ROLE OF LIPIDBODIESIN N,.FlXlNGROOT NODULES OF PEANUr (Arachis hypogaeaL.J
BY
©ABO. BJl.ItERH. SI DDIQUE , B. Be. (HoDa.), M.Be.
Athe.sillsubm.itte d to theSchool of Graduat e 8t uc1!ellin par ti a l fulf ilme n t of the
requirements fo r the 4e q r••of DoctorofPbilosophy
Departmentot Bioloq'y Kelllor!alunive r sityof New!o unc!1and
Jul y 1991
St. Jo h n ' lI Newfo u ndla nd ca ll ed e
ABSTRA CT
The pe a nutpla nt (Arachishypoga eaL.).unlikeother legumes, ca n sust ai n nitrogen fi xa tion when prolonged pe ri od s of darknessorde t op ing curtail th e supply of ph o t o s y nt ha t e to thenod ule.This ab ilityto withstandphotosy n thatestress is attributed to thepresenc eof lipidbo d i e s in infectednodule cells. In both dark-tre a ted and detopped pLents, the lipid bodies sho w a gr adua l decrease in numbers, whLc b suggests their utiliza t ion as a source of energy and carbon for ni t ro ge n fixation. Lipolyticactivitycan be localizedin the lipid bodi e s and the exist e nce of the B-oxictationpa t hway and gl yo xyla t ecycle is shownby therele aseof I~C02 from lino- leaylCoenz yme Abythe nodulehomog e na t e. Inad ditio n, the bLoche mfca I assayandcytoche mica l lo cal i z ati onof catalase and malate syn t hase also conf i rm li pid ca tabolism in the nodul e.
Ca t al as e fr om cUltured Bradyrhizobium sp . 32Hl, roo t nod ule s and see dsof pe a nut is act ive inawiderange of pH, hav ingtwopH opti ma.The enzyme activityisassociatedwit h bot hthe bacteroids andthe host cytoso l. Inis o lat ednodu le bac t eroids the pres en ce of catal ase is re str icted to the bac teroid sur fac e only, whe rea s in the rh iz obi a gro wn in cul ture, the activityre mains ir.si d e the cells . Tr i azole-
iii sensitive DAB reaction revealed microbodies in the host cyt o p l a s m, of te n lying close to the peribacteroidmembrane.
DAB- p osi t ive dense bodies ar e also found on the bacteroid sur f a c e at the host-symb iont interface.
Ine f fe c t ive nodules of peanut indu c e d by two nod·fix·
strains of Bradyrhizobium sp. were compared with the on e s ind u c e d by nod"fix "strain NC92. Both fix· strains (6 39 and 7091) ind u c e smallnoduleslackingleghemoqlobinandnitrogen- fixing activity. Ultrastructuralobservations revealed that the nodulesof 639have an enlarged peribactero id space and lackpersistenceofnod ule functi o n. The70 91- i nduc e d nodu les sho we danimpe d i me nt to bactero id rel e as e and differen t iation . Inth e ine f fe ctive nodules , lar ge r numbersof li p i d bodies were found to accumulate , co mpa r e d to th e e r rect.Lva NC92 nod u l e s. Th e large lipid accumu lation in the absence of nit r o g en fix ati onserves as further evidenceto confir mthat the nod u l elip i d bodies areut ili z e d as a supplementarysource of carbon and energy for nitrogen fixation Ln peanut root nod u l e s.
ToMyBelolledParents
iv
ACKNOWLEDGEMENTS
I ....oul dlike to expressmy deep senseofgratitud etoDr. A. 1<. Bal , Pro fesso r of Biology , for his constant guida nc e , wi se counci l and pa r t ial financ i al su pp o r t th rough out th e progra m. Than k s aredu e to mysuperviso r y commi t t e emembe r s , Dr . P. J. Sc ott an d Dr. T. R. Pa te l , fo r their val u a ble sug g estion sand frie ndlydis cus sions dur ing the research .I am grat e f u l totheSc ho olof Gr a dua te Stud ies and Department of Biology for pr ovi d ing the graduate student support and teaching as sLst.arrtah Ip throughoutthe program.
I am also thankf ul to the te c h nical st a ff of Bio logy De pa r t me nt , pa r t.Lcu Lar-Ly , C.Eme rs on, R.Flcken,E.Fit z ge r ald and W. Brownfor the irexcellent help. Thanksare due tomy fri ends Ja yaram, John, Ganpati, Ra v i and my Burton's pond room-mate Fid zi. Sh a r r and Shaf iq fortheir lo vely comp a ny.
A specialthanks to mybrother",Dr.Azim U.Mallik , for his wise adv ice and constant inspirat ion in my academi c caree r.. lowe an inte l l ect ua l debt to him.
Fin a lly I wouldliketo expressmy profoundthanks tomy spe cialfriend for her precious love and support.
vi
TABLE OF CONTENTS
ABSTRACT ACKNOWLEDGEMENTS TABLE OF coNT ENTS
LISTOFTABL ES LIST OF FIGURES I. INTRODUCTION 1.1.General Int r o d uc t i on 1. 2 . Ni t ro g en-fildngOrganisms 1. 3. Biologi calNitrogenFixatio n
I.J.!. Asymbiotic Nitr og en-f ixati o n 1.3.2.symbio ticNit r ogen-fi xdtion 1.3.J. Rhizobium-legume symbiosis 1.3.4.Rhizobium- theNitr o g en-f i x ing
Bac t e ri um •.••. •
pag e i i
vi
xi
!o3.5. TheInfection and Nodul at i onProcess . . 11 LJ.6 .Mec hanism of Ni t r o g e n Fixation 12 1.3.7 .Hemogl obi ninNitroge n Fixation 14 I.J.8 .Ammoni um Ass i mila t ionand Tra ns porta t ion 16 1.4. Photosyn the si s in Nitrogen-fi xa tion 17 1.5. Peanut (Arachishypoga ea ) , Ta xo nomy,
Dist ri but i onand Impor tanc e 21
1.5 . 1. Sp ecifi city of Pean ut-Rhi z ob ium symbiosis 22
vii
30 31 31 32 33 2J 24 25 27 28 I.6.Catalase in NitrogenFixation ... ..•
I.7.Lipaseand Li p i d Catabo lism I. 7.1. Lipid in NitrogenFixation 1.8. Objecti ves
1.9. Outline of Methods Empl oy ed .••• .• • II.MATERIALS AND METHODS
11.1. RhizobiumStrains and Medium ••..•.
11.2. Plant Mate r ials and Gr owing Conditions II. 3.{)l.lrkTreatment and Oetop ing of Plants II.4. ElectronMic ro s c opy (EM)of Samples 11.5. Nc d u Le Nu mbe r s
II. 6. NitrogenaseAssaybythe Acetylene
(C!H!) Red uctionTechnique 33
II. 7. Lip i d Bod yPreservation, Stai n i ngandCou nt i ng 34
11.8.Sample Preparation 35
II.8.1. Noduleand Bacteroid Fracti ons ... .. 35 11.8.2. Cell - f ree Ex tra ct of CulturedRhizobium 36
11.8.3. Se e dCytosol ...•• • 36
II.9.De t e r mina t i o n ofLe gh emoglo b i n (LHb) 37
11.10. Catalase 37
11.10 .1.Assa y ... ... •. • 37
11.10.2.As sayat Dif fer entTemperatu r e s ••• 39
II.1 0.3. Cytochemica l Lo c a lizat i o n 39
11.11. Mal ate Synthas e ••• •••.•. 40
46
57 viii
11.11.1.A_ssay 40
II.11.2. cy t oc hemi c a l Localizat i on 41 II.l2. Lino l eo y l Coenzyme A(LYL· CoA)ox i d a t ion .•.. 42
11. 12. 1. sa mp l e Preparation
II.12.2 . Determinationof LYL-CoA Oxid a tio n.. 4) 11.13. Loca li zat ionof Lipolysis •••••. 4) II.14.Gel Electrophores is ..•.. •... .• 44 11.14 .1. Preparationand Runningof Ge l ••. ..• 44 11.14. 2 . GelSt a ini ng .
rxr, RES ULTS
III.1. Acetylene Reductio nAs say (ARA) and Li p i d Bodies(LB) .. . .•.
III.2. Leghemoglobln (LHb), Total Protein (TP) and Catalase in Dark-stressedandDetopped Nonudles •.•...• ••••.•.••.• ..•
III.J.oa r x-an d Detopp- Stressed on Nod ule
Ult ra s t r u c t u r e. . .. .. . .• •• •• ••••. 57 III .4. Llno leoylCoenzyme A(LYL-CoA) Oxidati on •••.• 66
III. 4.10 In vivo Sample 66
111. 4 . 2. InvitroGrownBradyrhl zobiu msp. J2H1 . 69
III.s. LipaseLocalization 76
11 1.6 . Ma l a t e SynthaseAss ay and t.oceLdz et Lon 76 III.7.CatalaseAssay and Localizatio n •••.•.• 79 III. S.Gel Electrophoresis ••. .•.• •••.••. • 93
III.9. Ultr as truc t ure of Peanu t Nc d u LeuInduucedby Fi>( and Fix· Strai ns of eradyrhizobiumsp. 93 IV. DISCUSSION'
IV.l.Ace tyleneRe duc tion As s a y (AM) and
Lipi dBodies(LB) •.•••.. 111
IV.2. Lipas eIncaHzatio n •.•.••. 115
IV. 3. ~1alateSynt h ase ASl';ayand Loc al izat i o n . ...•• •• . 116 IV.4. Lino l eo yl CoenzymeA (LYL-CoA) Oxidation...•.• • 117 IV.5 . Leq h e moqlo b in (LHb)andTot a l Prote i n (TPj .. .•• 118 IV.6.Ca tal ase inStressedNodu les... 119 IV.7. Ul t ras tructu reofStr e s s ed Nod u l e s 12 1 IV.B . Catalase As s a y andLoca li zation
