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Two aspects of C-4 plants : 1. effects of light intensity on photosynthesis : 2. A nitrogen-fixing association 1974

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TWO ASPECTS OF M PLANTS E F F E C T S OF L I G H T I N T E N S I T Y ON P H O T O S Y N T H E S I S 2, A N I T R O G E N - F I X I N G A S S O C I A T I O N by A L A N L E E D H A M H A R T S c . ( H o n s . ) , U n i v e r s i t y o f C a n t e r b u r y , N . Z . , 1 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R O F P H I L O S O P H Y i n t h e D e p a r t m e n t o f B O T A N Y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A In 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 a d v a n c e d d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e 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 a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada ABSTRACT Part 1 concerns the e f fect of l i g h t in tens i ty on the leaves of Gomphrena globosa, a C-4 p lant . Plants were grown under three l i g h t i n t e n s i t i e s . The structure and function of the photosynthetic apparatus of newly matured leaves were examined by a number of techniques. These were electron microscopy, in f ra - red f luorescence photo- micrography, dye (TNBT) reduction and l k C 0 2 feedings?. Contrary to conclusions from the l i t e r a t u r e , l i g h t in tens i ty did not a f fect the membrane conf igurat ion of the ch io rop las ts . Metabolite leve ls seemed to vary with l i g h t i n t e n s i t y . With respect to i t s photosynthetic propert ies G. globosa seems to occupy an intermediate pos i t ion in a range of other C-4 pi ants. Part 2 describes some aspects of the re la t ionsh ip between a grass, Paspalum notatum, and a bacterium, Azotobaetev p a s p a l i which is found mainly on the root surface of the grass. These organisms form a nitrogen f i x i n g assoc iat ion in South- Eastern and Central B r a z i l . Both were studied separately because of the d i f f i c u l t y of estab l i sh ing the assoc iat ion in the laboratory. i i An apparatus was b u i l t in which photosynthesis and root function of P. notatum could be monitored. 1 1*C0 2 was fed to the leaves and  lhZ appearing in the roots and root exudate was analyzed. P. notatum appears to exude s u f f i c i e n t substrate to support nitrogen f i x a t i o n by A. paspali but the amount and nature of exudate proved d i f f i c u l t to ascer ta in . The assoc iat ion is found in acid s o i l s but nitrogen f i x a t i o n by A. paspali is inh ib i ted by low pH. It was found that roots of P. notatum were able to neutra l ize increases in a c i d i t y in the i r environment. A low pH inh ib i ted N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of A. paspali grown in continuous culture at high and low concentrations of carbohydrate and oxygen. Other nitrogen f i x i n g bac te r ia , e .g . Beigevinckia spp and Devxia gummosa,are found in r e l a t i v e l y low numbers in the rhizosphere of P. notatum. Nitrogen f i x a t i o n by A. paspali may be less prone to oxygen i n h i b i t i o n than i t is in these other bac ter ia , thus allowing A. paspali to compete more e f f i c i e n t l y for carbohydrate. It seems that A. paspali l i ves with P. notatum because of the favourable pH that is maintained in the root environment. The carbohydrate and oxygen concentration are probably also favourable for nitrogen f i x a t i o n . How can the assoc iat ion be eas i ly establ ished in the laboratory? Before further research can be done, a way must be found to answer th is quest ion. i v TABLE OF CONTENTS Page ABSTRACT t i LIST OF TABLES vi i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i i P A R T I E F F E C T 0F ILU'GHT (I'NTENSII'TM ON iH0T0S¥NTblES<ItS CHARTER 1. EFFECT * OF 'LIGHT INTENSITY"' ON THE TRHOTOSYNTHETIC APPARATUS OF GOMPHRENA GLOBOSA 1 Foreword 1 Introduction 1 Materials and Methods 3 Resul ts 9 Discussion . . . 2 7 Addendum 38 LITERATURE CITEDratura Cites 48 v Page P A R T 2 A N ITROGEN F I X I N G A S S O C I A T I O N : PASPALUM NOTATUM AND AZOTOBACTER P A S P A L I TERMINOLOGY. 53 LITERATURE CITED 54 PROLOGUE 55 LITERATURE CITED 71 CHAPTER 1. ROOT EXUDATION BY PASPA'LUM NO-TATUM 75 Introduction 75 Materials and Methods 77 Results 88 Discussion 109 LITERATURE Cf.TED.ratyre CUeci 120 CHARTER 2. THE IMPORTANCE OF pH TO THE PASPALUM NOTATUM-AZOTOBACTER PASPALI ASSOCIATION. . . . 124 Introduction 124 Materials and Methods 126 Results 133 Discussion 141 LITERATURE COEBratura Cited 144 vi Pag_e CHAPTER 3. THE INFLUENCE OF OXYGEN CONCENTRATION ON COLONY MORPHOLOGY OF AZOTOBACTER PASPALI. 146 Introduction 146 Materials and Methods 148 Results 149 D " s Discussion 162 LITERATURE CITED 154 EPILOGUE 155 LITERATURE CITED 164 v i i LIST OF TABLES Tab! e Part 1 Page Chapter 1 1 Bundle sheath ch loroplast c h a r a c t e r i s t i c s . Means and standard deviations 10 2 TNBT reduction in l i g h t by bundle sheath ch loroplasts from leaves grown at d i f fe rent l i g h t i n t e n s i t i e s 18 3 Ratio of ll*C in malic acid to that in aspart ic acid at d i f fe rent l i g h t i n t e n s i t i e s 26 4 Summary of temperatures recorded at the Plant Science weather s t a t i o n , Univers i ty of B r i t i s h Columbia, Vancouver during the summer, 1 970 (°C). 30 5 Summary of experimental r e s u l t s . 36 Part 2 Prologue 1 N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in the rhizospheres of t r o p i c a l grasses 59 Chapter 1 1 Oxygen so lut ion rates (mmole l _ 1 h r " 1 ) corresponding to p0 2 of root gas supply 82 2 Rates of photosynthesis of P. notatum under d i f f e r e n t root oxygen regimes 89 v i i i Tab! e Page 3 Comparison of r a d i o a c t i v i t y in samples of young nodal roots and o lder , f i n e , branched roots 94 4 Estimates of the s ize of the v o l a t i l e and non-vo lat i le f ract ions of the root exudate . . . . 99 5 D i s t r i b u t i o n of a c t i v i t y among f ract ions of soluble extract of roots of P. notatum dpm^C (root g.dry w t : ) - 1 . . 104 6 Percentage of total a c t i v i t y in each spot of chromatograms of neutral f rac t ions 105 7 Percentage of tota l a c t i v i t y in each spot of chromotograms of ac id i c f r a c t i o n . . . . 106 8 D i s t r i b u t i o n of r a d i o a c t i v i t y among f rac t ions of non-volati le residue of root medium 107 Chapter 2 1 Nitrogen-free medium used to grow A. paspali 'i. . . 127 2 Chemostat culture condit ions for A. paspali. . . . 132 3 Quantity of a c i d , umoles H + . ( g . r o o t dry wt- . )" 1 , required to lower pH and maintain the root medium at low pH for 30 mintites. . . 135 4 pH and O.D. of chemostat cultures of A. paspali approximating to steady state condit ions 138 5 The e f fect of low pH on C 2 H 2 reduction by A. paspali . . . . . . . . 139 i x LIST OF FIGURES Part 1 Figure Page 1 Mesophyll ch lo rop las t . 550,000 lux 12 2 Bundle sheath ch lo rop las t . 50,000 lux 13 3 Bundle sheath ch lo rop las t . 10 ,000 lux 14 4 Bundle sheath ch lo rop las ts . 2,000 lux 15 5 Bundle sheath ch lo rop las t . 50,000 lux 16 6 Bundle sheath ch lo rop las t . 10,000 lux 17 7. lelieaf1 f luorescence. 5O5OQOO0uil:ux. . . . 20 8 Leaf f luorescence. 1 0,000 lux 21 9 Beaf f luorescence. 2,000 lux 22 10 Bundle sheath. 2 ,000 lux 23 11 Leaf f luorescence 24 12 Leaf f luorescence 25 13 Fluorescence emission spectra of system I. Spinach 43 14 Fluorescence emission spectrum of system I I . Maize . . . . . . . . . 44 x Figure Page 15 Fluorescence emission spectra of system I. Maize 4 5 16 Fluorescence emission spectrum of system I I . Maize 46 17 Fluorescence emission spectrum of system I. Maize. 47 Part 2 Prologue 1 Growth curve for an N 2 - l im i ted culture of A. ohrooooocum. . 6'6 Chapter 1 1 Schematic diagram of open c i r c u i t apparatus . . . 79 2 E f fect of low root temperature on photosynthesis 90 3 Ef fect of low root temperature on •/?2 'u5^photosynthesis 9 1 4 Autoradiographs of root system. . 93 a5 Loss of 1 4 G from root medium 95 6 Accumulation of l l f C in root medium with time. . . 97 7 Accumulation of lkC in root medium 98 8 Ef fect of lowering pH to 4.0 on 1 I f C content of root medium 101 9 Effe&ttof"ethanol icdr i f^Gctrioroof n o mediumt 102 10 Chromatogram of acid f r a c t i o n of root ext ract . 108 xi Figure Page Chapter 2 1 Schematic diagram of chemostat 129 2 Increase in pH of root medium 134 3 Quantity of acid required to keep pH of root medium low 137 Chapter S 1 Morphology of A. paspali colonies . . . „ . . . » . . . . 150 Epilogue 1 Scheme for development of P. notatum var "Pensacola Bahia" having A. paspali in i t s rhizosphere 164 2 Scheme for development of a wheat rhizosphere containing modified stra ins of Azotobaoter spp 163 • i.d.ix xi i ACKNOWLEDGEMENTS I would l i k e to thank Dr. Bruce Tregunna, who made i t a l l poss ib le . During my work I en l i s ted the help of a number of people. I would l i ke to thank a l l of them and mention those whose help was espec ia l l y appreciated: Dr. Thana B i s a l p u t r a , who made the f a c i l i t i e s of his e lectron microscopy laboratory f r e e l y ava i lab le at a l l t imes. Dr. Iain Tay lor , for the use of a gas chromatograph. Dr. Bruce Bohm, for doing some glass-blowing during the construct ion of the continuous culture apparatus. L a s t l y , many part's of my work could not have been attempted without the assistance of Mr. Mel Davies, Mr. Charles Co l l inson and Mr. Ken Je f f r i ' es . And to a l l the people I have met at U.B.C . : I won't fo rget . x i i i P A R T I EFFECT OF LIGHT INTENSITY ON PHOTOSYNTHESIS Chapter 1 EFFEGT^iDF: L l 6 H T ' IWTEUSI TY "0N1THE rPHOTO'SYNTHETIC APPARATUS OF 6QMPHRENA GLOBOSA Foreword The work described in this chapter was done in 1970 and reported (21.) at a conference on photosynthesis and photorespirat ion held at Canberra, A u s t r a l i a , 23 November - 5 December 1970. It is discussed using the knowledge of C-4 photosynthesis ava i lab le at that time, which is contained in the proceedings of the conference. An addendum contains comments made on the basis of relevant papers which have appeared since the conference. Introducti on Gomphrena globosa is a C-4 plant (12). These plants are character ized by a number of properties which include C-4 d icarboxy l ic acids as the major products of short-term 1 2 photosynthesis and leaf photosynthetic t issue cons ist ing of mesophyll and bundle sheath c e l l s (11). The chloroplasts of the bundle sheath show various degrees of granal development (11). Downton (10) has divided C-4 plants into "malate formers" and "aspartate formers." The bundle sheath chloro- plasts of "malate formers" have no or rudimentary grana, and malic acid is a major product of short-term photosynthesis. When 1 1*C0 2 was fed to G. globosa for six seconds, 70% of the carbon f ixed into C-4 d icarboxy l ic acids was found in malic a c i d . The rudimentary grana of the bundle sheath chloroplasts consist of only two stacked thylakoids (10,33). Downton placed G. globosa in the "malate formers" group (10). There- is 'ev idence to suggest that bundle sheath chloroplasts which are agranal or have only rudimentary grana are de f i c ient in photosystem II (PS I I ) ; which would resu l t in a low capacity to reduce NADP (23,40) (the problem of the re la t ionsh ip between PS II a c t i v i t y and the presence of grana is not yet resolved; there are a number of reports (19,24,31) showing that PS II a c t i v i t y is not dependent on grana formation). It was expected therefore , that i f they lacked, or were d e f i c i e n t i n , PS I I , G. globosa bundle sheath chloroplasts would not be able to reduce the dye t e t r a n i t r o blue tetrazol ium (TNBT) as i t s reduction requires non-cyc l ic e lectron flow. However, prel iminary experiments in th is laboratory indicated 3 that bundle sheath chloroplasts of G. globosa were able to reduce TNBT in the absence of an exogenous electron donor. A possible reason for these unexpected resu l ts was that the plants used in the various experiments may have had funct ional d i f ferences because they were grown in d i f f e r e n t environments. The experiments discussed here were done to show whether the photosynthetic apparatus of G. globosa was modified by the l i g h t in tens i ty during growth. The structure and function of the. photosynthetic apparatus under the various condit ions were measured by TNBT reduct ion, e lectron microscopy, in f ra - red f luorescence photo- micrography and  lhZQz feedingi§s t Materials and Methods Gomphvena globosa var. globe amaranth was grown in vermicu l i te , and watered with ha l f -strength Hoagland's so lut ion (15). The plants were grown from seed in a growth chamber at 10,000 lux, 16 hour day, and 24°C/18°C day/night temperature. When approximately s ix leaves were present, some plants were transferred to a l i g h t in tens i ty of 2,000 lux in the growth chamber and some outside for three months during the summer where the average maximum l i g h t in tens i ty was 50,000 lux. 4 Leaves used in the experiments had grown more than 95% of the i r length in the environment indicated and had just stopped elongat ing. Electron microscopy. Samples from the mid-sections of leaves were taken at approximately 09.00 hours. Pieces 1 to 2 mm square were f ixed for one hour at 4°C in 2.5% v/v glutaraldehyde in 0.1 M sodium cacodylate buf fer , pH 6.8, and post- f ixed in 1% w/v Q 5 O 4 in 0.1 M sodium cacodylate buffer for one hour at 4°C. The pieces were dehydrated in a graded ethanol ser ies and embedded in Spurr's medium (38). Sections were cut with a glass knife on a Sorval l MT-1 u l t ra microtome, post-stained with uranyl acetate and lead c i t r a t e (35) and viewed in a Zeiss EM-9A microscope. Before u l t r a s t r u c t u r a l var ia t ion in c e l l s or organelles can be ascribed to some treatment, the var ia t ion must be s t a t i s t i c a l l y s i g n i f i c a n t . Unfortunately, the micrographs in th is study were not taken with the intent of doing a s t a t i s t i c a l a a n a l y s i s , making i t d i f f i c u l t to apply an analysis when the need for one was r e a l i z e d . To see i f there was any var ia t ion in bundle sheath ch lorop last u l t ras t ruc ture with l i g h t i n t e n s i t y , an analysis of variance (anova) was performed 5 on the terms: (a) grana per ch lo rop las t , (b) thylakoids per , . / \ p a r t i t i o n length x number of par t i t i ons granum and (c) ^ . = c h f o r o p 1 a s t a r e a Term (c) is an estimate of the total p a r t i t i o n length per thin sect ion of ch loroplast on a per unit area ch loroplast bas is . These factors were chosen for analysis because they should r e f l e c t any changes in ch loroplast conf igurat ion or u l t r a s t r u c t u r e . Dye reduction The a b i l i t y of hand-cut transverse leaf sections to reduce TNBT was studied using a test so lut ion containing 1 part TNBT (1 mg/ml) , 1 part 0.1M phosphate buf fer , pH 6.0, and 3 parts 0.33M sucrose (13). The three controls used were (a) darkness, (b) no TNBT, and (c) the addit ion of ascorbate- di chl orophenol indophenol, at 6 x 10 _ 3 M and 1.5 x 10 - I tM r e s p e c t i v e l y , as the a l ternate electron donor. The leaf t i ssue was examined as described by Downton et al. (13) A Zeiss Ultraphot II microscope was used. If reduction of TNBT was going to occur i t usual ly did so in f resh ly mounted sections within three to four minutes; i f i t had not occurred within 10 minutes reduction usual ly did not occur af ter a longer per iod. As Downton et al. (13) also found, controls stored in darkness reduced the dye more quickly when they were 6 returned to l i g h t (less than two minutes) as the dye had penetrated the sect ion during storage. Infra-red f luorescence photomicrography The technique described here was brought to our attent ion by Lynne El kin (16). Fluorescence from hand cut transverse leaf sections and from i so la ted bundle sheath strands was s tud ied . For comparative purposes the f luorescence from sections and bundle sheath strari-ds of the monocotyledons Zea mays. Sorghum sudanense, Saooharum sp and Panioum spp3 was observed. The bundle sheath strands were i so lated using a method s imi la r to that described by Berry (5). Sections from the mid-lamina were cut into 1 cm pieces and ground with a pest le in a mortar containing 5 ml of glass beads (0.25 - 0.30 mm) and 10 ml of grinding medium (0.1M Tr i s buf fer , 3 x 10 _ I fM EDTA, pH 7.0) . Grinding was continued unt i l the mesophyll became t rans lucent , leaving the green bundle sheath around the veins. The fragments of t issue were then transferred to a small Ten-Broeck homogenizer which was used to loosen the strands from the vascular t i ssue and remove any remaining mesophyll c e l l s . Both the bundle sheath strands and transverse sections were mounted on microscope s l ides in grinding medium. 7 Blue l i g h t was used to excite the pigments. The l i g h t source was a high pressure Ph i l ips HB0200 mercury lamp housed in a Zeiss Ultraphot II microscope. The l i g h t passed through a deep blue BG3 exc i ter f i l t e r before i t reached the specimen s l i d e . Dark f i e l d i l luminat ion was used. Before reaching the f i l m , the f luorescence passed through an X16 planar object ive and a yellow barr ie r f i l t e r (Kodak Wratten No. 12). The f luorescence was recorded on Kodak Ektachrome Infra-red Aero f i l m . Exposure time (usual ly two to three minutes) was determined automatical ly by a device in the microscope after i t was ca l ib rated using test exposures. It was found that G. globosa chloropdasts photobleached quite qu ick ly . Although the material was photographed as quickly as poss ib le , the length of exposure necessary to take a photograph was often s u f f i c i e n t to resu l t in photobleaching (see Discuss ion) . The f i lm was developed using the Kodak E-3 process for Ektachrome f i l m . Developing inst ruct ions were followed prec ise ly except that the developing tank was kept at room temperature and the water for washing came d i r e c t l y from the cold tap. A reverse exposure of 15 s, was given under a Sylvania No. 2 Superflood lamp. The response of the f i l m to red and fa r - red l i g h t was determined by placing a camera body, without the lens , 8 in the path of the sample beam of a Uni.cam SP800A UV spectro- photometer. Light from 440 - 750 nm, at 10 nm i n t e r v a l s , was used to expose successive frames of the f i l m . From 700 to 750 nm the f i l m was exposed with and without a deep-blue f i l t e r (Kodak Wratten No. 47B) in the beam path. lhC02 feedings Leaves were detached, recut under water, and i l l u m i - nated for at least 20 minutes. They were then placed in a P lex ig las chamber (120 ml) and i l luminated at 20,000 lux for a further 10 minutes while the chamber was f lushed with a i r . Approximately 10 yc of  lliC02 were then fed to the leaves and after s ix seconds they were k i l l e d in l i q u i d n i t rogen. The leaves were extracted in bo i l ing 80% v.yjv.aethanol and then in 0.2N formic acid in 20% v/v ethanol . Descending chromatography of the combined extracts were performed using 4 x 46 cm Whatman No. 1 paper s t r i p s with phenol ( l i q u e f i e d , 90% v/v), water, acet ic a c i d , IM EDTA (840: 1 60: 1 0: 1 ) as a solvent (5). In th is system malic and aspart ic acids are separated from one another and from other early products of photosynthesis, the acids having Rf values of 0.32 and 0.23 respec t ive ly . The 1 I f C content of malic and aspart ic acids was determined using autoradiography followed by l i q u i d 9 s c i n t i l l a t i o n counting or by the use of a radiochromatogram scanner. Results Plants at 10,000 lux showed the fastest growth. At 2,000 and 10,000 lux the leaves were bright green and about 10 to 15 cm long when mature. The plants grown outside (50,000 lux) had more l a t e r a l branches than the others and the leaves were leathery and shorter , about 4 to 8 cm. The plants grown outdoors also had a red pigment in many of the leaves mainly about the midrib and the edges of the lamina. Some of the leaves had pale t i p s . Electron microscopy The u l t ras t ructure of the photosynthetic t issue was s imi la r to that described by other workers (10,33). At a l l l i g h t i n t e n s i t i e s , the mesophyll ch loroplasts had prominent grana and peripheral r e t i c u l a . Many, though not a l l , mesophyll ch loroplast p r o f i l e s showed a few starch gra ins . A typ ica l mesophyll ch loroplast section (from 50,000 lux) is i l l u s t r a t e d i n Figure 1. The anova (Table 1) of the bundle sheath, chi bropl ast charac- t e r i s t i c s showed there was s i g n i f i c a n t var ia t ion (P < 0.05) 10 Table 1 Bundle sheath ch loroplast C h a r a c t e r i s t i c s . Means and standard deviat ions " ~ Lux 50,000 10,000 2,000 Grana/chloroplast Thylakoi ds/granum PLrx P R 1 Area 112.4 ± 7.2 2.5 ± 0 . 7 103.9 ± 134.0 11.8 ± 2.1 3.5 ± 1.1 38.6 ± 23.3 27.3 ± 17 4.0 ± 1.0 186.0 ± 212.8 P a r t i t i o n length x number of p a r t i t i o n s ch lorop last area S i g n i f i c a n t var iat ion (P < 0.05) among treatments for grana per ch loroplast but not for other terms. 11 among l i g h t i n t e n s i t i e s for grana per ch loroplast but i n s i g n i f i - cant var iat ion for thylakoids per granum and p a r t i t i o n length x number of par t i t ions J h e t h l a k o 1 d o v e r _ chloroplast area J laps (Figure 2) c h a r a c t e r i s t i c of G. globosa bundle sheath chTonopjhastss (10) were seen at a l l l i g h t i n t e n s i t i e s . Some chloroplasts had grana of more than two thylakoids p a r t i c u l a r l y at 10,000 and 2,000 lux (Figures 3 and 4) ; the mean number of thylakoids per granum for 50,000, 10,000 and 2,000 lux were 2.54, 3.48 and 3.96 respec t ive ly . Many of the chloroplasts at 50,000 lux showed d i l a t i o n of the in t ra - thy lako id space (Figure 5); th is was also sometimes present in ch loroplasts at 10,000 lux. A peripheral reticulum was present in a l l cases. Starch grains were a prominent feature of the bundle sheath ch lorop las ts , p a r t i c u l a r l y those from 50,000 and 10,000 lux. The starch grains in chloroplasts from 10,000 lux were sometimes so large as to almost completely exclude the internal membranes (Figure 6) . Dye reduction In a l l cases mesophyll ch loroplasts reduced TNBT in the l i g h t . No reduction of TNBT by e i ther bundle sheath or mesophyll ch loroplasts occurred in the dark. Table 2 shows the response of the bundle sheath chloroplasts to TNBT. 1 2 The black bar on each plate represents 1pm Figure 1. Mesophyll ch lo rop las t . 50,000 lux. Large granal stacks. X26,000. MZ. A 13 Figure 2. Portion of a bundle sheath ch lorop last showing thylakoid over laps. 50,000 lux. X54,800.  