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}VAITE INSTII'iJTEs. 8.1r+LItìRARY
ETFECTS OF BORO{ DSFISIEI{CY O{ RNA }ÍETABOLISM
TII Fhaseohn ørcuE ROÛT TIPS
by
Iønfr SAIIUEL ROy CHAPMAÛ, B.Ag.Sc. (Eo¡ts.)
A tbes{e gr¡bnftted for a.rrn{sefoa to ttre degree of
Doctor of Phflosorpþ
Deptútent of Ag?í,et&hpal Bí,ochemisttg
ltuiæ AgrianLhæal Reaeæh fnetítuteThe tlni.oe?sit7 of Ailelaide
South Austw,Lía
Deceúer 1972
I give consent to tk¡is copy of ny thesis, wiren tlepositetl i¡ theiiniversity lribrary, bein6 avajJable for loan anô photooopy,iag.
Dete SigDedl
WAITE INSTITUTE
LIBRARY
lABLE OF CONTENTS
Page
SIIMI{ARY
Declaratfon ...Ackno¡ledgeoentsAbbrevlatioas ...
I. INTROil'qTION
1.1
1,1)
1)
7r,.
l{orphologfcal and PhysíologÍcal Syuptone
1.1.1 luhLbltfon of root elongatlon ...L.L.z fhickeû1og of roots ...1.1.3 Necrosle of root tlps ...1.1.4 Lateral root growth ...
aaa
1
2
2
3
3
4
4
II
L.2
r.3
1.4
1.5
Sone Ge¡eral Hypotheses ...
Nuclefc Acfd Metabolisn ...1.3.1 Nuclefc acfd levels ...L.3.2 lacotporatLon of labelled precurs¡ors
lnto nucleÍc acf.ds ...1.3.3 NucleotideS ..¡1.3.4 Enzynes lnvolved io nuclelc acfd netabollso ...
1.3.4.1 RNase ..ot.3.4.2 ATPase aod other phosphatase
enzjn¡es . . .
1.3.5 Base analogue treatoent ...
Protein l{etabollsn ...1.4.1 Proteln levels ...L.4.2 ProtelE slmthesls
Plant Eo¡mnes
1.5.1 Ar¡xl-ns ...Ar¡xfn levels . o.Ar¡¡in degradatfon ...Relatl.onshlp betrrcen boron, atxlasand nuclefc acid metabollem ...
8
11
t2
l2
13
L4
15
15
16
18
18
18t9
2L
l'5.1.5,1.5.
1,1L.21.3
lab1e of Contente (Corúí.nted)
L.5.2 Gibberelllc acíd .. '
1.6 Outlfne of thls lavestigatLon
2. MAÎERIALS AI{D UETTIODS
Page
2L
22
24
24
26
28
28
24
2.1
2.2
Plant MaterLal ...z.L.L ctrol.ce of plant naterial .. o
2.L.2 Culture of. PhøeeoLug a¿neus ...2.1.3 Root elongatioo Deasurenents ...2.L.4 Eanresting of root tfps ...2.L.5 Incoraoratl.on of labelled Precutsors
into excised root tiPs ...
NucleLc AcLd Extractioû aod DeternlnatLon
2.2.1 Extractlofi ...lotal nuclel.c acldsNucleotides .. o
RNA ...DNA ...
2.2.2 Deterîninatfons
2.2.I.L2.2.L.22.2.L.32.2.I.4
2"2.2.32,2.2.4
2.2.2.L2.2.2.2
Absorptlon spectraRibose ...IÞo:ryribose ...Phosphate ...
28
29
29
29293031
32
32323333
34
34
34353637
39
3940
2.3 Analysfs of, Nucleic Aclds
2.3.1 RNA ...
J.ÈlAK chromatography ...Polyacrylamide-ge1 e lectrophoresLsBase coryositlon . ..DNA-RNA hybrldl-zatlon . r.
1.11.21.3r.4
3.
.3.
.3.
