologically quite different than oceanic species (Sunda and Hunstman, 1995). Pennate diatoms are more difficult to isolate and grow in the lab compared to centric diatoms, and are studied less frequently than their centric relatives. Most studies available on the physiology of pennate diatoms have focused on the diatom Phaeodactylum triconutum, a weedy, coastal species (for examples, see Geider et al., 1993; Johansen, 1991; Osborne and Geider, 1987; Glover, 1977). The Genus Pseudo-nitzschia: The two most famous genera of Nitzschoid diatoms are Nitzschia, a genus of single, benthic cells encompassing marine andfreshwaterspecies, and Pseudo-nitzschia, a genus of thinly silicified, planktonic and chain-forming marine species (Hasle 1995, 1994). The split between Nitzschia and Pseudo-nitzschia was suggested in the early 1900's by Paragallo on the basis of chain formation, raphe structure and motility (Hasle 1995, 1994). The split between these genera is widely debated and will not be discussed any further. Nitzschoid diatoms are the most common pennate diatoms on the coast of British Columbia, especially in the Strait of Georgia (Harris, 2001). Some diatoms are thought to be transported to the  7  west coast of Vancouver Island via the Vancouver Island Coastal Current (VICC), which is driven by the input of the Fraser River into the Strait of Georgia (Taylor and Haigh, 1996). Certain members of the genus Pseudo-nitzschia gained a measure of notoriety over the past decade because of their association with domoic acid, a causative agent of amnesic shellfish poisoning (Horner and Postel, 1993; Bates et al., 1989). In 1987, many people were poisoned in Prince Edward Island, Canada, after ingesting shellfish that had accumulated domoic acid in their tissues (Bates et al., 1989). Later studies and examination of water samples revealed a bloom of Pseudo-nitzschia in the area where the shellfish were cultured and harvested. In the early 1990s, sea lions and pelicans died because of domoic acid contaminated shellfish off the coast of California (Scholin et al., 2000). The threat of Pseudo-nitzschia is responsible for fuelling a flurry of scientific research that was specifically aimed at monitoring blooms of the genus and the physiological causes of domoic acid production (Bates and Leger, 1992). Most of this research has focused on identifying toxic species of Pseudo-nitzschia and monitoring blooms before they occur. Classifying species of Pseudo-nitzschia depends on minor morphological features indistinguishable under light microscopy, and requires careful examination under the scanning electron microscope (Hasle, 1994). Before samples are examined, careful techniques are applied to dissolve the organics that obscuresfinefeatures on the siliceous ffustule without damaging it (Hasle, 1995; 1994). Attempts have been made to develop an RNA-based, fluorescent marker to distinguish between toxic and non-toxic strains. This has been met with little success because RNA degrades quickly and because of cross-reaction of RNA markers between different strains and species (Scholin et al., 2000).  8  The exact rates of domoic acid production are unknown. Domoic acid is a nitrogenous compound, which recently has been shown to have a high Fe-binding constant (Maldonado, unpublished results). This feature may assist the cells of Pseudo-nitzschia to acquire Fe at OSP by acting as an Fe-chelators similar to those in bacteria and fiingi (Trick et al., 1983). Studies on the Fe-sequestering nature of domoic acid are still in their infancy. Some oceanic isolates of this genus, such as Pseudo-nitzschia granii, have been shown to produce trace amounts of domoic acid (Marchetti, A. and Trainer, V., unpublished results). It is unknown whether they have the ability to produce domoic acid at harmful levels, or whether this process is affected by Fe nutrition. Summary:  Iron (Fe) limits the growth of phytoplankton in many high nutrients low chlorophyll regions of the oceans. In high latitude, Fe-limited regions such as the NE subarctic Pacific (Ocean Station P, OSP) and the Southern Ocean, Fe-limitation is confounded with light limitation in the winter. Light and Fe interact through the process of photosynthesis, and cells under light limitation have a larger Fe quota and are often Fe deficient. Large diatoms are scarce in HNLC regions but when Fe is added, they grow efficiently and dominate the Feenriched assemblages. For example, species of the chain-forming, pennate diatoms, Pseudonitzschia dominate Fe-enriched phytoplankton community in the summers at OSP. In  addition, Pseudo-nitzschia is an important genus of diatoms in coastal temperate areas because its blooms are sometimes associated with domoic acid production, a causative agent of amnesic shellfish poisoning. Studies on Fe and light co-limitation, as well as studies on the physiology of pennate, oceanic diatoms are scarce.  9  Chapter 2  Pulse Amplitude Modulated Chlorophyll Fluorescence  Chlorophyll Fluorescence and The Kautsky Effect: C h l o r o p h y l l F l u o r e s c e n c e is a h i g h l y e x p l o i t e d property o f p h o t o s y n t h e t i c cells, e s p e c i a l l y i n b i o l o g i c a l oceanography and l i m n o l o g y , w h e r e it is u s e d as a p r o x y f o r p h y t o p l a n k t o n b i o m a s s ( F a l k o w s k i et a l . 1999). L i g h t absorbed b y photosynthetic m a t e r i a l encounters o n e o f three, c o m p e t i n g fates ( M a x w e l l a n d J o h n s o n , 2 0 0 0 ) : U n d e r n o r m a l c o n d i t i o n s , the m a j o r i t y (97%) is used i n p h o t o c h e m i c a l processes (photosynthesis a n d linear electron transport), a f r a c t i o n is directed towards n o n - p h o t o c h e m i c a l reactions ( x a n t h o p h y l l c y c l e , state transitions and photo i n h i b i t i o n ) and f i n a l l y a s m a l l f r a c t i o n (up to 3%) is remitted as l o n g w a v e l e n g t h ( - 6 9 0 n m ) , red fluorescence ( M a x w e l l and J o h n s o n , 2 0 0 0 ; H a l l and R a o , 1999; K r a u s e and W e i s , 1991). P h o t o c h e m i c a l q u e n c h i n g , n o n - p h o t o c h e m i c a l q u e n c h i n g and fluorescence compete w i t h each other, and, the accurate q u a n t i f i c a t i o n o f one or t w o o f these processes c a n indicate the e f f i c i e n c y o f the r e m a i n i n g process(es) ( K r a u s e a n d W e i s , 1991). F l u o r e s c e n c e can be easily q u a n t i f i e d u s i n g m o d u l a t e d and n o n - m o d u l a t e d fluorometers. O n a m o l e c u l a r l e v e l , w h e n c h l o r o p h y l l m o l e c u l e s absorb light, they acquire energy that elevates t h e m f r o m a g r o u n d l e v e l to a high-energy, e x c i t e d l e v e l ( T r i s s l e a n d L a v r a n g e , 1994), a n d i f this energy is not dissipated v i a p h o t o c h e m i c a l and n o n - p h o t o c h e m i c a l processes, it is remitted as fluorescence. T h e difference i n w a v e l e n g t h b e t w e e n absorbed and re-emitted fluorescence a l l o w s the differentiation and subsequent q u a n t i f i c a t i o n o f b o t h the light source a n d the fluorescence signal ( M a x w e l l and J o h n s o n , 2 0 0 0 ) . T h e intensity o f fluorescence depends o n the amount o f light (Watts, E i n s t e i n s , Q u a n t a . . .etc) per unit area,  10  and o n the w a v e l e n g t h o f the source light and the concentration o f c h l o r o p h y l l ( M a x w e l l a n d J o h n s o n , 2 0 0 0 ) . I f fluorescence intensity remains constant, the change i n f l u o r e s c e n c e y i e l d , d e f i n e d as the change i n the amount o f fluorescence at the same intensity o v e r t i m e , c a n be calculated ( M a x w e l l and J o h n s o n , 2 0 0 0 , F a l k o w s k i and R a v e n , 1997, S c h r e i b e r et al., 1995a; 1995b). W h e n a s a m p l e o f solvent-extracted c h l o r o p h y l l is transferred f r o m d a r k into light, the fluorescence s i g n a l f r o m the extract is constant o v e r the p e r i o d o f i l l u m i n a t i o n . H o w e v e r , w h e n a s a m p l e o f intact photosynthetic c e l l s is transferred f r o m dark i n t o light, the fluorescence signal has a distinct k i n e t i c shape, w h i c h is d i r e c t l y related to the p h y s i o l o g i c a l status o f the c e l l ( H a l l and R a o , 1999). W h e n dark-adapted samples are transferred to a c t i n i c (photosynthetic) light, c h l o r o p h y l l fluorescence rises f r o m a basal l e v e l ( F ) to a m a x i m a l 0  l e v e l ( F M ) and then it decays ( G v o i n d j e e , 1995, Schreiber et al 1986, B r a d b u r y and B a k e r , 1981). T h e rise and decay o f fluorescence is p o l y p h a s i c , and a b r i e f steady state l e v e l is reached after F M (Schreiber, 1994). These fluorescence k i n e t i c s were first o b s e r v e d i n 1931 b y K a u t s k y and c o - w o r k e r s . Therefore, the k i n e t i c change o f fluorescence y i e l d o v e r t i m e w h e n dark-adapted samples are i l l u m i n a t e d is c a l l e d the K a u t s k y effect ( F i g 2.1) ( G o v i n d j e e , 1995). T h e shape and v a r i o u s phases o f the i n d u c t i o n c u r v e are d i r e c t l y related to electron transfer t h r o u g h the photosynthetic unit and to the p h y s i o l o g y o f the o r g a n i s m i n v o l v e d . A t r o o m temperature, most o f plant fluorescence originates at p h o t o s y s t e m 2 ( P S 2 ) ( F a l k o w s k i and R a v e n , 1997; K r a u s e and W e i s 1991). U n l i k e p h o t o s y s t e m 1 ( P S 1 ) , w h i c h is quite efficient at c a p t u r i n g light energy, P S 2 is a s h a l l o w electron trap; the core c o m p l e x e s do not c o n t a i n the e x c i t a t i o n energy w e l l , and some o f that energy is returned to the antennae  11  p i g m e n t s r e s u l t i n g i n fluorescence ( F a l k o w s k i and R a v e n , 1997). T h e total f l u o r e s c e n c e o f plant c e l l s increases at 7 7 K (- 80°C) due to increased c o n t r i b u t i o n o f the core c o m p l e x e s o f P S 1 , a n d the contributions o f P S 1 fluorescence m a y also be t a x a s p e c i f i c ( H a l l a n d R a o 1999). W h e n a p h o t o n is absorbed b y P S 2 , P 6 8 0 (the core o f P S 2 ) b e c o m e s e x c i t e d a n d a n electron is transferred f r o m the electron carrier, p h a e o p h y t i n and then to the p l a s t o q u i n o n e p o o l t h r o u g h the carrier QA- A t this stage, P S 2 is c l o s e d , w h i c h means that it cannot accept any electrons u n t i l QA is r e o x i d i s e d . T h i s leads to the rise i n fluorescence to the p e a k l e v e l F . T h e subsequent q u e n c h i n g o f fluorescence is due to a c o m b i n a t i o n o f p h o t o c h e m i c a l M  q u e n c h i n g (the r e - o x i d i z a t i o n o f QA b y QB) a n d n o n - p h o t o c h e m i c a l q u e n c h i n g ( G o v i n d j e e , 1995, K r a u s e a n d W e i s , 1991). T h e d i f f e r e n c e b e t w e e n F a n d FM, referred to as v a r i a b l e f l u o r e s c e n c e , F y , indicates 0  the potential e f f i c i e n c y o f P S 2 . V a r i a b l e fluorescence is n o r m a l i z e d to FM ( E q u a t i o n s 1, 2) (Schreiber et a l . 1986), and the m a x i m u m , theoretical e f f i c i e n c y o f P S 2 is expressed i n terms o f FV/FM- U n d e r o p t i m a l c o n d i t i o n s , FV/FM ranges f r o m 0.83 f o r h i g h e r plants to 0.37 f o r some species o f algae, and the differences between t a x a are dependent o n their p h y s i o l o g i c a l m a k e u p ( M a x w e l l and J o h n s o n , 2 0 0 0 ; B i i c h e l a n d W i l h e l m , 1993). W i t h i n each group o f o r g a n i s m s , c o n d i t i o n s that decrease photosynthetic e f f i c i e n c y o f the c e l l w i l l l o w e r F v / F ( C a s p i et al., 1999; G e n t y et a l . 1989).  F* = F -F„ m  Y"  F  (D  m  Fig 2.1: A schematic of the Kautsky effect illustrating the kinetics of fluorescence observed when plants are transferred from darkness to high light. F is basal fluorescence, P is the 0  fluorescence peak after the sample is removed from the dark into actinic light, F M is maximal fluorescence, F is variable fluorescence and F is steady-state fluorescence. V  S  Time (min)  13  M e a s u r i n g F V / F M i n dark-adapted samples is s i m p l e . It requires a m e a s u r i n g l i g h t b e a m to measure basal fluorescence l e v e l F , and is i d e n t i c a l to the m e a s u r i n g b e a m i n m o s t 0  n o n - m o d u l a t e d fluorometers. F  0  s h o u l d be adequate to p r o v i d e a r e l a t i v e l y strong signal  w i t h o u t i n d u c i n g any a c t i n i c effects ( T i n g and O w e n s 1993; 1992). M a x i m a l f l u o r e s c e n c e , F M , is o b t a i n e d b y a p p l y i n g a saturating pulse to the dark-adapted s a m p l e ( S c h r e i b e r et al., 1986). F / F V  M  c a n also be determined u s i n g the t o x i n 3-(3,4-dichlorophenyl)-1,1 - d i m e t h y l  urea ( D C M U ) , w h i c h a r t i f i c i a l l y closes reaction systems b y b l o c k i n g the a c t i v i t y o f P S 2 ( E q u a t i o n 3) ( S a t o h and F o r k , 1983).  Q A Q B " + D C M U -> Q " D C M U + Q A  B  (3)  PAM fluorometry: A s m e n t i o n e d p r e v i o u s l y , q u e n c h i n g k i n e t i c s o f F M contains v a l u a b l e i n f o r m a t i o n o n p h o t o c h e m i c a l and n o n - p h o t o c h e m i c a l processes. T h e potential i m p o r t a n c e o f this has l e d to the d e v e l o p m e n t o f v a r i o u s techniques to decipher the fluorescence i n d u c t i o n c u r v e to express these processes i n m a t h e m a t i c a l terms. T h e m o s t s i g n i f i c a n t o f these techniques is the l i g h t d o u b l i n g m e t h o d i n t r o d u c e d b y B r a d b u r y a n d B a k e r (1981). T h e y p r o p o s e w h i c h photosynthetic processes are responsible f o r the o b s e r v e d fluorescence signals w h e n the sample is i l l u m i n a t e d w i t h photosynthetic " a c t i n i c " light, and their w o r k serves as the f o u n d a t i o n f o r Schreiber's P u l s e Saturation m e t h o d and the d e v e l o p m e n t o f the first c o m m e r c i a l P u l s e A m p l i t u d e M o d u l a t e d ( P A M ) fluorometers ( S c h r e i b e r , 1994; S c h r e i b e r et al., 1986). T h i s is c o m p a r a b l e to m e a s u r i n g F V / F M except that samples are i l l u m i n a t e d w i t h an actinic light source, so they are a c t i v e l y p h o t o s y n t h e s i z i n g ( w h i l e f l u o r e s c e n c e is at a steady state l e v e l F ) w h i l e the saturating pulses are a p p l i e d ( F i g s  2.2). T h e r e f o r e , the  m a x i m u m fluorescence peaks ( F ' ) M  14  Fig 2.2. The fluorescence trace generated using Pulse Amplitude Modulated fluorescence in dark-adapted Pseudo-nitzschia granii. F is basal fluorescence after dark-adaptation, F is 0  M  maximum fluorescence, F is steady-state fluorescence under actinic conditions, F ' is s  M  maximum fluorescence under steady-state conditions, F ' is the basal fluorescence after 0  steady-state.  1.2  1  Actinic Light  6  x  1 0  40  6  0x10  Scans  Saturating Pulse  15  obtained d u r i n g a c t i n i c i l l u m i n a t i o n p r o v i d e i n f o r m a t i o n o n n o n - p h o t o c h e m i c a l processes w h e n n o r m a l i z e d to the m a x i m a l fluorescence o f dark-adapted samples. N o r m a l i z i n g v a r i a b l e f l u o r e s c e n c e to the steady state l e v e l fluorescence p r o v i d e s v a l u a b l e i n f o r m a t i o n about the actual photosynthetic y i e l d o f the c e l l (the q u a n t u m y i e l d o f P S 2 , ^ P S 2 , equation 5) ( S c h r e i b e r et al., 1995a; 1995b; G e n t y et al., 1990; 1989; Schreiber et a l , 1986).  