14 Figure 3. Bundle sheath ch lorop las t . Grana of more than two thylakoids are arrowed. 10,000 lux. X37,000.  1 5 Figure 4. Bundle sheath ch lo rop las ts . Some of the grana of more than two thylakoids are arrowed. 2,000 lux. XI9,900.  16 Figure 5. Bundle sheath ch loroplast with d i l a ted thy lako ids . 50,000 lux. Large starch gra ins . X20,100.  17 Figure 6. Bundle sheath ch lo rop las t . 10,000 lux. Starch grains occupy nearly a l l ch lo rop las t . X20,400.  18 Table 2 TNBT reduction in l i g h t by bundle sheath ch loroplasts from leaves grown at d i f f e r e n t l i g h t i n t e n s i t i e s Light i n t e n s i t y (lux) Test Conditions 50,000 10,000 2,000 TNBT + l i g h t Infrequent darkeni ng Mosa i c Mosai c TNBT + l i g h t + , ascorbate - DCIP1 ^Mosaic Mosaic + DCIP: dichlorophenol indophenol. TNBT is co lour less and becomes blue-black i f i t is reduced within the ch lo rop las ts . "Infrequent darkening" means that reduction occurred in only some of the several t e s t s . "Mosaic" means that , c o n s i s t e n t l y , ch loroplasts reduced T N 8 T ; tin! ohly^sofne bel'l seofihtheh.bundl e sheath. 19 Infra-red f luorescence photomicrography Infra-red Aero f i lm portrays v i s i b l e red l i g h t as yellow and i n v i s i b l e far- red l i g h t as red (the hue varies somewhat according to the source) . In a l l cases, the mesophyll ch loroplasts appeared bright red as they emitted v i s i b l e red f luorescence. Depending on whether they f luoresced in the red or far - red region of the spectrum, bundle sheath chloroplasts appeared red or were almost i n v i s i b l e , and yellow or red on the f i l m respec t ive ly . Figures 7 to 10 show the f luorescence of G. globosa ch lorop las ts . Figures 11 and 12 show f luorescence from Panicum maximum and Sorghum sudanense and are representat ive of monocotyledon f luorescence. Bundle sheath chloroplasts in transverse sections from leaves of G. globosa grown at a l l three d i f f e r e n t i n t e n s i t i e s emitted far- red f luorescence (Figures 7, 8 and 9) ; some v i s i b l e f luorescence was d i scern ib le at times. Isolated bundle sheath strands from leaves grown at 50,000 lux showed a red f luorescence. Strands from leaves grown at 10,000 and 2,000 lux (Figure 10) emitted far- red f luorescence together with some red wavelengths. x l t C 0 2 feedings Table 3 gives the resul ts of the l l t C 0 2 feedings. 20 Figure 7. Photograph of f luorescence from leaf' of G. globosa T . S . 50,000 lux. Colour of f luoresenee from bundle sheath (arrow on protect ive f lap) is not much d i f f e r e n t from that of the mesophyll; d i f ference is more marked on o r i g i n a l colour s l i d e . 2 1 Figure 8. Fluorescence from leaf of G. globosa T . S . 1 0 , 0 0 0 lux. Bundle sheath f luorescence (arrow) is at far - red wavelengths. Mesophyll f luorescence i s v i s i b l e .  22 Figure 9. Fluorescence from leaf of G. globosa. T . S . 2,000 lux. Far-red f luorescence from bundle sheath (arrow), v i s i b l e from mesophyll. 23 Figure 10. Isolated bundle sheath strand of G. globosa T . S . 2,000 lux. The bundle sheath c e l l s (small arrow) emit mostly far- red wavelengths. The pure yellow areas (large arrows) are mesophyll c e l l s .  24 Figure 11. Fluorescence from leaf of Panicum maximum. The large bundle sheath c e l l s (arrow) emit only v i s i b l e f luorescence, as do the mesophyll c e l l s . 25 Figure 12. Fluorescence from leaf of Sorghum sudanense. Far-red f luorescence is emitted from the bundle sheath c e l l s (arrows); v i s i b l e f luorescence from the mesophyl1 . *S~A 26 Table 3 Ratio of  lhC in malic acid to that in aspart ic acid at d i f fe rent l i g h t i n t e n s i t i e s Light i n t e n s i t y (tux) 50,000 : il»0,000 2,000 Ratio 0.9 ± 0.2 2.4 ± 0.9 4.5 ± 0.5 27 Discussion Electron microscopy Reports of experiments in which the e f fect of l i g h t in tens i ty on the membrane structure of mature chloroplasts has been studied , indicate a tendency towards an agranal condit ion at high l i g h t i n t e n s i t y . Ba l lant ine and Forde (3) found that in leaves of soybean grown at high l i g h t in tens i ty (220 W.nr 2 in the 400-700 nm range, 420 W.rtr2 approximately equals 1 0,000 f t . c . , 1 f t . c . = 10 lux) the granal staeks'were reduced to two or three appressed thy lakoids; the e f fect was even more marked at low temperature (20°C/12.5°C day/night) the grana being confined to occasional thylakoid over laps. Amaranthus l i v i d u s (a C-4 dicotyledon (27))has granal chloro- plasts in both the mesophyll and bundle sheath (27). At high l i g h t in tens i ty (approximately 145 W.m - 2) only a few rudimentary grana were present in ch loroplasts from both t issues (27). The degree of granal stacking increased markedly at lower i n t e n s i t i e s . When maize was grown under continuous f luorescent l i g h t (60 W.m - 2) (32), mesophyll ch loroplasts had smaller grana than under f u l l natural dayl ight ( l i g h t in tens i ty not ac tua l ly measured). Under f u l l dayl ight the bundle sheath chloroplasts had the usual lamel lar structure but under continuous f luorescent l i g h t there was a repression of the " lamel lar condit ion" together with increased l i p i d 28 depos i t ion . It was expected then that chloroplasts from leaves grown at high l i g h t in tens i ty would show less granal development, and hence, assuming that agranal ch loroplasts are d e f i c i e n t in PS II funct ion , have a lower a b i l i t y to reduce NADP. Light in tens i ty did not seem to a f fect granal development of the mesophyll ch lo rop las ts ; large granal stacks were present at a l l l i g h t i n t e n s i t i e s . This is in contrast to the s i tuat ion in Amaranthus l i v i d u s (see above). There is some ind icat ion from the anova that bundle sheath chloroplasts from the two highest l i g h t i n t e n s i t i e s contained fewer grana but there was no s i g n i f i c a n t var ia t ion in the tota l p a r t i t i o n length. In view of this and the fact that the anova was performed on micrographs not taken for this purpose, I cannot conclude that there were dfilfferences in the a b i l i t y of bundle sheath chloroplasts to photo-reduce NADP among the three l i g h t i n t e n s i t i e s . Bundle sheath chloroplasts of C-4 plants have often been observed to contain large quant i t ies of starch (25). Judging by published photographs (25), the bundle sheath chloroplasts at the two highest l i g h t i n t e n s i t i e s contained unusually large amounts of s ta rch , espec ia l l y those grown at 10,000 lux. For the plants grown at 50,000 lux a possible reason for the large amount of starch in the bundle sheath ch loroplasts 29 has i t s o r i g i n in the fact that at low night temperature (10°C) transport of starch out of mesophyll ch loroplasts of Dig-ttaria deaumbens was severely inh ib i ted (22). D. deoumbens accumulates starch in both mesophyll and bundle sheath chloro- plasts whereas i t is usual ly in the bundle sheath of C-4 plants (17). In summer in Vancouver, minimum da i ly temperatures, which occur at night, sometimes f a l l below 10°C and more frequently below 15°C, Table 4. It seems probable that th is would resu l t in an i n h i b i t i o n of starch transport out of the bundle sheath, i f the extrapolat ion from C-4 monocotyledons to C-4 dicotyledons can be made. Such an i n h i b i t i o n of carbohydrate t rans locat ion may have contributed to the slower growth of these p lants . It might also be noted that the mean maximum and mean da i ly temperatures are only s l i g h t l y above the temperatures, 10°C to 15°C, at which photosynthesis and growth of maize and other t rop i ca l grasses are very low. They are well below the temperatures, 30°C to 40°C, which are considered to be optimum for C-4 photosynthesis (11). These low temperatures may also be a factor in the slower growth of plants outside compared to the ones in the growth chamber at 24°C. However, th is is only a tentat ive suggestion as a i r temperatures measured metero logica l ly are often considerably d i f f e r e n t from leaf surface temperatures (36). 30 Table 4 Summary of temperatures recorded at the Plant Science weather s t a t i o n , Univers i ty of B r i t i s h Columbia, Vancouver during the summer, 1970 (°C) Mean Max. Mean Min. Max. Min. Mean Daily June 20.0 12.2 30. 0 8.3 16.1 July 20.5 12.6 27/ 2 9.4 16.5 August 20.0 12.7 24 4 9.4 16.1 Sept. 16.3 9.4 21 .7 -1 .7 12.9 Number of days per month in which the temperature f e l l below: 10°C 1 5°C 18°C June 7 26 29 July 3 28 31 August 2 31 31 Sept. 17 30 30 31 The very la rge , unusually shaped starch grains in the bundle sheath chloroplasts from leaves grown at 10,000 lux are something of an enigma. I would not have expected an i n h i b i t i o n of starch t rans locat ion under the i r temperature regime (24°C/18°C) nor that photosynthesis would be so rapid as to produce such apparently large quant i t ies of s ta rch . The marked formation of B-cyanin in leaves grown at 50,000 lux may be due to the i r high starch content. Anthocyanin formation seems to be commonly associated with accumulation of sugars in plant t i s s u e s . Any environmental factor such as high l i g h t i n t e n s i t y , low temperature, drought or low nitrogen supply, which favours an increase in the sugar content of a given plant t i s s u e , often favours synthesis of anthocyanin in that t issue (29). In view of these comments i t is surpr i s ing that v i s i b l e pigment formation did not occur at 10,000 lux; 10,000 lux is also intermediate in the range of l i g h t i n t e n s i t i e s (approximately 3000 to 24,000 lux) used by Downs and Siegelmann (9) to study photocontrol of anthocyanin synthesis in Sorghum vulgare seedl ings. Dye reduction The resu l ts of the TNBT reduction tests ind icate that chloroplasts from leaves grown at 50,000 lux had a low non- c y c l i c e lectron flow which is associated with a low PS II 32 a c t i v i t y . This would f i t in with expectations about the e f fect of l i g h t in tens i ty on chloroplast structure and function derived from the l i t e r a t u r e but in this study electron microscopy did not provide concrete evidence for less granal development at high l i g h t i n t e n s i t y . Some reserve is needed in in terpret ing TNBT resul ts as the dye has d i f f i c u l t y penetrating in tact c e l l walls (the "mosaic" observed in some cases may be due to var iab le rates of TNBT penetration into d i f f e r e n t c e l l s ) . A l so , after th is work was completed i t was reported that TNBT is one of the least s a t i s f a c t o r y dyes for in vivo demonstration of PS I and PS II a c t i v i t y (25); "no explanation for th is statement was given. Infra-red f luorescence photomicrography El kin (16) found, using in f ra - red f luorescence photomicrography, that on exc i ta t ion with blue l i g h t , granal ch loroplasts emitted v i s i b l e red f luorescence and agranal chloroplasts emitted i n v i s i b l e far - red f luorescence. I decided to use the technique to see i f f luorescence from mesophyll and bundle sheath chloroplasts would change with the environment, and corre late with changes in the granal s t ructure . The hypothesis was that under high l i g h t i n t e n s i t y , bundle sheath chloroplasts would show more far- red f luorescence and under lower l i g h t i n t e n s i t i e s more red f luorescence. 33 For those monocotyledons where I had pr ior knowledge of the ch loroplast s t ructure , there was, as El kin found, a cor re la t ion for bundle sheath chloroplasts between the occurrence of grana and the type of f luorescence; two examples are shown in Figures 11 and 12. G. globosa bundle sheath ch loroplasts gave more var iab le resu l ts (see Figures 7 to 10). These might be due, to environmental d i f ferences but may also be due to photobleaching to which G. globosa ch loroplasts seem p a r t i c u l a r l y prone. El kin (16) noted too, that G. globosa bundle sheath chloroplasts seemed unusually sens i t ive to photobleaching. She also found that , depending on the species used, ch loroplasts with double or t r i p l e thylakoids or even a small number of well developed grana gave red or far - red f luorescence, i . e . the cor re la t ion between f luorescence and structure broke down. At the moment environmental studies using this technique should be confined to those species where there is a d e f i n i t e cor re la t ion between f luorescence and ch loroplast s t ructure . In viewofvEl k i n 1 s comments i t may not be su i tab le even in these cases, as in ch loroplasts where the granal s t ructure was ' C h a n g i n g , e . g . young Sorghum bioolor bundle sheath chloroplasts (14), the f luorescence may not r e f l e c t granal structure as i t does in mature ch lorop las ts . It would be in terest ing to see i f other C-4 dicotyledon chloroplasts behaved s i m i l a r l y to those of G. globosa. 34 What is the o r i g i n of the f luorescence seen using this technique? There is a c o r r e l a t i o n , for some spec ies , between the red and far-red f luorescence and the presence or absence of grana (my own and E l k i n ' s (16) observat ions) . Further, as noted previously in the Introduction and Discussio there is evidence for granal ch loroplasts having both PS I and PS II whereas agranal chloroplasts are d e f i c i e n t in PS I I . As a resu l t El kin (16) has considered that the far - red f luorescence is from PS I, the red f luorescence being from PS II (a conclusion which is not e n t i r e l y incompatible with the ample evidence for the two photosystems f luoresc ing at d i f f e r e n t wavelengths, e .g . (34)). Concerning the actual wavelength of the f luorescence from sections of C-4 leaves there are two pert inent papers. Boardman et al. (7) showed with subchloroplast fragments enriched in PS I or PS II that a f luorescence emission band at 735 nm (far-red) or ig inated mainly from PS I and bands at 683 and 695 nm (red) came from PS I I . It was then shown by Woo et al. (40) that mesophyll ch loroplasts of Sorghum bioolor had emission bands at 683, 695 and 735 nm whereas the agranal bundle sheath ch loroplasts emitted 95% of t h e i r f luorescence in the 735 nm band. E lk in (16) and also Laetsch (25) have apparently equated the 735 nm f luorescence, and hence PS I, with the long wavelength f luorescence from sections of C-4 leaves. However the spectra studied by Boardman et al. 35 (7) and Woo et al. (40) were obtained at 77°K, not at room temperature. Room temperature spectra do not show the large 735 nm band seen at 77°K; in f a c t , system I p a r t i c l e s are "weakly f luorescent" at room temperature (see Figure 13). Even i f i t is assumed that the v i s i b l e f luorescence seen in the leaf sections at room temperature is from PS I I , then i t seems that the f luorescence from PS I would hardly be s u f f i c i e n t to form an image of comparable in tens i ty on f i l m to that formed by the f luorescence from PS I I . I have to conclude that the nature and o r i g i n of the f luorescence from the leaf sections and i so la ted bundle sheath ch loroplasts i s s t i l l uncerta in , there being no d i rec t proof that the two kinds of f luorescence come from PS II and PS I. ^COg feedings The rat ios of malic acid to aspart ic acid show that at high l i g h t in tens i ty more l l fC was i n i t i a l l y f ixed into aspart ic a c i d , while at the lower i n t e n s i t i e s most of the l l fC was f ixed into malic a c i d . This was somewhat surpr is ing as electron microscopy (see above) showed there was no appreciable d i f ference in ch loroplast u l t ras t ructure between l i g h t inten- s i t i e s . The unexpected rat ios may be due to d i f ference in the pool s izes of the d icarboxy l ic acids at the time of feeding. 36 Assays of malic enzyme a c t i v i t y which catalyses the decarboxy- l a t i o n of malic acid (11) would have been more i l l u m i n a t i n g . Conclus ion There was no e f fec t of l i g h t in tens i ty on p a r t i t i o n length (an ind icator of PS I funct ion) of bundle sheath c h l o r o p l a s t s , a conclusion which is strengthened by the lack of an e f fect of l i g h t in tens i ty on mesophyll ch loroplast s t ructure . This was unexpected as the l i t e r a t u r e indicated there' would be greater granal development at lower i n t e n s i t i e s (3,27,32). There was some var ia t ion in metabolite levels among plants grown at d i f f e r e n t l i g h t i n t e n s i t i e s . Starch formation seemed to be contro l led by l i g h t in tens i ty and possib ly by s t r e s s , a l thoughthe importance of a t h i i i c Tatter > factor is.- not cl e a r c t c b ~ r * n . Var iat ion in metabolite levels was also indicated by g-cyanin formation at 50,000 lux and the rat ios of malic to aspart ic a c i d . I have co l lec ted the resu l ts of th is work together in Table 5. For the mesophyll ch loroplasts at a l l l i g h t i n t e n s i t i e s the a b i l i t y to reduce TNBT, the occurrence of grana and the red f luorescence c o r r e l a t e , as expected. At 10,000 and 2,000 lux the bundle sheath chloroplasts show the expected cor re la t ion between far- red f luorescence, thylakoid overlaps and high malic acid content, but the apparent capacity to reduce 37 Table 5 Summary of experimental resu l ts TNBT Fluorescence Malate Aspartate E.M. 50,000 lux Bundle sheath Mesophyll Infrequent reduction + Far-red; red in i so lated c e l l s Red 0.9 ± 0.2 Thyla koi d overlaps Grana 10,000 lux Bundle sheath Mesophyl1 Mosai c + Far-red Red 2.4 ± 0.9 Thyla koi d overlaps Grana 2,000 lux Bundle sheath Mesophyl1 Mosai c + Far-red Red 4.5 ± 0.5 Thylakoid overlaps Grana 38 TNBT does not agree. For the bundle sheath chloroplasts at 50,000 lux the low capacity to reduce TNBT, the mainly f a r - red f luorescence and the thylakoid overlaps corre late together but the high aspartate content does not. Perhaps with i t s lack of s t ructura l response to l i g h t i n t e n s i t y , grana of two stacked thylakoids only and lack of cor re la t ion between characters which are known to do so for other p lants , G. globosa occupies an intermediate pos i t ion between an extreme agranal type such as Sorghum b i c o l o r and a typ ica l granal type such as Amaranthus l i v i d u s . P y l i o t i s et al. (33) place G. globosa between t y p i c a l l y agranal and granal types in a table comparing total p a r t i t i o n length per unit area of ch loroplast and chlorophyl l a to chlorophyl l b r a t i o . G. aelosoldes was found to be intermediate in malic enzyme and aminotransferase a c t i v i t y between a group containing Sorghum sp and another including Amaranthus sp (20). Addendum Studying the e f fect of c l imat i c stress on Sorghum sp3 Taylor and Craig (39) found that when the night temperature was dropped to 10°C a pronounced increase in starch in bundle sheath chloroplasts occurred. After three days of treatment two-thirds of the sect ional plane through the ch loroplast appeared as starch gra in . In a l a te r paper (8) Brooking and 39 Taylor observed a greater proportion of l l f C f ixed into aspart ic acid at 6 s in Sorghum leaves af ter 6.5 hours at 10°C and at 30 hours nearly 80% of the l l f C remained in aspart ic acid after one minute of chasing with 1 2 C 0 2 . The higher proportion of 1 4 C found in aspart ic acid in leaves of G. globosa grown outside may bear some r e l a t i o n to th is observat ion, although the low temperature stress occurred at night, not during the day. There now seems to be a general consensus on the question of whether agranal c h l o r o p l a s t s , in p a r t i c u l a r bundle sheath ch lo rop las ts , contain PS II . In agranal bundle sheath chloroplasts an intact e lectron transport chain inc luding both photosystems has been demonstrated, mainly by the use of H i l l oxidants (1 ,24,37) , the dependence of cytochrome f and b oxidat ion on the wavelength of the exc i t ing red l i g h t (4,6) and by measuring f luorescence exc i tat ion and emission s p e c t r a . ( 4 ) . Isolated maize bundle sheath chloroplasts would only reduce NADP in the presence of added plastocyanin (37) ind icat ing that plastocyanin was los t during i s o l a t i o n of the ch lo rop las ts . These agranal bundle sheath chloroplasts are d e f i c i e n t in PS II however, there being about a three- fo ld higher PS I to PS II ra t io in these chloroplasts than in mesophyll ch loroplasts (4) . It is l i k e l y that the s ize of the def ic iency varies between spec ies . On the basis of experiments using d i f f e r e n t species (1 ,2,4 ,6 ,28,37) I would 40 put A t r i p l e x spongiosa (a granal type) G. globosa, D i g i t a r i a sanguinalis (crab-grass) , maize and Sorghum sp as a ser ies showing increasing PS II de f i c iency . To my knowledge no work has been published which gives a d e f i n i t e clue to the nature and or ig in of the far- red f luorescence seen in the bundle sheath ch lo rop las ts . An in terest ing paper on chlorophyl l f luorescence in a system I - chlorophyl l a - protein complex and system II p a r t i c l e s has been published by Mohanty .et al. (30). Using d i l u t e suspensions (chlorophyl l concentration 2.0 - 3.0 mg/ml) prominent f luorescence peaks were found at 685 nm in system I p a r t i c l e s as well as in system II p a r t i c l e s at 77°K (see Figures 14 and 15A). It has been considered, egg. (7) , that system I f luorescence at 77°K is predominantly at 735 nm. When a thick suspension (chlorophyl l concentration 40 mg/ml) was used the 685 nm band was present only as a small shoulder, most of the f luorescence being in the 735 nm band (see Figure 15B). This was thought to be due to reabsorption of the 685 nm band by neighbouring p a r t i c l e s . It should be noted that Boardman et al. (7) who found the majority of system I f luorescence at the 735 nm band at 77°K d i luted t h e i r f rac t ions to an absorbance of 0.1 at 436 nm to minimize reabsorption of emitted l i g h t (the chlorophyl l concentration as such as not g iven) . Either Mohanty et al. (30) d i luted the i r suspension even more, or there is a genuine d i f ference in r e s u l t s . 