IV.9.Ultra s truct u reand Li pidBodies of Fix'andFi x · Nod u l e s SUMMARY
VI. CONCLUSIONS VI:I.
123
l25 12"
l3 0 133
LIST OF TABLES
page Table 1. Leghemoglobinandtota l proteincontent
inth e root no d ule s of dark-treated
peanutplant ••• ••• •• ••••... ...• .. . 58 Tab l e2. Leghem o globinand to ta l pro t e i n content
in the rootnodu l e s of detoppedpe a nut plants
Table 3. Leghemoglobinandtotal proteincontent in therootnodu l e s of dark-treatedcow
59
peapl ants •••• •.•• •. •.• •.• • • . •• .. .•. •••.• 60 Table4.Catalaseactivityinpeanut andco ....pea
root nodu l ehomoge nateobt a lnedfr om plantssubject ed to diffe r ent periods
of darkness •• ••• ••••• .• •.••.• •• • •....•• • 61 Tabl e5. Catala s e ac t i v ity in diffe r ent fractions
ofbac t eroids and ba cteria Ta b le 6. Nu mber, sizeand te xtu reof peanut
no dule s producedbythe different str ains
of rhiz o bia .... .. ..•.•. 10 8
LIST OF FIGURES
Figu re1. Schematicdiagram of nitrogency cl e .. ... • . Fig ure2. Sc he ma t i c diagramshowing relationship
between photosynthes is and nitrogenfixati o n Fi g u r e3. Acetylenereductionassay (ARA) of
peanutand cowpe a nodules obtained from da r k - t r e a t e d plant s
Figure<I. Acetylene re du c ti o n assay (ARA) of peanutan d cowpea nodulesobt ai n e d from
detopped plants .
Figure5. Ac e ty l e n e reductionassay (ARA) of co wpe a nodul e s obtai ned from dark-treatedplants at constanttemperature .... . FiQU1'tl 6. Ac e t y l e n e reduce Len as s ay (ARA) of the
nodulesof dark-tr.eatedpeanut plants re v.EarlySpanish)
Fi gure 7. Lipidbody counts in 1.5- J'rn thick sections of nodules from dark-treated and detopped peanut plants .. .. . Figure 8.An electron micrograph of peanut
nod u l e t issuesobtained from 24 h dark-stressedplant.•.. . ... •... . Figure9.Anelectronmicrographof peanu t nodu le
xi
pag e
48
50
52
54
56
63
xii ti s s ue s obtained from 48 hand 72 h
dar k·stressed pl ant s . . Fig u re~O.Anelectro nmicrographof cowpea nodule
tissues obtainedfrom 24 hand 48h dark-stressed pl ants
Figu re11. I~COlreleasedfrom(I~C ] lino leoyl coenzyme A by the nod ulehomogenate
68
of peanutand cowpea ncduLes 71
Figure12.1~C02 re teesec from ('~Cl linoleoyl coenzymeAby pean ut nod u l e cytosol
and bacte r oids .
Figure 13. I~COIreleasedfrom[1~Cl lino 1eoy l
7J
75
78 coenzyme A by differentfractionsof
cultu re dBradyrhizobi umsp. 32H1 Figu re14.Cytochemicallocaliza tionof lipase in
peanut root nodu letissue . Figure15.cy t oc hemical loc a li z a t i cn of mej.etie
synthase in pean ut.no d uletis s ue s 81 Fig1lr e16.Gr ap h shOWi ng cacaraecactivityat
dif fere nttimes inin vitro culture ofBradyrhizobium sp , 32Hl growing in'iEM med i um .
Fi gure 17 .Graphsh owingcat alase ac t i v i t yofthe bacteria l cellconte n t at dif feren t pH valu es •..•.. •...•. • . .••... •.. . . .. . 85
FigurelaoGraph showingcata laseactivity of the peanutnod u l e cytosol at differentpH
values . .
xiii
87 Figure19.Graph showing catalase ac t i vity ofthe
is olated bacteroidfr a c tio n from peanut
nodule at dif ferentpH values 89
Figure20.Graphshowing catalase activityofthe J-daygerminated peanut seedcytosol at
different pH values 91
"
Figure21. cytochemical loc a li za ti o n (DAB positive) of cat a la s e inBradyrh i z obiumsp, 32H1 .... 95 Figu r e22. cytoc he mica l localiz ation(DAB positive ) of
ca t a l as e inrootnod u le tissues of peanut . 97 Figure23. DABst a i n e d nativepOlyacrylamide (10%) gel
sho wi ng three distinctca t a l ase bands in 3-daygerminatedseed and one band in nodule cytosol of peanut
Figure24.Photo··andelectron microgra phs of nodules (3 week post-i nocu lation) inducedby
Bradyrhizobiumstrains NC92 and 639 103 Figure 25.Photo-andelectronmicrographs of
nodules (4.5 and 6 weeks post-inoculation) induced byBr ady rh izobi um strain639 •••.. 105 Fi g ur e 26.Photo-and electron micrographsof nodules
(Sweekspost-inocu lation) induc e d by
110 x Lv
Bradyrhizobiumstrain 7091 107
Fig ure27.Agra phshowi ng lipid body counts in theinfec te d cells of the threenodul es formedby BradyrhizobiumstrainsNC92, 639and 7091
Fig ure28. Schematic diagramshowingtheroleof lip id bodiesin Nl-fix ing rootnodules
ofpe an ut .
"2
Chapter I INTRODUCTION
I.1 .General Intro d u ction
Amino acids are key building blocks of pr ote i ns which ar e basic tocellularfunctions.In proteins, ni tr oge n is present in its combined form.Although mole cularnLcz oqen isabundant in the earth's atmosphere , accounting for 78%byweight, its relative stability and inertness make i t unavailable to eUkaryoticorganisms.Nitrogenis combined with other elements like hydrogen and oxygen in an endergonic reaction called nitrogenfixation.
There are basically thr e e different ways by which atmospheric nitrogen is fixed into a combined form: i) atmosphericfixation -nitrogen reacts with oxygen (Ohdue to lightningdischarge or ul travioletradiationto form oxides, e.g . ni t ric oxide (NO]) which is carried to the soil with rain; i i) industria l fixation (Ha b e r-Bo s ch process ) ~ nitrogen and H1react at high temperature and pre s s ure to yieldammonia (NH.a); and iii)biological nitrogen fix a t i o n- atmosphericnitrogen isfixed intoNHJcatalysedby the enz yme complex,nit roge naseinprokaryoticorganismseithersymb ioti- cally or inassociation wit hhigherorganisms.
In nat ur e , nitrog en bala nc e is maintainedby itscycling through pl an t s , animals and microbes. This nitrogen cycle (Fig. 1) invol vestherec overyofcombinedni t r og e n from dead pl a nts and animalsbymic r o bia l action , its passage through th e soil in various forms and its final reabsorption by plants. Inth i s cycle, combined nitrogen can be lost tothe atmosphere by denitrifica t ion and gai ne dfrom it bynat u r al and artificia l (industria l) nitroge nfixation.
Host plants take up nitrogen in a combinedform,usually as nitrate or ammonia, via th eir root system, sometimes assisted by mycorrhizalfungi. Asmall amo u n t of ammoniafrom animal exc reme ntor othe rsources ma y VOlatilize from soil and be absorbed by the plant canopy (Sprent, 19 8 7). The nitrite ions (NOil are used by most plants but under ne u t r a1 alk a line conditio nsuseammon iu m ions (NH/l better.
Figure1. Schemat icdiagram of ni troge n cycle inna t u r e (Pelc z a r et al. , 1986).
Atm os phencN2
NNF-Nalu.a l~il''''I.~"Ial.""
BN.-B'oloqica lnit.oql!nh,OIOon 1111.-I~dutlrjalnilro,,_nIi. ol,on
Ar _.l."'mon, r;~ohon
.1.2. Nitroge n-fixingorganisms
Nitrogenfixation is confinedto certaingroupof prokaryote, whic h ar e distrib u tedamongEubacteriaandcyano bacte ria.The taxonomyof these organ ismsis continuous lyunderrev ision.
They aredifficu l t to classi fy as theymayhave chara c t ers which span many differe n t taxa.
1.3. BiologicalNitrogen Fixation
Biological nitrog en fixation involves the reduction of atmosph eric nitro gento enecnLe,wh i c his incor porat ed into the or ganic ni trog e n of thenitrogen-fixing organism orits host. The overall rea ct i on can be represen tedas
N1+3H20
---->
2NHJ+ (3/2)Ol 6GO= +340 KJnca-' 6GOis the standard freeenergy cha ng e ofthe rea ct i on (Gallon and Chapli n, 1987). Energy required by the reaction is ult imate l y derived from the oxidation of the pr odu c ts of phot o s ynt he s i s . Th ereactionis catalysedby ni t roge na s e , an enzyme uni qu e to nitrog e n-f i x ing organisms. Ni trogenase is ext r e melysensitiveto oxygen. Bi ologi c a l nitroge nfixationis the ma j or source of rer,ewable combined nitrogen in th e biosphe re (P oo t gate , 1982). About 12 2 X 106met ric tons of nitrogenisfi xedpe r annum(Bu r ris , 1980) whi ch is almost71t of the wor l d ' s fixed nitrogen in the soiland wa t er . rndus- trial nit rog e n'-f i x a tion contributes approximately 15% andenvironm.e nta l facto rs contribute the other 1o, of fixed n1t rogen (Bra y. 1983 ).
Al t hou qh. biol ogicalni t rogen-fixat ion is U.itedtc a few pr o karyo te. some higher pla nt s can also profit frolllthis pr oces s in looseas s oc ia ti onwith such prokaryote(a s s ociative symbios i s) or by harbouring thepro ka ryote insideroot nodules (sy mbios is) (Stew art ,1977 ).Biologica l nitrogen-f ixationcan occ ur in prokaryote when they ar e also fr-e a-LfvI nq (aSyll- biotic).