A r  =  PSI  F' -F rpi F M M  S  ( 5 )  '  v  U n d e r m o s t p h y s i o l o g i c a l c o n d i t i o n s , the q u a n t u m (or operational) y i e l d o f P S 2 i s l i n e a r l y correlated w i t h the y i e l d o f c a r b o n a s s i m i l a t i o n , expressed as m o l e s o f a t m o s p h e r i c C0  2  f i x e d per unit light absorbed (equation 6) ( G e n t y et a l . 1990; 1989). ^PS2 is also a  measurement o f the rate o f charge separation i n P S 2 ( B r a d b u r y and B a k e r , 1981). T h i s parameter contrasts n i c e l y w i t h the F V / F M ratio, w h i c h indicates theoretical photosynthetic efficiency.  A T  ,  Moles =  COi  Takenup y_  ( 6 )  Irradiance  A r e l a t i v e rate o f electron transport (J ) c a n be d e r i v e d f r o m 0 P S 2 t a k i n g into account e  incident light, f r a c t i o n a t i o n between P S l and P S 2 and other electron sinks w i t h i n the c e l l s ( M a x w e l l and J o h n s o n , 2000). F o r terrestrial plants, the relationship b e t w e e n 0PS2 and the rate o f c a r b o n a s s i m i l a t i o n is strongly l i n e a r ( G e n t y et al., 1989) and varies f r o m l i n e a r i t y under extreme c o n d i t i o n s , s u c h as a b n o r m a l l y h i g h light. In m a r i n e p h y t o p l a n k t o n , the linear rate o f electron transport competes w i t h non-photosynthetic e n z y m e s that use electrons, s u c h as nitrate reductase ( F l e m l i n g and K o r m k a m p , 1998; B u c h e l and W i l h e m , 1993). It is also affected b y heterogeneity i n P S 2 centers and the c o n t r i b u t i o n o f P S l to f l u o r e s c e n c e signals.  16  Therefore, absolute values o f electron transport rates cannot be estimated accurately f r o m ^PS2 i n m a r i n e p h y t o p l a n k t o n . T h e r e h a v e been a f e w attempts to test the c o r r e l a t i o n between aquatic p r i m a r y p r o d u c t i v i t y d e r i v e d f r o m fluorescence w i t h that d e r i v e d u s i n g  1 4  C . B o y d et a l . (1997) tested  this c o r r e l a t i o n i n the N o r t h A t l a n t i c u s i n g a Fast R e p e t i t i o n Rate F l u o r o m e t e r ( F R R F ) . T h e y f o u n d that the trend i n p r i m a r y p r o d u c t i v i t y d e r i v e d f r o m fluorescence (using F R R F measured o p t i c a l cross-section values) correlated w e l l w i t h those d e r i v e d f r o m  l 4  C . Spectral  n o r m a l i z a t i o n e x p l a i n e d a 3-fold difference between the absolute values d e r i v e d f r o m b o t h techniques. H a r t i g et a l . (1998) c o m p a r e d p r i m a r y p r o d u c t i v i t y d e r i v e d f r o m a P A M f l u o r o m e t e r w i t h those d e r i v e d f r o m  1 4  C i n benthic, freshwater c o m m u n i t i e s . T h e y m e a s u r e d  o p t i c a l cross-sections u s i n g a d u a l w a v e l e n g t h spectrophotometer. T h e trends b e t w e e n b o t h techniques correlated w e l l , yet absolute values o f p r i m a r y p r o d u c t i v i t y ( d e r i v e d f r o m fluorescence) w e r e overestimated at h i g h irradiance a n d underestimated at l o w irradiance. N o s p e c t r a f n o r m a l i z a t i o n was p e r f o r m e d i n their experiments. O t h e r parameters calculated f r o m c h l o r o p h y l l fluorescence i n c l u d e p h o t o c h e m i c a l q u e n c h i n g ( P , w h i c h measures light capture e f f i e c i e n c y , C h a p t e r 3) ( B e l k h o d j a et al., 1998), q  r e d o x pressure o n P S 2 ( 1 - P , w h i c h measured the a c c u m u l a t i o n o f electrons o n the acceptor q  side o f P S 2 , C h a p t e r 3) ( M a x w e l l et al., 1994; 1995a; 1995b) a n d n o n - p h o t o c h e m i c a l q u e n c h i n g (Q ). S e v e r a l components o f n o n - p h o t o c h e m i c a l q u e n c h i n g c a n be r e s o l v e d N  d e p e n d i n g o n their r e l a x a t i o n k i n e t i c s . T h e fast c o m p o n e n t o f Q  N  (QE) indicates the re-  e n e r g i z a t i o n o f t h y l a k o i d membranes due to the b u i l d up o f the proton gradient across t h y l a k o i d m e m b r a n e s ( M a x w e l l and J o h n s o n , 2000). T h i s triggers heat d i s s i p a t i o n o f absorbed l i g h t v i a the a c t i v a t i o n o f the x a n t h o p h y l l c y c l e , and is t h o u g h to be the m o s t  17  important m e c h a n i s m o f photoprotection i n c h l o r o p h y l l - c c o n t a i n i n g o r g a n i s m s ; i n diatoms, excess light triggers the d e - e p o x i d a t i o n o f the carotenoid d i d i n o x a n t h i n into d i a t o x a n t h i n ( O l a z i o l a and Y a m a m o t o , 1994). T h i s c y c l e is u n i q u e to diatoms. In h i g h e r plants, the x a n t h o p h y l l c y c l e is based o n the r a p i d d e - e p o x i d a t i o n o f v i o l a x a n t h i n into z e a x a n t h i n ( O l a z i o l a and Y a m a m o t o , 1994). T h e m i d d l e c o m p o n e n t o f Q , QT, relates to the p o o r l y understood p h e n o m e n o n o f N  state transition, w h i c h has not been d o c u m e n t e d i n diatoms ( M a x w e l l and J o h n s o n , 2 0 0 0 ) . T h e last and slowest c o m p o n e n t o f Q  P H  is due to p h o t o i n h i b i t i o n . T h e i n c l u s i o n o f a  c o m p o n e n t o f p h o t o i n h i b i t o n i n the d e f i n i t i o n o f QPH is c o n f u s i n g because whereas QE and QT are a p r o d u c t o f protective m e c h a n i s m s , QPH is an i n d i c a t o r o f damage.  Technical concerns regarding PAM fluorescence: T h e t e c h n i c a l and m a t h e m a t i c a l articulation P A M fluorescence fostered a b o o m i n the a p p l i c a t i o n o f the saturation pulse m e t h o d ( M a x w e l l and J o h n s o n , 2 0 0 0 ; G o v i n d j e e , 1995). M o d u l a t e d fluorometers can d i s t i n g u i s h between e x c i t a t i o n beams, saturation pulses, emitted fluorescence signals and b a c k g r o u n d light. T h e y are e q u i p p e d w i t h a light e m i t t i n g d i o d e adequately sensitive to differentiate between the signals, w i t h o u t b e i n g saturated. In a d d i t i o n to this, a w i d e variety o f c o m m e r c i a l , m o d u l a t e d fluorometers are a v a i l a b l e f o r f i e l d a p p l i c a t i o n , w h e r e b a c k g r o u n d sunlight is a p r o b l e m f o r n o n - m o d u l a t e d fluorometers. C o n s i d e r a t i o n s r e g a r d i n g a p p l y i n g c h l o r o p h y l l fluorescence i n the f i e l d and laboratory arise f r o m the f o l l o w i n g : instrumentation (sensitivity and settings), o r g a n i s m a l p h y s i o l o g y , nomenclature and the interpretation o f i n d u c t i o n curves.  18  The excitation beam and determination of Fn T h e basal l e v e l o f fluorescence F , to w h i c h almost a l l m e a s u r e d parameters are 0  n o r m a l i z e d , requires an e x c i t a t i o n b e a m that is l o w i n intensity to a v o i d a c t i n i c effects, yet h i g h e n o u g h to generate a strong signal ( S c h r e i b e r et al., 1995a; T i n g a n d O w e n s , 1992; 1993). T h i s dictates that the frequency o f the b e a m is l o w ( u s u a l l y a r o u n d 1.6 K H z ) , because h i g h frequencies o f e v e n the lowest l i g h t intensities m a y i n d u c e photosynthesis. T o a certain extent, this depends o n the p h y s i o l o g i c a l status and photosynthetic s y s t e m o f the o r g a n i s m probed. H i g h l y concentrated samples w i l l require a l o w intensity e x c i t a t i o n b e a m to g i v e a strong s i g n a l ( T i n g and O w e n s , 1992), w h i l e l o w c h l o r o p h y l l samples need a h i g h e r intensity b e a m than h i g h c h l o r o p h y l l cells, i n c r e a s i n g the chance o f photosynthetic effect ( S c h r e i b e r et al., 1995a; B u c h e l and W i l h e l m , 1993). T h e w a v e l e n g t h for the e x c i t a t i o n b e a m is important as w e l l because P S 2 is e x c i t e d at 680 n m and P S l is e x c i t e d at 700 n m ( H a l l and R a o , 1999), therefore, P A M measurements m a d e w i t h l i g h t above 700 n m m a y lead to s i g n i f i c a n t errors because o f P S l fluorescence ( G e n t y et al., 1989). D a r k - a d a p t i n g the samples is c r u c i a l f o r m e a s u r i n g F / F because it a l l o w s the v  m  reaction centers to r e - o x i d i z e and o p e n ( B u c h e l and W i l h e l m , 1993). I f this is not the case, F  0  w i l l be overestimated and F / F M w i l l be underestimated ( M a x w e l l and J o h n s o n , 2000). T h e V  length o f the dark adaptation p e r i o d s h o u l d be sufficient to a l l o w the f u l l r e - o x i d i z a t i o n o f sample a n d to a l l o w a l l p h o t o c h e m i c a l and n o n - p h o t o c h e m i c a l reactions i n the c e l l to cease ( T i n g a n d O w e n s , 1993; 1992). T h i s p e r i o d u s u a l l y ranges between 10 m i n u t e s to a n h o u r d e p e n d i n g o n the p h y s i o l o g y and the g r o w t h c o n d i t i o n s ( B u c h e l a n d W i l h e l m , 1993). F o r e x a m p l e , c h l o r o p h y l l - c c o n t a i n i n g o r g a n i s m s m a y require a longer dark adaptation t i m e than c h l o r o p h y l l - b c o n t a i n i n g o r g a n i s m s ( S c h r e i b e r et al., 1995a; T i n g and O w e n s , 1992; 1993).  19  D a r k adaptation s h o u l d not be l o n g e n o u g h to induce dark respiration because anaerobic c o n d i t i o n s change the fluorescence signals i n some m a r i n e algae (Schreiber et al., 1995a; S c h r e i b e r and N e u b a u e r , 1990). A m p l e care and t i m e s h o u l d be invested i n the correct d e t e r m i n a t i o n o f F , and once these c o n d i t i o n s are determined, they s h o u l d be kept constant 0  for a l l measurements p r e f o r m e d f o r a single species to a l l o w v a l i d c o m p a r i s o n b e t w e e n treatments.  Terminology and Nomenclature: In the years that f o l l o w e d the i n t r o d u c t i o n o f the light d o u b l i n g m e t h o d and the pulse saturation m e t h o d , there w a s a b o o m i n the a p p l i c a t i o n o f this technique, a n d v a r i o u s research groups m o d i f i e d the o r i g i n a l technique to suit their a p p l i c a t i o n and o r g a n i s m s o f c h o i c e . T h e nomenclature o f the parameters calculated w a s not u n i f o r m l y used, and m a n y researchers u s e d different t e r m i n o l o g y to describe what is essentially the same t h i n g , therefore r e n d e r i n g the early literature o n fluorescence rather c o n f u s i n g f o r the i n e x p e r i e n c e d reader ( M a x w e l l and J o h n s o n , 2 0 0 0 ; v a n K o o t e n and S n e l l , 1990). V a n K o o t e n a n d S n e l (1990) s u c c e s s f u l l y u n i f i e d the t e r m i n o l o g y a p p l i e d i n fluorescence studies. A l m o s t a l l papers, w i t h a f e w m i n o r exceptions, p u b l i s h e d o n c h l o r o p h y l l fluorescence i n the past decade h a d u s e d the n o r m a l i z e d nomenclature.  The Physiological status of the cell: T h e successful interpretation o f fluorescence i n d u c t i o n curves requires s u f f i c i e n t k n o w l e d g e o f the p h y s i o l o g y o f the o r g a n i s m i n v o l v e d . T h e g r o w t h c o n d i t i o n s o f the o r g a n i s m m a y affect the shape o f the i n d u c t i o n c u r v e and the strength o f the s i g n a l r e c e i v e d . T h e same o r g a n i s m w i l l g i v e a different signal i f it is g r o w n under c o n t i n u o u s light rather than a r e g i m e n t e d light: dark c y c l e (Strasser et a l . , 1999). In some m a r i n e algae, y o u n g e r ,  20  faster g r o w i n g c e l l s ( m i d d l e exponential) are healthier and h a v e a h i g h e r F V / F M ratio than older c e l l s ( i n e x p o n e n t i a l phase), w h i c h have a l o w e r F V / F M ratio. In the course o f a n experiment, P A M fluorescence s h o u l d be m e a s u r e d i n the m i d d l e o f e x p o n e n t i a l g r o w t h f o r a l l treatments to e l i m i n a t e o n t o l o g i c a l effects. N u t r i t i o n a l status also affects fluorescence i n d u c t i o n curves, e s p e c i a l l y nutrients that affect photosynthesis (such as trace metals and nitrogen) ( P a r k h i l l et al., 2 0 0 1 ; C a s p i et al., 1999, L i p p e m e i e r et al., 1999). T h e s e effects depend o n the a c c l i m a t i o n o f the c e l l s (balanced v s . u n b a l a n c e d g r o w t h ) ( P a r k h i l l et al., 2001). U n l e s s e x p e r i m e n t a l d e s i g n i n v o l v e s testing the effect o f v a r y i n g nutrient concentrations, nutrients and m e d i a s h o u l d be kept constant f o r a l l treatments and samples i n an experiment.  Specific concerns for phytoplankton samples: K a u t s k y ' s i n i t i a l observations w e r e m a d e u s i n g a s o l u t i o n o f the green, freshwater algae  Chlorella ( M a x w e l l and J o h n s o n , 2 0 0 0 ; G o v i n d j e e , 1995). T h e studies p r e c e d i n g  K a u t s k y ' s w o r k m o s t l y dealt w i t h higher plants, w h i l e the d e v e l o p m e n t o f P A M f l u o r o m e t e r y f o r algal suspensions w a s r e l a t i v e l y i g n o r e d , based o n the rather n a i v e a s s u m p t i o n that algae are m i n i a t u r e plants and they f u n c t i o n i n v e r y m u c h the same w a y (Schreiber et al., 1995a; Schreiber, 1994; T i n g and O w n e n s , 1992; 1993). In 1994, the first cuvette a s s e m b l y P A M attachment w a s i n t r o d u c e d , w i t h a h i g h l y sensitive detection s y s t e m m a d e s p e c i f i c a l l y f o r l o w c h l o r o p h y l l algal suspensions (at u M levels) and attempts w e r e m a d e to r e m o d e l P A M technique used i n h i g h e r plant to suit p h y t o p l a n k t o n research (Schreiber, 1994). E v e n w i t h the i n t r o d u c t i o n o f this h i g h l y sensitive system, m a n y reasons e x i s t as to w h y P A M f l u o r o m e t r y is d i f f i c u l t to a p p l y to algal cultures and field assemblages. F i r s t l y ,  21  the current s c i e n t i f i c understanding o f the photosynthetic process i n algae is l a c k i n g , w h i c h hinders the interpretation o f c h l o r o p h y l l fluorescence. In a d d i t i o n to p h y s i o l o g y , there is a requirement f o r s u f f i c i e n t b i o m a s s . H i g h concentrations o f c h l o r o p h y l l increase the s i g n a l to noise ratio a l l o w i n g for a stronger, m o r e r e l i a b l e fluorescence signal ( T i n g and O w e n s , 1993), a n d m o s t o c e a n i c concentrations o f c h l o r o p h y l l are too l o w and n o i s y f o r r e l i a b l e P A M signals. U n d e r laboratory c o n d i t i o n s , p r o d u c i n g e n o u g h b i o m a s s to p e r f o r m r e l i a b l e P A M measurements is not p r o b l e m a t i c . C o a s t a l and freshwater s p e c i m e n s often reach a h i g h c o n c e n t r a t i o n o f c h l o r o p h y l l w i t h o u t m u c h effort. O p e n o c e a n species are d i f f i c u l t to g r o w i n the lab, and do not often r e a c h h i g h c h l o r o p h y l l levels, i n w h i c h case, the samples are concentrated either b y f i l t r a t i o n and resuspension or b y centrifugation and r e s u s p e n s i o n . C o n c e n t r a t i o n techniques are c r i t i c i z e d because they m a y sometimes result i n d a m a g e d c e l l s i f they are p e r f o r m e d too v i g o r o u s l y o r i f the c e l l s are fragile, w h i c h results i n the u n d e r e s t i m a t i o n o f F V / F M ( B i i c h e l and W i l h e l m , 1993, Schreiber, 1994). H i g h l y concentrated samples m a y also result i n self-shading w h i c h also underestimates F V / F M ( B i i c h e l and W i l h e l m , 1993). It is best, therefore, to a v o i d c o n c e n t r a t i o n procedures, and i f this is not p o s s i b l e , it is r e c o m m e n d e d to d e v e l o p a n o n - d a m a g i n g c o n c e n t r a t i o n technique f o r each o r g a n i s m used ( T i n g and O w e n s , 1992).  