41 Returning to the resul ts of Mohanty e t al. (30), Figure 16 shows a room temperature emission spectrum of a d i l u t e suspension of system II p a r t i c l e s . There is a large F685 band with small i l l - d e f i n e d bands at 720 nm and 740 nm. A spectrum, Figure 17, obtained s i m i l a r l y for PS I had an F685 band with a shoulder at 692 nm and bands at 720 nm and 740 nm somewhat larger than the corresponding ones in PS I I . Intact chloroplasts may be regarded as "thick suspensions" and I propose that in agranal bundle sheath ch lorop las ts , which are de f i c ient in PS I I , any 685 nm fluorescence is absorbed and reappears with the F720-740 band (which at room temperature would s t i l l not be as large as the 735 nm band seen at 77°K). In granal ch loroplasts the F685 and F695 bands are probably strong enough to mask any far - red f luorescence on the f i lm and the chloroplasts would thus appear to f luoresce only v i s i b l y . In a paper published by Bazzaz and Govindjee (4) emission spectra of mesophyll and bundle sheath ch loroplast fragments at room temperature are p r a c t i c a l l y ident i ca l (and very s imi la r to the spectrum shown in Figure 17) both spectra showing a large band at 685 nm. This may well be evidence against my proposal as reabosrption of the F685 band obviously did not occur. However these spectra are from fragments in which t ransfer of f luorescence may not have been able to occur between PS II and PS I. 42 L a s t l y , to end on a somewhat dampening note, I would l i k e to quote from an a r t i c l e on f luorescence from the 1972 ed i t ion of the Annual Review of Plant Physiology (18) where i t is suggested that , . . . the increase in F72S due to cooling is caused by an incvease in energy transfer to a long wave chlorophyll form. This form is usually present in low concentration but has a high i n t r i n s i c fluorescence yield. It is probably not i d e n t i c a l to P7003 the energy trap of photosystem I. . . . Possibly the chlorophyll form emitting F725 acts as an energy sink, protecting the photochemical systems from photo-oxidation under unfavorable conditions. 43 Figure 13. Approximate emission spectra of system I f r a c t i o n of spinach chloroplasts at 293°K and 77°K. Drawn from Figure 3, Boardman et al. (7) . 44 Figure 14. Approximate emission spectrum of system II p a r t i c l e s of maize chloroplasts at 77°K. Chlorophyll con- centrat ion approx. 3.0 ug/ml . Drawn from Figure 3, Mohanty et al. (30).  45 Figure 15. Approximate emission spectra at 77°K of system I chl orophy/11' a-protei n complex of maize. A. Thin suspension, chlorophyl l cone, approx. 2.0 ug/ml. B. Thick suspension, chlorophyl l cone, approx. 50 ug/ml. Drawn from Figure 4, Mohanty et al. (30) 46 Figure 16. Approximate emission spectrum at room temperature of system II p a r t i c l e s of maize. Chlorophyll cone, approx. 3.0 yg/ml . Drawn from Figure 2, Mohanty et al. (30). 21 FLUORESCENCE .RELATIVE UNITS ' ' ~ FLUORESCENCE .RELATIVE UNITS 47 Figure 17. Approximate emission spectrum at room temperature of system I chlorophyl l a-protein complex of maize. Chlorophyll cone, approx. 2.0 ug/ml. Drawn from Figure 2, Mohanty et al. (30). — • — — • — ' — • • — i — • — • i i- 660 680 700 720 740 ' 760 A n m Figure 17 L I T E R A T U R E C I T E D Andersen, Kirsten S . , Joan M. Bain, D.G. Bishop and Robert M. Smillte. 1 972. Photosystem II a c t i v i t y in agranal bundle sheath chloroplasts from Zea mays. Plant Physiol . 49:461-466. Anderson, Jan M., C.K. Woo and N.K. Boardman. 1971. Photochemical systems in mesophyll and bundle sheath chloroplasts of C\ p lants . Biochim. Biophys. Acta. 245(2):398-408. B a l l a n t i n e , J . El izabeth M. and B .J . Forde. 1970. The ef fect of l i g h t in tens i ty and temperature on plant growth and chloroplast u l t ras t ructure in soybean. Amer. J . Bot. 57(10):1150-1159. Bazzaz, Maarib Bakri and Govindjee. 1973. Photochemical propert ies of mesophyll and bundle sheath chloroplasts of maize. Plant Phys io l . 52:257-262. Berry, J . A . , W.J.S. Downton and E.B. Tregunna. 1970. The photosynthetic carbon metabolism of Zea mays and Gomphrena globosa: the locat ion of the C0 2 f i x a t i o n and the carboxyl t ransfer react ions . Can. J . Bot. 48:777-786. Bishop, D.G., Kirsten S. Andersen and Robert M. S m i l l i e . 1972. Photoreduction and oxidat ion of cytochrome f in bundle sheath c e l l s of maize. Plant Phys io l . 49:467-470. Boardman, N.K., S.W. Thorne and Jan M. Anderson. 1966. Fluorescence propert ies of p a r t i c l e s obtained by d ig i ton in fragmentation of spinach ch lo rop las ts . Proc. Nat l . Acad. S c i . , USS.A. 56:586-593. 48 49 8. Brooking, I.R. and A.O. Tay lor . 1973. Plants under c l imat ic stress V. C h i l l i n g and l i g h t ef fects on radiocarbon exchange between photosynthetic intermediates of Sorghum. Plant Phys io l . 52: 180-182. 9. Downs, R.J . and H.W. Siegelman. 1963. Photocontrol of anthocyanin synthesis in Milo seedl ings . Plant Phys io l . 38:25-30. 10. Downtonij, W.J.S. 1970. Preferent ia l C ^-d i carboxyl i c acid synthes is , the post - i l luminat ion C0 2 burst , carboxyl t ransfer step, and grana conf igurat ions in plants with C-photosynthes i s . Can. J . Bot. 48:1795-1800. 11. Downton, W.J.S. 1971. Adaptive and evolutionary aspects of Ct photosynthesis. In: M.D. Hatch, C . B . Osmond ' and R.0. S la tyer , eds . ; Photosynthesis and Photo- r e s p i r a t i o n . Wiley Intersc ience, New York, pp. 3-17. 12. Dowri.to.'nji W.J.S. 1971. Checkl ist of Ch spec ies . In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . ; Photosynthesis and Photorespirat ion. Wiley Inter- sc ience, New York, pp. 554-558. 13. Downton, W.J .S . , J . A . Berry and E.B. Tregunna. 1970. Ct -Photosynthes is : non-cycl ic e lectron flow and grana development in bundle sheath ch lo rop las ts . Z. Pflanzenphysiol . 63:1 94-1 98. 14. Dowton, W.J.S. and N.A. P y l i o t i s . 1971. Loss of photosystem II during ontogeny of Sorghum bundle sheath ch lo rop las ts . Can. J . Bot. 49:179-180. 15. Dunn, Arnold and Joseph A r d i t t i . 1968. Experimental physiology. Holt , Rinehart and Winston, Inc . , New York, p. 265. 16. El k in , Lynne. 1 970. Personal communication. Botany Dept. , Univers i ty of C a l i f o r n i a , Berkeley, C a l i f o r n i a 17. Evans, L.T. 1971. Evolut ionary, adaptive, and environmental aspects of the photosynthetic pathway: assessment. In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . ; Photosynthesis and Photoresp i rat ion. Wiley Inter- sc ience, New York, pp. 130-136. 50 18. Goedheer, J . C . 1972. Fluorescence in r e l a t i o n to photo- synthes is . Ann. Rev. Plant Phys io l . 23:87-112. 19. Goodenough, U.W., J . J . Armstrong and R.P. Levine. 1969. Photosynthetic properties of ac-31 , a mutant s t r a i n of Chlamydomonas veinhardi devoid of cjj lprpplast membrane stack ing. Plant Phys io l . 44:1001-1012. 20. Hatch, M.D. 1971. Mechanism and function of the C\ pathway of photosynthesis. In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . , Photoshythesis and Photorespirat i on , Wiley Intersc ience, New York, pp.. 406-41 2. 21. Hart, A .L . and E.B. Tregunna. 1971. Some aspects of environmental control of the photosynthetic apparatus in Gomphvena globosa. In: M.D. Hatch, C.B. Osmond and R.0. S I a t y e r , . e d s . , Photosynthesis and Photo- r e s p i r a t i o n , Wiley Intersc ience, New York, pp. 413-418. 22. H i l l i a r d , Joe H. and S.H. West. 1969. Starch accumulation associated with growth reduction at low temperatures i in a t rop ica l p lant . Science 168:494-496. 23. Homann, P.H. and G.H. Schmid. 1967. Photosynthetic reactions of chloroplasts with unusual s t ruc tures . Plant Phys io l . 42:1619-1632. 24. Izawa, S. and N.E. Good. 1966. Ef fect of. sa l t s and electron transport on the conformation of i so lated chloro- plasts I I . Electron microscopy. PI ant Physiol . 41: 544-552. 25. Laetsch, W.M. 1971. Chloroplast s t ructura l re lat ionsh ips in leaves of Ch p lants . In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . ; Photosynthesis and Photorespirat i on, Wiley Intersc ience, New York, pp. 323-349. 26. Langridge, J . and J .R. McWilliam. 1967. Heat responses of higher p lants . In: A.H. Rose, e d . ; Thermobiology, Academic Press, London, pp. 231-292. 51. 27. L y t t l e t o n , J .W. , J . E . M . Bal lant ine and B.J . Forde. 1971. Development and environment studies on chloroplasts of Amaranthus l i v i d u s . Iri: N.K. Boardman, Anthony W. Linnane and Robert M. Smillie, eds . ; Autonomy and Biogenesis of Mitochondria and Chlorop lasts , North Holland Publishing Co. , Amsterdam, pp. 447-452. 28. Mayne, B .C. , G.E. Edwards and Clanton C. Black. 1971. Spec t ra l , physical and electron transport a c t i v i t i e s in the photosynthetic apparatus of mesophyll c e l l s and bundle sheath c e l l s of D i g i t a r i a sanguinalis (L .) Scop. Plant Phys io l . 47:600-605. 29. Meyer, B.S. and D.B. Anderson. 1952. Plant Physiology. D. Van Nostrand Co. (Canada) L t d . , p. 389. 30. Mohanty, P., Barbara Z i l i nskas Brawn, Govindjee and J . P . Thornber. 1972. Chlorophyll f luorescence charac- t e r i s t i c s of system I chlorophyl l a-protein complex and system 'I11 p a r t i c l e s at room and l i q u i d nitrogen temperatures. Plant and Cel l Phys io l . 13:81-91, 1 972. 31. Ohad, I . , P. S iekevitz and G.E. Palade. 1967. Biogenesis of ch loroplast membranes II . P l a s t i d d i f f e r e n t i a t i o n during greening of a dark-grown algal mutant {chlamydomonas veinhardi). J . Cel l B i o l . 35:553- 584. 32. Osipova, O.P. and N.I. Ashur. 1965. Chloroplast structure in maize grown under various condit ions of i l l u m i n a t i o n . Soviet Plant Phys io l . 12(2):217-224. 33 P y l i o t i s , N.A., K.C. Woo and W.J. S. Downton. 1971. Thylakoid aggregation corre lated with chlorophyl l a-chlorophyl l b rat io in some Ĉ  spec ies . In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . , Phytosynthesis and photoresp i rat ion , Wiley Inter- sc ience, New York, pp. 406-412. 34. Rabionowitch, E. and Govindjee. 1969. Photosynthesis. John Wiley and Sons, Inc . , New York, pp. 196-215. 52 35. Reynolds, E.S. 1963. The use of lead c i t r a t e at high pH as an electron opague sta in in electron microscopy. J . Cel l Biol . 1 7:208-21 2. 36. Sa l i sbury , F.B. and C. Ross. 1969. Plant physiology. Wadsworth Publishing Co. , C a l i f . , pp. 78-111. 37. S m i l l i e , Robert M., Kirsten S. Andersen, N.F. Tobin, Barr ie Entsch and D.G. Bishop. 1972. Nicotinamide adenine d inucleot ide phosphate photoreduction from water by agranal chloroplasts i so la ted from bundle sheath c e l l s ofi.maize. Plant Phys io l . 49:471 -475. 38. Spurr, A.R. 1 969. A low-v iscos i ty epqxy.y res in embedding medium for electron microscopy. J . U l t r a s t r u c t . Res. 26: 31 -43. 39. Tay lor , A.O. and A.S. Cra ig . 1971. Plants under c l imat i c stress 11. Low temperature, high l i g h t ef fects on chloroplast u l t r a s t r u c t u r e . Plant Phys io l . 47: 719-725. 40. Woo, K.C. , Jan M. Anderson, N.K. Boardman, W.J.S. Downton, C.B. Osmond and S.W. Thorne. 1970. Def ic ient photo- system 11 in agranal bundle sheath chloroplasts of p lants . Proc. N a t l . Acad. S c i . U.S.A. 67:18-25. P A R T I I A NITROGEN FIXING ASSOCIATION: PASPALUN NOTATUM AND AZOTOBACTER PASPALI T E R 111 N-0 L 0 G Y The term "nitrogen" is commonly used as a generic term for any of a var iety of nitrogen-containing compounds such as ammonia or n i t r a t e . "Nitrogen" is also used in the s p e c i f i c sense as re fer r ing to the gas, N 2 . Where confusion may ex ist in the intended meaning, the term "din i trogen" is used to refer to the N 2 molecule in accordance with recent chemical convention (2). I have used the fol lowing terminology of Hardy et al. (1) to ind icate the ana ly t i ca l o r i g i n of data on nitrogen f i xati on: " N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y " - the data have been derived from tests of the a b i l i t y of a system to reduce acetylene ( C 2 H 2 ) , and these data were interpreted as a measure of the system's a b i l i t y to reduce d in i t rogen. " N 2 a s e [ C 2 H 2 ] - a c t i v i t y " - th is term is reserved to indicate the a b i l i t y of a preparation of the d i n i t r o g e n - f i x i n g enzyme, nitrogenase, to reduce C 2 H 2 . Some authors have used "nitrogenase a c t i v i t y " or some s imi lar term to indicate the C 2 H 2 reduction a c t i v i t y of, for example, a r o o t - s o i l system. I feel th is is mis leading. 53 L I T E R A T U R E C I T E D 1. Hardy, R.W.F., R.C. Burns and R.D. Holsten. 1973. App l i ca- tions of the acetylene-ethylene assay for measurement of nitrogen f i x a t i o n . Soi l B i o l . Biochem. 5:47-81. 2. Postgate, J .R. 1971. Relevant aspects of the physio- log i ca l chemistry of nitrogen f i x a t i o n . In: D.E. Hughes and A.H. Rose, eds . ; Microbes and b i o l o g i c a l p roduct iv i ty , 21st Symp. Soc. Gen. M i c r o b i o l . , Univers i ty Press, Cambridge, England, pp. 287-308. 54 P R O L O G U E Al l known b io log ica l agents of nitrogen f i x a t i o n are prokaryotes (23). They are general ly thought of as one of the partners of a symbiosis or as free l i v i n g , i . e . able to function without a p a r t i c u l a r re la t ionsh ip with some other organism. Free l i v i n g nitrogen f i xers have been found in a number of t e r r e s t i a l hab i tats , p a r t i c u l a r l y the s o i l (23). The bulk so i l usual ly contains less bacter ia l a c t i v i t y than the rhizoplane or the rhizosphere (25). Nitrogen- f i x i n g a c t i v i t y has been found to be p a r t i c u l a r l y associated with the rhizosphere of a number of p lants , the degree of f i x a t i o n often being much higher than that usual ly associated with asymbiotic s o i l nitrogen f i x e r s . By way of comparison, nitrogen f i x a t i o n by so-ca l led free l i v i n g s o i l bacter ia has been estimated as f a l l i n g within the range 0.04 to 15.0 KgN.ha" x yr . " 1 ( 2 0 ) . Some examples are 1 KgN.ha.~*yr . - 1 and 2 KgN.ha."*yr ." 1 for a natural grassland ecosystem in Saskatchewan, Canada (27), 0-3 KgN.ha.~ 1yr .~ 1 for tussock grassland s o i l s in New Zealand (18), 4-5 KgN.ha.~  1yr."1 for non-rhizosphere f i x a t i o n in a wilderness s i t e at Rothamsted, England (15). The amount of nitrogen f ixed may be increased by amending s o i l s , i . e . by adding carbohydrate (1,13,19). A 55 56 notable exception to these r e l a t i v e l y low rates are Egyptian s o i l s which support dense populations of Azotobaoter spp and Clostridium spp and f i x large amounts of nitrogen p a r t i c u l a r l y when an adequate supply of carbohydrate is present (20,23). As a resu l t of a survey of research on nitrogen- f i x i n g bacter ia Mishustin and Sh i l 'n ikova (19) concluded that Azotobaoter spp occurred frequently in the rhizosphere. How- ever, they also noted Rovira's comment (26), that Azotobaoter spp const i tute not more than 10% of the population of the root zone. Facu l tat ive anaerobic nitrogen f i x i n g bacter ia of the Klebsiella-Aerobaoter group have been i so lated consistently, , from the surfaces of roots and nodules of soybean p lants ; s imi la r types of bacter ia were also i so lated from a l f a l f a and c lover (12). Nitrogen f i x a t i o n , in the near absence of legumes, occurred at the rate of 55 KgN.ha."*yr.~ 1 on a s i t e at Rothamsted, England, l e f t uncult ivated since 1882 (15). A survey of about 40 plant species showed considerable nitrogen f i x i n g a c t i v i t y associated with the roots of Eeraoleum sphondylium II. (hogweed), Anthrisous s y l v e s t r i s Hoff. (cow p a r s l e y ) , Merourialis perennis L. (dogs mercury), Rumex acetosa ( s o r r e l ) , Convolvulus arvensis L. (bindweed), Viola oanina IL. (dog's v i o l e t ) and Staehys s y l v a t i e a L. (hedge woundwort). Roots of 5. s y l v a t i c a , free of loose s o i l , gave a maximum rate of C 2 H 2 reduction of 1.31 ± 0.20 nmoles C 2 H 2 . g . - 1 h .- 1at p0 2 0.04 atm. Plants with an intact s o i l - r o o t system gave rates of C 2 H 2 reduction 57 per gram dry weight of root two to three times higher than t h i s . Three n i t rogen- f ix ing i so la tes were obtained, but the organisms were not i d e n t i f i e d . Assuming that there are about 5 g of roots (fresh weight) per dm2 of ground area, 1.31 nmoles C 2 H 2 . g • " 1 h:~ 1 is about equivalent to 0.5 K g N . h a . - 1y r . - I . This value i s about 0.01 of the 55 KgN.ha~ 1y r .~ 1 noted above, and is probably an overestimate as i t was calculated on the basis of continuous nitrogen f i x a t i o n every day of the year. The discrepancy between the amount of nitrogen f ixed estimated from N 2 [C 2 H 2 ]=f ix ing a c t i v i t y and that from nitrogen accumulated in the s o i l over many years needs further i n v e s t i g a t i o n . Nitrogen f i x i n g a c t i v i t y has also been found in the rhizospheres of a number of t rop ica l members of the Gramineae and Cyperaceae. Nitrogen is cont inual ly ava i lab le to r i c e (Oryza satioa L.) for many years in flooded so i l even in the absence of f e r t i l i z e r (30). Yoshida and Ancajas (29) showed that nitrogen was f ixed by bacter ia in the root zones of r i c e in flooded s o i l . In a f i e l d experiment (30) these authors found the fol lowing rates of so i l nitrogen f i x a t i o n in the wet and dry season. Wet season: planted, flooded so i l 51.7 K g N . h a . - 1 ; unplanted, flooded s o i l 22.2 KgN.ha ." 1 ; p lanted, upland s o i l k'at_s_eas_o_n 7.0 K g N . h a . - 1 ; unplanted, upland so i l 3.0 K g N . h a . - 1 . Dry season: p lanted, flooded so i l 63.3 K g N . h a - 1 ; unplanted, flooded s o i l 28.3 K g N . h a . - 1 ; p lanted, upland s o i l 58 5.0 K g N . h a . - 1 ; implanted, upland s o i l 2.8 K g N . h a . - 1 . Dommergues et al. (10) measured N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in the rhizospheres of r i c e , maize and a number of other t rop ica l grasses and sedges. The species and associated C 2 H 2 reduction rates are given in Table 1. The N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in the r i ce rhizosphere was one-tenth of that of symbiotic systems. An anaerobic bacterium, Enterobacter cloacae , was i so lated from the root system of maize (24) although in this case the degree of nitrogen f i x a t i o n was estimated to be less than 0.5 KgN.ha.-  1 y r . " 1 . Sugar cane, l i k e r i c e , can be grown for long periods without nitrogenous f e r t i l i z e r . In B r a z i l , sugar cane has been cu l t ivated for 100 years without f e r t i l i z e r (9) . Experiments with 1 5 N 2 in Hawaii showed that even when 165 K g N . h a . - 1 was applied to sugar cane as f e r t i l i z e r , 70% of the nitrogen in the crop came from other sources (9). It has been found that the obl igate aerobe Beij e r i n c k i a is cons is tent ly stimulated in the rhizosphere of sugar cane (5 ,9) . Dobereiner et al. (9) estimated that the rhizosphere of s o i l of one sugar cane s i t e produced 1.51 nmoles C 2 H l t . h . _ 1 g . - 1 s o i l which extrapolated to 67 K g N . h a . - 1 y r . - 1 f ixed in the top 20 cm of s o i l . B e i j e r i n c k i a is also stimulated in the rhizosphere of Ei.parrh.enia rufa3 D i g i t a r i a decumbens, Panicum maximum, Cynodon dactylon, Setaria sphacelata3 r i c e , Panicum purpurescens and Paspalum notatum (a l l t rop ica l grasses) and Cyperus rotundus although less cons is tent ly than in sugar 59 Table 1 N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in the rhizospheres of t rop i ca l grasses. These values are taken from Table 2, Dommergues et al. (10) Plant species Plant age (months) N 2 [ C 2 H 2 ]-fi.xing a c t i v i t y Rice 1 1800 •+ 274 1 1190 + 140 1 1430 + 205 0.3 6153 + 1516 Eleusine covacana 1 374 165 1 1.3 + 0.5 Paspalum virgatum 4 528 + 48 8 345 163 Tennisetum puvpuveum 2 4 * 0.7 Panioum maximum 2 10 + 1 .1 Cypevus z o l l i n g e v i 3 162 + 47 6 110 + 25 Cypevus obtusiflovus 6 352 + 66 nmoles C 2 H 2 . h . _ : i ( g . dry r o o t ) " 1 1000 n m o l e s C 2 H i t . h . - 1 g . _ 1 a r e a b o u t e q u i v a l e n t t o 20 K g N . h a . - V r . - 1 . 60 cane (5 ,9) . N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y was also associated with roots of i.Pennisetum ipurpurem''1 andc.Cymbopogon. o i i r a t u s (9) . In examining the rhizosphere of a number of t rop ica l plants for nitrogen f i x i n g a c t i v i t y , Dobereiner (4) found large numbers of a species of Azotobaotev associated with Paspalum notatum var. "batatais" in B r a z i l . Recent work (8) has shown that th is assoc iat ion may f i x up to 93 KgN.ha" xyr.~ 1 . Dobereiner's work is in teres t ing for a number of reasons. The species of Azotobaotev is separate from the A. ohvooooocxumyivinelandvi group. The c e l l form and colony type are d i f f e r e n t (4) as are the CG% values of itssDNA (6). It is found associated exc lus ive ly with broad-leaved, te t rap lo id (8) v a r i e t i e s of P. notatum, these being known as "Common Bahia Grass" or "batatais" (6). This grass, which is a poor pasture grass (6) , invades the sward in large areas of extremely poor la toso ls in South Eastern and Central Braz i l (7). The bacterium has also been i so la ted from P. notatum var. "batatais" in F l o r i d a , southern Braz i l and at the Amazonian mouth in Belem, Para ( B r a z i l ) . Only sporadic occurrence of the bacterium with narrow leaved, d i p l o i d v a r i e t i e s (which are better pasture grasses) of P. notatum such as "pensacola bahia" and with P. plicatulum, P. dilatatum and P. virgatum has been observed (6). It was never found in 200 root s o i l samples of 31 other species of Graminae, 8 species of 61 Leguminosae and several other un ident i f ied p lants . In view of i t s exclusive assoc iat ion with P. notatum var " b a t a t a i s , " Dobereiner has named the new bacterium Azotobaoter paspali (4). In c u l t u r e , rapid growth of A. paspali on N-free media was r e s t r i c t e d to a pH range of 6.7 to 7.0 although i t occurred abundantly in the rhizosphere of P. notatum in s o i l s at pH 4.9 to 7.8 (6). There is normally a higher pH on the root surface of P. notatum than in the surrounding s o i l (4) . The assoc iat ion has proved d i f f i c u l t to estab l i sh in the laboratory . Dobereiner (6) found that the bacterium did not develop well in the rhizosphere in s t e r i l e sand and she was unable to measure nitrogen f i x a t i o n by the a s s o c i a t i o n . Kass et al. (16) inoculated plants of P. notatum var "batata is" growing in i n i t i a l l y s t e r i l e sand, with A. paspali, 4 and 12 weeks after p lant ing . At 16 weeks A. paspali was i so la ted from nhizospheres in only two out of s ix jars (25 plants per j a r ) . Consistent establishment of A. paspali did occur in those jars where glucose (41 mg per ja r ) was added at the f i r s t inocu la t ion . The nitrogen gain associated with A. paspali establishment was quite smal l , about 0.5 mgN per jar or 0.6 ppm. Increases in nitrogen of the same magnitude were also observed in jars containing plants alone, and in jars containing neither plants nor added bacter ia . In plants from jars in which A. paspali had become estab l i shed , s i g n i f i c a n t increases in nitrogen were observed in the roots but not in the shoots, and 62 i t was suggested that the higher levels of root nitrogen resulted from an accumulation of A. paspali on the root surface rather than an uptake of f ixed nitrogen. Rates of C 2 H 2 reduction by s o i l samples containing the P. notatum-A. paspali assoc iat ion were very smal l : 67 and 59 pmole. h r . _ 1 (g. s o i l ) - 1 . In pure c u l t u r e , 10 8 c e l l s of A. paspali reduced 1.29 nmoles C 2 H 2 . h r - 1 . The low rates are possibly due to the s o i l s being incubated in 0.21 atm 0 2 . It was concluded that the assoc iat ion probably f ixed less than 10 KgN.ha" x yr .