2222
2.4 41
4L
42
43
2.3.2 Nucleotides ...2.3.2.1 DEAE-Sephadex2.3.2.2 Do¡¡ex-l ...
Enz¡rme Activity Determfaatfons
2.4.1 RNase o..2.4.2 Uridine kÍnase ...2.4.3 RNA polyner¿¡se .,.
lable of Contents ßorúiruted)
RadÍolsotope DeterrÍnaÈfons r..2.5.L Lfqutd sclntÍllatl.on .. r
2.5.L.1 General ...2.5.L.2 Dtral-label analysls2.5.L.3 Cerer,kov radl.atfou
2.6
2.7
2.5.2 Gas-flow analysls ...
Other Technfqueft ...2.6.1 Protein deterrinatfons2.6.2 CeI1 co¡¡rts ...Itlaterlals . . r
2.7.L Buffers ...2.7.2 Bfologfcal materials ...2.7.3 Nucleic acl.d conponeûts
2.7.4 Radiolsotopes ...2.7.5 Other materials ...
Page
2.5 44
44
444445
46
46
46
46
47
47
47
48
48
48
50
50
50
51
52
52
53
53
53
55
55
56
3. RESTILÎS AI{D DISCUSSION
Morphologfcal Synptms of Boron Deflcieocy3.1.1 Root elongatioo ...3.L.2 Thickentrig of roots .. r
3.1.3 Browniug of root tfps r..3.L.4 Lateral roots ...3.1.5 Syuptos ln aerl.al plant parts ...Physiologlcal Effects of Boron Deficleucy3.2.L Proteln3.2.2 RNA ...3.2.3 DNA
3.2.4 Acfd-soluble fractfon
3.1
3.2
3.3 Uptake and Incorporatioû of tabelled Precursorsof Nuclel.c AcLds .. o 57
3.3.1 Effects on total uptake and lncorporation 57
ble of Contents
3.3.2
NucleoËldes ...3.4. I Irhc]urraiue lncorporatioû3.4.2 [32Pr] incotporaËfon ...
Fractlonation of RNA ...3.5.1 lfAK chromatography ...
Page
UrldLae concentratlon in thelncubatlon medlr¡n 57
Incubatfon tfæ ... 57Boron deflcleacy ... 58Base dfstrlbutfon or [lr+c] 1n RNA ... 59Other trace element deflcfencLes ... 60
3.3. 1.23.3.1.3
3.3.1.1
Effecte of boron deflcleney ou thespecific actlvlty of labelledprecursor focorporatfon
3.3.2.L RNA ...3.3.2.2 AcÍd-soluble fractl.on ...
3.3. 1.43.3. 1.5
3.4
3.5
62
6263
63
65
67
70
70
70737476
77
80
80
83
88
88
89
90
3.53,53.53.5
.1. I
.L.2
.1.3
.L.4
RNase . r.Uridine kinase
RNA polymerase
Appralsal of the technfque¡ tLgluridÍoe íncorporation
Ilr:el-labelliae studles ...[ 32prl Íucotaãratlon . ..
3.5.2 Polyacrylamfde-gel el.ectrophoresls
3.6 Propertíes of RNA ...3.6.1 Base conposlÈ1on ...3,6.2 DNA-RNA hybrfdlzatfon
3.7 Eazyne Activtty Determfnations
3.7.L3.7.23.7.3
4. GENERAI. DISCUSSION ...
4.L liforphologfcal and Physlologfcal Effectsof Boron Deficienc¡r ...
4.2 RNA Degradatfon
92
92
93
Table of Coatents (Conþínued)
4.3 RNA Synthesls ...
4.4 Plant Eormones ...
4.5 Concluslons ...
5. BIBTIOGRAPEY
Page
95
101
107
110
1.
2.
3,
4
5.
U}fMAR
Mot-phologLcal s5rmptooe and aone phystologfcal effects of boron
deflcf.ency have beea recorded for whole plants of, PluseoLus
caÅreut. Effects of the deflcÍency on $.!De aspects of RNA
netabollsn rtere exæfned Ín the root tfp of thls plant.