Summary: C h l o r o p h y l l fluorescence p r o v i d e s an easy and n o n - i n v a s i v e m e c h a n i s m o f i n v e s t i g a t i n g photosynthesis  in vivo. R e s e a r c h i n the area is f u e l l e d b y the a v a i l a b i l i t y o f  h i g h l y sensitive, user f r i e n d l y and portable m o d u l a t e d fluorometers. R e c e n t l y , the i n t r o d u c t i o n o f a m o d u l a t e d f l u o r o m e t e r that measures photosynthetic e f f i c i e n c y u s i n g a cuvette s y s t e m sparked interest i n u s i n g c h l o r o p h y l l fluorescence to investigate  22  photosynthesis i n m a r i n e p h y t o p l a n k t o n . T h e interpretation o f fluorescence signals i s c o m p l i c a t e d and requires sufficient k n o w l e d g e o f p h y s i o l o g y as w e l l as instrumentation. H o w e v e r , P A M fluorescence remains a p r o m i s i n g t o o l f o r o c e a n o g r a p h i c research.  Thesis Objectives: 1. T o isolate Pseudo-nitzschia granii f r o m the N E S u b a r c t i c P a c i f i c , a n d to o p t i m i z e its g r o w t h under laboratory c o n d i t i o n s . 2. T o d e v e l o p a p r o t o c o l f o r m e a s u r i n g the photosynthetic e f f i c i e n c y o f P. granii u s i n g P u l s e A m p l i t u d e Modulated ( P A M ) Fluorescence. 3. T o investigate the effects o f l i g h t and F e c o - l i m i t a t i o n o n the p h y s i o l o g y o f P.  granii. 4. T o investigate the effects o f light and temperature o n the photosynthetic e f f i c i e n c y and g r o w t h o f P. granii under e c o l o g i c a l l y relevant irradiances and temperatures.  23  Chapter 3 Effect of Light and Fe on the Physiology of  Pseudo-nitzschia granii Isolated  From the NE Subarctic Pacific  Introduction P h y t o p l a n k t o n photosynthesis ( p r i m a r y production) i s a s i g n i f i c a n t c a r b o n s i n k f r o m the atmosphere into the ocean. O n e o f the most important c h e m i c a l factors l i m i t i n g p r i m a r y p r o d u c t i o n i n - 3 0 % o f the oceans is i r o n (Fe) ( B o y d et al., 2000; H u t c h i n s , 1995; G e i d e r and L a R o c h e , 1994; M a r t i n et al., 1990). In h i g h latitude F e - l i m i t e d areas, s u c h as the N E subarctic P a c i f i c and the Southern O c e a n ( T i m m e r m a n s et al., 2 0 0 1 ; M a l d o n a d o et al., 1999), p r i m a r y p r o d u c t i o n m a y also be l i m i t e d b y the a v a i l a b i l i t y o f light, e s p e c i a l l y i n the w i n t e r season w h e r e surface irradiance is l o w , days are short and the m i x e d layer i s deep. F e n u t r i t i o n and light absorption are strongly c o u p l e d through the process o f photosynthesis. I n the photosynthetic unit, F e is required as a structural b u i l d i n g b l o c k i n p h o t o s y n t h e t i c reaction centers (Photosystems 1 a n d 2 ( P S 1, P S 2 ) ) and as a n electron carrier i n c y t o c h r o m e s and f e r r e d o x i n because o f its r e d o x versatility ( G e i d e r and L a R o c h e , 1994). In order to m a x i m i z e l i g h t absorption, cells g r o w n under l o w l i g h t increase the c o n c e n t r a t i o n o f photosynthetic p i g m e n t s and reaction centers, thereby i n c r e a s i n g their F e quota (i.e. requirement). S o m e t i m e s light l i m i t a t i o n drives o r g a n i s m s to the point o f F e l i m i t a t i o n ( F a l k o w s k i et al., 1985). C h l o r o p h y l l synthesis is dependent o n F e a v a i l a b i l i t y , a n d F e l i m i t e d cells are often c h l o r o t i c , w h i c h hinders their a b i l i t y to absorb light ( K u d o and H a r r i s o n , 1997; G r e e n e et a l . 1992; 1991). Therefore, cells g r o w n under l o w l i g h t and l o w F e are c o n s i d e r e d to be under a "double stress". T h e i n d i v i d u a l effects o f p h o t o a c c l i m a t i o n and Fe l i m i t a t i o n o n the photosynthesis o f v a r i o u s p h y t o p l a n k t o n are w e l l d o c u m e n t e d i n the  24  scientific literature (Langdon, 1988). However, the combined effects of both light and Fe on the physiology of oceanic phytoplankton are rarely studied (Van Leeuwe and De Baar, 2000). There is preliminary evidence to Fe and light co-limitation in the field. During the winter season at Ocean Station P (OSP, 145°N 45°W), increasing irradiance along with a Fe addition in shipboard experiments results in a dramatic increase in chlorophyll concentrations and carbon uptake, more than when each factor is added separately (Maldonado et al., 1999). Similar results have been obtained in the Southern Ocean (Timmermans et al. 2001). It is especially important to study the effect of Fe and light co-limitation on the physiology of oceanic diatoms. Large diatoms are rare in Fe-limited regions (<6% of biomass, Harrison et al., 1999) because of their low surface area to volume ratio, which is thought to hinder their ability to compete with smaller, more efficient cells for Fe (Harrison et al., 1999). In Fe-limited regions, when Fe is added during shipboard experiments, large diatoms grow quickly and dominate the enriched assemblage (Boyd et al., 1995a; 1995b; Martin et al. 1994; 1990). The ability to survive Fe stress under limiting conditions, as well as opportunistic growth when Fe is added, indicates that diatoms are physiologically well adapted to Fe stress. Studies on the physiology of diatoms often focus on coastal, centric diatoms, which are easier to culture and maintain in the laboratory than either pennate or oceanic diatoms. Pennate diatoms account for 30% of diatomaceous carbon at OSP (Boyd and Harrison, 1999), and the genus Pseudo-nitzschia dominates Fe-enriched assemblages, especially in the summer (Boyd et al., 1995a; 1995b; M . Lipsen, pers. comm.). Some coastal strains of Pseudo-nitzschia produce domoic acid, which causes amnesic shellfish poisoning  25  and is r e s p o n s i b l e f o r the death o f h u m a n s and w i l d l i f e o n the eastern coast o f C a n a d a a n d the coast o f C a l i f o r n i a r e s p e c t i v e l y (Bates et al., 1999; H o r n e r and P o s t e l , 1993; Bates et a l . , 1989). It i s u n k n o w n whether  Pseudo-nitzschia granii o r any other o c e a n i c Pseudo-nitzschia  sp. p r o d u c e d o m o i c a c i d at e c o l o g i c a l l y relevant levels. T h i s study e x a m i n e s the effect o f light and F e o n the o c e a n i c d i a t o m ,  Pseudo-  nitzschia granii (isolated f r o m O S P i n June o f 2 0 0 0 ) . I n a d d i t i o n to p h y s i o l o g i c a l and b i o c h e m i c a l parameters ( g r o w t h rates, c h l o r o p h y l l , particulate o r g a n i c c a r b o n , particulate o r g a n i c n i t r o g e n a n d b i o g e n i c s i l i c a ) , photosynthetic e f f i c i e n c y w a s m e a s u r e d u s i n g p u l s e a m p l i t u d e m o d u l a t e d ( P A M ) c h l o r o p h y l l fluorescence. T h i s technique i s w i d e l y u s e d i n terrestrial plant p h y s i o l o g y , and has been o n l y recently adapted f o r l i q u i d p h y t o p l a n k t o n s a m p l e s (Schreiber, 1994). T h e e f f i c a c y o f fluorescence-based techniques i n m e a s u r i n g p r i m a r y p r o d u c t i o n i s w i d e l y debated, but the p o s s i b i l i t y o f e m p l o y i n g t h e m to measure photosynthetic e f f i c i e n c y i n the oceans is desirable because they are easy, i n e x p e n s i v e a n d n o n - i n v a s i v e ( H a r t i g et al., 1998; B o y d et al., 1997). C h l o r o p h y l l fluorescence p r o v i d e s m o l e c u l a r i n f o r m a t i o n about the p h y s i o l o g i c a l status o f the c e l l w i t h o u t the use o f i n v a s i v e , b i o c h e m i c a l techniques. I n this study, m a x i m u m photosynthetic e f f i c i e n c y ( F / F M ) , q u a n t u m V  photosynthetic e f f i c i e n c y ( 0 P S 2 ) , p h o t o c h e m i c a l q u e n c h i n g ( P ) and n o n - p h o t o c h e m i c a l q  q u e n c h i n g ( Q ) w e r e measured u s i n g P A M fluorescence (see C h a p t e r 2 f o r r e v i e w ) . n  26  Materials and Methods  Diatom isolation: Chains of Pseudo-nitzschia granii were isolated from Fe-enriched (2 nM FeCb in 10% HC1 stock) OSP samples collected from Go-Flo® bottles on board the CCGS J.P. Tully in June of 2000. The samples were kept at low light and temperature (4°C, 12:12 L:D cycle at 50 pmol photons m" s") and enhanced with ESAW vitamins (Harrison et al., 1980) and 2  1  AQUIL nutrients (Price et al., 1988/1989; Appendix 1) until the time of isolation (Chemicals purchased from Sigma-Aldrich). The cells were 60 um in length and 5 urn in width at the time of isolation, and each chain contained 5-6 cells. The isolation medium consisted of AQUIL nutrients (metal stocks at half the concentration of the recipe to avoid metal toxicity) in a base of filtered (0.2 urn, trace metal clean) OSP seawater. The chains were isolated using two techniques: picking individual chains (to isolate monoclonal cultures) and dilution (to isolate polyclonal cultures). Experimental cultures were polyclonal to facilitate sexual reproduction (Hiltz et al., 2000). The isolated cultures were maintained at the above conditions until growth rates stabilized (almost a month). Growth rates were monitored using fluorescence (Turner AU-10 fluorometer). The species of Pseudo-nitzschia was identified by Dr. Vera Trainer's group (University of Washington, Seattle) using scanning electron microscopy.  Culture Conditions: Semi-continuous batch cultures were maintained in Aquil/SOW medium (Appendix 1). There were two Fe treatments: Fe-replete (control, pFe 19.4, where pFe = - log [Fe ])  27  and F e - d e f i c i e n t ( F e - l i m i t e d or Fe-deplete, w i t h no added F e , b a c k g r o u n d c o n t a m i n a t i o n w a s <2 n M (total F e ) , p F e = 24-25). N u t r i e n t stocks a n d S O W were treated w i t h C h e l e x 100 i o n exchange r e s i n ( B i o - R a d , R i c h m o n d , C A ) to r e m o v e traces o f F e ( P r i c e et al., 1988/89). C u l t u r e s were g r o w n i n polycarbonate containers (30 m l ) , w h i c h were a c i d - w a s h e d to a v o i d F e c o n t a m i n a t i o n . T h e containers were first incubated i n 1 0 % H C 1 f o r o n e w e e k , r i n s e d w i t h Milli-Q® water, incubated i n 1 0 % Seastar® H C 1 (Seastar C h e m i c a l s , S i d n e y , B C ) f o r one week, r i n s e d w i t h Milli-Q® water again, a n d f i n a l l y incubated i n 0.1 N Seastar® acetic a c i d ( P r i c e et al., 1988/1989). C u l t u r i n g tubes were m i c r o w a v e - s t e r i l i z e d ( K e l l e r et al., 1983) i n Milli-Q® water before usage. T h e r e were 6 light treatments (166, 9 0 , 6 0 , 3 0 , 15 a n d 7 p m o l photons m" s" , c o n t i n u o u s light), s u p p l i e d b y c o o l w h i t e V i t a - l i t e l a m p s ( H O W ) a n d attenuated u s i n g neutral density screening. T h e experiments were c o n d u c t e d at 11.5°C. C u l t u r e s were g r o w n i n triplicate, a n d a c c l i m a t e d to their respective treatments before s a m p l i n g (between 10 a n d 2 0 generations d e p e n d i n g o n the treatment). A c c l i m a t i o n w a s a c h i e v e d w h e n g r o w t h rates stabilized. C e l l s were enumerated u s i n g a C o u l t e r C o u n t e r ( M o d e l Z ) . G r o w t h rates ( p , d o u b l i n g s day" ) were calculated as the slope o f the natural l o g 1  o f fluorescence  (in vivo c h l o r o p h y l l ) values o v e r t i m e ( G u i l l a r d , 1973). S a m p l i n g w a s  conducted during exponential growth.  Chlorophyll (Chl cell ), non-modulated chlorophyll fluorescence (FSLO per unit 1  chlorophyll (FSU Chl'*): B a s a l , n o n - m o d u l a t e d c h l o r o p h y l l fluorescence ( i n fluorescence unit, F S U ) w a s measured u s i n g 10 m l o f intact sample i n the T u r n e r A U - 1 0 fluorometer. C h l o r o p h y l l w a s extracted a c c o r d i n g to the standard, f l u o r o m e t r i c m e t h o d o f Parsons et a l . (1984); a 10 m l sample w a s f i l t e r e d through a G F / F filter, w h i c h w a s then extracted i n 9 0 % acetone f o r 2 4 h  28  in the dark in the freezer. The samples were then thawed for half an hour and the fluorescence before and after acidification was measured to correct for phaeopigments. These chlorophyll measurements accounted for chlorophyll a, and chlorophyll c was not measured. Chlorophyll per cell (Chi cell" ) was calculated by normalizing chlorophyll to cell number. Basal fluorescence 1  was normalized to extracted chlorophyll to calculate FSU chi  Photosynthetic Efficiency Using DCMU  (FV/FMJJTMM):  The fluorescence of a 10 ml sample was measured to obtain basal fluorescence, F . 0  The fluorescence was measured again after the addition 50 pi of 40 u M 3-(3,4dichlorophenyl)-l,l-dimethylurea (DCMU) to obtain maximal fluorescence, FM- FV/FM was calculated according to the equation in Table 3.1.  Biogenic Silica fBSi): A 10 ml sample was filtered through a 0.6 um polycarbonate filter. Biogenic silica was dissolved then measured spectrophotometrically following Parsons et al. (1984).  Particulate Organic Carbon and Particulate Organic Nitrogen (POC and PON): A 10 ml sample was filtered through a combusted (450°C for 4.5 h) GF/F Whatman® filter. Ample care was taken not to contaminate the samples with extraneous carbon or nitrogen. The filters were dried at 60°C for 24 h and stored until analysis using a Carlo Erba NCS elemental analyzer NA1500. Filter blanks and container blanks were subtracted from the samples.  Modulated Chlorophyll Fluorescence: Chlorophyll fluorescence kinetics were measured using a 101 P A M fluorometer (Walz, Germany) complete with a cuvette system (Schreiber, 1994). Fluorescence was  29  m e a s u r e d at m i d - e x p o n e n t i a l g r o w t h w i t h c h l o r o p h y l l concentrations b e t w e e n 2 0 - 4 0 p g L"  1  to m i n i m i z e n o i s e w i t h o u t affecting the integrity o f the s i g n a l ( T i n g a n d O w e n s , 1993; 1992). A 2 m l sample w a s a c c l i m a t e d f o r 15 m i n i n the dark before measurements c o m m e n c e d ( T i n g and O w e n s , 1992). B a s a l light intensity w a s c h o s e n so that it w a s strong e n o u g h to g i v e a s i g n a l (basal fluorescence, F  0  between 0.30 -0.4) w i t h o u t i n d u c i n g a p h o t o s y n t h e t i c  effect (otherwise k n o w n as an actinic effect). A pulse o f saturating l i g h t (> 2 0 0 0 p m o l p h o t o n s m" s" f o r 7 0 0 m s , C o l e - P a l m e r l a m p ) w a s a p p l i e d to measure m a x i m u m 2  1  fluorescence, F , w h i c h is analogous to F M DCMU)- A c t i n i c light w a s a p p l i e d to the s a m p l e at M  the same intensity as the g r o w t h light ( H a n s t e c h l a m p , attenuated u s i n g neutral d e n s i t y screening) (see F i g . 