1.3.1.As ym.biot i cHi troqen-f ixat ion
Fre e - living ni t r ogen-fixi ngorg anisms can be aerob ic(Az oto - ba cter). anaerobic (Clos t r i di um) or ph otos yn t he tic (Nostoe). They are foundinboth soil and aquati c envi r om nents.tHtro gen fixed asymbl ot ic a lly isnot directlyavailab letothepla nts unt i l the nitr oq en-fixinq organismsdieand decomposein the soil. These orqan i s Dls are app roximate ly 1000 ti mes les s ef f ectiveintheirco ntri b utionof fixedni trogentot~"'!soil tha n symbiotic bacte ria (Bray. 1983). There ar e sOlie tree- l.iving ni troge n-fi x i ng ba c t eria which are abunda n t in the rhizos ph e r e of different pla nt s (Vose . 19B3; Haahte l a and Ka r i, 1986). Suchan associa tionsuggestsa form of mut ua lism (YouandZhou, 1S1 88 ).Nelill\anandBowen(1974)establ is hedtha t as s ocia te d ba cte ria were ra ndomlydi stri buted on roots and th a t 4-10\ofa rcc"." s surfa c ewas coveredby micro-o r gani sms.
Coloniza t ion of the roo t int e r i o r has also been reported (patriquinand Do bere iner, 1978; Patriquin,1981 ).
1.3.2.Symbi oticHitroqen-tixa tion
Symbiotic nitrogen-fixation is another form of biologica l nit r og en-f ixation, whereth e bacteria in h a bi t innc d u Ie cells formedbythe host plant.A verycommon symbioticassociation occurs between leguminousplants andRhizobium spp.In nature, nod u l at i o n of Parasponia sp. (a member of Ulmaceae) by Rhizobium indicates the possibi l ityof extendi ng Rhizobium symbiosis beyondthe Leguminosae. Nod uleshave been induced on the non-legume, oilseedrape , fol10...ing enzyme treatment of the seedl ing roots and inoculationwithRhi z obi um sp. (Al- Hallahet;al., 1990 ). In the same way , nodule-likestructu res have also been induced on rice and wheat plants roots in recent years (AI -Ml!I ll l!lh ettil.,19 S9a ,b ), but litt l e is known abou t the eff iciency of their function in termsof nitrogen fixation.Although vast majorityof nodules occur onthe roots of the host plan t,cert a i n aquati candwa t e r-t o l era n t species of legumes deve Iop nodules on their stems (Alazard, 198');
Dreyfus andDomme r gu e s, 1981; Arora, 195 4) or trun k (Pri net al., 1991).Thesenod u l e s have beenshown tobe capableof a hig h ra tes of nitrogen -fixatio n (SUbb a Rao and Yatazawa, 19 8 4).
1.3 .3.Rhizobium-legUllleSYlJIbiosis
Legumes are placedin the family Lequmi nosae , rec en.tl y renamed as Fabaceae, with sub-fami lies Caesalpinoideae , Mimoso i deae andPapiliono ideae.The family Fabaceae cons i s t sof about 750 genera with16,0 00-19,0 00 species (AllenandAllen, 1981 ) and is widelydistr ibutedfrom thetr o p ics to th earc t ic. cu l ti- vated spec i e s suchas peas, beans, alfalfa, cl over, lu pi n, soybean , cowpea, peanut,etc.have the characteristi c le g u me fr u i t. Most me mb er s form a symbiotic association wit h a Rhi z obi u msp •andpl a y a crucial eco logica l ro l einma.int.eLn- ingadequatenitroqe ""-eso ur- ces inthe earth.About10\ofthe legumesha v e beenexa mi ne d for nodu lation. Amo n g those,85%of the species belong to the papL'lIon o Ld e ae and. 25% to the Mimosoideae. Nodulation is ra r e in the cae salpinoideae (Postgate, 198 2).
1.3.4.Rhizob i um- the Nitrogen -fix i ng Bac teria
Therhi z o bi a are a group of bacteria belongingto the genus Rh i z obi um that fix atmosphericni t rog en in symbiotic associ- ations wi thspecifichost plants, almostexclu s i velylegu me s . Theyare gram negative , rods (0.5 - 0.9 /LmX1.2- 3.0/-Lrn) , occur singly or in pairs and are genera lly motile. The flagella are either per!trichous, polaror SUb-po lar (Jo r d a n and Allen. 1974). Rhizobia usuallygrow in a wide rang of te mpe r atur e under low oxygen tension. They do not produce
endc a p cres.Glycogen and poly-a-hydroxy butyricacid ar e formed as storage granules. The free-living rhizobiaca n in fec t a specific host and producenod u l e s inwhi c h theyare trans- formed into bactero idsafterundergoingbothmor p h olog ica l and biochemical changes. -rne bacteroids, that fix atmospheric nit r o ge n and supp ly it tothe host, are characterized by a highlyir r e gul ar shapecausedbyoutercel l wallchanges (Va n Brussel , 1973;sutton and Peterson, 1979 ; Bal etal., 1980 ) . The electrophoretic profile of pz'crt.ein in bacteroids is dif f e r e nt from free-livingrhizob ia (Sen and Weaver, 1988) .
There aretwo majorgr ou p s of rhizobia,commonlyknownas fast- andslow-growingsp ecies. Th e q ener-atiIon timefor fast- growers is 2-4hwhileit"is 6-8 h for slow-growers (Vi n c ent, 1977) . The s e two groupsdiffer biochemicallyand physiolog i- cally and havebeen placed in two separategenera,Rhizobium and Bradyrhizobium (Jordan, 1982). UnlikeRhiz ob i um, Br.ady- rhi z o b i um does not produce a lo t of mucoids (extracellular polysaccharide) in agar .The two grou psalso diff erin several symbioticpr opertie s;for example,Bradyrhizoblumstrainscan be inducedto fix nitrogen in fr ee-livi ng cultures (Kurz and LaRue, 1975; McComb at al., 1975; Pagan at al., 1975). Rhizobium species fixni trogen on l y symbiotically .Thesefa r"- growing speciesge ner a l l yin f e c t onlya eev closely related le gume s wh e r e a s Bradyrhizobium speciesof the cowpea cross- inocul ation grou p caninf e ct a broadrangeof diverse le gume
10 hosts (El k a n , 19 81). Fa s t -g r o wing species al mos t inva ri abl y inf a c t thei rhost vte the root he Lr-s whereas slow- growi ng spe c i e s infect by roothairs (i n pigeonpea) ordi r ec t penetr a- tion of epider ma l ce lls (i n groundnut ), the so - ca lled 'crac k entry' (Ch a n d l e r. 1978; Dar t, 1977; Kapil and Kapil, 19 71 ). Many sugars , pol y o lsand organ i cacids ca nbe utilized by th e fast-g row i.n qspec i e s but the slow-growf'': saremo r especia lized in their re q u i r e me nts andge n e r a lly preree-pantases(Vince nt, 1977). Although oneor more vitamins are required bysevera l st r ain s fo r growth (Gr a ha m, 196 3). Bradyrhizob iu mis less vitamindemandingthan fast-growing·ones.Mi neralrequi r e ment for rhizobi a l growth is import antandop t ima l conce nt r ation s for di f fe rentminer als va ry amongthe strai ns.The viabili t y of a culture is redu ce d in theabs enc e of Mg and Ca. Mag- nesiu m- limi t ed cell sbecomeelongated and someti me s bran c hed . Cal cium- limited ce lls become irregular , swol len , or roughly spherical (Vinc ent ,1977).Salts of both Na andK element sare to xic at higher conc e n t r a t ion s but omiss i on of Kfrom the basal medium restricts th e gr O\olt h of R. tri fo l i i and R.
me1110 t i (Vi nc en t , 1977 ) .
Indoleaceti cacid (I AA) andgib b e r e l li n- li k esubst a nc es are produced by rh i zobia (Katznel son and Col e , 1965 ). Both fungicidesandin s ec t i c i d e s can be toxicto rhizobia(Vinc ent, 195 8) , but hormone herbicides generallydo nothave any effect on rhiz ob ia (FletcherandAler o n,19 5 8).Rhizobiaare suscep-
11
tible to br oa d spectr um ant ibi o tics and the ef fe ct of eucn antibiotics cuts across specificbou ntiaries so th a t strains and sub-st r ains withinone spec i e s show a conside rable ra nge of sens i tiv ityandresis ta nce .Mi c r o- o r ga nis ms fou nd in soil and in therhiz oi'"r-ilere ca nbesynl!'rg istic or antagon i s ti c to rh iz obialgr owth .
I. 3. S. The In f e c t i on abd No du lation Proc lls s
The legume-Rhiz obium in t e r a cti o n inv olves a se r i e s of plan t - bacterium signal s which activate the success i ve steps in nodulation(Fauc h eret ~l., 198 8;Ualvers o nan dst a c e y,19 8 6 ; Lero ug e etal., 19 9 0 ; Peters et al., 1986; Truch etata1., 19 8 0 ).Af terintro d u c t i on of thebac t e r i ato th eroot surface ofyoun gseedlings,mu l t i p lying bac t e r i a co lonize therootand especia llyadhe re to emerging roo t hairs. Duri ng subs eq ue nt grcwt hof the s eroothairs,dif fe r e nttype s of deforma t i o ns occur.ofwhichma r ke d cur lingor she pherd' scroo ks-type sare most nota ble. Following root d~formation. the ba c t.eria penetrate the rootceLt .swhere the yareimme dia t e l y surro un ded bya membrane. This lea d s to th efor mat ionof the infect i on threadwhich pen etrates the root cortic al cells. coincident withthe development of the inf e c t i o n threadand the passage of rh iz o b ia through, :,~timulates cort ical cell division re sulting in the fo rma t i o n of the nod u l e. The bacte ria ar e released intothe nod ule cellsbyaproc e ssof endocytosisbut
12 they remainsurroundedby a host cel l cytoplasm icmembrane, called theperibac teroid memb r a n e.The peribactero idmembrane protects the bacteroid from plantdefencemechan i sms (Va nc e , 1983) .Insid etheperibac teroidmembrane the bacteriaundergo morphologicaland biochemicalchangesand becometrans formed into bacteroids . At this stage nitroge n fixat ion begins . Meanwhile,the hostcel ls have synthesized a seriesofno d u l e- specific proteins,the nccui rne (Downieetal.,198 8 ;xou cm et al., 1989). Among these nodulins, the apoproteinsof the leghemoglob ins are mos t obvious. This sequence of steps is rep r e s e n t a ti ve for many pla nts a-id their corresponding bacteria. Howe v e r in some plants, lik e thepe a nu t s , in f e c t i on through rootha i r s or the fo rma t i on of inf ectionthreads is not observed. Instead, direct entry oc curs through weak places of the epidermis suc h as at the junction of lateral roots. The bacteria muLt. dpLy in the inter ce llula r space (Ch a nd l e r , 19 7 8 ) and from thereenter the root cells (c r a c k infection).