Changes ia freeh weLght, cell nr¡nber, cell sÍze a¡rd proteln
content resultlng from boron defLcieocy have been followed in
5 m root tips. ltrese paraseËers, on whleh physf.ologlcal anil
blochenLcal deterninatLons are usually based, are affected
dfffereutly by boron deflcÍency. Thls Lryortant asPect Ls
dLscuesed, Trrlth particular reference to contradictory rePorts
ln the lfterature conceTnfng the bLochernlcal effects of the
deficLency.
lte RNA coûtent of root tÍps decreases r¡nder boron-deflclent
conditLons, but ouly after at least 100 hr of groltth ln
deficlent nedfum. At the same tl-æ, the DNA conÈeat of root
tips and fndlvldual cells Ís Íncreased by the defLcleacy.
The decrease 1n Rl{A content nay be due to fucreased RNaee
actf.vlty ¡vhLch occurs at aPProxinately the saoe stage of
boron deffcieucy.
Boron deficiency results fn an lncrease fa both the total and
the epecl.flc actlvfty of precuraor f.ncorporatLon Ínto RNA
after planta are grotm for ooly 6 hr ta deffcteot medl-rm.
ThLs was the fLrst effect of deflclency noted, and it occurs
1-'1,
mrch earlier thar vlsl-ble sJrrytoæ (tnhLbltloo of root
elongatÍon at 2tr48 hr belng the first). These eff:ete oa
precursor lncorporatLon becone ûore pronotmced the longer the
plants are kept in boton-deflcLent ueditn.
6. Durlng the first 144 hr, boron deflciency fncreases the
lueoryoratfon of precursors lnto RNA without Íoduclag aûy
corresponding change ln the total precursor poo1. ltre
iucotaoratfoa fnto an acid-soh¡ble fractlon ls not affected.
Aftet nore than 144 hr of deflclency' incoraoration fnto
both fractions fs Lncreased.
7. IÞflcfencl.es of either f.ron, rn¿mganese or coPPer dld not
resr¡lt la sLmilar changes la the lncorporatfoa of nucLeic
acld precuraors fnto RNA or total precursor pools.
8. Different fractlons of RNA (separated by llAK ehrmatography
and polyactyl¡n{de-gel electrophoresfs) are affected at
dffferent stages of boron defl.clency. Inftfa[y (flrst 48 hr)
the order of lacreased epeciflc actfvfty of precursor
Lncozporation ts TB-RNA > r-RNA > s-RNA, but thls changes to
r-RNA > ÎB-RNA > s-RùifA (100-300 hr) and eventually to r-RNA >
s-RNA > TB-RNA (nore than 300 hr). RNase degradatloo ef RNA
(partieularly r-RNA) conpltcates the separatlon of these
specÍes afÈer long perlods of deflcÍeqcy (nore than 200 hr-).
9. DurÍng the ffrst 48 hr of boron deflcfeacy, no changes ln the
properties of uewly synthesl.zed (labelled) nNA could be
10.
11.
12.
L.1,1,.
detected by DNA-RI{A hybrl.dfzaËfoa or base coqrosltl-on
analysis. Changes could be detected at later sÈag¿s (144 hr
or nore).
the aetivftles of uridiae kl.nase and Ri{A pollmerase' enzJru¡es
lnvolved in the pathray of lncorporatlon of nucleosides Lnto
RNÀ, were sËudled Ln plant extracta. Sone evldence was
obtalued suggesting that boron deffciency Day result in a
small increase fn RNA pol¡rærase actl.vfty, but urldine
kloase was not affected after 48 ot 91 hr of the deflciency.
Slnilarttles betr¡een changes Ln nuclefc acld Eetebollsn
resultlng from boron defLclency and those lnduced by
treatfng plants ¡rlttr auxins and auxln-t¡rpe herblcidee are
dfscussed.