2.2 f o r trace k i n e t i c s o f P A M fluorescence). O n c e steady state 2  1  fluorescence w a s a c h i e v e d (Fs), saturating pulses (at 2 0 0 0 p m o l p h o t o n s m" s" , 3 0 0 m s ) were a p p l i e d e v e r y 2 0 s to measure F'M- F' w a s measured after the f l u o r e s c e n c e s i g n a l 0  s t a b i l i z e d w h e n the a c t i n i c light w a s turned o f f (see Chapter 2 for details, F i g . 2.2). T h e o r e t i c a l photosynthetic e f f i c i e n c y (FV/FM), q u a n t u m photosynthetic e f f i c i e n c y ( 0 P S 2 ) , linear rate o f e l e c t r o n transport ( J ) , p h o t o c h e m i c a l q u e n c h i n g ( P ) , r e d o x p o i s e ( 1 - P ) and e  q  q  n o n - p h o t o c h e m i c a l q u e n c h i n g ( Q ) w e r e m e a s u r e d and c a l c u l a t e d a c c o r d i n g to equations i n n  T a b l e 3.1.  Statistical Analysis: A n a n a l y s i s o f v a r i a n c e ( A N O V A ) , f o l l o w e d b y a Student's t-test w a s u s e d to determine the statistical differences between treatments ( a = 0.95) ( a s s u m i n g data w e r e n o r m a l l y distrubited). W h e n s p e c i f i e d , a linear regression w a s also used to determine the degree o f c o r r e l a t i o n between parameters (r ). A l l statistical analyses f o l l o w e d Z a r (1984).  Table 3.1: Equations of photosynthetic parameters calculated from chlorophyll fluorescence kinetics. Fy is variable fluorescence, FM, is maximal fluorescence, F is basal 0  fluorescence, F is steady state fluorescence, F'M is variable fluorescence during steady state, s  F' is basal fluorescence following steady state, QA is the phaeophytin A, I is incident light 0  intensity and DCMU, is 3-(3,4- dichlorophenyl)-1,1 dimethyl urea. References listed are examples of how these parameters are applied. Parameter  Maximum Photosynthetic Efficiency (DCMU)  FV/F ' M v  Reference  Equation  Symbol  F  F  MDCMU  Parkhill et al. (2001)  o  1  F  MDCMU  Schreiber et al. (1995a; 1995b); Bradbury and Baker(1981)  Maximum Photosynthetic Efficiency  Fv/F;  M  V' M  ~  F o  1  Quantum Yield of  PS2  F  ^PS2  F'u  F  M  ~  F  F V  Gentyetal. (1989)  F  A  F  \  </)PS2 * J  Rate of Linear Electron Transport  Photochemical  (p  Quenching  ( F ' Redox Poise  Maxwell and Johnson (2000); Gentyetal. (1989) Buschmann(1999)  \, u  F  )  s  - F ' o ) Maxwell et al. (1995a; 1995b)  Q ARED 1-P„  Non-photochemical  Q ARED  -Q  (FM FC)  Quenching Qn  (FM FO)  AOXD  FV  F)V  FV  Maxwell and Johnson(2000); Buschmann(1999)  31  Results Diatom Isolation: Pseudo-nitzschia granii maintained its chain-forming ability in the isolation medium. The cells were gradually acclimated to an AQUIL/Synthetic Ocean Water (SOW)  medium  (Price et al, 1988/1989), whereby the number of cells per chain decreased to 3 per chain. Abnormalities in the shape of the cell appeared in this medium shortly after transfer. However, the cells did resume their natural shape two months after acclimation (Figs. 1 and 2). This is not unusual in cultures of nitzschoid pennate diatoms (Suba Rao and Wohlgeschaffen, 1990) and is thought to be the result of metal concentrations higher than natural oceanic levels. The length of cells decreased to 25 urn (length) and -3.5 pm (width) by the time of the experiment.  Growth Rate and Biochemical Parameters: The growth rate of Fe-limited Pseudo-nitzschia granii (Figs. 1 and 2) was 10-50% (average 30%) lower than Fe-replete cells (Fig. 3.3A). Over the range of irradiances studied, the greatest differences between Fe treatments occurred in the middle range of irradiance (Fig. 3.3B). In general, growth rates decreased with decreasing irradiance; at the two lowest light treatments (7 and 15 pmol photons m" s"), growth rates stabilized at 0.35 for Fe-replete cells, and 0.2 for Fe-limited cells. The initial slope (•) of the growth irradiance (p-I) was higher under Fe-replete conditions than under Fe limitation (6.5 x 10" and 2.2 x 10" respectively). Whereas Fe-replete cells reached a pmax of 0.81 day" at 166 pmol photons m" 1  2  s", Fe-limited cells did not plateau under the range of irradiance used in this study. 1  32  1  1  9  U n d e r Fe-replete conditions, c h i cell" at 7 p m o l photons m" s" was 3-fold h i g h e r 9  1  1  than at 166 p m o l photons m" s" ( F i g . 3 . 4 A ) . C h i cell" was i n v e r s e l y related to l i g h t ( F i g . 3 . 4 A ) . U n d e r F e - l i m i t e d c o n d i t i o n s , the relationship between c h i cell" a n d l i g h t w a s not as 1  2  1  1  p r o n o u n c e d as f o r F e - replete cells, as c h i cell" at 7 p m o l photons m" s" w a s o n l y 2 - f o l d higher at l o w light than at h i g h light. H o w e v e r , cells under F e l i m i t a t i o n were severely c h l o r o t i c c o m p a r e d to F e - replete c e l l s ( P <0.05). C h l o r o s i s means c h l o r o p h y l l d e f i c i e n c y , o r b l e a c h i n g m o s t l y due to nutrient stress or extreme p h y s i c a l stress. N o r m a l i z i n g c h i to particulate o r g a n i c c a r b o n r e m o v e s the effect o f d i f f e r i n g c e l l u l a r v o l u m e s b e t w e e n treatments a n d a l l o w s a c o m p a r i s o n o f the response o f F e l i m i t a t i o n between v a r i o u s organisms. C c h i " ' was s i g n i f i c a n t l y higher under F e l i m i t a t i o n c o m p a r e d to Fe-replete c o n d i t i o n s and c h i cell" increased w i t h i n c r e a s i n g irradiance ( F i g . 3.4b). 1  B a s a l n o n - m o d u l a t e d fluorescence is a g o o d i n d i c a t o r o f p h y s i o l o g i c a l stress i n p h y t o p l a n k t o n . N o r m a l i z i n g F S U to c h l o r o p h y l l a l l o w s c o m p a r i s o n between o r g a n i s m s o f d i f f e r i n g c h l o r o p h y l l concentrations. F S U c h i " ' w a s generally h i g h e r f o r F e - l i m i t e d cells c o m p a r e d to Fe-replete cells ( P < 0.05), and was i n v e r s e l y related to irradiance i n F e - l i m i t e d cells ( F i g . 3.5). T h e C : N ratio is a useful i n d i c a t o r o f nitrogen stress i n m a r i n e p h y t o p l a n k t o n (see discussion). In this experiment, the C : N ratio ranged between 7 and 10, and w a s g e n e r a l l y higher, t h o u g h not s i g n i f i c a n t l y (P >0.05) under F e replete c o n d i t i o n s as c o m p a r e d to F e l i m i t e d c o n d i t i o n s , and C : N was i n v e r s e l y related to irradiance ( F i g . 3 . 6 A ) . C c e l l " was not 1  s i g n i f i c a n t l y h i g h e r i n Fe-replete cells (P>0.05) c o m p a r e d to F e - l i m i t e d cells ( F i g 3 . 6 B ) . C e l l s under F e l i m i t a t i o n also e x h i b i t a l o w e r N cell" than F e replete cells ( P < 0.05) ( F i g . 1  3.6B). T h e r e w a s no relationship b e t w e e n either c e l l u l a r P O C or P O N w i t h light.  33  In the field, the uptake ratio o f S i to N is measured to assess F e l i m i t a t i o n . T h e p r e m i s e o f the S i : N ratio as an i n d i c a t o r o f F e l i m i t a t i o n is that F e l i m i t a t i o n decreases n i t r o g e n uptake (due to decreased a c t i v i t y o f nitrate reductase), but does not affect S i uptake ( H u t c h i n s et al., 1998; T a k e d a , 1998). Therefore, F e - l i m i t e d cells have a h i g h e r uptake ratio o f S i to N than F e - s u f f i c i e n t cells. H o w e v e r , the m o l a r ratio o f b i o g e n i c S i to N is rarely studied. F u r t h e r m o r e , the b e h a v i o r o f this ratio has not been e x a m i n e d u n d e r c o n d i t i o n s o f b a l a n c e d g r o w t h . In this study, the m o l a r ratio o f B S i to P O N ( B S i : N ) u n d e r b a l a n c e d ( a c c l i m a t e d ) g r o w t h w a s measured. T h e B S i : N ratio w a s at least 2- f o l d h i g h e r i n Fe-replete c e l l s c o m p a r e d to F e - d e f i c i e n t c e l l s a n d the ratio d i s p l a y e d a h y p e r b o l i c r e l a t i o n s h i p w i t h light ( F i g . 3 . 7 A ) . B S i c e l l " w a s s i g n i f i c a n t l y h i g h e r (P <0.05) i n c o n t r o l c e l l s c o m p a r e d to F e - d e f i c i e n t 1  c e l l s , a n d l i k e B S i : N , it d i s p l a y e d a h y p e r b o l i c r e l a t i o n s h i p w i t h l i g h t ( F i g . 3.7b). It is c o m m o n l y h y p o t h e s i z e d that the proteinaceous m a t r i x for s i l i c a d e p o s i t i o n i n d i a t o m s is c o m p o s e d o f m a i n l y serine a n d g l y c i n e , t w o by-products o f p h o t o r e s p i r a t i o n . T h e r e f o r e , c e l l s w i t h h i g h e r l e v e l s o f photorespiratory a c t i v i t y w i l l have a t h i c k e r silicate frustule.  Chlorophyll Fluorescence Kinetics: FV/FM w a s m e a s u r e d u s i n g the P A M  fluorometer  (results not s h o w n ) a n d b y D C M U  treatment. U s i n g b o t h measurements, F V / F M w a s s i g n i f i c a n t l y h i g h e r u n d e r Fe-replete c o n d i t i o n s c o m p a r e d to F e - l i m i t e d c o n d i t i o n s (P <0.05) ( F i g . 3.8). FV/FM (DCMU) w a s not s i g n i f i c a n t l y affected b y light i n either F e treatments. R e s u l t s f r o m b o t h m e t h o d s correlated s t r o n g l y ( r = 0.69, P < 0.05) ( F i g . 3.9). 2  O v e r the entire range o f treatments i n this experiment, the r e l a t i o n s h i p b e t w e e n c h l cell" a n d F / F M 1  V  (DCMU) w a s non-linear ( F i g . 3.10); w h e n c h l cell" w a s h i g h e r than 5 p g c e l l " , 1  1  34  FV/FM s t a b i l i z e d at - 0 . 6 (as seen i n F e replete cells). A t concentrations b e l o w this t h r e s h o l d l e v e l , as seen i n F e - l i m i t e d cells, FV/FM decreased l i n e a r l y w i t h d e c r e a s i n g c h l c e l l " . 1  T h e q u a n t u m y i e l d o f photosynthesis, 0 P S 2 was m u c h h i g h e r under Fe-replete c o n d i t i o n s t h a n under F e l i m i t a t i o n ( P < 0.05), and was i n v e r s e l y related to irradiance ( F i g . 3.11). In this study, the relative rate o f linear electron transport, J , w a s c a l c u l a t e d b y e  m u l t i p l y i n g #JPS2 w i t h i n c i d e n t light w i t h o u t a c c o u n t i n g f o r f r a c t i o n a t i o n b e t w e e n the photocenters, the photosynthetic cross-section, or the f r a c t i o n o f electrons donated to n o n photosynthetic e n z y m e s . J was s i g n i f i c a n t l y higher i n Fe-replete cells than under F e e  l i m i t a t i o n ( P < 0.05) ( F i g 3.1 I B ) . U n d e r b o t h F e treatments, J h a d a strong, p o s i t i v e e  r e l a t i o n s h i p w i t h g r o w t h rate ( r = 0.95 - 0.96) ( F i g . 3.12). 2  P h o t o c h e m i c a l e f f i c i e n c y , P was not s i g n i f i c a n t l y different b e t w e e n F e treatments ( P q  >0.05), and w a s i n v e r s e l y related to irradiance ( F i g . 3 . 1 3 A ) . T h e r e d o x pressure o n p h o t o s y s t e m 2, 1-P , w a s not s i g n i f i c a n t l y different between F e treatments (P> 0.05), and q  was p o s i t i v e l y related to irradiance ( F i g . 3.13B). N o n - p h o t o c h e m i c a l q u e n c h i n g , Q , w a s N  higher, t h o u g h not s i g n i f i c a n t l y under F e - l i m i t e d c o n d i t i o n s than under Fe-replete c o n d i t i o n s (P >0.05). U n d e r Fe-replete c o n d i t i o n s , Q w a s s i g n i f i c a n t l y l o w e r at l o w l i g h t than h i g h N  light (P<0.05) ( F i g . 3.14).  35  Figure 3.1: A ) Scanning electron micrograph of Pseudo-nitzschia granii. Arrow points to diatoms (bar =10 urn). B) Deformed cell after three months of isolation. Arrow points to deformation (both bars = 10 urn), and C) close-up of surface features (bar =1 pm; Photos were taken by Brian D. Bill, University of Washington).  Figure 3.2: Light micrographs of P. granii (bar = 10 pm). Arrow points to diatom chain.  Figure 3.3: G r o w t h rate (p) vs. irradiance f o r P. granii g r o w n under Fe-replete c o n d i t i o n s ( b l a c k c i r c l e s ) and Fe-deplete c o n d i t i o n s (white circles) ( A ) and, ratio o f Fe-deplete to F e replete g r o w t h rates v s . irradiance (B). E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e .  1.0 •  0.8  A  0.6 0.4 0.2  I  °0  B  <D -4—<  0 Q. 0  i_ i <D LL  ~=L  0.6  I  0.4  0) -i—'  j 0) § 0.2 Q. 0 T3  d) LL  0.0  0  40  80 120 160  200  Light (Limol photons m s ) 2  1  38  3.4:  Figure  C h l cell" v s . irradiance ( A ) , and C c h l " ' vs. irradiance ( B ) f o r 1  P. granii g r o w n  under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete c o n d i t i o n s (white c i r c l e s ) . E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n invisible.  1  "CD  12 10  i "(D  8 6 4  ^=  2  o  O) Q.  o  O  200 — t  6 o  160 120 80 40 0  40 Light  80  (LUTIOI  120 160 200 photons rrv s- ) 1  1  39  Figure 3.5: FSU c h i v s . irradiance for P. granii grown under Fe-replete conditions (closed circles) and Fe-deplete conditions (open circles) (n=l).  0  40  Light  80 (LIITIOI  120  160  photons m- s- ) 2  1  40  Figure 3.6: 1  C : N m o l a r ratios v s . irradiance ( A ) , and ( B ) C cell" vs. irradiance and ( C ) N cell" 1  vs. irradiance f o r  P. granii  g r o w n under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete  c o n d i t i o n s (white circles).  0  40  80  120 160 200  Light (timol photons m s" ) 2  1  41  Figure 3.7:  B S i : N m o l a r ratios v s . irradiance (a), and B S i cell" v s . irradiance (b) f o r 1  P.  granii g r o w n under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete c o n d i t i o n s (white circles). E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the symbols when invisible.  0  40  80  120  160  200  Light (|imol photons rrr s- ) 2  1  42  Figure 3.8: F / F V  M  (DCMU) VS. irradiance for  P. granii g r o w n under Fe-replete c o n d i t i o n s  ( b l a c k circles) and Fe-deplete c o n d i t i o n s (white circles). E r r o r bars represent standard error f r o m triplicate cultures and are s m a l l e r than the s y m b o l w h e n i n v i s i b l e .  0.8 0.6 o  Q  0.4  0.0  0  40  Light  80 (LIITIOI  120  160 200  photons m s- ) 2  1  43  Figure 3.9:  L i n e a r r e g r e s s i o n between F / F V  M  (DCMU) a n d F / F V  M  m e a s u r e d u s i n g the P A M  fluorometer.  44  Figure 3.10:  T h e relationship between F / F V  CMU) and C h i cell" for P. granii g r o w n under 1  M (D  Fe-replete c o n d i t i o n s (black circles) and Fe-deplete conditions (white circles). S o l i d lines are linear regression lines.  0.8 r = 0.85 Fe-replete 2  o  • • •• • ^  0.6  . —— 0  o  >  0.4 o / ry  r 2  O  =  0  7  1  F e - limited  0.2 2  4  6  8  10  12  Chi cell- (pg cell ) 1  1  45  Figure 3.11: Q u a n t u m  photosynthetic e f f i c i e n c y (0PS2) v s . irradiance ( A ) , a n d relative 1  2 1  electron transport (Je) ( p m o l e" m" s " ) v s . irradiance  (B),  f o r P. granii g r o w n under F e -  replete c o n d i t i o n s ( b l a c k circles) a n d Fe-deplete c o n d i t i o n s (white c i r c l e s ) . E r r o r bars represent standard error from triplicate cultures a n d are s m a l l e r than the s y m b o l w h e n invisible.  0  40  80  120  160  Light (Limol photons m- s- ) 2  1  46  Figure 3.12:  L i n e a r regression o f  J a n d u. f o r P. granii g r o w n under Fe-replete c o n d i t i o n s e  (black c i r c l e s ) and F e - l i m i t e d c o n d i t i o n s (white circles).  '(/)  0 -I  ,  0.0  0.2  .  0.4  .  0.6  pi (day ) 1  .  1  0.8  1.0  Figure 3.13:  P h o t o c h e m i c a l e f f i c i e n c y ( P ) vs. irradiance ( A ) , and r e d o x pressure (1-P ) vs. q  q  irradiance ( B ) , for P. granii g r o w n under Fe-replete c o n d i t i o n s (black circles) and Fe-deplete c o n d i t i o n s (white circles). E r r o r bars represent standard error f r o m triplicate cultures a n d are s m a l l e r than the s y m b o l w h e n i n v i s i b l e .  