1.3.6 .Mec hani smof NitrogenFi:lc:atio n
The basic mechanism of nitr"gen fixation is similar in all cases stUdied: atmospheric nitrogen is reduced to ammo nia insidethe bacteroidof the nodulebythe followingreaction:
N2+nATP +6NADPH+2W -->2NH~'"+nADP+nPi +6N/l.DP+ee
13
where,n'" 6.5 (6.0 w 6.9) ATP/NH/ (Rawsthrone
ee
al. ,1980). This reaction re quires i) nit ro genase, ii) a strong red uci ngagen t, iii) anenergy source suchas ATPand iv) lo w cxyqe ntension.Nitr,:)genase is loc a t ed ins i d e the bacteroid and its product ionis coded bytheni f gene complex.The struc t ureof nitrogenasevar ieslittle among nitrogen- f i x i ng organisms.In al l cases there are twodi s ti nc t compo ne nts to the actIve enzyme complex; the Fe prote i nand the Ma-Fe protein. The Fe proteinconsists oftwo subunits(mol.wt •60,000 Ca) and the Ma-Fepr ote in cons is tsof four subunits (mo.wt. 200 , 0 0 0 ne). The Ma-Fe protein is reg ard e d as the true dinitrogenase becausethe reductionofni t r o g e n takes placeon this prote i n and the Fe protein is designated as thfl dini tr o g e na s e reductase (Hagemanand Burris, 1978, 1979 ). The functioning systemis refer to as 'n itroge na s e'.
De p e n d ing upon thenit~ogen-fixingorganism, the source and natureof elect rondonorsare variable.NADPHis utilized as a reducta nt (Wo ng atal. , 19 7 1 ; Yates,1977) inRhizobium.
Th e use of NADPH as an elect ro n donor in nodule metabolism becom es very clear by the pre sence of NADP+- s p ecific Lso- citr a tedehyd ro ge nase in th e bacte ro idof thenodules (Bra y, 1983).
14
Nitrogen;::.se requires a lot of energy (ATP is the major source) to function in the symbioticprocess. Each mole of nit rogenreduced to ammo n ia cons ume s 12- 15moles of ATP making biological nitrogen fi x at i o n a bioenergetically expensive process (Gu tschick., 1980 ).Photosynthesisand respirationof thehost plant pr o v i de the ATP.
Nitrogenase is ver y sensi t ive to oxygen so it is very impor tant that low level of oxygen be mai ntai nedin thenodule for the activation of the nitrogenase. Leghemoglobin, an oxygen-bind ing protein, controls the oxygen flow to the bactero i d inthe nodul e (Appl eby, 19 8 4 ). The noduleanatomy also pla ys an important ro le in the r-e q u LacLcn of oxygen concen trat ion (Huntet al., 1987; Wittyetai., 1987; Oakora andAtkins , 1989).
1.3 .7 . Hemoglob inin Nitrogen Fixation
The re d pigment, hemoglobin, found in all nitroge n-fixing nodu lesof bothleg umes andnon-legumesplants (Wittenberget ai., 19 74 ; Bergersen and Turne r, 1975; Appleby, 1983) is cal led leghemoglo bin (LHb) in the legume nodule. Recen tly, hemoglo b i n geneha s also be en reportedintheroottis s ue s of pl an ts wh i ch are no t inv o l v ed inany symbiotic associations ...ithbac tero i d s fornitrogen- fixatio n (Applebyet ai.,19 8 B). The oxyg en - bindingnature of LHb plays an importa nt role in nit r o gen fi xation by contro l ling the oxygen flux to the
15 bacteroidwhere oxygen-sensitivenitrogenase is present. The amino acid sequenceof LHbshowsextensive analog ie stothos e of myoglo bi n and hu man hemog l o b i n (Ellfolk, 1972) an d the tert iarystructure ofLHbbea rsa close res embla nc eto that of anima l myoglobin (Vai ns h teinet al., 1977 ). LHb s generally show structuraland functio nalheterogeneityfor thei roxygen- binding func t ions (Uhed a and Syono, 1982a,b; Hol1 et al., 1983;Kortt eta1., 1987; Kuhse an d puh Lez,19 8 7 ) except ina fewPhaseolus spp• (Lulsdo rf and Holl, 19 9 1 ). As withother oxygen-ca rryi ng hemop rotei ns, LHbis sensitive to autoxida- tion, which resultsin the formationof FerriLHb, the oxida- tion form of Lilb.Thisform of LHb is unabletobi nd oxygen and is inactive in thenitroge n fixation pro ces s (Wittenberg et: al., 19 74 ) . Duringthe aging of the nodules the LHb is ir r e vers i bly converted to the green non-funct iona l form and nitrogenfi x at i on ce ases .The totalamountof proteinand LHb ....asfou ndto behighin th e rednod u l e s , but both dwind l ewith the onsetof nodule se nesce nce (swara jandGarg,19 77 ).
DuringLHbsynt hesis , th e heme part of LHb is derived either fromth e bacte roidofthe infected cell (Ap pelb y ,19 84 ) or the mitochondria of an un infected cell (Dimitrijevic et a1.,19R9) whilethe gl ob i npa rt is sy nthesized in the ho st cytoplas m.Bot h heme andglobin are assembled in cytopla s m of the ho s t cel l (Verma et al . , 19 7 9) . The synthesis of fun c- tionalnitrogenase by legume nod ul e bacteroidsisde p e ndent on
16 the prior appeara nce of LHb(App leby. 19 8 4 ).
Pre vi ous l yitwasthoughtthatLHb was onlyre s t ricted to thecyt o p l asmof infect ed cells (Verllaand Bal, 1976 ;Berger·
se n and Appl e by,198 1 ; Rob e r tson et al•• 1984) but, rec e n t l y , LHb has be en localiz e d in the ground cyto p l a sm of both inf e c t e d and uninfected cells of soybea n nodules (Va nd e nBosch and Newcomb, 1988 ).
Numerou spr o cedure shave been used to est im a t e the LHb content of nodules. These ar e gen era lly modifications of clin icalte chnique sdevelopedfor hemoglob in an d are basedon the opticalabsorp tio n of deoxygen a t ed LHb (I<e l lin andWan g'.
1945 ;Johns on and Hume, 1973).pyridi nehe mochro mogen(Vi r t a- nert at al., 19 47) or cyanomet LHb (Wils on and Reis e nau er, 196 3 ).U1bcont enthas als obeenmeasuredtluoroftle t rically (La Rue andCh ild , 1979).
I.3.8.Ammon i Wll Assimila t i on and Tra nspo r tat ion
The firs t st a ble produc t of nitroge nase acti vity is the ammonium ion. Itisge ne r al ly ac c epted thatammoniumIlust be assimilatedquickly. In nitrogen-fi xing syste r,s;therear etwo reasons for this - ammonium is tox ic and it su ppr esses synthe s i s of nitrogenase.Althoughthere is so me evidence that rhizobia can metabolize a high concentration of ammonium (Di l wo r th andGl e nn , 1982), their hostcell sprobably ca nno t.
In the nodUl e, ammoni um is export ed frolllthe bacteroid and
17
assimilatedbythenod u l e -speci fic enzyme, gluta mi ne synthe- tase (GS) in the nodul e cytosol (Duk e an d Hens on , 198 5 ; Pate and Atk i n s ,198 3).The presence of GS in the bacteroidisal so re ported but there is no evidencethatthis plays a subs ta n- tialrole in the as simila tionof fixe dnitroge n. In summary, the bac teroids red uc enitrogen ga sinto ammon i umwh ich passed to the host cellfor as similat ion- agoo d division of la b ou r bet we e n thesymb iontandthe ho s t (sprene , 1987).
Followi ng the assi milationof ammoniumintoglutamine,it istra nsportedto othe r partsof the plantthrough the xylem (Wal s h et al., 198 9) . Th e le g ume nodule transp orts its ni t ro ge nous compounds ma i n l y as ami d e (as pa r agine and gl ut ami ne) or ureides (allantoin orallantoicaci d), depe nding onth elegumespe cie s.Usually ,temperate le g umesexp o r tami de (She l p andAtk i ns, 1984) and trop ical andsubtropic al ones export ureide (Schube rt, 1386; At k ins, 198 7 ) fro m their nodul e s. Apart from ami de sand ureides, man y other nitrogen- co nt a iningcompo un ds are exported, but insmalle r quantit ies (Pa t e and. Atkins, 1983 ) .