Effects of the accelerated rate of maturatfon and seneacence
of boron-deflcÍent root tfps on ¡ucleÍc acÍd netabolLsm are
also coneldered.
11)
DECLARATION
Tt¡e lavestlgatíoos descrlbed hereln ¡¡ere carrled
out by ryeelf beÈtreeû lfarch 1969 and JuIy L972. To the
best of uy knoiledge and belÍef, no materlal ln thls
thesis has been prevlously subnltted for a degree fn eny
rninersLtyr or reported by any other peraon, except r¡trere
dr¡e refereoce le nade ln the text.
T@TTfr CEAPT,TAil
1t.
ACKNO!üLEDGEUENTS
I an greatly lndebted to I)r. J. F. Jacksoa fot hfs suPer-
rrlslou, guldance and encouragenent; to Professor D.J.D. NLcholas
for pernfesÍoa to rndertake thia ltork Ln the lþPertoent of
AgrLcultural Blochenistry and for valuable advlce and dLscussion;
€nd to many other people at the Í{al.te Agrfcultural Research
Instftute for assistance duriug the course of thÍs lvork.
I gratefully acknoiledge fLnanclal aesLstæcê prowlded by
e Comonwealth Postgraduate Award'
ItL
ABBREVIATI oN s
Abbrewlatfons recoãnended by rThe Blochemlcal Joumalr (Bfochem. J.
L26t2) are used Ln thlE thesLs and only additional or dlfferent
forns are llsted belo¡r.
DNase
RNase
ATPase
fB-RNA
TIDPG
c-AllP
IAA
NAA
GA
2r4-D
Trfs
sDs
ssc
TCA
PCA
l,fAK
0.I).
A.R.
deoxyrlbøruclease
rlbonuclease
adenosÍne trLphosphatase
teaaceously bound RNA
urfdlne dtphosphate glucose
eyclfc 3r r5t-adenoslne monophosphate
lndoleacetfc acLd
naphthaleneacetf.c acld
gtbberellfc acfd
2, 4- dLchlorophenoxyacetl c acld
trts (hy Ãtoryne thyl ) ¡m{ ae¡g¡þaos
sodfi¡cn dodecyl sulphate
standard salfae cftrate (0.15 ÌtsNaCl,0.015 H-sodl.r¡o cftraÈe; pE 7.0)
trlchloroacetlc acfd
perchlorÍc acf.d
oethylated elblnfn coated KLeselguhr
optfcal density
analytl-eal reagent
-:1'--:
rlL'ì..
&a
hr
f .w.
p.É| .1.
-B
+B
uflltary
haur(s)
freeh weLght
pormds Per square lnch
boron-defÍclent nutrl.ent medl.r¡m ( or plantsgrolrû in boron-deflcle¡t nut¡tæt nedtrm)
eorylete autrient nedl.rn (or plaota grom1n coqrlete nutrlent nedlr¡n)
Despfte a greet deal of
boron deftcLeacy, the prlnary
stfll not knowD. Io cont:iasË
not beea denoastrated to be a
system.
\.\,,AIiE iNSTiTUTE
LIsP.ARY
1. INTRODÜqTION
work on physfologfcal aspects of
slte of actloû of thLs elenent Ls
";o other Èrace elemenËs, boron has
coüponent or activator of any eûz)tme
lfany hypotheses e:çlafnfng the role of boron have been
preseoted, but nost have been based oa differences beûreen nor¡¡41
and grossly deftcient planÈs. Einde et aL. (f966) harre Just1y
crlticised this approach. -
"... L¡r atteuptLng to detemLne the dfrect actlonof boron fn plant gronth, no rellance c¿ul be placed
on coqarisous beÈween nomal tfssue and tlssuewhlch is at, or past Ëhe point of beconLng mecro-
scopLcally deflcfent^ This criticism rsould apply
to the najority of such studies presected Ln the
lfterature to date.'¡
Receot r.¡ork has shor,¡n Ehat, even before the appearanee of visible
deficfency syqtoms, maÍ,y physlologfcal and bfoche¡rfcal changes
occur. Thus, only by deternlnfng the sequence of these changes
can the coryarlson approach be used to dete¡mlne the prinary
effect(s) of boron deflclency. Ttrls has been ¿¡¡enîted ln only a
few studies, ¡nd the rerrLel¡ of lLterature r¡111 eurphasize thl.s type
of work. Apart from the fniËlal cotme¡rts on deficLency s]ruptorc
1n root tips, thLs revLew will be conflned to bfochenlcal aspects
2,
of boron deflcieucy, ând only work dl.rectly releva¡¡Ë to the
e:rperloental sectlon of the thesis wfll be dl.scussed ln .loÌaÍl.