0  40 Light  80  (jLimol  120 160  200  photons m- s- ) 2  1  48  Figure 3.14: Non-photochemical quenching (Q ) vs. irradiance for P. granii grown under Fen  replete conditions (open circles) and Fe-deplete conditions (closed circles). Error bars represent standard error and are smaller than the symbol when invisible (n=3).  O  200  Light  (LIITIOI  photons m s ) 2  1  49  Discussion T h e o v e r a l l goal o f the experiment was to study the effects o f light and F e c o l i m i t a t i o n o n the p h y s i o l o g y o f  P. granii. T h i s study w a s one o f v e r y f e w o n the p h y s i o l o g y  o f a n o c e a n i c , pennate d i a t o m g r o w n under laboratory c o n d i t i o n s , and one o f the first to l i m i t the g r o w t h rate o f a native H N L C species u s i n g F e - l i m i t e d synthetic o c e a n w a t e r rather than e n r i c h e d natural seawater. F e l i m i t a t i o n was attained b y a c c l i m a t i n g  Pseudo-nitzschia granii  to a synthetic o c e a n m e d i u m w i t h o u t added F e . T h o u g h u s i n g natural seawater e l i m i n a t e d p o s s i b l e a r t i f i c i a l effects f r o m synthetic m e d i a , it d i d not a l l o w f u l l c h e m i c a l c o n t r o l o v e r the properties o f the m e d i u m selected. I n a d d i t i o n , most laboratory experiments o n o c e a n i c p h y t o p l a n k t o n e m p l o y e d g r o w t h temperatures higher than the e c o l o g i c a l l y relevant temperatures. In this experiment,  Pseudo-nitzschia granii was g r o w n at a temperature  relevant to temperatures measured at O S P (between 6 and 12°C) and under e c o l o g i c a l l y relevant irradiances. H o w e v e r , the e x p e r i m e n t was c o n d u c t e d under c o n t i n u o u s light to m i n i m i z e c i r c a d i a n changes i n p h y s i o l o g y that have been s h o w n to i n f l u e n c e results, e s p e c i a l l y c h l o r o p h y l l fluorescence ( A r m i n g et al., 2000; Strasser et al., 1999). F o r e x a m p l e , variations i n the l i g h t d a r k c y c l e have been s h o w n to affect sexual r e p r o d u c t i o n i n d i a t o m s ( H i l t z et al., 2 0 0 0 ) .  Growth rates: T h e r e l a t i o n s h i p between g r o w t h and irradiance (p-I curves) is a n important t o o l i n m o d e l i n g p r i m a r y p r o d u c t i v i t y i n the ocean. T h e h y p e r b o l i c p-I c u r v e c a n be d i v i d e d into t w o regions ( L a n g d o n , 1988): 1) l i g h t - l i m i t e d g r o w t h rates characterized b y the i n i t i a l slope o f the c u r v e ( a ) and, 2) light-saturated g r o w t h characterized b y the plateau (p ax)- L i g h t m  50  l i m i t e d g r o w t h is affected b y the a b i l i t y o f the c e l l to absorb and process light, whereas lightsaturated g r o w t h is affected b y factors w h i c h l i m i t the c a r b o n f i x a t i o n p a t h w a y , s u c h as respiration, e x c r e t i o n o f waste and temperature (Greene et al., 1991; L a n g d o n , 1988). T h e response o f g r o w t h to light m a y d i f f e r i n t e r s p e c i f i c a l l y , p r i m a r i l y due to differences i n p i g m e n t c o m p o s i t i o n between different t a x a o f p h y t o p l a n k t o n ( L a n g d o n , 1988). It is w o r t h n o t i n g that d i a t o m s are thought to use light m o r e e f f i c i e n t l y c o m p a r e d to other p h y t o p l a n k t o n ( L a n g d o n , 1988). A s expected, F e - l i m i t e d cells g r e w m u c h s l o w e r (between 10 and 5 0 % o f Fe-replete g r o w t h rates) than Fe-replete cells ( F i g . 3 . 3 A ) . T h e m o s t p r o n o u n c e d differences b e t w e e n treatments w e r e o b s e r v e d i n the m i d d l e range o f irradiance ( F i g . 3 . 3 B ) . F e - l i m i t e d coastal d i a t o m s t y p i c a l l y g r e w at 3 0 % o f Fe-replete g r o w t h rates (Greene et al., 1992; 1991; H u d s o n and M o r e l , 1990). It had been s h o w n that F e - l i m i t e d oceanic diatoms, s p e c i f i c a l l y those isolated f r o m H N L C regions, have a h i g h e r g r o w t h rate ( 5 0 % o f Fe-replete) than their coastal relatives ( M u g g l i et al., 1996). T h i s was due to a difference i n F e quotas b e t w e e n the species; coastal cells h a d m u c h h i g h e r Fe quotas than o c e a n i c strains and were m u c h m o r e sensitive to F e l i m i t a t i o n under laboratory settings ( S u n d a and H u n s t m a n , 1997). T h e fact that F e limited  P. granii m a i n t a i n e d r e l a t i v e l y h i g h g r o w t h rates w a s a testament to h o w w e l l  adapted these cells were to F e l i m i t a t i o n . F e - l i m i t e d cells e x h i b i t e d a 3-fold l o w e r a than Fe-replete cells ( F i g . 3 . 3 A ) . F e l i m i t a t i o n is thought to affect the i n i t i a l slope o f the p-I c u r v e b y d e c r e a s i n g the concentration o f photosynthetic p i g m e n t s and r e a c t i o n centers (Greene et al., 1991; 1992), therefore decreasing the a b i l i t y o f the c e l l to absorb light. It i n d i r e c t l y affects the m a x i m u m rate o f photosynthesis b y l i m i t i n g the s u p p l y o f A T P and N A D P H to c a r b o n f i x i n g e n z y m e s ,  51  or b y i n c r e a s i n g dark respiration rates (Greene, 1992; R. Strzepek, u n p u b l i s h e d results). It has also been s h o w n that p r o n o u n c e d changes i n the i n i t i a l slope o f the p-I c u r v e m i g h t be due to the change o f the o p t i c a l cross-section o f P S 1 as c o m p a r e d to P S 2 ( D u b i n s k y et al., 1986).  P. granii g r e w faster w i t h i n c r e a s i n g irradiance regardless o f its n u t r i t i o n a l status ( F i g . 3.3a). H i g h light resulted i n a s m a l l c o m p e n s a t i o n or increase i n g r o w t h rate under F e l i m i t a t i o n . U n d e r b o t h F e treatments, g r o w t h rates reached a l o w plateau under l o w light. R e s u l t s obtained b y I v a n o v et a l . (2000) demonstrated a s i m i l a r trend i n F e - l i m i t e d  Synechococcus w h e r e s h u f f l i n g o f c y t o c h r o m e / , due to F e l i m i t a t i o n , i m p a i r e d electron transport, w h i c h then d e c o u p l e d P S 2 and P S 2 . T h i s enhanced c y c l i c e l e c t r o n transport a r o u n d P S 1 despite a l o w e r concentration o f active P S 1 centers, leads to h i g h e r g r o w t h rates. S i n c e most photosynthetic cells g r o w n under sub-saturating irradiance have large F e quotas ( H u d s o n and M o r e l , 1990), d e c o u p l i n g between P S 1 and P S 2 is p l a u s i b l e under l o w l i g h t as w e l l as under F e l i m i t a t i o n . U n l i k e c y c l i c electron transport around P S 2 ( F a l k o w s k i et al., 1986), c y c l i c electron transport around P S 1 has not been o b s e r v e d i n diatoms. In a d d i t i o n , whereas c y c l i c electron transport a r o u n d P S 2 does not contribute to c a r b o n f i x a t i o n n o r to g r o w t h , c y c l i c electron transport around P S 1 produces A T P b y c o n t r i b u t i n g to the p r o t o n gradient ( I v a n o v et al., 2000). Interestingly, the same pattern o f g r o w t h at l o w l i g h t w a s c o m p a r a b l e under b o t h F e treatments, i n d i c a t i n g that it m i g h t have been i n d u c e d p r i m a r i l y b y l o w light. F i n a l l y , F e c o n t a m i n a t i o n was not a l i k e l y c u l p r i t i n i n c r e a s i n g g r o w t h rates at l o w light g i v e n that this increase was also observed under Fe-replete c o n d i t i o n s i n  P. granii.  Chlorophyll and chlorophyll fluorescence:  52  F e - l i m i t e d cells h a d a s i g n i f i c a n t l y l o w e r concentration o f c h l c e l l " than i n Fe-replete 1  cells ( F i g . 3 . 4 A ) . F e i s thought to p l a y a central role i n the f o r m a t i o n o f p r o t o p o r p h y r i n , one o f i n i t i a l steps o f c h l o r o p h y l l synthesis ( F a l k o w s k i and R a v e n , 1997; G e i d e r and L a R o c h e , 1994). I n a d d i t i o n to c h l o r o s i s , G r e e n e et a l . (1991) reported a decrease i n P S 2 proteins ( n a m e l y C P 4 3 , C P 4 7 and D I ) i n response to F e d e f i c i e n c y i n the coastal pennate d i a t o m  Phaeodactylum tricornutum. H o w e v e r , nuclear e n c o d e d light h a r v e s t i n g proteins w e r e not affected b y F e l i m i t a t i o n . P.  granii g r o w n under l o w irradiance h a d h i g h C h l c e l l " , w h i c h 1  was a c o m m o n response to l o w irradiance i n p h y t o p l a n k t o n i n order to o p t i m i z e l i g h t absorbency ( H u t c h i n s , 1995, G e i d e r and L a R o c h e , 1994; G r e e n e et al., 1992). T h e C c h l " ratio is important i n estimating s p e c i f i c g r o w t h rates o f p h y t o p l a n k t o n in 1  situ ( B o y d a n d H a r r i s o n , 1999; H a r r i s o n et al., 1999), and i s a n important i n d i c a t o r o f p h o t o a c c l i m a t i o n . T h e ratio is u s u a l l y between 10 and 2 0 0 i n the field, but h i g h e r values (up to 5 0 0 ) h a v e b e e n reported i n the laboratory ( B o y d and H a r r i s o n , 1999; M u g g l i a n d H a r r i s o n , 1996; L e C o u r t et al., 1994). T h e values o f C chl" obtained i n this study f e l l w i t h i n the range 1  p r e v i o u s l y reported f o r open o c e a n diatoms and c o c c o l i t h o p h o r e s (between 80 and 2 0 0 ) ( M u g g l i and H a r r i s o n , 1997; M u g g l i et al., 1996) ( F i g . 3.4B). C c h l " ' w a s p r i m a r i l y affected b y the rate o f c h l o r o p h y l l synthesis c o m p a r e d to g r o w t h rate and therefore, w a s l o w under l o w light a n d h i g h under F e l i m i t a t i o n . G e n e t i c v a r i a b i l i t y , nutrient status and factors i n f l u e n c i n g c a r b o n f i x a t i o n and linear electron transport are also affected b y the C c h l  _1  ratio  ( F a l k o w s k i and R a v e n , 1997; E p p l e y et al., 1973). U n d e r F e l i m i t a t i o n , F S U chl" was higher than Fe-replete cells and it w a s also 1  n e g a t i v e l y related to light ( F i g . 3.5). T h i s agreed w i t h p r e v i o u s l y p u b l i s h e d results f o r d i a t o m s ( L e C o u r t et al., 1994), and i n sugar beets ( B e l k h o d j a et al., 1998). T h e differences  53  i n F S U c h i " b e t w e e n h i g h and l o w light intensities were less p r o n o u n c e d under Fe-replete 1  c o n d i t i o n s . C e l l s w i t h i m p a i r e d photosynthetic m a c h i n e r y , h a d a h i g h basal f l u o r e s c e n c e since p h o t o c h e m i c a l processes were b l o c k e d and m o s t o f the light absorbed b y the c e l l s was re-emitted as fluorescence ( C h a p t e r 2). A s i m i l a r effect w a s o b s e r v e d i n f l u o r e s c e n c e v s . depth traces i n the f i e l d , w h e r e the fluorescence o f p h o t o i n h i b i t e d cells i n the upper layers o f the euphotic z o n e is h i g h c o m p a r e d to the actual c h l o r o p h y l l b i o m a s s ( K o l b e r et al., 1994). N o r m a l i z i n g fluorescence to c h l o r o p h y l l facilitated c o m p a r i s o n s between treatments o f different c e l l u l a r c h l o r o p h y l l concentrations.  Particulate Organic Carbon (POC) and Particulate Organic Nitrogen (PON): T h e ratio o f P O C to P O N ( C : N ) is a useful indicator o f nutrient stress a n d the p h y s i o l o g i c a l status o f m a r i n e p h y t o p l a n k t o n ( F a l k o w s k i , 2 0 0 0 ) ( F i g . 3 . 6 A ) . B e c a u s e o f differences i n c e l l u l a r c o m p o s i t i o n and i n nutritional stresses, field measurements o f the C : N ratio o f m a r i n e p h y t o p l a n k t o n range between 4 and 4 0 (Geider, 1987) and d i f f e r s l i g h t l y between taxa. T h e m a j o r i t y o f c a r b o n i n m a r i n e algae is i n carbohydrates, proteins and l i p i d s , and the m a j o r i t y o f nitrogen is i n a m i n o acids w i t h a s m a l l e r f r a c t i o n i n p h o t o s y n t h e t i c p i g m e n t s ( 1 % o f c e l l u l a r mass) (Parsons et al., 1961). P h y s i o l o g i c a l c o n d i t i o n s that l i m i t nitrogen m e t a b o l i s m ( f i x a t i o n or reduction) y i e l d h i g h e r C : N ratios. T h e effect o f F e o n the C : N ratio is c o m p l i c a t e d b y the fact that F e l i m i t a t i o n affects c a r b o n f i x a t i o n (through linear electron transport) as w e l l as nitrogen r e d u c t i o n t h r o u g h nitrate reductase, w h i c h has a h i g h F e requirement ( - 2 3 atoms per m o l e c u l e ) ( G e i d e r and L a R o c h e , 1994). O t h e r factors that affect c e l l u l a r c o m p o s i t i o n , s u c h as temperature, also i n f l u e n c e the C : N ratio ( G a o et a l . , 2000).  54  In this study, C : N ratios were generally h i g h under Fe-replete c o n d i t i o n s . T h i s effect was most l i k e l y due to l i m i t a t i o n s i m p a r t e d o n nitrate r e d u c t i o n due to F e d e p l e t i o n ( M u g g l i and H a r r i s o n , 1997; Doucette and H a r r i s o n , 1991). T h e average C : N w a s 8.7, w h i c h w a s s l i g h t l y h i g h e r than what was p r e v i o u s l y reported f o r diatoms (~7) ( T h o m p s o n et al., 1991; R. Strzepek, pers. c o m m ) . T h i s was not unexpected since d i a t o m s v a r y w i d e l y i n c e l l u l a r c o m p o s i t i o n , a n d pennate diatoms have a h i g h e r concentration o f carbohydrates and p i g m e n t s than m o s t d i a t o m s (Parsons et a l . 1961).  Biogenic Silica (BSi): It has been h y p o t h e s i z e d that silicate uptake, u n l i k e nitrate uptake, does not require F e ( D e L a R o c h e et al., 2 0 0 0 ; T a k e d a , 1998). T h e uptake ratio o f silicate to nitrate is u s u a l l y 2-fold h i g h e r i n F e - l i m i t e d cells (-2.5) c o m p a r e d to Fe-replete cells ( - 1 ) ( H u t c h i n s et al., 1998; T a k e d a , 1998). T h e B S i : N ratio i n this study was m u c h h i g h e r i n Fe-replete cells c o m p a r e d to F e - l i m i t e d cells ( F i g . 7 A ) . B S i : N ratios i n Fe-replete cells were c o m p a r a b l e to what has been reported i n the literature ( D e L a R o c h a et al., 2000). A s m e n t i o n e d p r e v i o u s l y , the B S i : N ratio measured i n this study is not an uptake ratio and it w a s m e a s u r e d d u r i n g balanced g r o w t h as o p p o s e d to u n b a l a n c e d g r o w t h . These results indicate that the interpretation o f B S i : N as an i n d i c a t o r o f F e stress m a y be dependent o n the c o n d i t i o n s o f the experiment, and that the B S i : N responds differently to F e d e p e n d i n g o n whether it is an uptake ratio or a steady state ratio. T h e results o f this study also p r o v i d e p r e l i m i n a r y e v i d e n c e that silicate m e t a b o l i s m c o u l d be Fe-dependent. T h e h y p e r b o l i c relationship observed b e t w e e n B S i cell" and irradiance i n this 1  e x p e r i m e n t supported the hypothesis that silicate uptake is p o w e r e d b y p h o t o r e s p i r a t i o n ( L i p p e m e i e r et al., 1999) ( F i g . 