~ 1 . (Cff„ estimate of 93 KgN. h a . ~ x y r . " 1 obtained by Dobereiner et al. (8) .) The assoc iat ion has been establ ished in f i e l d experiments but only af ter about a year. S i g n i f i c a n t numbers of A. paspali were found in the rhizosphere of P. notatum var "batatais" a f ter 10 months fol lowing a) t ransp latat ion of P. notatum from aolong establ ished lawn into s o i l free of A. paspali and b) transplantat ion of P. notatum with or without A. paspali into s o i l containing A. paspali (7). The N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y associated with the roots is i n d i r e c t l y re lated to photosynthesis. Shading a lawn of P. notatum var "batatais" establ ished for many years resulted in a decrease in the number of micro-colonies of A. paspali i so lated from the rhizosphere (7). The N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of an in tact shoot- root -so i l system almost ceased af ter 45 hours in darkness. When the system was returned to the l i g h t , a c t i v i t y started qu ick ly , returning almost to the o r ig ina l rates after 15 hours (7). 63 Ce l l s of A. paspali appear to be concentrated on the root surface. Unwashed roots and rhizomes had only a s l i g h t l y higher N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y than when they were washed; in another experiment vigorous washing under a strong j e t of tapwater removed only half the a c t i v i t y (8). Light microscopy of f i e l d grown roots (8) showed a prominent mucigel layer associated with colonies of b a c t e r i a . The mucigel layer may prevent the bacter ia from being washed off and also help provide a su i tab le gaseous environment (see below) for nitrogen f i x a t i o n (8). Experiments with detached roots and rhizomes (8) showed that maximum rates of C 2 H 2 reduction occurred at a p0 2 of about 0.04 atm. In contrast to roots freed from s o i l , the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of intact r o o t - s o i l systems was not inh ib i ted by an external p0 2 of 0.20 atm. This indicates that at the s i tes of C 2 H 2 reduction within the r o o t - s o i l system there was a favourable gaseous environment. As a resu l t of the work of Postgate and others ( e . g . 21,922? 23?231)3j i t has become c lear that the p0 2 can be a very important factor in nitrogen f i x a t i o n by the Azotobacteraceae. Nitrogen f i x a t i o n is a reductive process, and the nitrogenase enzyme is severely inh ib i ted by oxygen (23). Extensive inves- t igat ions of the physiology of the Azotobacteraceae have shown that in v ivo , the nitrogenase is probably protected from oxygen in two ways (21): 64 1. r e s p i r a t o r y p r o t e c t i o n : a s s u m i n g t h a t s u b s t r a t e s a r e n o t l i m i t i n g , r e s p i r a t i o n a c t s t o keep t h e o x y g e n c o n c e n - t r a t i o n w i t h i n t h e o r g a n i s m v e r y l o w . T h i s i s r e f l e c t e d i n t h e h i g h Q o 2 and m a i n t e n a n c e c o e f f i c i e n t s c h a r a c t e r i s t i c o f t h e A z o t o b a c t e r a c e a e ( 2 1 ) a r i s i n g f r o m d i v e r s i o n o f c a r b o n compounds f r o m b i o s y n t h e s i s t o r e s p i r a t o r y p r o t e c t i o n ( 2 1 ) . 2. c o n f o r m a t i o n p r o t e c t i o n : when r e s p i r a t i o n i s n o t a d e q u a t e t o p r o v i d e a low p 0 2 w i t h i n t h e c e l l , t h e n i t r o g e n a s e enzyme a s sumes a c o n f o r m a t i o n i n w h i c h i t i s i n s e n s i t i v e t o o x y g e n . In t h i s c o n f o r m a t i o n i t i s n o t a b l e t o f i x n i t r o g e n . The c o n f o r m a t i o n can q u i c k l y c h a n g e t o t h e d i n i t r o g e n f i x i n g ( and o x y g e n s e n s i t i v e ) s t a t e . D r o z d and P o s t g a t e ( I I ) we re a b l e t o show a " s w i t c h o n - s w i t c h o f f " e f f e c t by v a r y i n g t h e o x y g e n s u p p l y t o a c o n t i n u o u s c u l t u r e o f A. chvoococoum. Work with c e l l - f r e e preparations of nitrogenase has shown that i t is a complex composed of two proteins (23). One has a r e l a t i v e l y high molecular weight (200,000-300,000) and contains i r o n , molybedenum (10 or 20:1) and sulphur; th i s protein is r e l a t i v e l y insens i t i ve to oxygen. The other is smal ler , has less iron and sulphur and no molybdenum. It is i r r e v e r s i b l y damaged by oxygen. The ideas that have developed about the e f fec t of oxygen on nitrogenase, and the protect ive mechanisms, have enabled comments to be made about the nut r i t iona l status of 65 cultures of Azotobaoter spp. With chemostat cultures of A. ohrooooooum and A. v i n e l a n d i i , Dalton and Postgate (3) found that , in media containing an excess of all soluble nutr ients and free dissolved oxygen, the bacter ia l density was inversely re lated to the d i l u t i o n rate (see Figure 1) . They explained the shape of the curve by proposing that the y i e l d was l imited by a gaseous component of the environment. It was concluded that the gas was d in i t rogen , and that the organism was i n t r i n - s i c a l l y l i m i t i n g the rate at which i t f ixed n i t rogen. This i n t r i n s i c l i m i t a t i o n was considered to be part of the resp i ratory protect ion mechanism; i t seems that at a given p0 2 only as much of the nitrogenase that can be protected by resp i ra t ion i s in the oxygen s e n s i t i v e , d in i trogen f i x i n g state (21). (Presumably, the probab i l i ty of any one molecule being in the dinitrogen f i x i n g conformation is the same throughout the population of nitrogenase molecules.) Such a population is said to be " N 2 - l i m i t e d . " A non-nitrogen f i x i n g populat ion, l imited by the supply of, for example, Nh\ + , is said to be "N- l imi ted . " So-cal led "ordinary" laboratory cultures are N 2 - l i m i t e d ; in the f i e l d organisms are l i k e l y to be carbon l imited (21). A carbon l imited (C- l imited) culture may be defined operat ional ly as one in which the organism concentration is halved i f the carbon concentration in the inflowing medium is halved, and the organism concentration is not a l tered when 66 Dilution rate (hr" 1) Population densities, contents of fixed N and growth rates of Azotobacter chroococcum m continuous culture, o: mg dry wt. organisms/ml culture; • mg N/ml culture A theoretical curve for an ideal, 'maintenance'-free, carbon-limited chemostat culture of Mmax - 0.31 h _ 1 is dotted in (•--). Figure Growth curve for A. chvoococoum. an N 2 - Ta ken l imi ted cul ture of from Postgate (21 ) . 67 the concentration of a l l other medium components is doubled (3). C - l im i ted , nitrogen f i x i n g cultures are d i f f i c u l t to estab l i sh as they are hypersensit ive to oxygen (3); resp i ra t ion is not adequate to protect the nitrogenase. Phosphorus l imited (P- l imited) cultures are defined in a s imi la r manner (3). They are also hypersensit ive to oxygen when f i x i n g nitrogen (3). There is evidence to suggest that resp i ra t ion in A. ohroococcum i s contro l led by the ATP/ADP ra t io (28). On the basis of this Dalton and Postgate (2) proposed that i f resp i ra t ion were suddenly increased to overcome an increase in p0 2 a l l the ava i lab le ADP would be converted to ATP. The ATP/ADP r a t i o would then be sh i f ted to favour decreased r e s p i r a t i o n , so negating the resp i ratory protect ion . However there is also evidence for a forked e lectron transport pathway in Azotobaoter (23). One path is associated with resp i ratory phosphorylat ion. In the other, e lectrons are t ransferred from substrates to oxygen without, or with l i m i t e d , phosphorylation. Under condit ions of high oxygen, the non- phosphorylating pathway could use up the oxygen without excessive generation of ATP (23). If this is the case, there seems to be no reason why P-l imited organisms should be unduly hyper- sens i t i ve to high oxygen. There is a need for further c l a r i f i c a t i o n of the problem. Ni t rogen-f ix ing cultures may also be 0 2 - l i m i t e d . In these the biomass is inversely related to the d i l u t i o n rate , 68 due to the increased synthesis of polyhydroxybutyrate and polysaccharide at the lower d i l u t i o n rates (17). When the oxygen supply was increased, 0 2 - l i m i t e d cultures were able to increase the i r resp i ra t ion to keep the level of d issolved oxygen low, and became N 2 - l im i ted cultures (17). The Azotobacteraceae c h a r a c t e r i s t i c a l l y form a lo t of slime (2) . Polysaccharide formation is typ ica l of most types of N-limited bacter ia (21) and Postgate suggested that the slime of Azotobaoter spp was an expression of the i r usual nut r i t i ona l s tatus , one of N 2 - l i m i t a t i o n (21). Ge l , such as the s l ime, has a high oxygen d i f f u s i o n res i s tance . Consequently the slime may have survival value in l im i t ing the d i f f u s i o n of oxygen to c e l l s . It would be of p a r t i c u l a r advantage to C- l imited populations (21). The mucigel layer which is present on the roots of P. notatum var "batatais" is possibly involved in preventing rapid e q u i l i b r a t i o n of so i l oxygen at the s i tes of nitrogen f i x a t i o n . Both the plant and A. paspali may contr ibute to the layer . Using axenic and non-axenic plants Greaves and Darbyshire (14) found that the mucilaginous layer on plant roots was derived both from micro-organisms and the p lant . When the work reported in this thesis was s ta r ted , l i t t l e was known about the physiology of the re la t ionsh ip between A. paspali and P. notatum. In view of the importance 6 9 of carbohydrate and oxygen concentration to nitrogen f i x a t i o n by Azotobaotev spp i t was decided to study root exudation under d i f fe rent root oxygen regimes. As the work continued, the emphasis of my experiments eventual ly sh i f ted away from exudation to a study of the buffer ing capacity of P.nnotatum roots and of the e f fect of pH and oxygen concentration on the growth of A. paspali in c u l t u r e . For two organisms such as these i t is obviously preferable to study them when they are assoc iated. However, in view of the d i f f i c u l t y of estab l i sh ing the assoc iat ion in the laboratory i t was necessary to grow and study the organisms separate ly . It was also decided to work with non-s ter i l e plants of P. notatum. The reason was a pragmatic one in that , as they were not already a v a i l a b l e , i t was f e l t to be too com- p l i cated to bui ld f a c i l i t i e s for gnotobiot ic culture of the large plants used here. There are some advantages in using non-s ter i le plants in that s t e r i l e plants are in the nature of an experimental a r t i f a c t , plants in the f i e l d always funct ion- ing in the presence of mi crosoijgann" sms. The mai n-,disadvantage, which admittedly outweighs many of the advantages, is that the presence of micro-organisms makes i t p r a c t i c a l l y impossible to study root exudation unaltered by microbial a c t i v i t y . 70 This t h e s i s , then, is concerned with some aspects the physiology of the re la t ionsh ip between -A. paspali and notatum var. "batata i s . " L I T E R A T U R E C I T E D Abd-El-Malek, Y. 1971. F r e e - l i v i n g n i t rogen- f ix ing bacter ia in Egyptian s o i l s and the i r possible contr ibut ion to s o i l f e r t i l i t y . Plant and So i l Spec. Vol:423- 442. Dalton, H. and J .R. Postgate. 1 969. Ef fect of oxygen on growth of Azotobaoter chroococoum in batch and continuous cu l tures . J . Gen. M i c r o b i o l . 54:463-473. Dalton, H. and J .R. Postgate. 1969. Growth and physiology of Azotobaoter ohrooooooum in continuous c u l t u r e . J . Gen. M ic rob io l . 56:307-319. Dobereiner, Johanna. 1966. Azotobaoter paspali sp .n . Uma bacteVia f ixadora de nitrogenio na r i z o s f e r a de Paspalum. Pesq. Agropec. Bras. 1:357-365. P~ ;-.v.v ... • . . ^ 1 ; : " : : . 1 968. Non-symbiotic nitrogen f i x a t i o n in t rop ica l s o i l s . Pesq. Agropec. Bras. 3:1-6. b r c b c " . 1 970. Further research on Azotobaoter paspali and i t s var iety s p e c i f i c occurrence in the rhizosphere of Paspalum notatum Flu'gge. Zent ra lb la t t fur Bakter io log ie , Parasitenkunde (Abt2) 124:224-230. Dobereiner, J . and A.B. Campelo. 1971. Non-symbiotic nitrogen f i x i n g bacter ia in t rop ica l s o i l s . Plant and Soi l Spec. V o l . , 457-470. Dobereiner, Johanna, J .M. Day and P . J . Dart. 1972. Ni t ro- genase a c t i v i t y and oxygen s e n s i t i v i t y of the Paspalum notatum-Azotobaoter paspali a s s o c i a t i o n . J . Gen. M i c r o b i o l . 71:103-116. 7-r 72 9. Dobereiner, Johanna, J .M. Day and P .J . Dart: 1972. Nitrogenase a c t i v i t y in the rhizosphere of sugar cane and some other t rop ica l grasses. Plant and S o i l . 37:191-196. 10. Dommergues, Y . , J . Balandreau, G. Rinaudo and P i e r r e t t e Weinhard. 1973. Non-symbiotic nitrogen f i x a t i o n in the rhizospheres of r i c e , maize and d i f f e r e n t t rop ica l grasses. Soi l B i o l . Biochem. 5:83-89. 11. Drozd, J . and J .R . Postgate. 1970. Effects of oxygen on acetylene reduct ion, cytochrome content and resp i ratory a c t i v i t y of Azotobaotev ehvobeoooum, J . Gen. M i c r o b i o l . 63:63-73. 12. Evans, Harold J . , N.E.R. Campbell and Susan H i l l . , 1972. Asymbiotic n i t rogen- f ix ing bacter ia from the surfaces of nodules and roots of legummes. Can. J . Microbiol . 18:13-21 . 13. Fehr, P . I . , P.C. Pang, R.A. Hedlin and C M . Cho. 1972. Some factors a f fec t ing asymbiotic nitrogen f i x a t i o n in s o i l s as measured by 1 5 N enrichment. Agron. J . 64:251-254. 14. Greaves, M.P. and J . F . Darbyshire. 1972. The u l t r a - structure of the mucilaginous layer on plant roots : Soi l B i o l . Biochem. 4:443-449. 15. H a r r i s , D. and P .J . Dart. 1973. Nitrogenase a c t i v i t y in the rhizosphere of Stachys s y l v a t i c a and some other dicotyledenous p lants . Soi l B i o l . Biochem. 5:277- 279 . 16. Kass, Donald L. , Matthew Drosdoff and Martin Alexander. 1971. Nitrogen f i x a t i o n by Azotobaotev paspali in assoc iat ion with Bahiagrass {Paspalum notatum) S o i l . S c i . Soc. Amer. Proc. 35:286-289. 17. Lees, H. and J .R . Postgate. 1973. The behaviour of Azotobaotev ohvooooooum in oxygen - and phosphate - l imited c u l t u r e . J . Gen. M i c r o b i o l . 75:161-166. 73 18. L ine , M.A. and M.W. L o u t i t . 1973. Studies on non-symbiotic nitrogen f i x a t i o n in New Zealand tussock-grassland s o i l s . N .Z . J . Agr i c . Res. 16:87-94. 19. Mishust in , E.N. and V.K. S h i l ' n i k o v a . 1971. B io log ica l f i x a t i o n of atmospheric n i t rogen. Macmillan Press L t d . , London, pp. 184-250. 20. Nutman, P.S. 1971. Perspectives in b io log i ca l nitrogen f i x a t i o n . S c i . Prog. Oxf. 59:55-74. 21. Postgate, John. 1971. F ixat ion by f r e e - l i v i n g microbes: physiology. In: J .R . Postgate, e d . , the chemistry and biochemistry of nitrogen f i x a t i o n . Plenum Press, London, pp. 161-190. 22. -?4r6 . 1971. Relevant aspects of the physio- log i ca l chemistry of nitrogen f i x a t i o n . In: D.E. Hughes and A.H. Rose, eds . ; Microbes and b i o l o g i c a l p roduct iv i ty , 21st Symp. Soc. Gen. M i c r o b i o l . , Univers i ty Press, Cambridge, England, pp. 287-308. 23. . 1 972. B io log ica l nitrogen f i x a t i o n . Merrow Publishing Co. , L t d . , England. 24. Raju, P .N. , Harold J . Evans and Ramon J . S e i d l e r . 1972. An asymbiotic n i t rogen- f ix ing bacterium from the root environment of corn. Proc. Nat l . Acad. S c i . USS.A. 69:3474-3478. 25. Rovira, A.D. and Barbara M. McDougall. 1967. Microbio log ica l and bi and biochemical aspects of the rhizosphere. In: A. Douglas McLaren and George H. Peterson, eds . , Soi l Biochemistry, Marcel Dekker, I n c . , New York, pp. 417-463. 26. Rovira, A.D. 1965. Ef fects of Azotobaotev, Bacillus and Clostvidium on the growth of wheat. In: Plant Microbes Relat ionship , Prague, Czechosl . Acad. S c i . , 193-200 (in Eng l i sh) . 27. Vlassak, K., E.A. Paul and R.E. H a r r i s . 1973. Assessment of b io log i ca l nitrogen f i x a t i o n in grassland and associated s o i l s . Plant and S o i l . 38:637-649. 74 28. Yates, M.G. 1970. Control of resp i ra t ion and nitrogen f i x a t i o n by oxygen and adenine nucleotides in N 2 - grown Azotobaoter ohrooooooum. J . Gen. M i c r o b i o l . 60:393-401. 29. Yoshida, T. and Ancajas, R.R. 1970. Nitrogen f i x a t i o n by bacter ia in the root zone of r i c e . Proc. So i l S c i . Am. 35:1 56-ili57. 30. Yoshida, Tomio and Rosabel R. Ancajas. 1973. The f i x a t i o n of atmospheric nitrogen in the r i c e rhizosphere. Soi l B i o l . Biochem. 5:153-156. 31. Nagai, S . , Y. Nishizawa, M. Onodera and S. A iba . 1971. Ef fect of d issolved oxygen on growth y i e l d and aldolase a c t i v i t y in chemostat culture of Azotobaoter v i n e l a n d i i . J . Gen. M i c r o b i o l . 66: 197-203. Chapter 1 ROOT EXUDATION BY PASPALUM NOTATUM Introduction Paspalum notatum var "batata is" and A. paspali are in close associat ion with one another in the f i e l d (11) and therefore i t is reasonable to assume that A. paspali makes use of organic compounds exuded from the roots of the grass; i t is also possible that sloughed off c e l l u l a r material is a source of nut r ients . In the f i e l d the carbohydrate supply to A. paspali may usual ly be high enough for the bacter ia to be N 2 - l i m i t e d . If the carbohydrate supply were low, then the bacter ia might be C - l i m i t e d , in which case nitrogen f i x a t i o n would be prone to oxygen i n h i b i t i o n . As the carbon and oxygen supplies are of cruc ia l importance to nitrogen f i x a t i o n i t was of in teres t to examine root exudation at d i f f e r e n t root oxygen regimes. Since the oxygen environment of the roots could a f fec t leaf funct ion (16) a prel iminary invest igat ion was made of the response of photosynthesis to the root oxygen regime. 7-5 76 In the course of th is i n v e s t i g a t i o n , i t was unexpectedly found that root anoxia had no e f fect on photosynthesis. Consequently the oxygen treatments were combined with low temperature t reat - ments to demonstrate that i n h i b i t i o n of root function would be re f lec ted in C0 2 uptake measurements. Photosynthesis was measured in .an open c i r c u i t apparatus, which was also used in the exudation experiments. The amounts of material exuded by plants within convenient experimental time periods are minute but can be estimated and analyzed by rad io- t racer techniques. In th is study 1'*C0 2 was fed to the leaves of the plant and the lhC in the roots and root medium was measured and analyzed. A s imi la r approach has been used by several other workers (23,24,26,27,28). Seedlings have often been used to study root exudation (e .g . 23,24,26,28) but i t was decided to use adult p lants , p a r t i c u l a r l y as f i e l d and laboratory experiments (10,11) involv ing the occurrence and establishment of A. paspali have been done using adult p lants . A number of experiments were done in which exudation occurred but in further experiments, exudation occurred at very low leve ls for no apparent reason. The p0 2 seemed not to be re lated to the amount of exudation, so an attempt was made to determine at least some of the other factors c o n t r o l l i n g exudation. This caused the emphasis of the work to move away from the ef fects of d i f fe rent root oxygen regimes. 77 A study was made of the ro le of young nodal roots in exudation, and of the e f fect of the pH of the root medium, ethanol and the composition of the root medium. These l a s t three factors were studied for the i r e f fec t on root permeabi l i ty . Ethanol was chosen as data (32) indicated that ethanol would increase the permeabil ity of the root c e l l membranes. The pH of the root medium proved to be important (although not, apparently, with respect to permeabi l i ty) ; th is is discussed further below and in the next chapter. Materials and Methods Germi nation Seeds of P. notatum Fltigge var "batatais" are d i f f i c u l t to germinate. Two methods of germination were used [1] . Seeds were placed in sulphuric acid (S.G. 1.84) for 15 minutes, then washed thoroughly with d i s t i l l e d water, care being taken not to l e t the temperature r i s e (9). [2] The hul l (lemma and palea) was removed using sharp-pointed forceps (1) . The s c a r i f i e d seeds or seeds without the hull were then placed in s t e r i l e sand in an incubator at 35°C, with d i s t i l l e d water being applied every day because germination is inh ib i ted by the presence of inorganic ions (9). The germination percentage was very low. In l a te r work, due to the d i f f i c u l t y of ger- minating seed, plants were obtained by c lon ing . 78 Growth condit ions Young seedlings were transferred to pots of sand in„ a growth room at 9 x 101* ergs . cm. — 2 sec . - 1 , 16 hour day and 30°/30°C day/night temperature. Light was provided by 400W Deluxe White GE mercury f luorescent lamps. The plants were watered every day, and Hoagland's so lut ion (12) was given twice weekly. Plants were used in experiments when 4 to 5 t i l l e r s were present; plants grown from seed were about 3 months old at th is stage. Experimental apparatus Figure 1 is a schematic diagram of the apparatus used. It was designed with three main functions in view: [1] monitoring photosynthesis, [2] c o n t r o l l i n g the root oxygen regime and [3] monitoring root exudation and c o l l e c t i n g the exudate at the end of the experiment. The plant was placed in the system as shown in Figure 1. The. root medium was c i r c u l a t e d between the root chamber (P) and the mixing tower (X). The d i rec t ion of flow of medium in the tower was opposite to that of the root gas. After leaving the mixing tower, the root gas passed into the root chamber via the overflow l ine (Z) and then into the shoot chamber (C). The level of medium in the root chamber was maintained by use of the valve ( H c ) . The root medium c i r c u l a t i n g system enabled the oxygen regime to be rep l i cated from day to day. 79 Figure 1 . Schematic diagram of open c i r c u i t apparatus used to study photosynthesis and root exudation. 