I.". Pbotosynthesisllnd Nitroqen-fixation
Photosynthates provide the energy and carbonske let ons for nitrogen fixation (Fig. 2). In additionto this, the nod u l e uses energy for itsgrowth and maintenance.The total cost is 15-30\ of the totalass im ilation ca p a ci t yof the plant ; for
18 example, 12 9 of carbohydrate is requiredfor everygramof nitrogen fixed (Gutschick, 19 8 0) . Aco n t inuou s supp l y of photosynthate to the nodule is essential for cont inued l"itrogen fixa ti o nin intact plants (Ba c h atal ., 1958 ).The clos e link betweennitrogen fixat ion and photo s ynthesi shas been investigatedin differentlegumes(Lawn and Burn, 1974;
Streeter, 1974; Hardy an d Havelka, 19 7 6; Rainbi rdet al., 19 8 3a ; ching et aI., 1975; swarajet al.,1986, 1988;Go r d o n et al., 1986 ; Ryle et al., 198 5a ; Pfeiffer et aI., 198 3;
Whe eler , 19 7 1 ). All these stud i es indicate that nitrogen fixation gradually decrea s es under photosyntheti c st r ess condit ionssuchas darkness , defoliation , detoping, shading and cloudiness. ch i n g et al. (1975) observed a r-eductLon of ATPby 70%, sucrose by60%, AOP by 60%, nitrogenase activity by 50 %andenergy ch a r g e d by15%insoybe a n rootnod uleskept in the dark for oneda y. The leg h emog lo bi n and total prote in of the nod u l e also decrea'se under photosynthet ic str ess (Pf e i f f e ret al., 1983 ; Swarajat al., 1986).Reservecarbohy- drates of th e nodule may also be used in the absence of photosynthesistosupport nitrogenfixation (Gers o n atal., 1978; Hostak at al . , 19 87 ) . Nodular disi ntegrationdue to photosynthetic stresshas alsobeen demonstrated inso yb e a n (Coh e n at al., 1986) and in whiteclo v e r (Gordon et al., 1986).
19
Figure2.Sc hematicdiagram showingrelationshipbetween photosynthesisandn~troge nfixation (Bidwell , 19 7 4)
2 '
N'- "--- - - --->
Figure 2
21
1.5.Peanu t (Arach i s hy poga e a L.):Taxonomy,Distributionand Import ance
The qenusArachis belongs toth e family Fabaceae(Le g umi no s a e j and sub -fam ily papLj.LonoIdeae and occurs in tropical and aubtn-opica I regions. Basedon morphology andcross-compatibi l- ityth e genusArach i shas be e n divided intoseveral sectio ns (wyn ne and fial ....ard, 198 9 ) . Th eyare nativeto SouthAmerica buthavebeen introducedint o many other areas. A. hypogaea, calledbydifferent pop ular name s such as pea nut,groundn ut, gooberetc, is the onlyspecies inCUltivation.According to archaeological data, peanuts have be e n cultivated for over 3,500years, during whichti me numerousmorphologica l forms have evolved (Hammons, 1982). Plants areannual or perenn ial he r bs and have a well-developed taproo t system with many la t eral roo t s emergingfr om the hypocotyl and aeria l branches. Tile rootsare soft,cylindr icaland lack roothairs, but roo t hair- l ikestructurewerefou nd by Nambiar et al. (1983). The depthof primary rootscan be 90-120cm with extensive late r al roo ts.Peanuts ar e 'Warm-seasonplants,preferring50-100cmof rainfal l/ year, and arebest suitedto well- drai ned, friable , loamy soil containingadequateamounts of phosphates, potas h and calc ium. propagationby cu tt i ng s is possible, but the plant is usual lyqr-own from seeds.
Peanu tsare important tohuman s as a source of nutrition.
Its fre sh fol iageis fe dtohogs and cattle, produ c e s hi gh
22 quality hayand hasva l u easa greenmanu refor soilimp rove- me n t.The flo wers furnishrichnecta r lo r bees. These eds are a ri c h sou r ce of vit allli n B co mpl ex. especially thiallin.
riboflavinand nicotinic acids, andare a source of pr otein and oil (Ahmed and Young. 1982 ). Peanuts rank se c ond to soybean in c01lllllElrc!al impo r t anc eas asourc e of highquality oil characterhedbyth epres en c eof arach idicand legnoc eric acid s as ....ell as glycerideof oleicand lei nole l c acids.The crop yield va ri esfrom742 to 44 0 0 kg/ha (DUke, 1981).
I.5.1.specifioityofPe anut -Rh i z obi umSymbi ollis
Unlik ema ny legum e sthatarenodulated onlyby sp e c ifi cgroups of rh i zo b i a, pe an uts are nodulated by rh izob i a that also nodulatese ve ra l et he r spe ciesof tropic a l leg umi nouspla n ts (AllenandAll en ,1981 ). The follo wing specialfea tur esof the pe anut-Rh izobjumsymbiosisma k e pea n u t ro o t nodul e sdis tin ct;
i) Lec t i ns Ar El generallyimportan t in th espe c if i cRhizobi ulII recogni tion for thehost(Dazzo , 1982).In peanut,lecti n is not important (Pueppkeetal., 1980).
ii) Normalroothairs are absent in peanaes .Instead,tuf ted clusters or ro s ettesof hairs are fr e qu ent l y found in th e junctio nof ro otax ile s (Gorbetand Burton, 1979 ). iii) Noin f e c t i o nthr e a d s are observedinpeanu ts (Ch and ler ,
1978). Peanut nodu les are beli eved to deve lo p from a single infected cell by SUbsequent ce ll divisions. In
24 Ls o a yrae a (CAT- I , CAT- 2and CAT-Jl are enc od ed on the three unli nk ed struct ura lgenes,Cat- I,Cat-2 andCat.-J ,respec tive- ly (Chand lee atal., 19B3) an dtheexpression of thesege nes is hig hl yre g ulat e d both tempora llyandspatially (Redinbaugh at al., 1998).
Virtanen (1956 )hypo thesized th a t catalaseactivity is responsible for pr ev enting the accumulat ion of HlOH whi c h othe rwisema y result in the oxidatio nand inhibitionof th e synthesis of some of the essential substa nces forni t rogen fixat i o n. Recent studies ha ve showntha t leghemoglo blnappe a rs to be suscep tibleto breakdown by H20l (puppo and Ha l liwel l , 1989).Ahi g hle v el of catalase isfound in theheterocystsof Cy a nobacter ia (Henry etal •• 1978) and in theFrankiavesicles (puppoet al., 1989) whereit protectstheoverallnitrogen - fixation pro c e s s aga in st H101. Francis and Alexander (1972) tr i e dtoclar i fyth e role of catalase in nitrogen-fix ingro o t nodules of wh ite clover and soybean and fo u nd a positive corre la t i o nbetwe en ni t r oqenfixat i o n andcatala seactivity.
1. 7 . Lip ase and Lipid Catabolism
Li pa s e , the fi rstenzyme inlipid catabolism, cata l yses th e hyd r o l y s is of res erve triac ylg l ycerol to fatty acids and gl ycero l (Beevers, 1969; Hu tto n and Stumpf, 1969 ). Dependi ng onth e plantspecies, the lipas emay be loc a lize d, ei t he r in
23 contrastwith otherle g ume s, no unlnfected interstitial cells are present in the infected zone (Chandler, 1978) . tv) The bacteroids are large and spherical (Staphorst and Strijdom, 1972) with lipid anddense bodies(Ba l et al., 1989).
I.ll: . Catalase inNitr oge:bPix.t ion
The enzyme, catalase (EC. 1.1 1.1 . 6 ; "101oXidoreductase), is widely distributed in nature and is fou nd in all aerobic micro-organismsas well as pla ntandanima l cells containing a cytochrome systems (Deiss~rothand Oounee,1970).All forms of catalase isolated to date have been shown to consist of four SUbunits of about 60,000 Da each,whichgivesa protein of approximately240,000 Da(Ae b !, 19 8 3 ). The enzyme functions as a scavenger of toxicH102produced by the cellsin various metabolic reactions. The targets of this "202are enzymes, lipids, aembranes and nucleic acids (Morgan et al., 19 7 6 ; Chanceat al., 1979 ). Multiple forms of catalase have been identified in several plants, such as tobacco (Havir and McHale, 1987), cotton (Kunce and Trelease, 1966), barley (Kendall et al., 1983) and also in some bacteria (Heir and YagU, 1984; Seah and Kaplan, 1973). The physiological significance of these mu ltip leisoenzymes is not yet clear. In maize (Zea mays L.), th reebiochemically distinct catalase
25 themembr ane of lipid bodies,or in othersubcellular compart- ments. with few exceptions, lipase activity is absent in ung ermi na t e d seeds , bu t increasesrapidlyduring the germina- tion pe rio d. The mechan isms of lipolysis vary greatlyamong different oil seeds. The oilseed lipases that have been studieddifferwidelyin characteristicssuch asth e optimal pHformaxima lact i vity,molecularwe Lqht;,substra tespecific- ity and subcellularlocation (Huang, 19 8 3 ).Lf p a .se s are widely distributed in animals, plants, fungi and bacte r ia l cel l s (Huang, 19 8 3 ).
The fa tty acids rele a s e d by lipa s e acti vityare metab- olized by the B-oxidation sequence to produce acetyl-coA, Whichis processed throughth e glyoxylate cycle.The enzymes of the e-cxteatrcn seque nceand gl y o xy l atecy cle s arelocal- ize d exclusively inth egl yo x y s ome s , which arespecial types of peroxisomes (Beevers, 1969; Hutton and Stumpf, 19 6 9).The lipid bod Le aandglyoxysomesarein physical contact with one another (Fr e d er i c k etal., 197 5 ),whichfacilita tes tran s p o rt ofac y lg l y c e r o l s or fattyacids from the lipidbod ies to the glyoxysomes.Succinate,generatedintheglyoxyla tecycle,is releasedfrom the glyoxysomes and isconv e r t e d eventuallyto sucroseand othe r metabolites (Huang, 1983).