7 B ) . P h o t o r e s p i r a t i o n was triggered b y h i g h l i g h t because o f  55  an increase i n r e s p i r a t i o n substrate p r o v i d e d b y h i g h rates o f p h o t o s y n t h e s i s ( H a l l a n d R a o , 1999). B e c a u s e p h o t o r e s p i r a t i o n generated a m i n o acids that acted as a m a t r i x for the s i l i c a frustule ( L i p p e m e i e r et al., 1999), d i a t o m s g r o w n under h i g h l i g h t m i g h t be h e a v i e r than those g r o w n under l o w light. I n c i d e n t a l l y , depressed #)PS2 at h i g h l i g h t m i g h t p r o v i d e a d d i t i o n a l support f o r increased p h o t o r e s p i r a t i o n a c t i v i t y at h i g h l i g h t (see F i g . 3 . 1 1 A a n d the d i s c u s s i o n b e l o w ) . In a d d i t i o n , there was e v i d e n c e that l o w rates o f p h o t o s y n t h e s i s were correlated w i t h decreased s i n k i n g rates, i n d i c a t i n g that under energetic stress, c e l l s ceased silicate uptake i n d u c i n g d i s s o l u t i o n o f their frustules ( C u l v e r a n d S m i t h , 1989). T h e relationship between B S I c e l l  - 1  and irradiance c o u l d have been a result o f the r e l a t i o n s h i p  between i r r a d i a n c e and l i g h t ( F i g . 3.3), i n d i c a t i n g that faster g r o w i n g d i a t o m s (at h i g h light) r e q u i r e d m o r e silicate than s l o w e r g r o w i n g d i a t o m s (at l o w light).  Photosynthetic efficiency  (FV/FM):  A s m e n t i o n e d i n chapter 2, the h e r b i c i d e D C M U b l o c k s the transfer o f electrons f r o m the acceptor side o f P S 2 to the electron carrier Q . In other w o r d s , it b l o c k s p h o t o c h e m i c a l A  q u e n c h i n g o f fluorescence. FV/FM denotes m a x i m a l , or theoretical, p h o t o c h e m i c a l q u e n c h i n g o f cells. W h e r e a s i n t e r s p e c i f i c differences i n F / F M between groups o f p h y t o p l a n k t o n are V  p r i m a r i l y due to different p i g m e n t c o m p o s i t i o n , photosynthetic unit arrangement a n d m e c h a n i s m s that alter F  0  (e.g. dark r e d u c t i o n o f P S 2 ) ( B u c h e l a n d W i l h e l m , 1993; J u n e a u  ( u n p u b l i s h e d results)), i n t r a s p e c i f i c differences i n F / F M are m a i n l y due to d a m a g e i n P S 2 V  centers. FV/FM h a d b e e n a p p l i e d i n the f i e l d to assess nutrient l i m i t a t i o n o f p h y t o p l a n k t o n w i t h m i x e d results. P a r k h i l l et a l . (2001) s h o w e d that under c o n d i t i o n s o f u n b a l a n c e d g r o w t h , FV/FM p r o v i d e s a g o o d i n d i c a t o r o f nutrient stress, s p e c i f i c a l l y nitrate stress. H o w e v e r , once  56  cells a c c l i m a t e d to nutrient stress (balanced growth), F v / F rose to c o n t r o l levels despite M  nutrient l i m i t a t i o n . A s i m i l a r trend w a s o b s e r v e d b y L i p p e m e i r et al. ( 1 9 9 9 ) f o r silicatel i m i t e d Thalassiosira weissflogii. H o w e v e r , FV/FM is an excellent i n d i c a t o r o f F e stress since it i n v a r i a b l y m a k e s c e l l s m o r e sensitive to p h o t o i n h i b i t i o n ( K o l b e r et al., 1994). I n this study, FV/FM (DCMU) values were reported, and P A M values were used m a i n l y to c o m p a r e b o t h techniques to test whether the pulses o f l i g h t e m p l o y e d i n P A M studies w e r e saturating. F o r Fe-replete cells o f Pseudo-nitzschia granii, F / F M (DCMU) was - 0 . 6 ( F i g . 3.8), V  w h i c h w a s w i t h i n the range reported f o r diatoms ( B u c h e l a n d W i l h e m , 1993). F V / F M (DCMU) w a s h i g h e r i n Fe-replete cells c o m p a r e d to F e - l i m i t e d cells, w h i c h w a s i n agreement w i t h p r e v i o u s l y p u b l i s h e d results ( B o y d et al., 2000). F /FM(DCMU) d i d not v a r y w i t h light under V  Fe-replete c o n d i t i o n s , w h i c h indicated that despite the damages i n c u r r e d b y the photosynthetic apparatus due to F e l i m i t a t i o n , the range o f irradiances used i n this e x p e r i m e n t w a s not h i g h e n o u g h to cause p h o t o i n h i b i t i o n . A c o m p a r i s o n between F v / F  M  obtained b y the P A M f l u o r o m e t e r and b y D C M U  revealed a strong correlation between b o t h ratios ( r = 0.67) ( F i g . 3.9). T h i s w a s v e r y s i m i l a r 2  to what P a r k h i l l et a l . (2001) observed, and w a s a g o o d i n d i c a t i o n that the pulses e m p l o y e d were saturating ( T i n g and O w e n s , 1993). D e v i a t i o n s f r o m a 1:1 relationship between the ratios were p a r t i a l l y due to o p t i c a l differences between m o d u l a t e d and n o n - m o d u l a t e d fluorescence ( P a r k h i l l et al., 2 0 0 1 ; Juneau, u n p u b l i s h e d results). T h e relationship between F v / F  and c h l cell" w a s different f o r each F e treatment 1  M  ( F i g . 3.10). F e - l i m i t e d cells f e l l w i t h i n the linear range o f the curve, where there w a s a strong relationship between c h l a n d FV/FM (r = 0.71). Fe-replete cells f e l l o n the plateau where i n c r e a s i n g c h l o r o p h y l l concentrations d i d not affect FV/FM (r = 0.85). O n e suggestion i s that  57  p h o t o i n h i b i t i o n m i g h t have been triggered o n l y b e l o w a t h r e s h o l d o f c h l o r o p h y l l concentration o f ~ 5 p g c h l c e l l " . A b o v e this threshold c h l o r o p h y l l concentration, FV/FM 1  s t a b i l i z e d at a v a l u e o f ~0.6. H o w e v e r , m o r e research o n the r e l a t i o n s h i p b e t w e e n p h o t o i n h i b i t i o n and c h l cell" is necessary before c o n c l u s i v e results c a n be d r a w n . 1  PAM Fluorescence Results: Quantum yield of PS2 (^PS2): U n d e r Fe-replete conditions, there was an inverse relationship b e t w e e n light and 0 P S 2 ( F i g . 3.11 A ) , a trend also o b s e r v e d b y F l a m e l i n g and K r o m k a m p ( 1 9 9 8 ) f o r a v a r i e t y o f p h y t o p l a n k t o n species. M a n y factors cause the r e d u c t i o n o f ^PS2 under h i g h light, s u c h as c y c l i c a n d p s e u d o c y c l i c electron transport processes that compete w i t h linear e l e c t r o n transport ( I v a n o v et al., 2000), as w e l l as respiratory m e c h a n i s m s , s u c h as m i t o c h o n d r i a l respiration and photorespiration ( F l a m l i n g and K o r m p k a m p , 1998). ^JPS2 was s i g n i f i c a n t l y l o w e r under F e l i m i t a t i o n than under Fe-replete c o n d i t i o n s ( F i g . 3.11 A ) . T h i s w a s most l i k e l y due to the sluggishness o f electron transfer i n d u c e d b y d a m a g e d r e a c t i o n centers and electron carriers i n the photosynthetic unit. T h e adverse effects o f F e l i m i t a t i o n o n p r i m a r y p r o d u c t i v i t y and o x y g e n e v o l u t i o n has been w e l l d o c u m e n t e d i n the laboratory (Greene et al.; 1991). A t O S P and the Southern O c e a n , the rate o f c a r b o n f i x a t i o n was l o w e r f o r  in situ p h y t o p l a n k t o n species as o p p o s e d to those to w h i c h F e was  added d u r i n g on-deck i n c u b a t i o n experiments ( M a l d o n a n d o et al., 1999). It w a s interesting that 0 P S 2 does not v a r y w i t h light under F e l i m i t a t i o n ; this was m o s t l i k e l y due to d a m a g e to c o m p o n e n t s o f the photosynthetic unit w h i c h render the cells p h o t o i n h i b i t e d e v e n under l o w light intensities, or to increased levels o f respiration under l o w e r light caused b y F e limitation.  58  T h o u g h c e l l s were m o r e efficient under l o w light than h i g h light, they w e r e nonetheless g r o w i n g faster under h i g h light. T h i s w a s m o s t l i k e l y due to a n increase o f the rate o f linear e l e c t r o n transport (J ) e  w i t h i n c r e a s i n g irradiance ( F i g s . 3.1 I B , 3.12).  Photochemical quenching (P ), redox poise ( 1 - P ) and Non-photochemical q  q  quenching (Q ): n  P h o t o c h e m i c a l q u e n c h i n g ( P , T a b l e 3.1) is the measure o f the f r a c t i o n o f l i g h t used q  i n p h o t o c h e m i s t r y (the fraction o f o x i d i z e d QA). In other w o r d s , P denotes the f u n c t i o n a l i t y q  o f the P S 2 . U n l i k e FV/FM, w h i c h indicates damage o f the p h o t o c h e m i c a l system, or the fraction o f damaged PS2's, P  q  indicates the e f f i c i e n c y o f P S 2 centers regardless o f their  number. In  P. granii, P was not s i g n i f i c a n t l y affected b y F e l i m i t a t i o n , but rather, it was o n l y q  affected b y light ( F i g . 3 . 1 3 A ) . T h i s agrees w i t h B e l k h o d j a et a l . (1998) w h o demonstrated that i n sugar beets, F e l i m i t a t i o n affected photosynthesis b y decreasing the n u m b e r o f active P S 2 centers rather than altering their e f f i c i e n c y . It w a s interesting that the o n l y s i g n i f i c a n t differences b e t w e e n F e treatments were i n intermediate irradiances that c o r r e s p o n d to the transition b e t w e e n l i g h t - l i m i t e d g r o w t h and light-saturated g r o w t h . T h e m o s t drastic differences i n c e l l u l a r c h l o r o p h y l l and g r o w t h rates between F e treatments were also observed i n the m i d d l e range o f the irradiance. T h i s was the r e g i o n o f the D-I c u r v e w h e r e cells w e r e i n transition between l i g h t - l i m i t e d g r o w t h and light-saturated g r o w t h ( F i g . 3 . 3 A ) , and w a s p r o b a b l y where most o f the c e l l u l a r m a c h i n e r y is dedicated to i n c r e a s i n g c e l l u l a r resources a n d a c t i v a t i n g x a n t h o p h y l l p i g m e n t s needed for light-saturated g r o w t h ( O l a z i o l a and Y a m a m o t o , 1994).The results o f P  q  and 0 P S 2 s h o w e d that F e and light affected the  photosynthetic apparatus differently, a n d whereas F e affected the q u a n t u m y i e l d o f  59  photosynthesis ( b y a f f e c t i n g the n u m b e r o f reaction centers), light affected p h o t o s y n t h e t i c e f f i c i e n c y . H i g h photosynthetic e f f i c i e n c y and h i g h c e l l u l a r c h l o r o p h y l l were m o s t l i k e l y a c c l i m a t i o n s to m a x i m i z e light absorption at l o w light. T h e r e d o x poise (or e x c i t a t i o n pressure) o f the cells, 1 - P , represents the f r a c t i o n o f q  c l o s e d QA- It is an i n d i c a t o r o f a c c u m u l a t i o n o f electrons o n the acceptor side o f P S 2 that s l o w s electron transfer ( M a x w e l l et a l , 1995a). 1-P acts l i k e the c l u t c h o f the p h o t o s y n t h e t i c q  c e l l , and affects v a r i o u s p h y s i o l o g i c a l processes, such as m o d u l a t i n g the p r o d u c t i o n o f m R N A , light h a r v e s t i n g proteins and the a c t i v a t i o n o f photoprotective m e c h a n i s m s (the x a n t h o p h y l l c y c l e ) ( M a x w e l l et al., 1995a; 1995b). T h e r e d o x poise, 1 - P , was h i g h under F e stress and under h i g h light w h e r e c e l l s were q  b o m b a r d e d w i t h photons faster than they c a n absorb t h e m , c a u s i n g electrons to a c c u m u l a t e o n the acceptor side o f P S 2 ( F i g . 3.13B). T h i s was supported b y e x a m i n i n g the trend i n Q  N  (Table 3.1; F i g . 3.13). N o n - p h o t o c h e m i c a l q u e n c h i n g , Q , is defined as the measurement o f the f r a c t i o n o f N  c h l o r o p h y l l fluorescence quenched b y n o n - p h o t o c h e m i c a l , u s u a l l y p h o t o p r o t e c t i v e means ( K r a u s e a n d W e i s , 1991). S e v e r a l components o f Q  N  c o u l d be deciphered f r o m c h l o r o p h y l l  fluorescence signals d e p e n d i n g o n the k i n e t i c s b y w h i c h they relax ( M a x w e l l and J o h n s o n , 2000). D u e to the l i m i t a t i o n o f the P A M s y s t e m e m p l o y e d b y this experiment, o n l y the overall Q  N  w a s measured. It has been s h o w n that the Q  N  correlated w e l l w i t h i n c r e a s i n g the  a c t i v i t y o f the x a n t h o p h y l l c y c l e i n diatoms. U n d e r h i g h light, Fe-replete cells o f  P. granii h a d a h i g h e r  Q  N  than under l o w light'.  T h i s was p r o b a b l y due to the a c t i v a t i o n o f the x a n t h o p h y l l c y c l e to protect the cells f r o m photodamage. U n d e r F e l i m i t a t i o n , n o n - p h o t o c h e m i c a l q u e n c h i n g w a s h i g h e r u n d e r l o w light  60  than it w a s f o r Fe-replete cells due to the damage i n c u r r e d o n the p h o t o s y n t h e t i c unit that i m p a i r e d l i g h t a b s o r p t i o n and caused cells to be m o r e susceptible to p h o t o i n h i b i t o r y stress e v e n under l o w light intensities. Therefore, whereas h i g h light compensated f o r g r o w t h rates under F e l i m i t i n g c o n d i t i o n s , it was not a n ideal situation f o r the cells that i n c u r p h y s i o l o g i c a l costs i n the f o r m o f enhanced n o n - p h o t o c h e m i c a l activity. I n a d d i t i o n , under b o t h F e treatments, g r o w t h rates were a f u n c t i o n o f the rate o f electron transport ( F i g . 3.12). T h e differences b e t w e e n the magnitude o f g r o w t h rates between F e treatments w e r e m o s t l i k e l y due to the a c t i v a t i o n o f n o n - p h o t o c h e m i c a l m e c h a n i s m s (such as Q ) that c o m p e t e w i t h n  photosynthesis and g r o w t h under F e l i m i t a t i o n .  Conclusions Pseudo-nitzschia granii i s a pennate d i a t o m isolated f r o m O S P , and F e - l i m i t e d station i n the N E S u b a r c t i c P a c i f i c . In this study,  P. granii was s u c c e s s f u l l y g r o w n i n a r t i f i c i a l  seawater culture m e d i u m . T h e effect o f light and F e c o - l i m i t a t i o n o n the p h y s i o l o g y o f P.  granii w a s tested w i t h the f o l l o w i n g c o n c l u s i o n s : 1.  P.granii was r e m a r k a b l y a c c l i m a t e d to F e stress and to light stress.  2.  G r o w t h rates o f  P. granii were light dependent for light l e v e l s b e t w e e n 2 0 - 100 p m o l  photons m" s" . Fe-stress was m o r e evident i n cells i n transition between l i g h t - l i m i t e d and light-saturated g r o w t h . 3.  Fe-limited  P. granii g r e w s l o w l y , w a s c h l o r o t i c and f l u o r e s c e d at a m u c h h i g h e r l e v e l  than Fe-replete cells. 4.  F e - l i m i t e d P. granii s h o w e d signs o f silicate l i m i t a t i o n ( l o w B S i c e l l ) . U n d e r F e -1  l i m i t a t i o n , the steady-state B S i : N ratios were different than the uptake B S i : N ratios.  61  U n d e r h i g h light,  P. granii was m o r e s i l i c i f i e d than under l o w light p o s s i b l y due to  increased photorespiratory a c t i v i t y at h i g h light. 5.  P h o t o i n h i b i t i o n m i g h t have been triggered b y l o w c e l l u l a r c h l o r o p h y l l concentrations.  6.  F e l i m i t a t i o n affected photosynthesis b y decreasing the n u m b e r o f photosynthetic centers ( l o w FV/FM) rather than altering their e f f i c i e n c y ( P ) . P h o t o c h e m i c a l e f f i c i e n c y was q  m o d u l a t e d b y light. 7.  T h o u g h ^PS2 was higher at l o w light, J w a s still s i g n i f i c a n t l y higher under h i g h light. e  Therefore cells s t i l l g r e w faster at h i g h light despite their l o w e r photosynthetic e f f i c i e n c y .  