80 Key to Figure 1 A - 1 amps B - h e a t sh ie ld C - p lex ig las chamber D - shoot gas input E - pump F - f1ow gauge G - shoot gas output H - valves: H a _ i n g o i n g chamber gas H - outgoing chamber gas H c - intake for foot medium - drain for root medium I - water vapour trap J - in f ra- red gas analyzer K - s t r i p chart recorder L - cooling c o i l M - fan N - heating element 0 - leaf temperature probe f - root chamber Q - root temperature probe R - telethermometer S - temperature contro l led water bath T - pH electrode U - pump c i r c u l a t i n g root medium V - sample port W - cooling jacket X - mixing tower Y - oxygen electrode Z - overflow l ine from mixing tower a - root gas input The connecting tubes of the root medium c i r c u l a t i n g system are represented by s o l i d black l i n e s . The d i rec t ion of medium flow is shown by the s o l i d arrows. The d i rec t ion of flow of the root gas is shown by the dotted arrows. The small arrow-heads show the d i rect ion of flow of the shoot gas. The arrowhead jus t above the root-shoot junct ion indicates that , at th is po int , the root and shoot gases merge. c?3A 8̂1 With the gas for the roots flowing into the apparatus at ' a ' a gas mixture of 0.99 atm N 2 , 0.01 atm C0 2 d i lu ted with N 2 or 0.98 atm N 2 , 0.02 atm 0 2 was l e t into the shoot chamber via the path D * E -> F -> H to give an ingoing C0 2 concentration of 450-500 ppm, as measured by the in f ra - red gas analyzer ( J ) . The temperature of the root medium was contro l led by a water bath (S) and a l s o , when necessary, by cool ing jackets (W) around the pump (U) and mixing tower. Shoot chamber tempera- ture was adjusted using the cool ing c o i l (M) and heating element (N) so that the temperature shown on the telethermometer (R) connected to the leaf temperature probe (0) was 30°C. Three 300W Coolbeam GE lamps provided an average l i g h t in tens i ty of 3.8 x 10 5 e r g s . c m . - 2 s e c . - 1 at the face of the shoot chamber. An oxygen electrode (Y) was used to determine the concentration of dissolved oxygen in the root medium because the p0 2 of the so lut ion w i l l not be equal to the p0 2 of the gas supply. Table 1 gives the oxygen solut ion ra tes , determined by the method of Cooper et al. (6) , corresponding to the p0 2 of the root gas supply. It shows that the nitrogen used to provide oxygen-free condit ions contained traces of oxygen below the s e n s i t i v i t y of the oxygen e lectrode. 82 Table 1 Oxygen Solution Rates (mmole 1 - 1 h r - 1 ) Corresponding to p0 2 of Root Gas Supply. Gas Flow Rate = 1;2 l . m i n - 1 p0 2 (atm) 0.0(N 2 ) 0.02 0.04 0.20 Solut ion rate 0.9* 2.3 5.0 15.3 This value is at the l i m i t of detection In the open c i r c u i t system described above the rate of plant photosynthesis is given by PPS = C i F i - C 2 ( F i + F 2 ) where PPS = rate of plant photosynthesis Ci = ingoing C0 2 concentration C 2 = outgoing C0 2 concentration Fi = rate of flow of ingoing shoot gas F 2 = rate of flow of ingoing root gas Photosynthesis under d i f fe rent root oxygen regimes Photosynthesis was monitored for up to 8 hours when the p0 2 in the ingoing root gas was 0.20 atm, 0.02 atm and 0.01 atm. Nitrogen was the otherccomponent of the root gas. Hoagland's so lut ion (12) was used as root medium. The root temperature was varied between 30°C and 5°C. The p a r t i c u l a r sequences and combinations of p0 2 and temperature to which the roots were subjected are indicated in the Results sec t ion . 83 1 ' t C 0 2 feedings and root exudation The design of the open c i r c u i t apparatus was such that part of any v o l a t i l e exudate was los t due to ag i tat ion of the root medium. In order to estimate the degree of l o s s , the apparatus was set up without a p lant . With the medium in the root c i r c u l a t o r y system at pH 6.8, 2 uc of N a H ^ C C ^ ; were injected into the medium, and samples were taken every 15 minutes for 9 0 minutes to determine the a c t i v i t y remaining in s o l u t i o n . The pH was then lowered to 3 . 9 and samples taken for a further 90 minutes. For experiments on root exudation, the apparatus was set up with the plant in the chamber. I n i t i a l l y Hoagland's so lut ion ( 1 2 ) was used as root medium; la ter 0 . 5 mM CaSOi t was used (see Resu l ts ) . The temperature of the medium was adjusted to 3 0 ° C ; the gas regimes are given in the r e s u l t s . Photosynthesis was allowed to come to steady s tate , a f ter which the shoot chamber was i so la ted from i t s gas supply and 50 uc of l l f C 0 2 were fed to the shoot. After f i v e minutes, unfixed l l f C 0 2 was f lushed from the chamber, and the chamber was reconnected to the gas supply. At hourly i n t e r v a l s , 1 ml of root medium was taken at the sample port (V) and placed in a s c i n t i l l a t i o n v ia l with approximately 1 5 ml of Aquasol l i q u i d s c i n t i l l a t i o n f l u i d . In some experiments a further 1 ml of root f l u i d was 84 a c i d i f i e d with approximately 0.05M HC1 and bubbled with nitrogen for 15 minutes before the Aquasol was added. At the end of the experiment, normally eight hours af ter feeding the 1 I t C 0 2 , the root medium was co l lec ted and ethanol was added to make the f i n a l ethanol concentration 20%. The medium was stored at 5°C unt i l analyzed. In la ter experiments the medium was treated to estimate the s ize of the v o l a t i l e f r a c t i o n before adding the ethanol . It was a c i d i f i e d with 2M HC1 and bubbled with nitrogen for 20 minutes, the carbon dioxide being trapped in 10 ml of IM NaOH. Then, 5 ml of 0.1M Na 2 C0 3 was added to the NaOH as a c a r r i e r and 5 ml of 0.1M BaCl 2 was added to p rec ip i ta te BaC0 3 . A l iquots (3.5 ml) of the BaC03 suspension were counted as a gel in Aquasol 1iquid sc int i11at ion f l u i d . The roots were washed twice, cut from the shoot and both shoots and roots were k i l l e d in l i q u i d n i t rogen. Shoots and roots were f reeze-dr ied and stored at -15°C. A comparison of the d i s t r i b u t i o n of l i f C in young nodal roots and the main root mass was made in two ways: [1] roots were blotted dry and placed between two layers of Saran-wrap (Dow Chemical C o . ) , and then against a sheet of Kodak No-Screen Medical NS54T X-ray f i l m . The f i l m was stored at -15°C and developed after three days to obtain an auto- radiograph. [2] short lengths of roots from the main root mass and yoting nodal roots were placed in l i q u i d s c i n t i l l a t i o n 85 v i a l s with Aquasol and counted. Correct ion for quenching was made using the channels rat io method (30). In order to study the e f fect of lowppH on exudation, the pH of the root medium was lowered to 4 .0 , four hours af ter feeding 1 4 C 0 2 to the shoot. The pH was kept at 4.0 ± 0.2 by the addit ion of d i lu te HC1 for the remainder of the experiment. 1 ; • e•• The ef fects of ethanol were studied by placing plants in the open c i r c u i t apparatus and four hours af ter feeding 1 I f C 0 2 to the shoot, exposing the roots to ethanol . The root medium was bubbled throughout with nitrogen conta in- ing 0.20 atm 0 2 ; in order to ensure that the gas supply to the roots was saturated with ethanol , the gas was f i r s t passed through ethanol solut ions of the appropriate concentrati ons. In the f i r s t experiment, the ethanol concentration in the root medium was brought to 2.5% at four hours, and increased through 5% and 7.5% to 10% at hourly i n t e r v a l s . In the second experiment, the ethanol concentration was brought to 7.5% at four hours. At 5.5 hours the root was removed, and replaced with medium without ethanol . The amount of lkC in hourly, 1 ml samples of the root medium was taken as a guide to the degree of root exudation in both experiments. 86 Analysis of root t issue and exudate. Roots from each plant were divided into two samples, and extracted in 75 ml of b o i l i n g solvent for one hour. The extract ion solvent contained 95% v/v ethanol: water: 90% v/v formic acid (33: 7: 2) (2) . The extract was separated from insoluble material by f i l t r a t i o n through Whatman No. 1 f i l t e r paper. The residue was washed with 3 x 5 ml lots of extract ion so lvent , 2 x 5 ml lo ts of 95% v/v ethanol and 2 x 5 ml lots of water. The washings and extract were transferred to a round-bottomed f lask and evaporated to dryness in vacuo at 50°C. Residual formic acid was removed by passing a stream of nitrogen gas over the dried ext ract . Pr ior to analys is the dried extract was taken up in 1 to 2 ml of water. The extracts were f ract ionated on cation (Dowex-50W, 50 x 8 - 400) and anion (Dowex - 1 , 1 x 8 - 400) exchange r e s i n s . Preparation of the resins and f rac t ionat ion of the extract 87 followed the procedure of Atkins and Canvin (2) except that only three f ract ions were obtained: the neutral f r a c t i o n (eluted with water), t h e b a s i c f r a c t i o n (eluted with 2M N H i f O H ) and the ac id i c f r a c t i o n (eluted with 2M HC1 ). The r a d i o a c t i v i t y of the f ract ions was determined by l i q u i d s c i n t i l l a t i o n counting. Amino acids in the basic f rac t ion were separated by two-dimensional thin layer chromotography. Samples were applied to plates coated with Avicel (19 x 19 cm, 250y thick layer) and developed in phenol ( l i q u e f i e d 90% v/v): ammonium hydroxide: water (178:0.6:21.4) . After drying for 18 hours the plates were developed in n-propanol: ethyl acetate: water (140:20:60) at r ight angles to the f i r s t solvent (4) . Sugars in the neutral f r a c t i o n were separated by one-dimensional descending chromatography on 46 x 57 cm Whatman No. 1 paper with ethyl acetate: acet ic a c i d : water (14:3:3) for 30 hours (2) . Organic acids in the acid f rac t ion were separated by two-dimensional paper chromatography. Samples were appl ied to 20 x 20 cm Whatman No. 3MM paper and developed in ethanol: water: ammonium hydroxide (140:52:8). After drying for 12 hours the papers were run in ethyl acetate: acet ic ac id : water: sodium acetate (100:56:50:240 mg sodium acetate) (22). Auto- radiography was used to locate rad ioact ive compounds. Spots were i d e n t i f i e d from standard maps. Most of the 1'*C in the medium from the root chamber was v o l a t i l e , and i t proved to be d i f f i c u l t to obtain 88 q u a l i t a t i v e and quant i tat ive i d e n t i f i c a t i o n of the non-vo lat i le f r a c t i o n . The fol lowing procedure gave the best recovery of 1 I f C in the non-vo lat i le f r a c t i o n . The volume of f l u i d co l lec ted from the root chamber was about 300 ml; th is was evaporated in vacuo at 40°C leaving a residue cons ist ing part ly of a white deposit (presumably CaSO^). The residue was taken up in 10 ml of 70% v/v ethanol and f i l t e r e d through 1 g of c e l i t e to remove the white deposit . The c e l i t e was washed with 2 x 10 ml lots of 70% v/v ethanol and 10 ml of acetone. After being quant i ta t ive ly t ransferred to a round-bottomed f l a s k , the f i l t r a t e was evaporated in vacuo at 40°C. The dried extract was taken up in 1 to 2 ml of water and f r a c - tionated using ion-exchange resins as described prev ious ly . Attempted to chromatograph the various f rac t ions were not successful because of inadequate amounts of l l f C - Results Photosynthesis under d i f f e r e n t root oxygen regimes Table 2 compares the photosynthetic rates obtained at d i f fe rent p a r t i a l pressures of oxygen in the root gas supply. 89 Table 2 Rates of Photosynthesis of P. notatum Under D i f ferent Root Oxygen Regimes P02(atm) 0(N 2 ) 0.02 0.04 0.20 mg C O 2 . h r . - 1 (g.dry w t ) ' 1 mg CO .hr.~ 1 p l a n t - 1 26.0 ± 5.1 50.7 ± 5.5 29.5 1 61 .4 ± 11.7 19.3 ± 5.8 43.9 ± 9.4 16.2 ± 3.0 57.7 ± 15.£ one determination only The rates r.va.ri edabetweentitKeatmentsrpebGi.t s photosynthesi s was not inh ib i ted by a low p0 2 around the roots . Figures 2 and 3 i l l u s t r a t e the e f fect of the temperature of the root medium. Photosynthesis became very low or stopped when the temperature was lowered to 5 ° C , or when the roots were immersed d i r e c t l y in medium at 5 ° C . Root exudation: Whether Hoagland's s o l u t i o n , pH 5.4, or 0.5 mM C a S O i j s o l u t i o n , pH 6.9, was used did not seem to have any e f fect on exudation. Once this knowedge was estab l i shed , CaSOi* was used as i t is the so lut ion of choice for studies of root exudation (13). 90 Figure 2. E f fect of low root temperature on photosynthesis. Roots at 5°C throughout. Roots supplied with ni trogen.  91 Figure 3. E f fect of low root temperature on photosynthesis. RoRoots cooled as indicated by arrows. Roots supplied with n i trogen.  92 The root system consisted mainly of branched, f ibrous nodal roots and the seminal roots . (No seminal roots were present, of course, in plants obtained by c l o n i n g ) . When young, the nodal roots were white, unbranched and of larger diameter than the o lder , branched, f ine roots which had turned a yel lowish colour . (New nodal root formation was stimulated by the presence of moist sand at the stem nodes.) The presence of several of the younger, unbranched roots was necessary to obtain detectable leve ls of exudation. Figures 4a and 4b are copies of autoradiography of the root system. Figure 4a shows young nodal roots , some of.. which, have developed la te ra l branches. Figure 4b shows the rest of the root system. It is c lear that the younger roots contain more l l f C than the older ones. Table 3 compares the 1 4 C content of samples of young nodal roots (as in Figure 4a) and of older ones. The mean a c t i v i t y of the young roots is s i g n i f i c a n t l y d i f f e r e n t (P < 0.01)ffromtfchatoof the older ones. Figure 5 shows the loss of l i f C from the root medium after in jec t ion of NaH l l *C0 3 . . The loss can be described by f i r s t order reaction k i n e t i c s . When the pH of the root medium was lowered to 3.9, the 1 J f C content dropped rap id ly to very low l e v e l s . A complex c a l c u l a t i o n would be required to correct for th is loss in estimates of the amount of l l f C exuded from the roots . However i t is c lear that graphs (see 93 Figure 4. A. Autoradiography of young nodal roots . The two lower roots have developed some l a t e r a l branches. The branches have a high 1 I f C content. B. Autoradiograph of the bulk of the root system (comprised of f i n e , branched nodal roots and the seminal r o o t s ) . The l l f C tends to be concentrated in the apices of the l a t e r a l branches of the older roots; the arrows point to some of the apices .   94 Table 3 Comparison of r a d i o a c t i v i t y in samples of young nodal roots and o lder , f i n e , branched roots . A c t i v i t y is dpm of l l f C per g. fresh wt. x 10 6 Young Roots Older Roots 1 .28 0.24 1 .46 0.12 0.64 0.14 Mean: 1.13 ± 0.43 0.17 ± 0.06 95 Figure 5. Graphs of the loss of 1 4 C from the root medium after i n j e c t i o n of NaH^COs • (No plant present) . When the percentage of l l f C remaining in so lut ion is plotted l i n e a r l y against time, i t shows an exponential decl ine (A). When the log of the percentage is plotted against time, a straight l i n e is obtained confirming that the loss of l l f C follows f i r s t order k inet ics (B). i i . i i— i i i i t, 1 2 3 4 5 6 7 8 9 TIME min x10 Figure 5 96 below) where the amount of 1 4 C in the root medium is p lotted against time underestimate the amount of l l f C exuded,from the roots . A representat ive graph of the accumulation of  lhC in the root medium with time is shown in Figure 6. The -̂ C was detected in the medium one hour af ter feeding l l f C 0 2 to the shoot. The amount increased with time although i t some- times decreased between s ix and eight hours. Curve A in f igure 6 i s . d e r i v e d from roots supplied with gas containing 0.15 atm 0 2 ; curve B is derived from roots supplied with 0.04 atm 0 2 . The maximum in curve A represents about 0.3% of the  lhC in soluble compounds of the root eight hours af ter feeding ^ C O ^ and the maximum in curve B represents about 1.4% of the soluble ^C of the root . Figure 7 shows the accumulation of  ll*C in the root medium as indicated by a c i d i f i e d and non-ac id i f ied samples. The maximum of the dotted l i n e (total a c t i v i t y in the root medium) represents about 2.5% of the  lkC in the soluble compounds of the root , and the s o l i d l i n e (non-vo lat i le l l f C ) represents about 0.3% of the soluble compounds of the root . Some estimates of the s ize of the v o l a t i l e f rac t ion of root exudates are in Table 4. Direct measurements of the s ize of the v o l a t i l e f rac t ion made by trapping C0 2 in NaOH (plants Y and Z, Table 4) gave values considerably lower than those 97 Figure 6. The accumulation of 1 I f C in the root medium with time. Each curve i s derived from a separate plant Curve A: Roots supplied with 0.15 atm 0 2 . B: Roots supplied with 0.04 atm 0 2 .  9 8 Figure 7. The accumulation of 1 4 C in the root medium. Dotted l i n e : Total a c t i v i t y in medium (from non-ac id i f ied samples). So l id l i n e : Non-volat i le f r a c t i o n (from a c i d i f i e d samples). c —i U C IN ROOT MEDIUM -1 ^ dpm.(g.root d r y wt . ) x10 to CO —1 zr »-• m cn cn 00 ro o 4S O co o oo o o o o 99 Table 4 Estimates of the s ize of the v o l a t i l e and non-vo lat i le f ract ions of the root exudate Plant i n t-ai .Sir* I ' v i t - V Total A c t i v i t y A c t i v i t y in non-vo lat i le f r a c t i o n A c t i v i t y in B a ^ C O a V W X Y Z 75500 57700 22900 88200 10500 12400a (83.6) 2400a (95.7) 20800a ( 9.2) 14000b (84.2) 55200b ((,5055) 23000 (26.3) 3360 (32.0) 'Total a c t i v i t y ' is the a c t i v i t y (dpm l l fC) in root medium at end of experiment. The f igures in brackets in the th i rd and fourth columns are the percentages of the a c t i v i t y in the vo lat i1e f r a c t i o n . a. The a c t i v i t y in the root medium extract a f ter evaporation and f i l t r a t i o n of the bulk medium. B. The a c t i v i t y has been ca lcu lated from the a c t i v i t y remaining in a 1 ml sample of the root medium af ter a c i d i f i c a t i o n , and bubbling with n i t rogen. The React iv i ty in the v o l a t i l e f rac t ion for a . and b. was ca lcu lated from: 100 A c t i v i t y in residue Total a c t i v i t y x 100 1 00 obtained by i n d i r e c t estimates. The assumption behind measure- ment of the v o l a t i l e f rac t ion by trapping C0 2 is that a l l the v o l a t i l e f rac t ion is C 0 2 . If th is assumption is r ight then the low values may have resulted from incomplete removal of the C0 2 from the bulk medium. A l t e r n a t i v e l y , a l l the v o l a t i l e f r a c t i o n is not C0 2 ; some other v o l a t i l e compounds were present which were not trapped by the NaOH. Lowering the pH of the root medium caused a sharp drop in the l l f C content (Figure 8) . The 1 I f C content increased only very s l i g h t l y during the remainder of the experiment, q In the f i r s t experiment with ethanol , exudation was low and remained low throughout, the ethanol apparently having no e f f e c t . The resu l ts of the second experiment are shown in Figure TO. After the addit ion of 7.5% ethanol , the amount of  lkC in the root medium did not increase. The  lkC content of the fresh root medium f i v e minutes a f ter being introduced into the system was low, although well above background l e v e l s , but appeared to r i s e in both instances over the next 30 minutes. For the remainder of the experiment, a dec l ine in l l f C content was observed for plant 1 and an increase for plant 2. In both cases the l l f C level remained below that reached before the addit ion of ethanol . At the end of the experiment, the amount of  lkC in the soluble compounds of the roots of plant 1 (3.5 x 10 6 dpm. (g. root dry w t . ) " 1 ) was s imi la r to that of other plants not treated with 101 Figure 8. The e f fect of lowering the pH to 4.0 (at arrow) on the l l f C content of the root medium.  1 02 Figure 9 . The e f fect of 7 .5% ethanol on ' l l f C in the root medium of P. notatum. The ethanol was added at 4 hours (so l id arrows). The ethanol ic so lut ion was replaced by fresh medium at 5.5 hours.  103 ethanol (see Table 5, Chapter 1) . The 1 4 C in the root medium of plant 1 when the ethanol was added was about 3.4% of that in the soluble compounds. W i l t i n g , and a decrease in the rate of photosynthesis, occurred 1.5 hours after the addit ion of ethanol . Analysis of root t issue and exudate. Table 5 gives the d i s t r i b u t i o n of r a d i o a c t i v i t y among the f ract ions of the soluble extract of several root samples. Glucose, fructose and sucrose appeared cons is tent ly in chromatograms of the neutral f r a c t i o n ; no other labe l led compounds were present (Table 6) . Control analyses showed that the glucose and fructose did not resu l t from acid hydrolysis of sucrose during evaporation of the eluate from the cation exchange column. Up to seven compounds were found in the a c i d i c f r a c t i o n . Compounds i d e n t i f i e d as malic a c i d , c i t r i c acid and a compound tentat ive ly i d e n t i f i e d as g l y c e r i c acid were always present; the occurrence of the other compounds was Table 5 D i s t r i b u t i o n of a c t i v i t y among f ract ions of soluble extract of roots of P. notatum dpm l f fC (root g.dry w t . ) - 1 . Each value of a c t i v i t y is the mean of two samples Neutral Basi c Ac id ic Plant No. Total A c t i v i t y A c t i v i ty % Total A c t i v i ty % Total A c t i v i ty % Total Percent Recovery 1 2 3 4 5 6 1.11 x 10 7 2.35 x 10 7 3.48 x 10 7 5.25 x 10 6 0.67 x 10 7 0.61 x 10 7 0.84 x 10 7 1 .78 x 10 7 1 .82 x 10 7 0.43 x 10 7 0.52 x 10 7 0.46 x 10 7 75.1 76.0 52.2 81 .9 77.5 75.1 0.18 x 10 6 0.04 x 10 6 0.97 x 10 6 0.26 x 10 6 0.46 x 10 6 0.56 x 10 6 6.39 0.72 2.77 4.38 6.8 9.1 0.29 x 10 6 0.43 x 10 6 1.31 x 10 6 0.44 x 10 5 0.51 x 10 6 0.71 x 10 6 10.6 7.67 17.4 7.35 7.6 11 .6 92.1 84.4 72.4 93.6 91 .9 95.8 105 Table 6 Percentage of tota l a c t i v i t y in each spot of chromatograms of neutral f r a c t i o n s . Total a c t i v i t y is that found on the chromatogram Plant No. Sucrose Glucose Fructose 1 26.2 38.7 33511 2 41 .0 30.3 28.7 4 15.0 43:8 41.2 5 32.1 34.0 33.9 6 17.3 42.3 40.4 Table 7 Percentage of tota l a c t i v i t y in each spot of chromotograms of ac id ic f r a c t i o n s . Total a c t i v i t y is that found on chromatogram Plant No. Or ig in Xi Ci t rate Mai ate Glycerate? x 2 Succi nate Glycolate? 