I.7.1. Lipid.in Nitro gen Fixati o n
Lipids are gene ra lly important components of cellul ar
I ,
l
2.
org a nellesand arealso used for energysto r a g e inec e t;ofthe seed s, es pe c ia lly oil seeds. Lip ids are synthes i ze d in plas tidsandstored in special structurescalled lipidbodi e s (Ching , 1972; Linand Huang ,19 83), oil bodies (Rou g h a n and Slack, 1982; Herman, 19871.sphe ros o mes (Muhleth ale r, 1955;
Frey-wys sling at 0111., 19 63; Hrsel , 1966; sorokin, 1967 ) or oleo somes (Yatsuet al., 1971 ) . A lipid body ha s a matrixof tr i acyl g lyc ero l su r r o un de d by II ha it-u n!t memb r a ne of one pho s p h o lipid la y er (Huang,198 5; Yats uandJack <:.,19 72).The y are sph er i c al bo d i e s about 1 Jlttl in diamete r and highly ref r acti leunder the light mi cro scop e (Yats uet0111., 1971). Al tho ugh the syn thesisoflip i d s (triacylglyr:erol)inpla s tid s is well known, th e format ionof lipidbodies is still contro- versial. It has beenproposedthatlip i d bodiesoriginate fr o m the ERby the accumulationof triacylglycerol between the two laye r s of the ERunit membr a n e andfinally pinch off tofor lA the lipid bodies (Wa nn e r and 'rneteer, 1978 ; Wanner ata1., 1981). Alternatively, lipidbodies may arisein the cytoplasm by an accu mulationof triacy lg lycerolfollo....ed byth e fo rma- ti on of the memb ra ne (Be rg f i e l d et a!., 19 78 1. Theformation and functi on of lipi d bodies have been well studi ed in differentse e ds (Warnneret al., 1981 ;Huangat al.,198 6). It ha s been we ll document e d th a t lipid bod ies in se e d s provide ene r g yand ca rbon ske letonsfor the gr owth and deve l opme nt of newcells during germination (Huang at al., 198 6 ). But the
a funct i o nof lipid bodies innitrogen-fixingroot nodules has not bee nelucidatedsi n c e th e ywe r e reported in the peanut (Ha meed and Bal, 1985), in arctic le gu me s Oxytropis.lIIa y d e- lli anaTrautv. andAstragul au5alplnusL. (Newcomb and Wood, 1986; Pre vostand eei, 1989) and in the su b-arct ic legume, Lathyrus lIIarl t lmus (Bal and Bari mah-As a r e, 1991) . In the peanu troot nod ul e. lipid bodi e s are present inboth infected and uninfec t ed cells of al l deve l opmentll l st ag e s of nodul es, inclUd i ng tho se Wh i c h ar e senesce nt . Compa red with matu r e nodu l e s (active inNJ fixa t i on). immatureone s (tho~1 ewhi ch la ck legh e mog l o bin l conta i ne d the maximum number of lipid bodies(Jay aramandBell, 1991).ThisIs proba blydue to ala ck of Nl fixation in th e absence of leg hemoglobin (Berger s e n, 1982).It ha s beenspeculatedthat lipidsmaysu p po r t nitrogen fixa t i o n and/orhelp in increasingth e tempe r a t u r e withinthe cellto facilitategrowth and develop ment atco l d temperatures in arctic le gume s (Newcomband Wood,1986 ) . In peanu t nodules , th ey may providea supplementaryenergyso u r c e for nit r og e n fixation(Ba l eeal . , 1989).
I.B. Obj e o t ive s
This study was und e rtaken to determine the ro l e of lipid bodie s innitrog e n fixa tio n in the root nodul e s of the peanut.
ae I . '. Ou tline ot' MethodsEmployed
Nodulated plantswere sUbjectedto stress conditions so as to crea te an extra demands for energyand carbonrequired in ni tro ge nfixation.In effective no d ul es were used to investi- gate the stat usof lipid bod i es wi tho u t nit rogen fix at i o n.
Stressconditionswere imposedbykeepingpla ntsin dark and bydet.o pLnq,
at intervals during the periodof st r e s s , the rat e of ni t r ogen fixatio n (as measured by C1H1reduction ), the numberof lipid bodies, leghemoglobinandtotal protein contents were meas ured to determineanyco r r elat ion with nodule runcetcn,
theultrastructuralchanges ofnod ule s due tostress were observed to determine i fstructura l integrity islinked to nodu lefunct ion.
catalase activityat different inter val.s of darkness, oxidationof exo genouslinoleoylco e nzymeA(LYL-CoA) and mal ate syn thase were assayed in nodul e homo genate to determineoperat i on of a-oxid ation pa t hwa y and glyoxy late cycle.
2 .
cytoche micallocal izationof lipa se,ca ta la s e andmala te sy nt ha s e vas conducted in nodule tis s ues as further ev iden c eat' a-oxida t ion pathway and glyo xylat ecyc l es.
mor phome t ric cOllpa riso n of lip id bodies wa s do n e in nitrog en- !i xing (fi x ·)an d non- fixing (fix") nodules to clar ify the involvemen t of lipi d bodies in nit roge n fi xa t i on .
Chapter II MATERIALS AND METHODS
II.1.Rh izob i um strainsand Medi um
Bradyrhizobium sp , strain 32Hl....as obtained from Nitraqin, Milwaukeeand strains NC92, 639 and 7091 were obtained from Dr. P. T.C. Nambiar, InternationalCrop Research Institute forSemi-Arid Tropics (ICRISATj,Patencheru, India.The strain 639is a kanamycine resistant Tn5 mutantotthe wild type Ncsz (Wi l s o n et al., 1987). The strain 7091 is an ineffective bradyrhizobial iso l a t e fromICRISAT. Bath NC92 and 7091 ....ere found to be kanamycin sensitive. All strains were culturedin broth yeast extract mannitol (YEM) medium at pH 6.8 - 7.0 (Vincent, 1970) withconstan t shaking (140- 150 rpm) at room temperature in an orbitEnviron-shaker(Lab-lineInstitutions Inc). Six-dayold cultureswere used in all experiments. 'the YEMme d i ulII consists of ma nni t o l (10 9), K~HP04 (0.5 g), MgS04'7H 20(0.2 9),yeast extract (0.4 9) and distilled water (1 litre). Rhizobial cul tures were maintained on YEM agar slantsinculture tubes and stored at 4°C (or lyopt. ilizedthe cul ture) for future use.
31 11.2. Plant Haterialsand Growi nq Condi t ions
Seeds of peanut(ArachishypogaeaL.cv , 'J umbo virginia'and 'Earlyspanis h') and cowpea (Vi gna unguicula taL. cv. 'Cal i- fornia Blackeye ') were purchased from w. Atlee BurpeeCo., Warmi nster , PA, USA. The pe anut seeds we r e placed onmoist pa pe r towel in a trayfor 5- 6 daysfor germinationand then the seeds were inoculatedwit hBradyrhizobiumsp.from a broth cul tu r eas describedb~'Senand We a v e r (1980).In th e case of cowpea, the seeds wer ewash ed (3 - 4 times) and soaked in dist i lledwater for approximatelytwelvehou r s be:l:'oreinocula- tion. The inoculated seeds of both peanut and cowpea were planted individuallyin pots (6" STD) withaucccf.evedvermicu- li te. The potted plants were ke pt in an enviro nment chamber W'ithapproximately700 J.Lmolemole''PPFD (photosy nthe ti cphot on flux density)under day/night conditio nsof 16h/Bh, 27'C/22'C and 70%/50% relativehumi d i t y and irrig a t edW'ithani t r og en·
free nutrie ntsolution (Ellfolk, 1960),
II.3. Dar ktreatllle n t andDetopi nq of Pl a n t s
After 45 daysfor peanutand28 days for cowpea plan ts, the li ght s in th~environmentchamberswere turne d off. Half of the pla nts were detopped (th e stemwas cut off at ground level). Nodul atedplan tswere samp ledfromdarkand detopped tre a t ments after0h, 3n, 6h, 12 hI24 h, 48h,72hand 96
h for themeasuremen t of acetylene reduction, catalase assay, total protein andleghemogl obin . Sampleswere alsoprocessed for cytologicaland ultrast ructuralstudies. Three plants were used at eachti me interva l foreach sample.
IX... E:~ectrobMicroscopy (EM)of"aamp~es
Nodules from three plants were slicedand fixedin a mixture of 4%paraformaldehydeand 2. 5 % glutaraldehyde (Karnovsky, 19 6 5) in O.1M Sorensen's buffer for 60 min at 4·C. After fixing , the slices were washed withthesame buffer (pH7.2) at lea s t 3 times for 15 - 20 min at 4·C. Thewashing was followed by post fixati onin1%osmiumtetroxide(O S O~ )in the same ')uffer for 60 minat 4·C.The samples were washed again inthe buffer and dehydratedin an ethanolseries (351:,501:, 70%, 80%, 95% and absolute ) with 5 min exposures in each concentration except 20 and 60 min in 95% and absolute respectively. After dehydration , the sections were passed thro ugh the series1: 1,1: 2 and 1:3 (v /v)of absolute ethanol and spurr 'sembeddingresinmixture (Sp u r r ,19 6 9 ),le f t for 30 min in each mixture under vaCUUI". Th e sections were then placed in 100% resin for approximately twe l v e hours under vacuum, and embedded and polymerized at 70uCfor atle a s t 8 hours.
J3
Ultrat.hinsect i ons were cut witha Sorvall MT-l ultra- mi crotome .The sections werepostwstained ...it h leadcitrate anduranyl aceta te be f o r e obser vation under a zeiss 109 transmissi on electron mi c r osc o p e.
XI.S. No cSu le NUmber
Nodules ind uced by dif f e r ent st r a ins (f ix" and fix "') of Br adyrh i zobium sp, werecounted on42-day old plants .Fifteen healthyplantswere selectedfor each samplingand the nodules were counted . Mea n value s and standard er r or s ofmean were calcul a t ed.