62  Chapter 4 Effect of Irradiance and Temperature on the Physiology of Pseudo-nitzschia granii Introduction  D e s p i t e c i r c a d i a n and seasonal fluctuations o f oceanic and coastal surface temperatures, the effects o f temperature o n p h y t o p l a n k t o n p r o d u c t i v i t y have not b e e n w e l l studied. T e m p e r a t u r e affects the a c t i v i t y o f c e l l u l a r e n z y m e s , and b y c h a n g i n g m e m b r a n e f l u i d i t y , and it also affects the a c t i v i t y o f m e m b r a n e - b o u n d proteins (see F a l k o w s k i a n d R a v e n , 1997 and D a v i s o n , 1991 f o r r e v i e w s ) . Temperature is thought to affect e n z y m a t i c a c t i v i t y l i n e a r l y u n t i l a n o p t i m u m is reached, and the degree o f dependence o n temperature, as d e r i v e d f r o m the A r r e n i u s equation, i s d e f i n e d as Q i o ( D a v i s o n , 1991). T h e r m o a c c l i m a t i o n , the process b y w h i c h o r g a n i s m s a c c l i m a t e to temperature, i s a c o m p l i c a t e d process to study f o r a m y r i a d o f reasons. C e l l u l a r e n z y m e s often have temperature o p t i m a independent o f those f o r g r o w t h rates ( G a o et al., 2 0 0 0 ; D a v i s o n , 1991). T h e r m o a c c l i m a t i o n is i n f l u e n c e d b y g r o w t h irradiance, stage o f a c c l i m a t i o n (balanced v s . u n b a l a n c e d g r o w t h ) and b y genetic constraints o n m e t a b o l i c a c t i v i t y ( T h o m p s o n , 1999; S u z u k i and T a k a h a s h i , 1995; D a v i s o n , 1991). T e m p e r a t u r e - i n d u c e d responses i n m a r i n e p h y t o p l a n k t o n are also t a x a specific and sometimes, species s p e c i f i c d e p e n d i n g o n the geographic r e g i o n f r o m w h i c h they were isolated. It has been s h o w n that A n t a r c t i c species o f p h y t o p l a n k t o n have l o w e r temperature o p t i m a than equatorial species ( D a v i s o n , 1991). Therefore, extrapolating thermal responses f r o m one group o f p h y t o p l a n k t o n to another i s m i s l e a d i n g , a n d t h e r m a l responses s h o u l d i d e a l l y be studied o n a species-by-species basis.  63  In a d d i t i o n to the p h y s i o l o g y , changes i n temperature have b e e n s h o w n to i n f l u e n c e . the species c o m p o s i t i o n o f p h y t o p l a n k t o n assemblages ( S u z u k i and T a k a h a s h i , 1995). F o r e x a m p l e , changes i n oceanic, surface temperatures associated w i t h E l - N i n o / L a - N i n a events are a c c o m p a n i e d b y changes i n p r i m a r y p r o d u c t i v i t y i n the N E S u b a r c t i c P a c i f i c ( B o y d a n d H a r r i s o n , 1999) a n d o n the west coast o f V a n c o u v e r Island ( H a r r i s , 2 0 0 1 ) . A single p h y t o p l a n k t o n species faces a w i d e range o f temperatures because o f h o r i z o n t a l a d v e c t i o n between different regions and because o f seasonal and d a i l y changes i n temperature. Therefore, understanding h o w temperature affects p h y t o p l a n k t o n p h y s i o l o g y is essential f o r understanding fluctuations o f p r i m a r y p r o d u c t i o n i n the oceans. R e c e n t e v i d e n c e suggests that p h o t o a c c l i m a t i o n and thermal a c c l i m a t i o n are opposite faces o f the same p h y s i o l o g i c a l c o i n ( M a x w e l l et al., 1995a; 1995b). A s m e n t i o n e d i n p r e v i o u s chapters, irradiance affects the concentrations o f c e l l u l a r p i g m e n t s , p h o t o s y n t h e t i c e f f i c i e n c y and m - R N A p r o d u c t i o n o f v a r i o u s c o m p o u n d s . Temperature also affects p h o t o c h e m i c a l e f f i c i e n c y b y a f f e c t i n g electron transfer t h r o u g h p h o t o s y s t e m 2 ( P S 2 ) ( R a v e n , 1988) and photosynthetic cells g r o w n under l o w temperature are often c h l o r o t i c ( G e i d e r , 1987). M a x w e l l and colleagues ( M a x w e l l et al., 1995a; 1995b) s h o w e d that l o w temperatureacclimated,  Dunaliella salina is b i o c h e m i c a l l y i d e n t i c a l to D. salina a c c l i m a t e d to h i g h light.  T h e same response is s h o w n f o r Chlorella b y W i l s o n and H u n e r (2000). T h e s e researchers argue that temperature and irradiance interact to co-modulate the r e d o x pressure (poise) o f the c e l l , a n d that cells g r o w n under different temperatures, but the same irradiance, have different r e d o x statuses a n d cannot be c o m p a r e d . Therefore, the effects o f temperature o r irradiance o n p h y s i o l o g y cannot be studied independently o f each other. T h e r e d o x pressure ( 1 - P ) is the a c c u m u l a t i o n o f electrons o n the acceptor side o f P S 2 , a n d c a n be e a s i l y q  64  measured u s i n g c h l o r o p h y l l fluorescence (Chapter 3). B e c a u s e s t u d y i n g the effects o f temperature and irradiance is c o m p l i c a t e d , o n l y a f e w studies have been attempted. U n f o r t u n a t e l y , m a n y o f these studies have used irradiance and temperature ranges that are e c o l o g i c a l l y irrelevant to the m a j o r i t y o f p h y t o p l a n k t o n species. B e c a u s e studies o n species isolated f r o m l o w temperature r e g i o n s s u c h as the N E Subarctic P a c i f i c are rare, this study attempts to elucidate the c o m b i n e d effects o f temperature and irradiance o n the p h y s i o l o g y o f the o c e a n i c d i a t o m (Chapter 3).. I n this study, the p h y s i o l o g y o f  Pseudo-nitzschia granii  P. granii was e x a m i n e d under f i v e temperatures  (8, 10, 14, 17 and 20°C) and f o u r irradiances (sub-saturating: 2 0 , 5 0 , 100 p m o l p h o t o n s m" s" a n d saturating: 150 p m o l photons n f ' s " ) . B e c a u s e P S 2 i s the most t h e r m o - l a b i l e 1  1  1  c o m p o n e n t o f photosynthesis ( D a v i s o n , 1991), and the m a j o r i t y o f c h l o r o p h y l l fluorescence originates f r o m P S 2 under n o r m a l p h y s i o l o g i c a l c o n d i t i o n s ( F a l k o w s k i and R a v e n , 1997), c h l o r o p h y l l fluorescence i s a n ideal t o o l to measure the response o f p h o t o s y n t h e t i c e f f i c i e n c y to changes i n temperature. T h e f o l l o w i n g questions w e r e addressed i n this study: what i s the o p t i m u m g r o w t h temperature o f  P. granii? H o w do temperature and irradiance i n f l u e n c e  g r o w t h rates o f this species? H o w do temperature and irradiance affect p i g m e n t c o m p o s i t i o n and h o w d o they interact to affect photosynthetic e f f i c i e n c y ? F i n a l l y , does c o l d temperature a c c l i m a t i o n m i m i c h i g h irradiance a c c l i m a t i o n i n  P. granii?  65  Materials and Methods  S e m i - c o n t i n u o u s 30 m l batch cultures o f  Pseudo-nitzschia granii were g r o w n i n p F e  19.4 A Q U I L m e d i u m (Chapter 3, and A p p e n d i x 1). T h e e x p e r i m e n t w a s c o n d u c t e d i n a graduated, a l u m i n u m temperature bath (8-24°C) f o l l o w i n g the d e s i g n o f S t r e z p e k and P r i c e ( 2 0 0 1 ; A p p e n d i x 2). C o n t i n u o u s light w a s p r o v i d e d u s i n g H O W V i t a - l i t e c o o l l a m p s . L i g h t intensities were attenuated u s i n g neutral density screening to achieve f i n a l g r o w t h irradiances (20, 50, 100 and 150 p m o l photons m" s" ). C u l t u r e s were g r o w n i n triplicate and a l l o w e d to 2  1  a c c l i m a t e to respective treatments for at least 10 generations, or u n t i l g r o w t h rates s t a b i l i z e d . A t m i d - e x p o n e n t i a l g r o w t h , c e l l u l a r n u m b e r s and c h l o r o p h y l l were m e a s u r e d f o l l o w i n g the m e t h o d d i s c u s s e d i n Chapter 3. P h o t o s y n t h e t i c e f f i c i e n c y w a s measured u s i n g a 101 W a l z P A M f l u o r o m e t e r f o l l o w i n g Chapter 3 as w e l l . Q i o f o r g r o w t h rates, Qio^ was c a l c u l a t e d as Qion = ( 1 0 / ( O P T I - 8 ) ) * ( L n ( p O P T I / p 8 ) ) w h e r e O P T I is the o p t i m u m g r o w t h temperature f o r each treatment and u O P T I and p 8 are the s p e c i f i c g r o w t h rates at the o p t i m u m g r o w t h temperature and 8°C, r e s p e c t i v e l y . Statistical analysis consisted o f A N O V A testing f o l l o w e d b y Students t-test f o l l o w i n g Z a r (1984)(oc = 0.95) ( a s s u m i n g data were n o r m a l l y distributed).  66  Results Growth rate and photosynthetic pigments: G r o w t h rates increased w i t h i n c r e a s i n g g r o w t h irradiance (between 150 a n d 2 0 p m o l photons m" s" ) ( F i g . 4.1 A ) . O p t i m u m g r o w t h temperature w a s 14°C, a n d Qio^ increased 2  1  l i n e a r l y w i t h d e c r e a s i n g light intensity ( F i g . 4 . I B ) . O p t i m u m g r o w t h temperatures were independent o f g r o w t h irradiance. B e y o n d 14°C, g r o w t h rates decreased w i t h i n c r e a s i n g g r o w t h temperature. There w a s no detectable g r o w t h at 20°C a n d 2 0 p m o l photons m " s" . 2  1  C h i c e l f w a s h i g h ( P > 0.05) at l o w light c o m p a r e d to h i g h light ( F i g . 4.2). T h e 1  m a x i m u m c h i cell" w a s observed at 10°C. C e l l s g r o w n at 8 a n d 20°C were c h l o r o t i c . 1  Chlorophyll fluorescence: T h e theoretical y i e l d o f photosynthesis, FV/FM, w a s s l i g h t l y depressed at 8 a n d 20°C, e s p e c i a l l y at h i g h light ( F i g . 4.3). T h i s w a s m o r e p r o n o u n c e d at 8 than at 20°C. I n the m i d d l e temperature range, FV/FM w a s not s i g n i f i c a n t l y different ( P > 0.05) b e t w e e n different light and temperature treatments. T h e q u a n t u m e f f i c i e n c y o f photosynthesis, ^ P S 2 , w a s l o w e r 20°C than i n the m i d d l e range o f temperature a n d w a s s i g n i f i c a n t l y h i g h e r ( P < 0.05) at l o w light c o m p a r e d to h i g h light ( F i g 4 . 4 A ) . T h e rate o f linear electron transport, J , increased w i t h i n c r e a s i n g irradiance. A t h i g h e  light, J reached a m a x i m u m at o p t i m u m g r o w t h temperature (14°C). H o w e v e r , at l o w light, e  J  e  w a s unaffected b y g r o w t h temperature ( F i g . 4 . 4 B ) . A t h i g h light, p h o t o c h e m i c a l e f f i c i e n c y , P , w a s the highest at 8°C a n d it decreased q  w i t h i n c r e a s i n g temperature ( F i g . 4 . 4 A ) . A t l o w light, P w a s u n affected b y changes i n q  67  temperature. In general, P , was s i g n i f i c a n t l y higher at l o w light c o m p a r e d to h i g h l i g h t (P< q  0.05). T h e r e d o x pressure, 1-P , had was i n v e r s e l y related to temperature a n d p o s i t i v e l y q  related to irradiance ( F i g . 4 . 5 B ) . N o n - p h o t o c h e m i c a l q u e n c h i n g , Q , w a s s i g n i f i c a n t l y affected b y light o n l y at l o w n  temperature (8 and 10°C) where Q Q  n  n  was h i g h at h i g h light ( F i g . 4.6). A t h i g h temperatures,  was higher at h i g h light c o m p a r e d to l o w light.  68  Figure 4.1: A ) G r o w t h rate (u) f o r Pseudo-nitzschia granii g r o w n at 150, 100, 50 and 2 0 u m o l photons m" s" v s . temperature. E r r o r bars are standard error ( n = 3) and are s m a l l e r 2  1  than the s y m b o l w h e n i n v i s i b l e .  B) Qiop. f o r P. granii  1.0 0.8  •  •  > , 0.6 \  V  O  CO ^ 0 . 4  •  o  o  O  150 100 50 20  0.2  -o-  0.0  10  15  25  20  Temperature (°C)  O  o  o  40  80  120  160  200  Light |umol photons m" s" 2  1  69  Figure 4.2: C h i c e l l for P. granii g r o w n at 150, 100, 50 and 20 p m o l photons m " s" vs. - 1  2  1  temperature. E r r o r bars are standard error (n = 3) and are s m a l l e r than the s y m b o l w h e n invisible.  C  8 2  r (D O Q_ O O  O  18 16 14 12  «  10  V  •  8 6 4 2 0  O v  150 100 50 20  O 9  6  8  10 12 14 16 18 20 22 24 Temperature (°C)  70  Figure 4.3: F / F f o r P. granii g r o w n at 150, 100, 50 and 2 0 u m o l photons m " s" v s . 2  V  1  M  temperature. E r r o r bars are standard error (n= 3) a n d are smaller than the s y m b o l w h e n invisible.  0.8 0.7 0.6 LL  •  v  V  0.5  •  O  0.4 0.3 0.2 6  8  10 12  14  16  18 20 22  Temperature (°C)  24  150 100 50 20  Figure 4.4: A) ^PS2, and B) J of P. granii grown at 150, 100, 50 and 20 pmol e  photons rn s" vs. temperature. Error bars are standard error (n= 3) and are smaller than the 2  1  symbol when invisible.  0.5 0.4  o 1  CM  </> 0.3 CL  0.2  »  0.1  4 ;  •  0.0 28  V  •  O  24  x  20 CD  16  O  12  E i  CD  150 100 50 20  B  t  8  o  o  0  4 0 6  8  10 12 14 16 18 20 22 24 Temperature (°C)  72  Figure 4.5: A) P and b) 1 -P of P. granii grown at 150, 100, 50 and 20 umol photons q  q  m" s" vs. temperature. Error bars are standard error (n=3) and are smaller than the symbol 2  1  when invisible.  0.8 0.6 \ CL." 0.4  •  0.2  V  •  0.0 0.9  O  150 100 50 20  B 0.6  V  0.3  0.0  6  8 10 12 14 16 18 20 22 24  Temperature (°C)  73  Figure 4.6: Q for P. granii grown at 150, 100, 50 and 20 pmol photons n i s" vs. 2  1  n  temperature. Error bars are standard error (n=3) and are smaller than the symbol when invisible.  1.0  •  0.8  V  •  o  0.6 0.4 0.2 0.0  6  5  i  8  10 12 14 16 18 20 22 24  150 100 50 20  v V  Temperature (°C)  74  Discussion Temperature is one o f the most important p h y s i c a l factors i n f l u e n c i n g p r i m a r y p r o d u c t i o n a n d c o m m u n i t y c o m p o s i t i o n o f p h y t o p l a n k t o n i n the oceans. R e c e n t e v i d e n c e suggests that t h e r m o a c c l i m a t i o n and p h o t o a c c l i m a t i o n are essentially the same processes i n green algae ( W i l s o n and H u n e r , 2 0 0 0 ; M a x w e l l et al., 1995a; 1995b). H o w e v e r , n o s u c h evidence exists f o r temperate, o c e a n i c p h y t o p l a n k t o n . T h i s study e x a m i n e d h o w light and temperature interact to i n f l u e n c e g r o w t h rates, p i g m e n t c o m p o s i t i o n and p h o t o s y n t h e t i c e f f i c i e n c y o f the o c e a n i c d i a t o m  Pseudo-nitzschia granii o v e r an e c o l o g i c a l l y m e a n i n g f u l  range o f irradiances and temperature. T h e results o f experiments, s u c h as this one, are c r u c i a l f o r m o d e l s o f p r i m a r y p r o d u c t i v i t y i n the ocean ( T h o m p s o n et al., 1999).  Growth rates and Cellular Chlorophyll: T h i s study c l e a r l y demonstrated that the g r o w t h o f  Pseudo-nitzschia granii w a s  m o d u l a t e d b y light and temperature ( F i g . 4.1 A and I B ) . A s c o m m o n l y reported i n the literature ( K u d o et a l . , 2 0 0 0 ; T h o m p s o n , 1999), g r o w t h rates increase l i n e a r l y w i t h i n c r e a s i n g g r o w t h temperature u n t i l an o p t i m u m i s reached (~14°C) ( F i g . 4.1 A ) . B e y o n d this o p t i m u m , g r o w t h rates decreased. T h e o p t i m u m g r o w t h temperature o f  P. granii was s i m i l a r to  temperature o p t i m a reported f o r other temperate species ( S u z u k i and T a k a h a s h i , 1995). L i g h t intensity not o n l y i n f l u e n c e d the m a g n i t u d e o f g r o w t h rates, but also the degree to w h i c h g r o w t h rate depended o n temperature such that Qio^ decreased w i t h d e c r e a s i n g i r r a d i a n c e ( F i g . 