1 1 6 . 7 1 0 . 5 1 6 . 7 4 9 . 4 - 3 7 8 - 6 . 8 2 1 7 . 0 1 3 . 3 1 8 . 9 3 5 . 8 - 3v8 2.1 9 . 0 3 9 . 9 1 8 . 9 1 5 . 2 4 6 . 3 9 . 7 - - - 7 5 . 5 1 1 . 7 1 7 . 2 4 6 . 4 1 0 . 2 5 .5 3 . 6 - 8 9 . 7 7 . 9 1 0 . 7 5 0 . 9 9 . 4 4 . 0 3 .1 4 . 3 107 more v a r i a b l e . Table 7 shows the d i s t r i b u t i o n of r a d i o a c t i v i t y among the compounds of the ac id i c f r a c t i o n . Figure 10 shows the posit ions of the compounds on a chromatogram. In the basic f r a c t i o n s , radioact ive aspart ic and glutamic ac ids , alanine and some un ident i f ied compounds, were always present. Sometimes asparagine was present as w e l l . Judging from autoradiograph spot density glutamic acid and alanine contained the largest amounts of l l f C . The unknown* compounds contained only nominal amounts of 1 4 C . As noted in the Methods sect ion i t was d i f f i c u l t to obtain a s a t i s f a c t o r y analysis of the non-vo lat i le f r a c t i o n of the root medium. In some cases i t was not possible to elute s i g n i f i c a n t quant i t ies of  lhC from r e s i n s , and even in the cases, see Table 8, where this was achieved, adequate chromatograms of the f ract ions were not obtained. Compounds were detected by autoradiography only in the basic f r a c t i o n of one sample. The spots on the autoradiograph did not match with any of the spots on standard maps. Table 8 D i s t r i b u t i o n of Radioact iv i ty Among Fractions of Non-volat i le Residue of Root Medium - Plant Neutral Basic Ac id i c V 39.0 27.3 11 .7 X 42.8 9.1 30.7 1 08 Figure T;0. A composite chromatogram of the acid f r a c t i o n of the soluble root extract . S U C C I N A T E £ )GLYCERAT ' E C I T R A T E _ G L Y C O L A T E ? M A L A T E X 2 1 u r e 10 1 09 Discussion P. notatum plants were able to funct ion , at least for eight hours, when the roots were provided with very low amounts of oxygen. There is l i t t l e j u s t i f i c a t i o n for extrapo- la t ing these resul ts to longer periods of time but i t is probable that the grass can grow for long periods of time under low root oxygen. In an analys is of the oxygen require- ments of roots , Greenwood (17) concluded that aerobic metabo- lism would be unaffected by oxygen def ic iency when the p0 2 at the root surface was 0.01 atm (provided the d i f f u s i o n c o e f f i c i e n t in the roots was greater than 1.2 x 10 - l f cm. 2 s e c ~ 1 ) . It has been shown (17,18) that mesophytic plants (as well as, for example, bog plants) can meet at least some of the root oxygen requirements by oxygen d i f f u s i o n from the aer ia l parts . In my experiments th is is un l ike ly to have occurred as the p0 2 in the shoot chamber was less than 0.02 atm, and there was a pos i t ive gas pressure in the root to shoot chamber d i r e c t i o n . A small value for oxygen so lut ion rate (0.9 mmole. l ~ 1 h r . " 1 ) was obtained when nitrogen was used ind icat ing that traces of oxygen were ava i lab le to the root . If th is amount of oxygen is below that necessary to support aerobic resp i ra t ion then P. notatum may be able to function for short periods of time under root anoxia. In view of the n o many observations (16) of the importance of an adequate root oxygen supply (although as pointed out above the p0 2 can be very low) for shoot and root growth, i t is un l ike ly that P. notatum can function adequately under prolonged root anoxia. With respect to exudation, anaerobiosis probably resul ts in loss of compounds from the root; i t was observed to do so in cotton rad ic les (5). However th is represents an abnormal s ta te . It is important that at low oxygen concen- trat ions exudation be f u l l y under control (a lso , A. paspali is an obl igate aerobe). The i n h i b i t i o n of photosynthesis at a root temperature of 5°C is probably due to reduced water absorption leading to a reduced leafe water content. Retardation of water absorption is a c h a r a c t e r i s t i c resu l t of low root temperature (21). Ehrler (14) found that whether a l f a l f a roots were placed in a precooled water bath or whether the root temperature was lowered over two hours, a 5°C treatment caused wi l t ing and a 70% reduction in water uptake in 24 hours. A rapid transient drop in the water content of maize leaves occurred when the root temperature was lowered to 5°C (20). The autoradiographs of roots , Figure 4, and Table 3 show greater amounts of  lhC in the young nodal roots than in the rest of the root system. This w i l l be due part ly to the i r proximity to the shoot but the i r fast growth rate implies a greater demand for photosynthate. Radioact iv i ty can I l l sometimes be seen to be concentrated at the apices of l a t e r a l roots (arrows in Figure 4b); a s imi la r d i s t r i b u t i o n was seen in wheat seedlings by McDougall and Rovira (24) who considered that the bulk of exudation occurred from l a t e r a l root apices . Some l l f C was present in the root medium one hour a f ter feeding l l f C 0 2 to the shoot. The movement of 1 I f C appears to be faster than in wheat seedl ings , where t rans locat ion of l l f C to the primary root t ips took two hours, exudation taking place one to two hours l a te r (23). The absolute amount o f ' 1 I f C in the root medium did not vary much (8 to 10 x 101* dpm. (g. root dry w t . ) - 1 ) between root oxygen regimes, although there were d i f ferences in the amounts of l l f C exuded expressed as a proportion of the l l f C in the soluble f r a c t i o n of the roots . Unfortunately, because of the var ia t ion in exudation from plant to plant, i t was not possible to demonstrate an e f fect of p0 2 on exudation. The absence of an increase in  lkC content of the root medium in the presence of ethanol was unexpected. The high soluble  lkC content of the roots of plant 1 at the end of the experiment indicates that the ethanol did not extract compounds from the roots . Apparently, the e f fect of the ethanol was not to increase permeabi l i ty; i t changed the roots in such a way as to prevent further exudation. The wi l t ing observed in the second experiment suggests that the e f fect of ethanol on the roots was the same as that observed by 112 Al lerup (31) in barley seedl ings. Transpirat ion showed a t rans ient increase followed by a steep decl ine within 30 minutes when the barley roots were exposed to nutr ient so lu - t ion containing 10% ethanol ( i t follows there may have been a transient increase in permeabil ity of p. notatum roots on exposure to ethanol) . A less steep decl ine occurred at 5% ethanol but t ransp i rat ion containued to be affected at 3%, 2% and 1% ethanol . These changes in t ransp i rat ion were a t t r i - buted to changes in the water permeabil ity of root c e l l s . At 5% and 10% ethanol the changes were thought to be i r r e - v e r s i b l e . The increase in  lhZ content observed in the fresh root medium may indicate that the changes in P. notatum roots were not wholly i r r e v e r s i b l e . On the otherhharid, the lhZ of the fresh root medium may have been l l f C adhering to the root surfaces , a f ter removal of the ethanol ic s o l u t i o n . It was thought that lowering the pH of the root medium would increase the permeabil ity of the roots , causing an increase in the amount of  lhZ in the medium but th is did not happen in my experiments (Figure 8) . Most of the decrease in  lhZ is due to the loss of 1 1*C0 2 from the medium but no increase in the non-vo lat i le l l f C occurred. The two most l i k e l y sources of the l l f C 0 2 are the root and microbial r e s p i r a - t ion u t i l i z i n g exudedn non-vo lat i le compounds. Rovira and 113 Ridge (28) reported a 65% decrease in r a d i o a c t i v i t y of the so lut ion around the roots of non-s ter i le wheat seedlings fed 1 1*C0 2 compared with the so lut ion around s t e r i l e roots . The decrease in ' lkC content of the root f l u i d observed in some cases after s ix hours, Figure 6, may be due to the cessation of  1'*C exudation combined with loss of 1 I f C 0 2 due to microbial resp i ra t ion and ag i tat ion of-the root medium. In order for the amount of 1 I f C in the root medium to increase, exudation had to occur at a s u f f i c i e n t rate to overcome losses due to ag i tat ion and microbial r e s p i r a t i o n , and thus the l l f C exudation was larger than indicated by Figures 6, 7 and 8. In sp i te of the discrepancies between d i r e c t and ind i rec t estimates of the s ize of the v o l a t i l e f r a c t i o n (Table 4) there is l i t t l e doubt that most of the exudate is v o l a t i l e . Most of the exudate from wheat seedlings was also v o l a t i l e ; McDougall (23) found that a c i d i f i c a t i o n and aeration of the exudate so lut ion removed approximately 80% of the r a d i o a c t i v i t y . In the soluble extract of the roots 70 to 90% of the  lkC was in the sugars and organic acids which is com- pat ib le with carbohydrate making up the bulk of the root exudate. It does not necessar i ly fol low that these compounds w i l l be represented to the same degree in root exudate, although in those instances where an estimate of the s ize of 114 the exudate f rac t ions was obtained 51 to 74% of the label was in the neutral and ac id ic f r a c t i o n s . About 50% of the  lhC in the a c i d i c f r a c t i o n of the root t issue was in malic a c i d . It is possible that some of th is was formed by f i x a t i o n of resp i ratory  lkC02 (29). An inducible g lucose-spec i f i c uptake system is present in membrane ves ic les of A. v i n e l a n d i i (3). The uptake of glucose is coupled to an FAD-linked L-malate dehydrogenase since the addit ion of L-malic acid to a preparation of membrane ves ic les resulted in a 25-fold st imulat ion of both the rate and steady- state level of glucose accumulation. The e f f i c i e n t uptake of carbohydrate is l i k e l y to be of c r i t i c a l importance to A. paspali and i t is tempting to speculate that i f malic acid is also present in the root exudate i t is involved in the uptake of carbohydrate by A. paspali. Amino acids are commonly found in root exudates (26). Rad ioact iv i ty was found in the basic f r a c t i o n of the exudate solut ions analyzed, so i t is possible amino acids are present in exudate of P. notatum. It is d i f f i c u l t to say i f they would have any funct ion in re la t ion to A. paspali. The amount of carbon exuded from roots is smal l , and the non-vo lat i le f r a c t i o n is even smal ler . Rovira and Ridge (28) concluded that the total r a d i o a c t i v i t y of the root f l u i d around wheat seedlings was probably less than 0.5% of- the applied l l tC0 2^/, Some examples of the amounts of carbon 11 5 exuded by various plants are given by Rovira (26); these include corn: 8.2 and 8.4 mg of reducing sugars over 20 days and wheat: 2.6 to 22.5 mg over two months. These f igures and the small amount of exudate found in my experiments ra.ise the question as to how much non-vo lat i le exudate as opposed to sloughed off c e l l u l a r material is ava i lab le to A. -paspali under f i e l d condi t ions . Assuming that A. paspali can compete s u f f i c i e n t l y well so that the numbers of other micro-organisms are low, there may be more non-vo lat i le exudate ava i lab le to i t than is indicated here. The a v a i l a b i l i t y of adequate levels of soluble carbohydrate is suggested by the re la t ionsh ip between photosynthesis and N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in an in tact P. notatum-A. paspali assoc iat ion (11). Reduction of C 2 H 2 was unaffected by day- night changes but was much diminished after 45 hours of darkness, increasing rapid ly upon a return to l i g h t . This indicates a dependence of nitrogen f i x a t i o n on r e l a t i v e l y recent exudation. Ca lcu lat ions based on rates of C 2 H 2 reduction and photosynthesis, and on the carbohydrate requirements of Azotobaotev for nitrogen f i x a t i o n also suggest that P. notatum is able to provide s u f f i c i e n t exudate to support nitrogen f i x a t i o n by A. paspali. Dobereiner et al. (11) found the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in two s o i l cores to be: 116 143.2 nmole C 2 H 2 . h r . " 1 and 39,8.3 nmole C 2 H 2 . h r . " 1 assuming an equivalence between 3 C 2 H 2 and 1 N 2 . These rates are equivalent to 1334 yg N /hr and 3.72 yg N 2 /hr. An N 2 - l i m i t e d cul ture of A. ehroooocoum f ixed N 2 with an e f f i c i e n c y of 38 mgN f ixed per g mannitol consumed (8) . Hence, the mannitol required to f i x the amounts of nitrogen above are 0.035 mg/hr and 0.098 mg/hr. If a photosynthetic . rate of 57.7 mg C O 2 . h r . " 1 p l a n t " 1 (Table 2) is taken for P. notatum, and i t is assumed that 0.5% (27) of the carbon f ixed is exuded as glucose (M.W. 180 c f . mannitol M.W. 182) then the amount of glucose exuded is approximately 0.2 mg g l u c o s e i h r . " 1 pi a n t " 1 . This quantity is s u f f i c i e n t to meet the carbohydrate requirement of the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of the s o i l cores. By extrapolat ing the N 2[C 2 H 2 . ] - f i xi ng a c t i v i t y of s o i l cores to larger areas, Dobereiner et al. (11) obtained values for nitrogen f i x a t i o n by the P. notatum-A. paspali assoc iat ion inc luding one of 93JKgN.har~ xyr.~ 1 Kass et al. (19) found the nitrogen content of P. notatum tops to be 0.4% onaa dry weight bas is . If A. paspali is taken to be the sole source of nitrogen 'for P. notatum, then the dry weight of P. notatum corresponding to 93 KgN.ha"  1yr.~ 1 is 23250 KgDW.ha." x yr." 1 a . An N 2 - l im i ted culture of A. ohroooocoum 1 1 7 f ixed N 2 with an e f f i c i e n c y of 38 mg N f ixed per gram of mannitol consumed (8) . Plant dry matter is about 40% carbon, hence the amount of carbon that has to be exuded from the plant to f i x 93 KgN . ha ."" * y r . - 1 is 2450 KgDW.ha.~ 1 yr . - 1 b. If 30% of the carbon f ixed in photosynthesis is l os t as r e s p i r a t i o n , then resp i ratory losses are equivalent to 9964 KgDW.ha. _ x yr ." 1 c. The total product iv i ty of the a e r i a l parts in terms of dry weight is therefore 33664 KgDW.ha . _ 1 yr . " 1 (the sum of a , b and c ) . Expressed as a percentage, the amount- exuded for nitrogen f i x a t i o n , 2450 K g D W . h a . - 1 y r . - 1 , is 6.9%. The conclusion that can be drawn from th is c a l c u l a t i o n is that exudation has to be higher than the 0.5% of the f ixed carbon mentioned prev ious ly . A l t e r n a t i v e l y , i f exudate is only about 0.5%, then A. paspali can act only as a p a r t i a l suppl ier of the nitrogen requirements of P . notatum. In a review of root exudates Rovira (26) s ta tes , In studying the e f f e c t s of plants upon s p e c i f i c micro-organisms . . . i t is u n l i k e l y that the ubiquitous sugars, amino acids, and organic acids w i l l make a s i g n i f i c a n t contri- bution, but rather i t w i l l be the balance of these compounds and/orrthe presence of the rarer exotic compounds peculiar to a p a r t i c u l a r plant species that w i l l be important. A. paspali, however, is able to grow quickly in a simple medium and i t seems un l ike ly that i t is dependent on a p a r t i c u l a r balance of compounds or some exotic molecule. I suggest that 118 i t makes r e l a t i v e l y unspeci f ic use of whatever soluble car- bohydrate is a v a i l a b l e , except possibly malic a c i d , i f i t appears in the exudate. My experiments show that P. notatum plants absorb lhZZ,z and quick ly t ransfer the f ixed  lhC to the root environ- ment by root exudation. Theeexperiments using low root oxygen provide c ircumstant ia l evidence that contro l led exudation can occur at oxygen leve ls su i tab le for nitrogen f i x a t i o n by A. paspali. The analys is of the nature and amount of the labe l led compounds in the roots ind icate that these were of a type compatible with a carbohydrate based exudate for A. paspali. In ret rospect , the d i f f i c u l t y of c o l l e c t i n g non- v o l a t i l e exudate under non-s ter i le condit ions was underestimated. There was also a certa in incompat ib i l i ty between exudation studies and the method of control of oxygen concentrat ion. Unti l a carbon and nitrogen budget is drawn up for the P. notatum-A. paspali assoc iat ion i t w i l l not be possible to decide whether A. paspali in the rhizosphere is normally C- or N 2 - l i m i t e d (to this end i t i s . imperative that a convenient means be found for estab l ish ing the assoc iat ion in the laboratory) . Whether they are P- l imited is also a question of prime i n t e r e s t . Of relevance to th is question is the observa- t ion by Dobereiner et al. (11) of hyphae and spores of Endogone, a mycorrhizal fungus, in the outer cortex of P. notatum roots . Its presence becomes fasc inat ing in view of a report 119 (25) on the ef fects of Endogone on growth of P. notatum var "batata i s . " In two B r a z i l i a n s o i l s , d e f i c i e n t in ava i lab le phosphorus, in fec t ion of the grass with Endogone improved growth almost to the extent of added phosphate. Lime was added in some cases, as some Endogone s t ra ins do not grow well in acid s o i l s (the pH of the unlimed s o i l s was 4.5 and 5 .2) , and the improvements in growth of the grass were greater in these than in unlimed s o i l s (25). In the f i e l d , Endogone can be envisaged as mobi l iz ing phosphorus both for P. notatum, which without an adequate phosphorus supply would not respond to nitrogen (25), and for A. paspali. The buffer ing capacity of P. notatum roots (see next chapter) may also be of benef i t to Endogone as well as to A. paspali. Presumably the external supplies of oxygen.and d i - nitrogen are not l i m i t i n g . There is a need for a descr ipt ion of the gaseous phase in the rhizosphere. A comment by Da'lton and Postgate (7) is relevant to the problem of the n u t r i - t ional status of A. paspali populations: The concept of l i m i t a t i o n by s i n g l e nutrients in chemostats thus becomes rather involved in a circumstance in which oxygen concentration is c r i t i c a l and access of nitrogen to nitrogenase is l i m i t e d by some i n t r a c e l l u l a r mechanism. L I T E R A T U R E C I T E D Andersen, Alice M. 1 953. The ef fect of the glumes of Paspalum notatum Flu'gge on germination. Assoc. of O f f i c i a l Seed Analysts . Proc. 43rd Annual Meeting, pp. 93-100. Atk ins , C.A. and D.T. Canvin. 1971. Photosynthesis and C0 2 evolution by leaf d i s c s : gas exchange, extract ion and ion-exchange f r a c t i o n a t i o n of 1 ^C-label1ed photosynthetic products. Can. J . Bot. 49:1225-1234. Barnes, Eugene M. 1972. Respirat ion-coupled glucose transport in membrane ves ic les from Azotobaoter v i n e l a n d i i . Arch. Biochem. Biophys. 152:795-799. Berry, Joseph Andrew. 1970. The 3-carboxylation pathway of photosynthesis. Ph.D. t h e s i s , Univers i ty of B r i t i s h Columbia, Vancouver, B .C . , Canada. Chr i s t iansen , M.N., H.R. Cams and Dolores J . S l y t e r . 1970. St imulation of solute loss from rad ic les of Gossypium hirstutum L. by c h i l l i n g , anaerobios is , and low pH. Plant P h y s i o l . 46:53-56. Cooper, C M . , G.A. Fernstrom, and S.A. M i l l e r . 1944. .Performance of agitated g a s - l i q u i d contactors . Ind. Engng. Chem. 36:504-509. . Dalton, H. and J .R . Postgate. 1969. Ef fect of oxygen on growth of Azotobaoter chrooooccum in batch and continuous cu l tures . J . Gen. .M ic rob io l . 54: 463-473. S a l . 1969. Growth and physiology of Azotobaoter ohrooooocum in continuous c u l t u r e . J . Gen. M i c r o b i o l . 56:307-319. 120; 1 21 9. Dobereiner J . 1971. Personal communication. I .P . E . A . C . S . , Km 47, Campo Grande, ZC-26, Guanabora, Brazi1 . 10. Dobereiner, J . and A.B. Campelo. 1971. Non-symbiotic nitrogen f i x i n g bacter ia in t rop ica l s o i l s . Plant and Soi l Spec. V o l . , 457-470. 11. Dobereiner, Johanna, J .M. Day and P . J . Dart. 1972. Nitrogenase a c t i v i t y and oxygen s e n s i t i v i t y of the Paspalum notatum-Azotobaotev paspali- a s s o c i a t i o n . J . Gen. M i c r o b i o l . 71:103=116. 12. Dunn, Arnold and Joseph A r d i t t i . . 1968. Experimental physiology. Holt , Rinehart and Winston, I n c . , New York, p. 265. 13. Epste in , Emanuel. 1972. Mineral n u t r i t i o n of p lants: p r i n c i p l e s and perspect ives . John Wiley and Sons, Inc . , New York, pp. 103-150. 14. E rh le r , Wi l l iam, L. 1 fit 9 6 3 . Water absorption of a l f a l f a as af fected by low root temperature and other factors of a contro l led environment. A g r o n . ' J . 55:363-366. 15. F loyd, Robert A . , and A . J . Ohlrogge. 1971. Gel forma- t ion on nodal root surfaces of Zea mays. Some observations relevant to understanding i t s action at the r o o t - s o i l i n t e r f a c e . Plant and S o i l . 34:595-606. 16. Grable, Albert R. 1966. Soi l aeration and plant growth. Adv. Agronomy, 18:58-106. 17. Greenwood, D.J . 1969. Ef fect of oxygen d i s t r i b u t i o n in the so i l on plant growth. In: W.J. Whitt ington, e d . , Root growth. Butterworths, London, pp. 202- 223. 18. Healy, M.T. and W. Armstrong. 1972. The ef fect iveness of internal oxygen transport in a mesophyte {Pisum sativum L.) Planta ( B e r l . ) 103:302-309. 1 22 19. Kass. Donald L . , Matthew Drosdoff and Martin Alexander. 1971. Nitrogen f i x a t i o n by Azotobaoter paspali in assoc iat ion with Bachiagrass (Paspalum notatum) Soi l S c i . Sco. Amer. Proc. 35:286-289. 20. Kle inendorst , A. and R. Brouwer. 1970. The e f fec t of temperature of the root medium and of the growing point of the shoot on growth, water content and sugar content of maize leaves. Neth. J . Agr. S c i . 18:140-148. 21. Kramer, P . J . 1955. Water re la t ions of plant c e l l s and t i s s u e s . Ann. Rev. Plant Phys io l . 6:253-272. 22. L i s t e r , G. 1973. Personnal communication. Biology Dept. , Simon Fraser Un ivers i ty , Burnaby, B.C. Canada. 23. McDougall, Barbara, M. 1 970. Movement of 1 I f C-photo- synthate into the roots of wheat seedlings and exudation of 1 < f C from in tac t roots . New Phytol . 69:37-46. 24. McDougal1 , Barbara , M. and A.D. Rovira. 1970. Sites of exudation of l l f C - l a b e l l e d compounds from wheat roots . New Phyto l . 69:999-1003. 25. Mosse, Barbara. 1972. E f fect of d i f fe rent Endogone st ra ins on the growth of Paspalum notatum. Nature 239:221-223. 26. Rovira, A lbert D. 1969. Plant root exudates. Bot. Rev. 35:35-57. 27. Rovira, A.D. and Barbara M. McDougall. 1967. Microbio- log i ca l and biochemical aspects of the rhizosphere. In: A. Douglas McLaren and George H. Peterson eds . , So i l Biochemistry, Marcel Dekker, I n c . , New York, pp. 417-463. 28. Rovira, A.D. and E.H. Ridge. 