J:I.6. Nitr og e nase As say by th e Acety lene (ClH1) Reduction Te chnique
Nitrogenase activity of nodules ....as assayed by mea s uri ng reduction ofacety l e ne to ethylene(Hardy at a1.. 1968). Eac h root system with nodules (abo ut 100 ncdu Les] was enclosed singly in13 mlva cutai n e r euc e efitte d withairti g ht rubber stoppe rs, Using a pressure lock gas syringe (pr e c i sion samplingcorporation ) ,O. 1 atmofai r wasreplacedbyO.1at m of acetylene (fresh lygener<>.ced in th e laboratoryfrom ca l cium carbide ) , Th enod u l at ed root was incubated for 3 h at 23± 2'C,Controltubes : (a) 0. 1atm acet ylenewithout the nodulat- ed rootand (b) nod ul ate d root without acetylene. Gassa mpl e s
,.
were ana l y z e d in a BasleT\( Gas Chromatography GC 9700.
Nitrogenase activitywascalculated by using standard curves for acetylene and ethyl ene an d was expressed as ~moles of et hyl ene h-I g-I fresh weightof nodul e . Each assaywas donein tripl ica t e .
II.7 . Lipid Body Preservation. st ai ni nq and countinq For l ip i d body preservation , nodu l e sliceswe r e fixed and pr oce sse d as described for EM (in I I.4 )except samples were tr ea ted withU p- Phe ny l enediamlne(pPD) in70t et han o l for 1 h duringdehydratio n(enblocs tain i ng,Boshier etal.. 1964; Bal et 81.t 1989 and Bal, 19 9 0 ). For controls, the samples were treated with hexane for 45 min after fixation and dehyd ration (Bal, 1990). After dehydration , the samples were embeddedin spurr 's med i umas mentionedabove.
The semi -thin sect ions (1-2 ~m) were cut from both tr eat e d and contr olblocks, viewedunderlight microsco peat 40xand the number of lipi dbo d i es pe r unitarea wa s de t er- mined. For ea chtr e at ment , several sections were used from five differe n t tissuebloc ks.
A new techni q u einvolving stai n ingof the sections (from blocksnot enblocstained) "'it hpPOwas also developed. Thick se ctions we recut from the tissueblock,pla c ed ona sl i deand dried by ge n tle he ating. Aft ercooling, the sections were
J5 dipped in1%pPD in70\ ethanol for about 3-5 min in the dark and then washed, first with 70% alcohol and then with distilledwater. The slidewas passed over a fLameto dry the section and then mounted witn one drop of histoclad and a cover slipto mak.e a permanentpreparation.
:U.8. Sample preparati on
rI •••a, Nod.u l eand. Ba c t e r oid Fract i o ns
Fresh and healthyroot nodules were removed from 45-day old plants, washedlrii t h diEtilledwater and homogenized gently with0.05M buffer containing 5mMEDTAat desired pH in a mortar. The homogenatewas then centrifuged at 265 xg for 10 minand the supernatant was further centrifuged at 14,000 xg for 10 min. The reeuftent; pellet and supernatant were con- sidered bacteroid and nodule cytosol fractions, respectively after microscopical observation. Before using the bacteroid fraction,it was washed with buffer until the supernatant from washing showed no catalase activity. To solubilize the peribacteroid membrane (PBM),the pellet was treatedwith0.1t Nonidet P""(Bal et al., 198 0 ). washed and resuspended in buffer.
The bacteroid fraction was thenpassed through a modlt'led Hugh'spress (Model9AB Biotec, Sweden) at 2.6metric tons/cm1 to break the cells. The cell walls werepelletedat 200,000 xg
for 1 h in a scrveti OTO-5ultracentrHuge and washed with bUffer to produce a cell-freeextract. Throughout the experi- ments, phosphate and glycine-sodiumhydroxide buffers were used and every step ....as carried out at4·C.
11.8. 2 . Ce ll- f r ee ExtractofCu 1tu re d Rhizobium
The6- d a y old cultured cellswere collected by centrifugation, washed with buffer and disrupted by sonication for)-5 min at short intervalsor with the modifiedHugh'spress(a s above).
The sample was centrifugedat 200, 0 0 0 xg to se para t e cell walls (pellet) and cellcontents (s up e r nata nt).
:n . 8. 3. seed.cytoso l
Peanut seeds were prepared fo r germinati on (see II . 2) and after three days the testa and embryo were removed . One co tyl e d o n wae ground in 5ml of appropriate bufferat the va r i ous experimental pH. The homogenate was centrifuged at 6500rpm (regular centrifuge )for 10min. The centrifugation produced three distinct layers: a thick lipid layer (t o p), seed cytosol (middle)and cell debris (bottom) from which the seed cytosol was removed witha glass pipette and wasus e d for the catalase assay and gel electrophoresi s . Phosphate and sodium hydroxide buffers (0.0 5 M) were used throughout the experiments.
37 II.' .D.terminatioDof Leqhamoqlobin ILHb)
A co lorime t r i c assay of LHb in fr esh nodul e s ....as carriedout using the procedure of Wils o n and Reisenauer (1963 ) . The nodul es (O.OSq) weregroundinJml Drabk in 's solu tion(52 mq potassi umcyanide,19 8mg pota s s ium fer ricya n i de and1 9 sodiu mbicarbonateto 10 0 0 ml of dist illed wat er ).Thenodule sampl ewas centr ifuged (15minat500 xg) andthe super natant was saved. The LHb....as extracted twic e more fro m the nodu le tissue (pellet), th e supernatan ts were comb inedandthe volume adjus tedup to10 ml with Drabki n 's solution. The resu lting supernat an t was centrif ugedat20, 00 0 xgfo r 30 min,pro du cing a clear sol u tionof host cellcytoplasmthatwas used for LHb determination.
A col o r irnetr ic assay of HLbwas performed in a 1.0em cell at 540 nm against Dr abkin' s solutionwith a Spec t r onic 1001 taa us c n &Lomb) . A standa rdcurve was pre paredwi t h known amountsof hemog lob in (Rb) in Dra bk i n ' s so lu tion (0.5, 1.0, 2.0, 4. 0and 6.0 mg per 10 mI) so that the absorbanceof the sampl e readings cou ld be extr ap o l ated fr om the sta ndardcurve tofind theamount of LRbinthe samp le. Th e fina l resul t was expressedin mg of LHb/gm fresh weigh t of nodul es.
11.10. Cata1aaa 11.10 .1.Cata l aseAssay
The enzyme, cat a l ase, wa s as sayed spe ctr ophotometric ally
J8 accordingto the method of Beersand Sizer (1952 ).During the assay procedure,a cons t a nt amountof prot e i n(0. 5mg) in the sampl e (in 50mMphosphate buffer) wa s takeninJ ml cuve tte and made up to 2 ml with the same buffer. Incase of acti vi ty inhibition, 0.2 M aminotria zol in phosphatebuff e r was used along with the sample. To this samp le, 1ml of 59 mMH101in the same buffer was addedjust before starti ngthe rea c ti on (zerot ime).As aco nt r o l,onl y bUf f erwasus ed . There acta nts were mixed thor oughly and the chang e in absor ban c e was recorded ev e ry10 sec o nd s intervals for 70 sec ondsat 24 0om on a Spectronic 1001 (Ba uch and Lomb). Th e specificac t iv ity ofcat a lase wasca lculatedas units/ mgof pr otein:
changes in absorbanc e / mi nutes x 10 0 0 Specificac tivity ""~---~---~--- 43.6x mg protein / mlofrea c t i on mixture
where 43. 6""Molar Absorban ce Index for H20 ] and one enzyme unit is equ a l to one ~mole of HP 2 decomp ose d per mi nut e (Worth ingt on Enzyme Manual ,1972 ).Pr o t ei nwasmeasuredin the sample byLowr y1s me thod (1 95 1 ) calibrated withbo vin e serum albumin (BSA).
39 11.10 . 2. 1r.s sa yat Differ e n t Temper.atures
Th esonicated ba ct e ri al cells(in 0.05Mphosphat e buffer, pH 7.0)we r et reeuedat different tempe ra tures(70"C, 65°C,GO"e, 5S"C, SOoCand 40"C) for 5 minin a wa t er bat h.After co oling toro om temperature,theca t a las e activitywas measu redalong withacontrol (withouttreatme nt).
II.l 0.3. Cyt oobemica lLoca liza tion
cytochemical loc ali zat ionof catalase in peanutrootnod u l e s and in Bl-adyrhizobium sp. 32Hl was done by using the 3,3- diaminobenzidine (DAB) rea c t i o n fo l l owi n g the pr oc e d u re de s c ribedbyFred erickand Newcomb (1969).Thesamp les(small segments of noduletissues and bacteria l pe l l e t ) werefix e d in a Kar no v s ky in 50 mMpotassi umphosphate bu f fer atpH a.0 (l(ar n ovsk y , 1965) for 1 h at4°C and thenwashed thoro ug hly withthe sa me buf f e r for at le a st20 min (3 - 4 times) at 4·C . Aft e r washing, thesamples were pr e-incu b ate d in DAB solut ion (2 mgjmlof 50mM2-amlno-methyl -l,3-propan ediolbufferat pH 9.5) . Pre incubation wa s followedbytheincubationof samples ina H10 ! contai ning DAB sol utio n (0.1 ml of 3%H202in 5 ml) for 60 min at J7'c with cons ta nt shaking. Aft e r this, the sampl e s were was hedwithbuff er (3- 4times) and post-fixed in 2t osmiumte t ro x i de in50mMpo t a s siu m phosphatebuffer (pH 6.S) for 2 h, wa shed wit h bu f f er andprocessed for el e c tr on
microscopy (se c t ion IIo4).