4. I B ) . A t h i g h light, some o f the c e l l ' s energy w a s allocated towards a l l e v i a t i n g lighti n d u c e d stress, rather than r e s p o n d i n g to increased g r o w t h temperature ( A n n i n g et al., 2 0 0 1 ) , as demonstrated b y a s l i g h t l y elevated Q„ at h i g h light, e s p e c i a l l y at l o w temperature ( F i g  75  4.6). Short-term responses o f diatoms to l o w temperatures i n c l u d e d increased concentrations o f c e l l u l a r carotenoids ( A n n i n g et al., 2 0 0 1 b ) . S a v i t c h et a l . (2001) s h o w e d that a n increase i n Q at l o w e r temperature was due to x a n t h o p h y l l p i g m e n t a c t i v i t y a n d not to the M e h l e r n  reaction i n  Arabidopsis thalina. T h e increase i n Q at h i g h light was p r o b a b l y due to n  increased p r o t e c t i v e a c t i v i t y o f the x a n t h o p h y l l p i g m e n t s ( O l a z i o l a a n d Y a m a m o t o , 1994). T h e concentration o f c e l l u l a r c h i w a s h i g h at 10°C and l o w o n either side o f the temperature gradient. T h o u g h c h i cell" was generally h i g h at l o w light (Chapter 3 1  discussion), s o m e b l e a c h i n g w a s o b s e r v e d for cells g r o w n at l o w l i g h t and at temperature > 10°C. T h e r e are c o n f l i c t s i n the literature regarding the response o f c h i cell" to temperature; 1  w h e r e some researchers reported a decrease o f c h i cell" at l o w temperature ( S t r z e p e k and 1  P r i c e , 2 0 0 1 ) , others reported a n increase ( T h o m p s o n , 1999). D a t a f r o m these e x p e r i m e n t s s h o w that this c o n f l i c t i n the literature m i g h t , i n fact, be due to the non-linear r e l a t i o n s h i p between temperature and c e l l u l a r c h l o r o p h y l l concentration. T h e response o f c h i cell" to 1  temperature m a y also depend o n the geographic range f r o m w h i c h the species i n study w a s isolated. It h a d been s h o w n that the g r o w t h o p t i m a for species isolated f r o m c o l d regions were w e l l b e l o w those isolated f r o m w a r m e r regions ( S u z u k i and T a k a h a s h i , 1995). T h o u g h the l o w c h i cell" values obtained f r o m 1  P. granii g r o w n at 8°C supported the p o p u l a r  hypothesis that l o w temperature a c c l i m a t i o n m i r r o r e d h i g h light a c c l i m a t i o n , the o v e r a l l response o f c h i cell" to temperature i n d i c a t e d that this c o m p a r i s o n m i g h t not b e as s i m p l e as 1  o r i g i n a l l y thought. A n in-depth e x a m i n a t i o n o f the response o f c e l l u l a r c h l o r o p h y l l concentrations to temperature was needed before c o m p a r i n g l o w temperature and h i g h light a c c l i m a t i o n strategies i n m a r i n e p h y t o p l a n k t o n .  76  Chlorophyll Fluorescence: F V / F M w a s s l i g h t l y depressed at b o t h ends o f the temperature gradient (8 a n d at 20°C). T h i s w a s m o s t l i k e l y due to c h l o r o s i s - i n d u c e d p h o t o i n h i b i t i o n ( C h a p t e r 3). H o w e v e r , c h l cell" 1  at 20°C w a s c o m p a r a b l e to that at 16°C, w h i c h h a d a m u c h h i g h e r F / F M v a l u e . T h e V  m e a s u r e d decrease i n F V / F M at 20°C w a s m o s t l i k e l y the result o f a c o m b i n a t i o n o f p h y s i o l o g i c a l processes i n d u c e d b y supra-optimal temperature. Strasser (1997) p r o p o s e d that stress i n d u c e d b y s u p r a - o p t i m a l g r o w t h temperature i n d u c e d changes i n the k i n e t i c s o f the o x y g e n e v o l v i n g c o m p l e x (donor side) o f P S 2 , thereby r e d u c i n g F M (and F V / F M ) i n  Scenedesmus obliquus. U n f o r t u n a t e l y ,  donor side k i n e t i c s o f  P. granii  w e r e not e x a m i n e d i n  this study. I n a d d i t i o n , the e x p e r i m e n t p e r f o r m e d b y Strasser (1997) represented a n effect o f short-term temperature stress (~2 hours) at v e r y h i g h temperature (40°C;  S. obliquus  was  g r o w n at 30°C). W h e t h e r a s i m i l a r response c o u l d be i n d u c e d i n d i a t o m s i s u n k n o w n . F V / F M decreased at l o w l i g h t at b o t h 8 a n d 20°C. B e t w e e n 8 a n d 20°C, there w e r e n o s i g n i f i c a n t differences b e t w e e n v a r i o u s l i g h t a n d temperature treatments. H o w e v e r , F V / F M w a s s l i g h t l y h i g h e r at 10°C than at h i g h e r temperatures, consistent w i t h w h a t w a s reported i n the literature about l o w temperature a c c l i m a t i o n i n c r e a s i n g F V / F M ( D a v i s o n , 1991). T h e s e observations further strengthened the argument p r o p o s e d i n the p r e v i o u s s e c t i o n r e g a r d i n g the care w i t h w h i c h the results o f e x p e r i m e n t s o n temperature s h o u l d be interpreted. T h e r e l a t i o n s h i p between ^)PS2, the q u a n t u m y i e l d o f photosynthesis, a n d temperature indicate that $ P S 2 w a s s l i g h t l y depressed at 8°C a n d m o r e severely depressed at 20°C. S a v i t c h et a l . (2001) s h o w e d that photosynthetic rates o f low-temperature a c c l i m a t e d A  thalina w e r e  c o m p a r a b l e to, i f not s l i g h t l y h i g h e r than high-temperature adapted plants. T h e  same results w e r e o b s e r v e d i n m a r i n e p h y t o p l a n k t o n ( D a v i s o n , 1991). I n l o w temperature,  short-term incubations seemed to d r a m a t i c a l l y decrease the q u a n t u m y i e l d o f photosynthesis i n d i a t o m s ( A i m i n g et a l . 2 0 0 1 b ) . T h e r m a l a c c l i m a t i o n often m o d u l a t e d c e l l u l a r concentrations o f R u b i s c o a n d v a r i o u s C a l v i n c y c l e e n z y m e s ( D a v i s o n , 1991). O t h e r results s h o w that the r e l a t i v e l y u n c h a n g e d photosynthetic rates o b s e r v e d at l o w temperature w e r e due to increased rates o f electron transport ( D a v i s o n , 1991) rather than elevated R u b i s c o activity. A n e x a m i n a t i o n o f the effect o f temperature o n Je, the linear rate o f electron transport, r e v e a l e d that J w a s l o w e r at 8 a n d 20°C c o m p a r e d to J i n the m i d d l e o f the e  e  temperature spectrum ( F i g 4 . 4 B ) . T h i s i n d i c a t e d that the o b s e r v e d decrease o f ^ P S 2 at 8 a n d 20°C m i g h t be due to a decrease i n J (due to i m p a i r e d light a b s o r p t i o n created b y l o w e  c h l o r o p h y l l concentrations). U n l i k e , J , there w a s n o s t r i k i n g temperature o p t i m u m f o r ^ P S 2 . e  In general, ^PS2 w a s higher at l o w light c o m p a r e d to h i g h light (see C h a p t e r 3 f o r discussion). A t l o w light, p h o t o c h e m i c a l e f f i c i e n c y , P , w a s s l i g h t l y h i g h e r at 8°C a n d decreased q  w i t h i n c r e a s i n g temperature. A t l o w light, P , w a s m a x i m u m at 14°C a n d decreased o n either q  side o f the temperature spectrum. T h i s result disagreed w i t h p r e v i o u s l y o b s e r v e d trends i n l o w temperature a c c l i m a t e d at h i g h light than  Dunaliella salina ( M a x w e l l et a l , 1995b), w h i c h h a d h i g h e r P  q  D. salina a c c l i m a t e d to h i g h temperatures. T h e exact m e c h a n i s m b y w h i c h  temperature affects p h o t o c h e m i c a l q u e n c h i n g is not understood. P u r e l y p h o t o c h e m i c a l reactions are independent o f e n z y m a t i c a c t i v i t y , a n d are theoretically independent o f temperature. H o w e v e r , the p h o t o c h e m i c a l reactions o f photosynthesis take place a l o n g the t h y l a k o i d m e m b r a n e , w h i c h is, l i k e other p h o s p h o l i p i d bilayers, v u l n e r a b l e to temperature. A t l o w temperature, often c a l l e d the c r i t i c a l temperature, b i o l o g i c a l m e m b r a n e s l o o s e their  flexibility  and become crystalline ( F a l k o w s k i and R a v e n ,  78  1997). W i t h the loss o f f l e x i b i l i t y , the f u n c t i o n o f m e m b r a n e proteins i s also lost. I n a d d i t i o n , the d i f f u s i o n o f electron carriers is temperature dependent ( M a x w e l l et al., 1995b). I n a d d i t i o n , p h y t o p l a n k t o n c e l l s f r o m different t a x a a n d different e n v i r o n m e n t s h a v e b e e n s h o w n to have different responses to temperature. P is l i n e a r l y related to ^PS2 ( M a x w e l l a n d q  J o h n s o n , 2 0 0 0 ) . Increasing p h o t o c h e m i c a l e f f i c i e n c y at l o w temperature p r o v i d e s c e l l s w i t h a p h y s i o l o g i c a l m e c h a n i s m to m a x i m i z e photosynthetic capacity at l o w temperature i n order to keep electron rates at h i g h e n o u g h l e v e l s capable o f supporting g r o w t h y i e l d and c a r b o n f i x a t i o n . T h i s also e x p l a i n s w h y i n d i a t o m s i n contrast to green algae ( M a x w e l l et a l . 1995b), the r e d o x pressure o n the acceptor side o f P S 2 is l o w at l o w temperature. T h i s strategy is advantageous f o r p h y t o p l a n k t o n native to the N o r t h e a s t P a c i f i c w h e r e the average temperature is b e t w e e n 8 and 9°C ( F r e e l a n d et al., 1997). H o w e v e r , green algae, s u c h as D.  salina, h a v e m u c h h i g h e r g r o w t h temperature o p t i m a (30°C) and have n o need f o r s u c h a strategy. P w a s also h i g h at h i g h light and l o w at l o w irradiance (see chapter 3 f o r q  discussion).  Are Thermoacclimation and Photoacclimation similar processes in P. graniP. B y comparing,  P. granii g r o w n at 8°C a n d 2 0 p m o l photons m " s' (8/20) to P. granii 2  g r o w n at 17°C and 150 p m o l photons m" s 2  l  (17/150), it cannot be c o n c l u d e d that l o w  temperature a c c l i m a t i o n m i m i c s h i g h light a c c l i m a t i o n i n P  q  ]  P. granii. In fact, F v / F , ^PS2 and M  were a l l h i g h e r at 8/20 c o m p a r e d to 17/150. T h i s i s s t r i k i n g l y different than w h a t i s  o b s e r v e d i n green algae ( W i l s o n and H u n e r , 2000; M a x w e l l et al., 1995b) w h e r e c e l l s g r o w n under 5/20 and 2 7 / 1 5 0 are b i o c h e m i c a l l y i n d i s t i n g u i s h a b l e . T h e r e are m a n y reasons f o r the differences b e t w e e n the results o f this study and those o f W i l s o n and H u n e r ( 2 0 0 0 ) a n d M a x w e l l et a l . (1995b). D i a t o m s ( c h l o r o p h y l l c based) and green algae ( c h l o r o p h y l l b based)  79  are different p h y s i o l o g i c a l l y a n d f u n c t i o n a l l y so it is not s u r p r i s i n g that they e m p l o y different t h e r m o a c c l i m a t o r y strategies. I n addition, m o s t green algae have m u c h h i g h e r temperature g r o w t h o p t i m a than temperate p h y t o p l a n k t o n (~ 27°C c o m p a r e d to ~15°C) a n d l i v e i n e n v i r o n m e n t s that are o n average m u c h h i g h e r i n temperature than the o p e n ocean. S i n c e temperatures fluctuate between 6 a n d 12°C at O S P ,  P. granii m a y benefit b y e m p l o y i n g a  t h e r m o a c c l i m a t o r y strategy that i s better suited f o r c o l d e n v i r o n m e n t s rather t h a n w a r m ones.  80  Conclusions: T h e g r o w t h o p t i m u m for  Pseudo-nitzschia granii w a s ~ 14°C and w a s not affected b y  g r o w t h i r r a d i a n c e ( F i g 4.1). Qion o f  P. granii depended o n the p h o t o a c c l i m a t o r y status o f the c e l l a n d cells g r o w n at  l o w irradiance were m o r e temperature-dependent (higher Q j o n ) than those g r o w n under h i g h light. C h a n g e s i n the q u a n t u m y i e l d o f photosynthesis, 0 P S 2 , m i g h t have been due to changes i n the rate o f electron transport, J , t h r o u g h the cells ( F i g 4 . 4 A and B ) . C h a n g e s i n J e  e  were m o s t l i k e l y due to i m p a i r e d light absorption due to l o w concentrations o f c e l l u l a r chlorophyll. Increased p h o t o c h e m i c a l e f f i c i e n c y , P , at l o w temperature was, perhaps, a strategy to q  m a i n t a i n h i g h J , ^PS2 and g r o w t h rates. c  F i n a l l y , the results o f this study p r o v i d e p r e l i m i n a r y e v i d e n c e that w h e t h e r T h e r m o a c c l i m a t i o n m i m i c s p h o t o a c c l i m a t i o n m a y depend o n the p h y s i o l o g y a n d the g r o w t h temperature o p t i m u m o f the o r g a n i s m i n question.  Future Research  T h i s thesis p r o v i d e d basic i n f o r m a t i o n o n the p h y s i o l o g y o f the o c e a n i c d i a t o m , Pseudo-nitzschia granii, isolated f r o m O S P i n the N E Subarctic P a c i f i c . T h e effects o f i r o n (Fe) and light, as w e l l as the effects o f light and temperature o n the p h y s i o l o g y o f P. granii were investigated. T h i s study w a s b y no means c o m p r e h e n s i v e and there are m a n y d i r e c t i o n i n w h i c h future research m a y proceed. H e r e are some questions that s h o u l d be addressed i n future studies: 1) H o w is the F e quota o f P. granii affected b y F e and light c o - l i m i t a t i o n ? 2) H o w does the F e quota o f P. granii c o m p a r e to those measured i n other d i a t o m s ? 3) Is the p h y s i o l o g i c a l response o f P. granii F e and light c o - l i m i a t i o n c o m p a r a b l e to the response o f coastal, pennate d i a t o m s ? 4) H o w does F e and light c o - l i m i t a t i o n affect nutrient uptake i n P. granii"? 5) W h y are F e - l i m i t e d P. granii cells t h i n l y s i l i c i f i e d ? 6) W h a t is the exact effect o f Fe d e f i c i e n c y o n nitrogen m e t a b o l i s m i n P. granii? 7) W h a t is the effect o f F e and light o n the p r o d u c t i o n o f d o m o i c a c i d i n P. granii? 8) W h a t is the effect o f F e and light o n sexual reproduction i n P. granii? 9) H o w does temperature affect F e quota i n P. granii? 10) H o w does 0PS2 c o m p a r e to C - b a s e d q u a n t u m y i e l d measurements i n the 14  laboratory? 11) H o w does ^PS2 relate to the a c t i v i t y o f photorespiration i n P. granii?  82  Bibliography  A N N I N G , T., G . 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Prentice-Hall.  93  Appendix 1  Media used in this study  Concentration (M)* Na CI S04 K Br B03 HC03 F Cations Mg Ca Sr Nutrients P04 N03 Si03  Anions  Metals  Zn Mn Co Cu Mo04 Se03  EDTA Vitamins Thiamine  B12 Biotin  p  4.81E-01 5.60E-01 2.88E-02 1.02E-02 8.40E-04 4.85E-04 2.38E-03 7.14E-05 5.46E-02 1.05E-02 6.36E-05 1.00E-05 3.00E-04 1.00E-04 4.51E-07 4.00E-09 2.30E-08 2.50E-09 9.97E-10 1.00E-07 1.00E-08 5.00E-06 2.97E-07 1.47E-10 4.09E-10  Concentration (M) Fe 19.4*  P  Concentration (M) Fe 24- 25  4.81E-01 5.60E-01 2.88E-02 1.02E-02 8.40E-04 4.85E-04 2.38E-03 7.14E-05 5.46E-02 1.05E-02 6.36E-05 1.00E-05 3.00E-04 1.00E-04 4.51E-07 2.00E-09 1.15E-08 1.25E-09 4.99E-10 5.00E-08 5.00E-09 5.00E-06 2.97E-07 1.47E-10 4.09E-10  4.81E-01 5.60E-01 2.88E-02 1.02E-02 8.40E-04 4.85E-04 2.38E-03 7.14E-05 5.46E-02 1.05E-02 6.36E-05 1.00E-05 3.00E-04 1.00E-04 0.00E+00 2.00E-09 1.15E-08 1.25E-09 4.99E-10 5.00E-08 5.00E-09 5.00E-06 2.97E-07 1.47E-10 4.09E-10  * Recipe from Price et. Al. 1988/1989 ** Recipe for iron replete medium used in this thesis *** Recipe for iron limiting medium used in this thesis **** T contamination of Fe estimated at < 2 nM *****Recipe from Harrison et al. (1980) r a c e  94  Appendix 2 Schematic of Temperature Block Top View  Cold Water (4°C) Culture Chambers  ooo o c ooo o oo ooo c ooo o oo ooooooo o ooooocoooo  d  Temperature  8 10 v  12  14  16 18  24  Hot Water (30°C)  95  Appendix 2- continued Temperature block - side view  Block  Hot Water  Cold Water Screening Light Unit  Lamp  96  

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