1973. Exudation of Re- l a b e l l e d compounds from wheat roots: inf luence of nut r ients , micro-organisms and added organic compounds. New Phyto l . 72:1081-1087. 1 23 29. T i n g , Irwin P. and W.M. Dugger, J r . 1967. C0 2 metabolism in corn roots . I . K inet ics of carboxylat ion and decarboxylat ion. Plant Phys io l . 42:712-718. 30. Wang, C.H. and David L. W i l l i s . 1965. Radiotracer methodology in b io log ica l sc ience. P r e n t i c e - H a l l , Inc . , New Jersey, pp. 104-143. 31. A l l e r u p , S. 1962. Changes in t ransp i rat ion induced by ethyl a l c o h o l . Nature 194:1193,1194. 32. S t e i n , W.D. 1967. The Movement of Molecules Across Cel l Membranes. Academic Press, New York, pp. 65-125. Chapter 2 THE IMPORTANCE. OF PH TO THE PASPALUM NOTATUMr AZOTOBACTER P A S P A L I A S S O C I A T I O N Introduction Species of Azotobaotev are capable of growing within a pH range o f . 4 . 5 to 9.0 i f the medium contains f ixed n i trogen, but nitrogen f i x a t i o n occurs only within a range of 5.5 to 7.2. They are not usual ly encountered in s o i l s with a pH below 5.6 (10). A. paspali was observed to occur abundantly in the rhizosphere of P. notatum in s o i l s ranging in pH from 4.9 to 7.8 (4) . In culture medium, however, good growth occurs only above pH 5.5 (4) , and nitrogen f i x a t i o n is r e s t r i c t e d to a narrow range of pH 6.7 to 7.0 (5) . A. paspali produces acid i t s e l f , both in l i q u i d and on s o l i d , n i t rogen-free media (4); in a l i q u i d medium with sucrose, growth stopped when the pH reached 5.2 because of acid production (4). Dobereiner (4) found a higher pH on the root surface than in surrounding acid s o i l , and suggested that the occurrence of A. paspali 1:24) 125 in the rhizosphere of P. notatum in acid s o i l s was due to a buffer ing e f fect of the plant roots (6). In studies of root exudation (previous chapter) I observed that when the pH of the root medium was lowered, i t rose again f a i r l y qu ick ly . A l s o , i f the pH of the root medium at the beginning of an experiment was, for example, 6.6 then the pH normally increased to about 6.9. In such cases, s i g n i f i c a n t leve ls of  lhC were found in the root medium. When the increase in pH did not occur, less 1 4 C was found in the root medium. In one experiment, the pH dropped to 5.4 over four hours from an i n i t i a l pH of 6.6. When the pH was lowered further to 4.0 by the addit ion of a c i d , the pH remained at 4.0 for the remainder of the experiment (another four hours). Part of the reason for the low X1*C leve ls at the lower pH values is due to increased loss of the l l f C 0 2 from the root medium, but there seemed to be a cor re la t ion between root exudation and the capacity to buffer the root medium. In view of these observations and the f indings of Dobereiner and other workers on the response of Azotobaoter spp. to pH, experiments were carr ied out to determine more prec ise ly the buffer ing capacity of P. notatum roots . In a d d i t i o n , chemostat cultures of A. paspali were set up to determine the e f fect of low pH and other n u t r i t i o n a l condit ions on the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y . 1 26 Mater ials and Methods Buffering capacity of roots Plants of P. notatum were set up in the experimental apparatus described previously (Chapter 1 ) . The root medium at 30°C was bubbled with compressed a i r at the rate of 1.2 l . rn in" 1 . Photosynthesis was allowed to come to steady s ta te . After 30 minutes to an hour of steady state photo- synthes is , the pH of the root medium was lowered to 5.0 by the addit ion of 0.05M HC1. The subsequent increase in pH was noted for about an hour. The pH was then brought to 6 .9 , and af ter 30 minutes, the pH was lowered to either 4.0 or 5.0, and the increase in pH noted again. Control experiments were done in which the apparatus was set up without a plant and the r i s e in pH noted after lowering the pH of the medium in the c i r u l a t o r y system. In further experiments, the ' a l k a l i ' causing the increase in pH was t i t r a t e d with d i l u t e HC1. To do t h i s , the pH was lowered to 5.0 from 6.8 and maintained between 5.0 and 5.1 for 30 minutes by the addit ion of 0.05M HC1. The pH was then brought back to 6.8 for 30 minutes, by the addit ion of NaOH, before repeating the procedure. After th is had been done three times, the pH was lowered to 4.2 and kept between 4.2 and 4 .3 . The resu l ts of these experiments indicated that 127 the buffer ing capacity of the roots decreased with continued s t r e s s , so in some experiments the pH of the medium was lowered to 4.2 from the beginning. The pH of the root medium was returned to 6.8 for 30 minutes between successive t r i a l s as before. pH and N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of A. paspali Azotobaoter paspali, s t ra in Ax 4, was kindly provided by Dr. Dobereiner. It was maintained on a yeast extract and sucrose medium. For experiments, i t was grown on the ni trogen- free medium described in Table 1. Table 1 Nitrogen-free medium used to grow A. paspali Components Concentration (g/1) K2HP0i» 0.05 0.<15 MgS0i».7H20 0.20 FeEDTA 0.027 NaMo0/(.2H20 0.002 Sucrose 20 or 2 For s o l i d media, bromothymol blue (5 ml/1 of a 1% alcohol so lut ion) and 15. g/1 agar were added. The pH of s o l i d and l i q u i d media was adjusted to 6.9 ± 0 .1 . 1 28 Figure 1 is a schematic diagram of the simple chemostat used (a more s a t i s f a c t o r y and elaborate chemostat i s described in a paper by Baker (1) ) . The culture vessel (C) was a round-bottomed 500 ml Pyrex f lask with ports for an oxygen electrode (H,0) and a weir-type overflow (D). The cul ture volume was 200 ml. A Q u i c k f i t RF28/3/500 f i t t i n g , modified to allow the entry of the medalum (A), gas (L) and thermocouple probe ( J ) , was attached to the top of the f l a s k . The cul ture medium was run into the vessel through an i n t r a - venous in jec t ion set (B), the rate of flow being contro l led by the height of the medium reservo i r and a screw clamp on the in jec t ion set . Detai ls of the oxygen electrode port are shown at 0 in Figure 1. A piece of m i l l i p o r e membrane f i l t e r (b) , pore s ize 0.22 y, was glued to the end of a Beckman oxygen electrode cap (c) which was then glued into the electrode port (a) of the culture v e s s e l . A Beckman oxygen electrode (d) was screwed into the cap af ter s t e r i l i z a t i o n of the v e s s e l . It was necessary to determine the response of the electrode to oxygen in so lut ion with and without the f i l t e r on the cap. The chemostat was housed in a temperature contro l led cupboard. The culture vessel containing 190 ml of medium and a s t i r r i n g bar, and with the gas l i n e , e f f luent l i n e (D) and thermocouple probe in p lace, was s t e r i l i z e d by autoc lav ing. The medium and e f f luent (F) vessels were autoclaved separate ly . 129 Figure 1. Schematic diagram of chemostat. 130 Key to Figure 1 A - medium reservo i r B - S a f t i s e t , Volutro le intravenous in jec t ion set . Cutter Labs, I n c . , Berkeley, C a l i f . , U.S.A. C - culture vessel D - e f f luent l i n e E - sample port F - e f f luent receiver G - magnetic s t i r r e r H - Beckman oxygen electrode I - Beckman oxygen ampl i f ier J - temperature probe K - YSI telethermometer L - gas supply M - flow meter N - Whatman Gamma-12 i n l i n e f i l t e r unit with 12-03(3u) f i l t e r tube. 0 - deta i l of oxygen electrode port: a - wall of culture vessel b - m i l l i p o r e f i l t e r glued to electrode cap c - electrode cap glued to vessel d - oxygen electrode Figure 1 131 The s t e r i l e medium r e s e r v o i r , intravenous in jec t ion set and e f f luent vessel were attached under s t e r i l e condi t ions . To s tar t a culture in the chemostat, 10 ml of bacter ia l suspension, O.D. 6 oo = 0.12, was made by mixing l i q u i d medium with bacter ia grown on s o l i d medium for four days. The suspension was injected into the culture v e s s e l , the magnetic s t i r r e r s ta r ted , and the bacter ia grown as a batch culture for approximately 24 hours (prel iminary experiments showed that 24 hours corresponded to about the middle of the exponential phase of growth under the condit ions used in these experiments). Medium was then allowed to dr ip in from the reservo i r at the rate of 40 m l . h r . - 1 (D = 0.2 h r . - 1 ) , and the gas flow was started at a flow rate of 1.2 l . m i n " 1 . The temperature in the cu l ture , throughout the batch and continuous phases, was kept at 35 ± 1°C. Dry weight of bacter ia was determined by cor re la t ion with measurements of the opt ica l density at 600 nm of cultures grown at 0.04 atm 0 2 , 20 g/1 sucrose. The oxygen so lut ion rate was determined by the m method of Cooper et al. (2). One of the primary reasons for cu l tur ing organisms in a chemostat is to maintain populations in a steady s ta te . When they are in th is s ta te , the nut r i t i ona l s tatus , for example, can be studied without complications caused by the population going through the growth curve typ ica l of batch cu l tures . With the chemostat used in these experiments i t was d i f f i c u l t to keep the cultures in perfect steady state 1 32 as judged by changes in opt ica l dens i ty . However every e f fo r t was made to keep the opt ica l density constant for some hours before doing experiments with samples of the cu l tu res . The e f fect of lowering the pH on the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of cul tures grown under the condit ions shown in Table 2 was measured. Sucrose at 20 g/1 was used as th is was the concentration used by Dobereiner; one-tenth of th is amount was taken as const i tut ing a low carbohydrate concentrat ion. Dobereiner et al. (7) found that a p0 2 of 0.04 atm was optimal for N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y . This was taken as a low oxygen concentrat ion. Table 2 Chemostat culture condit ions for A. paspali N-free Dobereiner's Medium (Table 1) T = 35 ± 1°C, D = 0.2 hr . " , i n i t i a l pH = 6.9 ± 0 .1 , cu l ture volume - 200 ml 0.04 atm 0 2 , 0.96 atm N 2 , 20 g/1 sucrose 2 g/1 sucrose 0 2 so lut ion rate = 5.9 mmole l " 1 h r . " 1 0.20 atm 0 , 0.80 atmN , 20 g/1 sucrose 2 g/1 sucrose 0 2 so lut ion rate = 24.4 mmole l ^ h r . - 1 1 33 Erlenmeyer f l a s k s , 58 ml in volume were f i t t e d with rubber caps, f lushed with argon and used to hold 15 ml samples of cu l ture . In some of the samples, the pH was lowered to 5.2 by the addit ion of 0.05M HC1. Acetylene and oxygen were then injected to give a f i n a l pC 2 H 2 of 0.1 atm, and a p0 2 of 0.04 or 0.20 atm. The f lasks were incubated at 35°C for one hour in a water bath with a shaking amplitude of 3.5 cm and a frequency of 0.7 Hz. Every 15 minutes, a 1 ml sample of the gas phase was removed and 1 ml of the o r i g i n a l gas mixture was in jec ted . The ethylene content of the gas samples was determined using a Wilkins Aerograph gas chromatograph f i t t e d with a flame ion izat ion detector and a 182.5 cm x 0.63 cm od Porapak R column. The column was operated at 45°C with helium as c a r r i e r gas flowing at 30 m l . m i n " 1 . Results Buffering capacity of plant roots Figure 2 shows the increases in pH, for two p lants , a f ter lowering the pH of the root medium. Table 3 shows the quantity of acid required to lower, and maintain the pH at ei ther 5.0 or 4 .2. 134 Figure 2. The increase in pH of the root medium after lowering the pH of the medium from 6.9 to the value given in the f i g u r e . Plant A had a root dry wt. of 0.2g. Plant B had a root dry wt. of 0.8g. For each plant the data for the lower curve was obtained after that for the upper. 134- A F igu re 2 135 Table 3 Quantity of a c i d , ymoles H + . (g . root dry wt- . )" 1 , required to lower pH and maintain the root medium at low pH for 30 mihutesT The data in each column were obtained success ive ly . I n i t i a l pH was 6.8 Acid required to lower pH Acid, required to maintain pH Plant C to pH 5.0 225 211 92 1 08 1 31 97 to pH 4.2 478 492 769 369 306 28 . D to pH 5.0 316 183 50 163 23 137 to pH 4.2 1 588 1388 960 67 E to pH 4.2 1171 1092 1327 471 667 157 F to PH 4.2 534 964 686 672 1 34 Contro l 1 to pH 5.0 35 2 0 to pH 4.2 223 3 70 C a S O i j so lut ion without roots . 27-70% of the absolute amount of acid required to lower pH with the plant present. 25-57% of the absolute amount of acid required to lower pH with the plant present. 1 36 The a b i l i t y to r e s i s t the lowering of pH seems to decrease with continued stress in the form of low pH. In- Figure 2 the increases in pH af ter lowering the pH for the second time are smaller than after the f i r s t decrease i n . p H ; for plant B the smaller increase may also resu l t from the pH being lowered to 4.0 rather than 5.0. The data in Table 3 show a s imi la r e f f e c t . With successive t r i a l s the amount of acid required to lower the pH to 5.0 decreased, as did the amount needed to keep the pH low. S i m i l a r l y , the amount of acid needed to keep the roots at 4.2 decreased with repeated s t r e s s . After three t r i a l s in which the pH had been lowered to 5.0, the amount of acid needed to lower the pH to 4.2 was less than that required to lower the pH to 4.2 in plants which had not been stressed. Most of the acid needed to lower the pH to 4.2 was needed in the region 4.5 to 4 .2 . A s imi lar e f fect was seen in control experiments in which the amount of acid required to lower the pH of C a S O i j in the absence of a plant was measured, so i t seems the CaSOtt so lut ion used as root medium has buffer ing propert ies of i t s own in th is reg ion. For plants C and D, the absolute amount of acid required to maintain the pH at 5.0 and 4.2 has been plotted against the time at which the acid was added af ter lowering the pH (Figure 3) . Most of the acid had to be added within 137 Figure 3. The absolute amount of acid required to maintain the pH of the root medium at 5.0 and 4.2 for plants G and D (see text and Table 3) . Sol id l ines represent amount of acid required to keep pH at 5.0; dotted l ines the amount required to keep pH at 4.2. 190 100 80 ytxmoles 60 HC l 40 20 j j F igure 3 l l I I . i l l ! 10 20 30 TIME min 138 10 minutes of lowering the pH; the same trend was observed for other pi ants. pH and N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of A. paspali Table 4 gives the pH and O.D. of the various cu l tures . The bacter ia produced acid in culture and continued to do so while being tested for N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y . A l l the cultures appeared to contain dissolved oxygen at a concentra- t ion of about 1OuM, except for the ones at 0.20 atm 0 2 , 20 g/1 sucrose where the concentration was about 30uM. Table 5 shows the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of samples at the control pH and low pH. A low pH inh ib i ted N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in a l l cultures except those grown at 0.04 atm 0 2 , 2 g/1 sucrose. Table 4 pH awd 0/D/ of Chemostat Cultures of A. paspali Approximating to Steady State Conditions 0.04 atm 0 2 , 2 g/1 sucrose 0.04 atm 0 2 , i 20 g/1 sucrose )0220aa1tm002 , 2 g/1 sucrose 0.20 atm 0 2 , 20 g/1 sucrose PH 5.80 ± 0.12 1 6.24 ± 0.13 5.60 ± 0.17 5.22 ± 0.13 O.D. 0.24 ± 0.02 0.24 ±0 .04 0.34 ±0 .10 0.53 ± 0.05 mg dry wt.ml~ 1 1 .2 1 .2 1 .7 2.7 Standard dev ia t ion . 1 39 Table 5 The e f fec t of low pH on C 2 H 2 reduction by A. -pas-gall The amounts of C 2 H 2 reduced are nmoles C 2 H 2 .(mg dry w t ) " 1 . Rates are nmoles C 2 H 2 . (mg dry wt)".1 m i n " 1 . Means with the superscr ipt ' a ' are s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from the correspond- ing control means. Means without a superscr ipt are not s ign i fy i cant ly d i f f e r e n t . Culture conditions 0.20 atm 0 2 , 20 g/1 sucrose Contro l 1 pH 5.2 30 min 60 min 30 min 60.mi n Amount Rate 211 ± 90 7.0 461 ±295 7.7 166 ± 87 a 5.5 331 ± 205 a 5.5 0.20 atm 0 2 , 2 g/1 sucrose Cont ro l , pH 5.6 pH 5.2 30 min 60 min 30 min 60 min Amount Rate 181 ±58 6.0 407 ± 209 6.8 104 ± 35 a 3.5 252 ± 129 a 4.2 0.04 atm 0 2 , 20 g/1 .sucrose Contro l , pH 6.2 pH 5.2 30 min 60 min 30 min 6 0 m i n Amount Rate 295 ± 119 9.8 641 ± 94 10.6 229 ± 41 a 7.6 346 ± 76 a 5.8 0.04 atm 0 2 , 2 g/1 sucrose Control , pH 5.8 pH 5.2 30 min 60 min 30 min 60 min Amount Rate 362 ± 354 12.1 578 ± 522 9.6 255 ± 127 8.5 671 ± 409 11.2 •i CONTINUED 140 Table 5 (Continued) As the pH of the culture was already 5.2, the pH of the control samples was brought to 6.9 by the addit ion of NaOH. A t - t e s t was used to test the d i f ferences between means, the formula for paired comparisons being used where there was large var ia t ion between successive experiments under the same condi t ions . 141 Discussion Buffering capacity of plant roots It is c lear from the resu l ts that P. notatum has the a b i l i t y to neutra l ize increases in a c i d i t y in the root environ- ment, at least in the system used here. The decrease in buffer ing a b i l i t y under repeated stress suggests that 30 minutes was not long enough for the buffer ing mechanism to recover. The pH changes induced in these experiments were r e l a t i v e l y large and abrupt. In the f i e l d , the plant may be concerned with neutra l i z ing lower rates of addit ion of acid over longer periods of time. However in the acid s o i l s where the associat ion is found, large changes in the pH may occur from time to time; a f ter a ra in shower, for example. There are a number of mechanisms by which the root might r e s i s t increases in a c i d i t y . Protons may exchange with + 2 + cat ions , e . g . Ca , K bound to carboxyl groups of the c e l l w a l l . Figure 3 which shows the most of the acid necessary to keep the pH low had to be added within a short time, provides supportive evidence for an exchange s i t e mechanism. The protons may be neutra l ized by OH' ions transported a c t i v e l y out of the c e l l in exchange for counter-ions, e . g . PO^ - 3 , SOzj"2. A l t e r n a t i v e l y , the protons may be taken into 142 the c e l l in exchange for ions giving r i s e to an a l k a l i n e pH in aqueous s o l u t i o n . The use of radio- isotopes under appropriate conditions would be a su i tab le approach to d i s t ingu ish between passive and act ive processes. It has the advantage of allowing the study of both organic (e .g . malate) and inorganic ions. pH and N 2 [ C 2 H 2 ] - f i'xi ng a c t i v i t y A low pH inh ib i ted the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of samples taken from cultures grown at three of the four sets of condit ions used. The lack of i n h i b i t i o n in samples from cultures grown at 0.04 atm 0 2 , 2 g/1 sucrose was unexpected; further experimentation may have shown an i n h i b i t i o n . It seems that nitrogen f i x a t i o n in continuous cul ture is inh ib i ted by low pH, as i t is when A. -paspali is grown on s o l i d medium or in l i q u i d batch culture (4). This conclusion could have been stated with more cer ta inty had s i g n i f i c a n t i n h i b i t i o n been found under a l l condi t ions . Bearing in mind th is proviso, the i n h i b i t i o n of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y at low pH emphasizes the importance of the buffer ing capacity of the plant roots . Dobereiner noted that good growth of A. paspali occurred only above pH 5.5 (4). In my experiments (see Table 4) the pH of cultures was as low as 5.2 and N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y took place at th is pH (see Table 5) . Dobereiner 143 used batch cu l tures ; in chemostat culture the continuous supply of nutr ients may have mitigated the e f fect of low pH? Cultures grown at low carbon concentrat ions, p a r t i c u l a r l y 0.20 atm 0 2, q/1 sucrose, might be hyper- sens i t i ve to oxygen and thus unstable. However the cultures were stable and not so sens i t ive to oxygen as to be washed out from the culture v e s s e l . If the amount of carbon ava i lab le to these cultures had been increased, then the i r biomass may have been larger but under the condit ions used in my experiments the supply of carbon was s u f f i c i e n t to support nitrogen f i x a t i o n at atmospheric oxygen concentrations at both optimal and low pH. These observations indicated that A. paspali may be r e l a t i v e l y res i s tant to oxygen i n h i b i t i o n of nitrogen f i x a t i o n , p a r t i c u l a r l y with respect to other nitrogen f i xers such as Beij e r i n c k i a spp and Devxia sp found in the rhizosphere of P. notatum. It is shown in the next chapter that A. paspali i s less sens i t i ve to oxygen than Derxia gummosa. L I T E R A T U R E C I T E D Baker, K. 1968. Low cost continuous culture apparatus. Lab. Pract ice 17:817-824. Cooper, C M . , G.A. Fernstrom, and S.A. M i l l e r . 1944: Performance of agitated gas- l iqu id contactors . Ind. Engng. Chem. 36:504-509. DJalton, H. and J .R . Postgate. 1 969. E f fect of oxygen on growth of Azotobaotev ohvooooocum in batch and continuous cu l tu res . J . Gen. M i c r o b i o l . 54:463- 473. Dobereiner, Johanna. 1966. Azotobaotev -paspali sp .n . Uma bacter ia f ixadora de nitrogenio na r i z o s f e r a de Pasp alum. Pesq. Agropec. Bras. 1:357-365. . 1970.' Further research on Azotobaotev paspali and i t s var iety s p e c i f i c occurrence in the rhizosphere of Paspalum notatum. Fliigge. Zent ra lb la t t fur Bakter io logie , Parasitenkunde (Abt. 2) , 124: 224-230. Dobereiner, J . and A.B. Campelo. 1971. Non-symbiotic nitrogen f i x i n g bacter ia in t rop ica l s o i l s . Plant and S o i l , Spec. V o l . , 457-479. Dobereiner, Johanna, J .M. Day and P . J . Dart. 1972. Nitrogenase a c t i v i t y and oxygen s e n s i t i v i t y of the Paspalum notatum-Azotobaotev paspali a ssoc ia t ion . J . Gen. M i c r o b i o l . 71:103-116. H i l l , Susan. 1971. Influence of oxygen concentration on the colony type of Devxia gummosa grown on n i t r o - gen-free media. J . Gen. M i c r o b i o l . 67:77-83. 144 145 9. Lees, H. and J .R. Postgate. 1973. The behaviour of Azotobaoter ohvoooooeum in oxygen - and phosphate - l imited chemostat cu l tu re . J . Gen. M i c r o b i o l . 75: 161-166. 10. Mishust in , E.N. and V.K. Shi 1 1 nikova . 