Three centrolswere run alongwith the tre a t men ts: a) 0.02 M 3-amino-l,2,4-t riaz ol e (a competit ive ca talase inhib ito r ) was add e dtothe DABsolut iondu ri ngpre -i ncu ba tio n time, b)pre-dncubat.Ion andinc ub atio n were bo t hdon e without DAB, c) inc ubati onwithout thesubs t r a t e, H10 1•
:II.11 . Malate Syn tha s e
:II.l~.~.Assa y
Th e presenceof mala t e synt h asewa s dete rmi n e d inth e nodule homogenate us ingtheme t h odof Dixon andKor n b ery (1959) . This me thodmea s u res the de cr ea s e in the rate of optica l den s ity (00) at 232nm dueto bre a ka ge ofthethioe ster bon d of acet yl Co en z ymeA inthepres ence of glyo xyla te.The reactionmixtu r e co nsis ts of 3ml Tri s HCl buffer (0.1 M, pH8.0), 0.15 ml Mg2Cl (0.1M), 0.10 pI acetyl CoA (10 roM) , (1.10 ml nodul e homog e n a t e (contained 0. 4 -0 .5 mqpr o t ein) and 0.0 2 ml Na- gl yoxylate (0.02 M). The reactio n mixt ure , exc e p t for na- glyoxy late , was pl ac e d in a 3 ml cu v e t t e and the spectrophotometerwasse t at zero andall owed t.ostab ili zefo r several minu tes .Then gl yo xy l ate wa sadde d inth e cuve t t eand 0.0 . changeswere recordedfor 2 minutesat 30sec inter va ls.
specificactivity is expressed asun i t s of ena yme smqprotein. Oneunitot enzyme is definedas theamount thatca t a lyse sthe
41 cleavage of 1 ,umole of acetyl coenzyme A permi nut e .
11. 1 1.2. cytocbemical Localization
cytochemicallocalizationof malate synthaseinnod ul eti s s ue s was do ne accordingto Tre lease et al. (1974). Mature nodule slices were fixed in a mixtureof 4%paraformaldehyde and 1%
gl u t ara ldehydein 50 roMphosphatebuffe r (pH6.8) for20-3 0 min at 4·C . Afterwashing thorough lywith 3-4 changes of the above buffer (20 roM) for 15- 20min, the samples were pre- inc u b a t edwith 3mMpotassiumferricyan idein washing buffer for 30min atroomtemperature. The sampleswere washedagain for 15-20min withthesame bufferand the n incubated in an incubation medium for 40 min at 37°C in a water bath with co n stant shaking. The incubationme d i umwa s preparedimmedi- atel yprior to use byadding the fo llowi n g compo und s inth e order indicated belowwith consta nt stirring between each addition: 0.30 mlX-pho sphatebUffer(65mM,pH 6.9), 0.20 ml copper-tartrate solutio n (pH 6.9), 0. 25 ml distilledwater, 0. 0 3 ml potassium ferricyanide (150mM)and 0.10ml acetylCoA (1.0mM) . As a control, sampleswereincubatedin media from whic h eit her acety l CoA or glyoxyla te or both had be en omitted . Af t e r the incu b ation,sampleswe r e rinsedfor 20-30 min with several changesof th ebu ff er and po s t-f ll<:e d in OsO.
(2% solutionmade up in50mMX-phosp hatebUffe r, pH 7.0) for
42 1 h at room tempe ratu re. They we r e wa s h ed in buffer and dehydratedthroughan alcohol series and embedde dinSpurr' s medium (section Il.4 ). Ultra-t hin secti o ns were cut and observed unde r TEMwit h or without be ing post-staine d in uranyl acetateand lead citrate.
Allsolutionswe r e prepa redoneday beforeuse and stored (4" C) , except for ferrocya nide which wa s made up in a dark battle immediately beforeuse. Th e cop p e r - ta r tr a t e soluti on was preparedbyslowlyadd i ng coppersul p h a tecr yst a l s (0.624 g) to50 0mM Na,K-tartratesolutionand thepHwas adjusted to 6.9\<lithINNaOH.Th e volume was broughtup to 50mlwith distilled water . The final con cent r ati on of the copp e r- tartrate solutionwas 50 mM coppersulphate and500 mM Na, K- tar t r ate.
II. 12 . Linoleoy lCoenzymeA (LYL-CoA) oxid at ion II.12.1.samp l e preparati on
oxidationof LYL-Co A wasdet e r mi n e d indifferentfracti onsof the nod ule (eq,no d u l e homog ena t e, nodule cytoso l, bacteroid, bacteroidwithPBM, bacteroidal cell content and bacteroidal cell wall) andin vitrogrow nBradyrhizobiumsp. 32Hl (wh ole cell, cell wall and ce ll cont e n t) , indi v i d ually . All the sa mp les were prepa red as described in sections II.8. 1 and 11.8.2, exceptbuffer,which was us ed at pH7.0. Each sample was standardized so that constant amou nts of protein
4J (0.5 mg / ml) inthe samples were usedthrough outthe experi- ment s.
XI.12.2. Determination of LYL-CoA Ox i d atio n
One ml of sample was placed ina 60-m l rea cti on flask with 0.4ml of 1 N KOR in a center cup suspended fromth e rubber st o p pe r.The reaction was startedbyinjecting0.1~Ciof [I'e) LYL CoA (Dupo n t NEN products) .Incubationwas carried out for
0, 20, 40, 60 and 80 min at JO'c in a Oubnoff me tabolic shaker .Three controlswereus e d . The reactionmixtureswere as descr ibedaboveexc e pt that they (a) wer e kept at 0 - 4'C, (b) wereke p t atJO'Cbutdidno t contain(l~C)LYL CoAor (0) co ntai ne dablankwith only the KOH. Inc ubat i onwas terminated wi th 0.4ml of 4 N Hel.Th e«co,wasallowe d to be absorbedby the KORovernight and radioact iVitywasmea s uredinaBeckman LS-J150TSc in t i llat i o n Cou nter. Protein fr omthe sampleswas measured by the methodof Lowry et al. (19 51).
II.13. Locali zat i on of Lipolysis
Li po l ytic activity in the lipid bodie s of nod ule ce lls was demonstrat.ed by a method modified fromthat of Blanchette- Mackie and Scow (1981). Nodule slices we r e fix e d with a aldehydefixative (Karnovsky , 19 6 5) inca c od yl a t e buffer (p H 7.2) at 4'Cfor 1 h, washedthoroug h l y in buffer at 4'Cand
"
incubatedat 37'Cfor18 h in1%tannicacid dissolvedin the buffer.Th e controlwasno dul esl i c es incubated in the ta nnic acid/buffer solutionat 4·C . Th e samples were the n treated with 1%OsO~ in K-phosphate bu ffer (pH 7.0) for1 h at <1·C fo ll owe dbyrout i ne dehydrationand embedd ing (section II.4) .
II .14. GelElectrop horesis
11.14 .1 . Preparationand Runninq ofGel
Th i s was performedaccording tothemethodof La e mmli (1970) withafe w modifi c ation s.Slabgel (15 X12em)was used with 4%acrylamidefo r stack ing and10%acryl amideforthe separa t- in g gel. The fi n a l co nc e nt r a ti o n s inth e separating gel were as follows: 0.375 M Trig -Hel (p H7.0) , 0.0 2 5 % (v o l u me) of te t r a me thy l - e t hy l e ne d i a mi n e (TEMEO) and ammonium persu lphate (APS) . Th e stackinggelcontai ned 0.12 5 MTri s-Hel (pH7.0) and0.02 5\ TEMED and APS.Th e runn i ngbuffe r ,Tri s-glyc ine (pH 8.3), contained0.0 625 M Tris-Hel (pH7.0) . 10\glycerol and
o.oon bromophenolblue as the trackingdye.50 p,gof prote in was loaded ineac h well.Electrophores iswas ca r r i e d out with a current of 15\lIAfo r stackinggeland 30rnAfor run ninggel untilthebromophenol bluemarker reachedthe endof the gel.
Theto t al time required for runningwas about7h.
.5 II.U. 2.GelStai ni n g'
Nat ivegel was stained according to the method de sc ri bed by Cl a r e et a!. (1984). The gel was soak ed in hors e radish pe ro x idase (50 1l9/IIII in0.05Hpota s s i u mpho sphate buffer (pH7.0) for 45min."102wa s the n addedtoII conc e nt r ationof 5.0 mMand soakingwas con ti n u edfor 10p'oin.High e r lev els of HIOJred uc e dthe se nsi t i v i t y ofthestain. The ge ls we rethe n quicklyrin s ed twicewith disti lle d water and placed inDAB (0.5 1IIg/ml) in pota s siumphosphate bUfferuntil stainingW3S comp l e ted. DABsolutio n was freshl y prepa re d and keptin the dark.
Cha pter III RESULTS
111.1.Acetyl. n . Red uc t i o DAs.ay lARA) andLipi d B04 ie s (LBI Pe anutplan t s mai nt ai ned a normal rateof A.RAva l ues in the nodules for up to 48h in the dar k. Afte r 48h therewasI'l
dr o p in AMva l ue s (Fig.3). Simila rtrends in ARA valueswere observ ed indet o p pe d peanut nodules (Fi g. 4). In nodula te d cowpe a plan t s, the AM activ i ty startedtodec linewithin 3h of the begin n ingof da rk n e s s and therewas a 40 \ redu ct i onof AM va luesafter 12 h ofdarkn e s s (F i g. 31. Oeto p ped cowpe a nodul esbehavedina similar fa sh i on(Fig.4). Cowpe a plan ts main t ainedat constant temp era t u r e (d a y / nig h t ) alsoshowed a gr a dual decline of ARA ac tivi t y (Fig. 5).
Anothe r se t ofexpe r ime ntswasconductedwith the pe anu t var i e ty 'Ea rl y spa n ish' (F ig. 6) to confirm the res ul t s obt ai ned with the var i e t y 'J umbovirginia ' (Fig. J). The results pr psented in Figure 6 indicate tha t the re was no reduction in ARAva l u e s until after 48 hof darkness . The number of lip id bodies in the nodul e ti s su esof dar ktreat.ed and de t o p p ed peanut plants gradually declined under the experimentalcond i t i ons (Fig. 7).