11971. B io log ica l f i x a t i o n of atmospheric n i trogen. Macmillan Press L t d . , London, pp. 184-250. Chapter 3 INFLUENCE OF OXYGEN CONCENTRATION ON COLONY MORPHOLOGY OF AZOTOBACTER PASPALI Introduction When grown in a i r on N-free agar medium A. paspali forms copious quant i t ies of s l ime. Growth is very rap id , and i f the inoculum is large , raised mucilaginous streaks cover nearly a l l of the petr i dish surface within 24 to 48 hours. Slime formation i s c h a r a c t e r i s t i c of the Azotobacteraceae and i t has been suggested that the slime plays a ro le in keeping (the oxygen concentration below leve ls inh ib i to ry to nitrogen f i x a t i o n (5) . In some of the B r a z i l i a n s o i l s from which A. paspali was i s o l a t e d , another member of the Azotobacteraceae was found, Devxia gummosa (1). D. gummosa is sens i t ive to the oxygen concentration in l i q u i d culture (3,4) and i t s colony morphology is markedly influenced by the oxygen concentration (3) . 1.46 147 In a i r on N-free agar medium, D. gummosa formed smal l , " th in" colonies af ter two days. These colonies did not reduce C 2 H 2 . After four to nine days, scattered "massive"ccolonies appeared e i ther from indiv idual " th in" colonies or from confluent growth of " th in" co lon ies . "Massive" colonies were mucilaginous and reduced C 2 H 2 . The formation of "massive" colonies was stimu- lated by lowering the concentration of oxygen in the gas phase. At 0.05 atm 0 2 a l l the colonies were of the "massive" type. When plates incubated at 0.20 atm 0 2 were transferred to 0.05 atm 0 2 , the " th in" colonies developed into the "massive" type. The low growth and lack of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of the " th in" colonies was considered to be due to oxygen i n h i b i t i o n ; the "th in" colonies were thought to develop only to the l i m i t of nitrogenous impurit ies in the medium. Formation of the few "massive" colonies in a i r was thought to be due to the i n i t i a t i o n of nitrogen f i x a t i o n e i ther af ter a period of growth on nitrogenous impur i t i es , or , in cases where the " th in" colonies were densely packed, af ter competition between the organisms for oxygen had lowered the local oxygen concen- t ra t ion s u f f i c i e n t l y to allow some of them to i n i t i a t e nitrogenase a c t i v i t y . It was suggested that the viscous slime of the massive colonies protected the c e l l s from oxygen. When colonies grown at 0.05 atm 0 2 were transferred to 0.20 atm 0 2 , they did not show i n h i b i t i o n of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y . 148 It was c lear from my own observations that , in a i r at least at high carbohydrate concentrations (20 g/1 sucrose A. paspali did not form "th in" and "massive" colonies l i k e those of D. gummosa. Although, as discussed in the previous chapter, 2 g/1 sucrose may have been s u f f i c i e n t to prevent oxygen i n h i b i t i o n in chemostat cu l ture under the condit ions used, the apparent lack of i n h i b i t i o n of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y at 0.20 atm also suggested that the oxygen response of A. paspali may be d i f f e r e n t from D. gummosa. A. paspali was incubated at high and low oxygen concentrat ion, using techniques s imi la r to those described by H i l l (3) , in order to gain more information about i t s oxygen response and to compare i t s colony morphology to that of Derxia gummosa. Methods and Materials A. paspali, s t ra in 4, was grown on Dobereiner' medium (2) containing sucrose at 20 g/1 or 2 g/1 , and on modified Burk's medium (3) containing sucrose or glucose at 1 g/1. Bromothymol blue was included in both Burk's and Dobereiner's medium. Dobereiner's medium w i l l be referred to as D medium and the modified Burk's medium as B 5 medium (3). 149 Bacteria were grown in a i r at 35°C for four days on D medium containing 20 g/1 sucrose. A d i l u t e suspension was then made of these bacter ia in l i q u i d D medium. An inoculat ing loop was dipped into the suspension and used to streak agar plates containing the medium given above. The plates were incubated at 35°C in polyethylene bags which were f lushed continuously with nitrogen containing 0.04 atm or 0.20 atm oxygen. Results On B 5 medium the bacter ia grew q u i c k l y . At three days the colony morphology was ident i ca l on a l l plates whether incubated at high or low oxygen or whether sucrose or glucose was the carbon source. Figure 1 shows photographs of typ ica l plates incubated under various cond i t ions . Most of the colonies were convex and mucilaginous, the c e l l s within the mucilage being grouped into a ret iculum. (The reticulum was easy to see because of the yellow colour produced by the bromothymol blue on react ion with the acid produced by the b a c t e r i a ) . Some smaller colonies were present: which were hemispherical and mucilaginous but did not contain any ret icu lum, being e n t i r e l y t rans lucent . S imi lar resu l t s were obtained using D medium con- ta in ing 2 g/1 sucrose. Under both oxygen concentrat ions, the 150 Figure 1. A. paspali grown on s o l i d i f i e d B 5 medium with sucrose or glucose at 1 g/1, and at 0.04 atm or 0.20 atm 0 2 . A. 0 .04 atm ° 2 , sucrose B. 0 .20 atm ° 2 , sucrose CC . 00 .04 atm o 2 , glucose D. 0 .20 atm o 2 , glucose  1 51 growth at three days on a l l plates was i d e n t i c a l , the c e l l s being grouped into r e t i c u l a within the convex mucilaginous co lon ies . At four days the small translucent colonies were apparent. When these were streaked onto plates of D medium containing 20 g/1 sucrose and incubated in a i r , large mucilaginous streaks formed by seven days. The c e l l s were not grouped into r e t i c u l a . Colonies with r e t i c u l a were also formed on medium containing 20 g/1 sucrose. Some in terest ing observations were made in one instance when the bacterium was incubated at 0.20 atm 0 2 . At s ix days large translucent colonies were present, a r i s ing from underneath and breaking through the older colonies or growing on top of them. Gram sta in ing of the two types of colony showed that the r e t i c u l a were made up of densely packed gram negative rods with many macrocysts. The translucent colonies consisted of long, thin rods sparsely d i s t r ibuted among the mucilage. Formation of translucent colonies of this nature were not observed in other experiments. In older colonies the r e t i c u l a general ly became more concentrated towards the centre of the mucilage, thus resu l t ing in a more extensive mucilaginous s h e l l . 1 52 Discussion From these resul ts i t appears that A. paspali is not as sens i t i ve to oxygen i n h i b i t i o n as D. gummosa. One possible explanation for th is is that i t is able to make e f f i c i e n t use of the carbon a v a i l a b l e , 1 g/1 of sucrose or glucose being s u f f i c i e n t to provide adequate resp i ratory protect ion . A l l the colonies were probably N 2 - l i m i t e d . In comparison, D. gummosa showed oxygen i n h i b i t i o n at a mannitol concentration of 10 g/1 (3). Postgate (6) has made the comment, "It seems l i k e l y that Devxia has r e l a t i v e l y i n e f f i c i e n t resp i ratory or conformational protect ion mechanism compared with Azotobaotev. " ........ The growth rate of A. paspali .is higher than that of D. gummosa. A. paspali always formed large colonies within three days whereas D. gummosa took up to 13 days to produce what appears (from H i l l (3) , Figure 1) to be s imi la r amounts of growth. The nature and occurrences of the translucent colonies of A. paspali are d i f f i c u l t to exp la in . They are possibly slow growing mutants. As they were formed under both high and low oxygen, t h e i r presence does not seem to be re lated d i r e c t l y to the oxygen concentrat ion. More work needs to be done on the oxygen response of A. paspali. In p a r t i c u l a r , cultures should be grown for 153 longer periods of time, and the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of each colony type determined. The experiments reported here estab l i sh the high growth ra te , and lack of response of the cultures as a whole to oxygen concentrat ion, in contrast to the response of D. gummosa. L I T E R A T U R E C I T E D Dobereiner, Johanna. 1968. Azotobaoter paspali sp .n . Uma bacter ia f ixadora de nitroggnio na r i z o s f e r a de Paspalum. Pesq. Agropec. Bras. 1:357-365. Dobereiner, Johanna, J .M. Day and P . J . Dart. 1972. N i t ro- genase a c t i v i t y and oxygen s e n s i t i v i t y of the Paspalum notatum-Azotobaoter paspali a s s o c i a t i o n . J . Gen. M ic rob io l . 71:103-116. H i l l , Susan. 1971. Influence of oxygen concentration on the colony type of Derxia gummosa grovrn or\ nitrogen-free media. J . Gen. M i c r o b i o l . 67:77-83. H i l l , Susan, J.W. Drozd and J .R . Postgate. 1972. Environmental ef fects on the growth of n i trogen- f i x i n g b a c t e r i a . J . Appl . Chem. Biotechnol . 22: 541-558. Postgate, John. 1971. F ixat ion by f r e e - l i v i n g microbes: physiology. In: J .R . Postgate, e d . , The chemistry and biochemistry of nitrogen f i x a t i o n . Plenum Press, London, pp. 161-190. — . 1972. B io log ica l nitrogen f i x a t i o n . Merrow Publishing Co. , L t d . , England, pp. 25-32. 1 54 E P I L O G U E Why is the d i s t r i b u t i o n of A. paspali in the f i e l d r e s t r i c t e d to an exclusive assoc iat ion with the rhizophere of P. notatum? A. paspali can grow, div ide and f i x nitrogen in a simple medium, so obvious hypotheses such as a require- ment for an exotic nutr ient are apparently not app l i cab le , although at low carbohydrate concentrations malic acid produced by the plant may be important as a st imulator of carbohydrate uptake. The work reported here shows that the roots of P. notatum provide a favourable pH for nitrogen f i x a t i o n in the rhizosphere. Growth and nitrogen f i x a t i o n by A. paspali are inh ib i ted by low pH. The capacity of P. notatum roots to neutra l ize increases in a c i d i t y in the root environment would prevent decreases in pH due to acid production by the bacterium. It also explains the d i s t r i b u t i o n of the assoc ia- t ion i n a c i d s o i 1 s . The bacterium probably u t i l i z e s material recent ly formed by photosynthesis as a carbon source (3) and the demonstration of rapid t rans locat ion and exudation of photo- synthate (Chapter 1) is c i rcumstant ia l evidence for t h i s . 1.55 1 56 Unfortunately I did not succeed in estimating accurately the amount of non-vo lat i le material exuded. An exact descr ipt ion of the gaseous phase of the rhizosphere does not ex ist but the mucigel probably maintains oxygen concentrations su i tab le for nitrogen f i x a t i o n in the rhizosphere. Some other members of the Azotobacteraceae, Beij erinokia indiea, B. d e r x i i , B. fluminensis and Derxia gummosa are found in the same s o i l s as the P. notatum-A. paspali assoc iat ion (1) . In some instances B e i j e r i n c k i a spp and Derxia sp have been found in the rhizosphere of P. notatum although A. paspali apparently always predominates, the numbers of A. paspali being very large (greater than 10,000 micro-colonies per gram of s o i l adhering to the roots (2) ) . B e i j e r i n c k i a spp and Derxia sp have a greater tolerance to low pH than A. paspali ( i t is in terest ing that B. d e r x i i , which appears to be the species found in the rhizosphere of P. notatum (13) is the most sens i t i ve to low pH of the three species of B e i j e r i n c k i a found in B r a z i l i a n s o i l s ) . I think the reasons why they do not form an equally large proportion of the bacter ia l population of P. notatum are as fo l lows . Provided the pH is o p t i o n a l , A. paspali has a higher growth rate than Derxia sp (see Chapter 3) or B e r i j e r i n c k i a (9) , as i t is able to make more e f f i c i e n t use of ava i lab le carbohydrate. This w i l l be p a r t i c u l a r l y important at low carbohydrate concentrat ions. A.- paspali also has a higher 1 57 optimal temperature for growth than B e i j e r i n c k i a , though not Derxia (1). Consequently i t is able to compete success fu l ly against other bacter ia in the rhizosphere of P. notatum. In a d iscuss ion of the rhizosphere e f fect Macura (8) noted that bacter ia adapt to a set of condit ions in such a way that the ir growth is as fast as poss ib le . In an ecosystem l i k e the rhizosphere i t is the most rap id ly growing bacter ia that are se lec ted . The d i f f i c u l t y of estab l ish ing A. paspali in the rhizosphere and the length of time required to do so, is a puzzl ing problem. If A. paspali is a good competitor against other organisms by v i r tue of i t s e f f i c i e n t use of carbohydrate, i t seems un l ike ly i t would take a long time to become estab l i shed . Perhaps in low numbers, i t faces heavy compe- t i t i o n from heterotrophic b a c t e r i a , and cannot compete success fu l ly for i t s niche in the rhizosphere u n t i l , eventual ly , i t s numbers increase. The exclusive assoc iat ion of A. paspali with P. notatum, the locat ion of the bacter ia in the mucigel , the re la t ionsh ip between photosynthesis and N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y , the demonstration of root exudation, the buffer ing of the rhizosphere and the pH requirements of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y a l l suggest at least a p a r t i a l l y symbiotic assoc ia- t i o n . I do not think i t can be regarded as wholly so, as 158 the bacterium can l i v e and f i x nitrogen in the absence of the p lant . Further, as y e t , no uptake of b a c t e r i a l l y f ixed nitrogen by the plant has been demonstrated. Parker (12) has proposed that the stages in the evolution of nodule symbioses are as fo l lows: casual assoc ia- tions of N 2 - f i x i n g bacter ia and plants loose associat ions on plant surfaces -* symbiosis within the plant in c o r t i c a l t issues -> symbiosis in organized, highly adapted t i s s u e s . Light micrographs (3) show A. paspali embedded in mucigel on the root surface. There is no apparent penetration of the bacter ia into the cortex. The assoc iat ion can hardly be described as " loose ," so i t seems to represent a stage about midway between a loose assoc iat ion and the penetration of the bacter ia l partner into the c o r t i c a l t i s s u e s . Beijevinekia and the plants in the rhizospheres of which i t is sometimes found, represents a stage equivalent to the loose assoc ia t ions . Its d i s t r i b u t i o n does not appear to be re lated to a p a r t i c u l a r plant but to those rhizospheres where there is an abundant supply of carbohydrate. For example, large populations of B e i j e r i n c k i a have been observed in the rhizosphere of sugar cane which has a sucrose-r ich exudate (1 ) . Many of the t rop ica l plants which have been noted as having r e l a t i v e l y high rates of N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y in the ir rhizosphere have the C-4 pathway of photosynthesis (5). 159 Plants with th is pathway have a high photosynthetic product iv i ty ( 4 ) , espec ia l l y in the condit ions preva i l ing in the savannas (high solar r a d i a t i o n , high temperatures). Consequently nitrogen f i x i n g bacter ia in the rhizospheres may have access to r e l a t i v e l y large amounts of carbohydrate. In temperate zones, N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y has also been found in the rhizosphere of. Calvin cycle plants (6) but the a c t i v i t y is not as high. The P. notatum-A. -paspali assoc iat ion h ighl ights the importance of the rhizosphere to so-ca l led asymbiotic nitrogen f i x a t i o n , p a r t i c u l a r l y in t rop i ca l ecosystems. It is poss ib le that , in unamended s o i l s , high rates of nitrogen f i x a t i o n only occur within the rhizosphere. F ie ld surveys to estimate nitrogen f i x a t i o n should be done with cognizance of th is p o s s i b i l i t y . Where high rates are obtained e f for ts should be made to ident i fy the plant concerned so that tests of the N 2 [ C 2 H 2 ] - f i x i n g a c t i v i t y of the rhizosphere can be made, p a r t i c u l a r l y at sub-atmospheric oxygen l e v e l s . There is a desperate need to increase the produc- t i v i t y . o f these ecosystems underpinning mans' welfare. Since one of the factors l i m i t i n g product iv i ty is the a v a i l a b i l i t y of f ixed nitrogen any associat ion of organisms that is capable of f i x i n g atmospheric nitrogen is of considerable i n t e r e s t . In i t s e l f , the P. notatum-A. paspali assoc iat ion is not very promising as a system which could be incorporated eas i l y 160 into a g r i c u l t u r e , p a r t i c u l a r l y intensive a g r i c u l t u r e . P. notatum var "batatais" is a poor pasture grass. It is not a cereal , so advantage cannot be taken of grain with a high nitrogen content. The assoc iat ion is slow to es tab l i sh and the e x c l u s i v i t y of the assoc iat ion mitigates against the inoculat ion of the rhizosphere of other plants with A. paspali. Further research should be concerned with modifying the assoc iat ion tocmake i t more su i tab le for a g r i c u l t u r a l use. A l t e r n a t i v e l y , i t represents a p o t e n t i a l l y valuable source of components which could be incorporated into other systems to enhance the i r n i t rogen- f ix ing capac i ty . I w i l l out l ine some poss ib le , and highly specu lat ive , s t rateg ies for carry ing out these broad a l t e r n a t i v e s . Subst i tut ion of a s ing le chromosome pair in c u l t i v a r s of spring wheat markedly changes the nature of the rhizosphere population (10,11). Perhaps there is a chromosome pair '('for buffer ing of the rhizosphere?) in P. notatumm var "batatais" which contributes to a rhizosphere favourable for growth of A. paspali. ' If th is pair could be incorporated into the genome of those v a r i e t i e s of P. notatum which are good pasture grasses, e . g . the d i p l o i d Pensacola Bahia types, then A. paspali might grow- in-the i r - rh izospheres . The problem s t i l l remains of the slowness of estab- lishment of A. paspali. Bacteria of the A. chvooooocum-A. v i n e l a n d i i growth readMyrin':many rhizospheres (9) . Jackson & 161 Brown (7) induced A. ehroococcum to mult ip ly in the rh i zo- spheres of wheat and peas. It is possible to t ransfer genes from one bacterium to another by conjugation, transformation or transduction (14). If the gene(s) responsible for good growth of A. ehvooeoccum-A. v i n e l a n d i i in rhizospheres could be located , perhaps they could be transferred to A. paspali, so resu l t ing in i t s rapid establishment in the rhizosphere of "Penscola Bahia-grass." Figure 1 is a summary of th is s t rategy . Another approach might be to work with a cereal such as wheat. Var ie t ies could be chosen, or developed, which have high rates of photosynthesis and exudation and the a b i l i t y to buffer the rhizosphere. The rhizosphere could then be inoculated with A. paspali Havingthe "broad host range" factor (see Figure 1) . A l t e r n a t i v e l y bacter ia of the A. ohrooooaoum-A. v i n e l a n d i i group containing a "symbiont" gene derived from A. paspali could be used as an inoculum. I have speculated on the existence of a "symbiont" gene since A. paspali i s capable of forming a c lose assoc iat ion with P. notatum whereas members of A. ohrooooooum-A. v i n e l a n d i i group do not appear to form associat ions of a symbiotic nature. Figure 2 summarizes these ideas. 162 Figure 1. Hypothetical scheme for development of P. notatum var "Pensacola Bahia" having A. -paspali in i t s rhizosphere. "Broad host range" gene from A. v i n e l a n d i i or A. ohrooeocoum Chromosome pair from "batatais" for A. paspali occurrence - increased exudation? - buffer ing of rhizosphere? - induction of tetraplo idy? A. paspali Pensacola Bahia-Grass Good pasture grass with A. paspali in rhizosphere 1 63 Figure 2. Hypothetical scheme for development of a wheat rhizosphere with modified s t ra ins of e i ther A. paspali or A. v i n e l a n d i i - A . chvooeooeum. Wheat Variety with high photosynthesis, exudation and buffer ing capacity Wheat rhizosphere A. -paspali A. vinelandii or A. ehvoooooQum "Broad host range" gene from A. v i n e l a n d i i or A. elavoooooeum "Symbiont" gene A. paspali L I T E R A T U R E C I T E D Dobereiner, Johanna. 1968. Non-symbiotic nitrogen f i x a - t ion in t rop ica l s o i l s . Pesq. Agropec. Bras. 3:1-6. . 1970. Further research on Azoto- baoter paspali and i t s var iety s p e c i f i c occurrence in the rhizosphere of Paspalum notatum Fliigge. Zent ra lb la t t fur Bakter iol ogi e, Paras i tenkund.e (abt. 2) 124:224-230. Doberiner, Johanna, J .M. Day and P . J . Dart. 1972. Nitrogenase a c t i v i t y and oxygen s e n s i t i v i t y of the Paspalum notatum-Azotobaoter paspali a s s o c i a t i o n . J . Gen. M ic rob io l . 71:103-116. Downton, W.J.S. 1971. Adaptive and evolutionary aspects of C 4 photosynthesis. In: M.D. Hatch, C.B. Osmond and R.0. S l a t y e r , eds . , Photosynthesis and Photo- r e s p i r a t i o n . Wiley Intersc ience, New York, pp. 3-17. C : . - - - t c n , " „ J — . 1971. Check l i s t of Ci* spec ies . In: M.D. Hatch, C.B. Osmond and R.0. S la tyer , eds . , Photosynthesis and Photoresp i rat ion. Wiley Intersc ience, New York, pp. 554-558. Har r i s , D. and P . J . Dart. 1973. Nitrogenase a c t i v i t y in the rhizosphere of Staohys s y l v a t i o a and some other dicotyledenous p lants . Soi l B i o l . Biochem. 5:277-279. Jackson, R.M. and McE. Brown. 1966. Behaviour of Azotobaoter ohrooooooum introduced into the plant rhizospheres. Ann. Inst . Pasteur 111(3), Supp l . : 103-112. 1 64 165 8. Macura, J . 1971. Some b io log i ca l and ecologica l aspects of the rhizosphere e f f e c t . Fo l ia M i c r o b i o l . 16: 328-336. 9. Mishust in , E.N. and V.K. S h i l ' n i k o v a . 1971. B io log ica l f i x a t i o n of atmospheric n i trogen. Macmillan Press L t d . , London, pp. 251-259. 10. Neal , J . L . , J r . , T .G . Atkinson, and Ruby I. Larson. 1970. Changes in the rhizosphere microf lora of spring wheat induced by disomic subst i tut ion of a chromosome. Can. J . M i c r o b i o l . 16:153-158. 11. Neal, J . L . J r . , Ruby I. Larson and T .G. Atkinson. 1973. Changes in rhizosphere populations of selected phys io logica l groups of bacter ia re lated to sub- s t i t u t i o n of s p e c i f i c pairs of chromosomes in spring wheat. Plant and S o i l , 39:2094212. 12. Parker, C.A. 1957. Evolution of n i t rogen- f ix ing symbiosis in higher p lants . Nature (Lond.) , 179:593-594. 13. Ruschel, Alaides Puppin and^Dirce Pinto Pacca De Souza B r i t t o . 1966. Fixacab assimbidt ica de nitrogenia atmosfe>ico em algumas Gramfneas e na T i r i r i c a pelas bacte*rias do genero B e i j e r i n c k i a Derx. Pesq. Agropec. Bras, 1:65-69. 14. S ta iner , Roger, Y . , Michael Doudoroff and Edward A. Adelberg. 1970. The microbial world. 3rd e d . , Englewood C l i f f s , N . J . , Prentice H a l l , p. 44.

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