1.1 tbrphological a¡rd Physlological Syqrtons
For reasone wtrLch are dLscussed later (Sectfon 2.1,1), root
tfps were used as the e:çerimental rnatetfal in the work reported fu
this thesis. A brLef discusslon of the motphologLcal s¡rupÈoms of
deffcf.ency in root tlps ls therefore televant.
1.1.1 Inblbltfon of root elongatÍon
Ttre earllest vislble syqËons of boron deffcleney have
been obserr¡ed f.n roots (Odhnoff 1957 r Ï{hltÈlngton 1959, Sarfn &
Sadgopal 1965). Conplete Lnhibltlon of root elongaÈ1on ln pea and
touåto planËs has been reported within 24 hr of transfer to boron-
defl-cfent nediun (Neales 1960, Albert & WLlson 1961' Yih e Glark
1965), buÈ varlations between species are large.
thfs rapfd effect f.s probably due to the absence of any
red{stributl-on of boron wlthln the plant. Albert & T{11son (f961)
used splft-plant orperiments to demonstrate that boron supplled to
one half of the plant does aot stimulate root elongatfon in the -.'.' .'_.,)ð' - -
other,half. Vlanls & Ì[illians (f970) have suggested that bortc i¡,r'
acld exists largely Ln the rndÍssoclated foru, utrlch moves
passlvely 1n the transpfratl.on strea¡n to accr¡nulate 1u the leaves.
Eoroo fs not ffxed 1n plant tlssues, but 1s soluble and f-mobile
because of the r:nLdfrectlonal flo¡¡ of water (Oertll e Alroed l97l).
3.
If the flor¡ f-s artlfícia11y rerærsed, the movemerrt of boron ls also
reversed. Under boron-defLcfeot growth condltíons, therefÐre, one
wor¡ld enpect the roots to be the flrst part of the plant deprf.ved
of boron, and the leaves the last to be affected. ThLs nay explain
why the flrst vfslble syeptone (and physlological and blochenlcal
effects -
Sectlon 1.3.2) of deficlency occt¡r Ln root tl.ps.
1.1.2 ThLckeafng of roots
Ylh & Clark (1965) found that, after 72 hr ín boron-
deflcieat uedium, the root tips of tonato plants lrere thicker thari
those growo Ín coqlete nedir¡n. The dry wetght of the roots ltas
also hLgher thari that of the controls, due to enlargement of
parenchyua and cambial cel1s (l{arÍngton 1926, Saria & Sadgopal
f965). A rapfd cessatloa of cell dlvtsLon occurs fo boron-deffcf.ent
field beau seedllng radicles, but this 1s preceded by an lnhlbftion
of cell elongation (Whitttngton 1957, odhnoff 1961, Slack &
WtrLttfngtoo. L964). Cell dirrlslon and elongation ceaÉte, but radial
e:<pæs1on cootlnues.
1.1.3 Necrosis of root tips
lhe brorsn colour of root tips has been reported nany
Ëlmes (tlHttfngton 1957, Albert & I{1lson 1961, Tih e Clark 1965,
Lee & Aronoff L967), æd as early as 24 hr after transfer of plants
to boroo-defLcient medfr¡m. Pol.yphenolic conpounds, especfally
caffelc and chlorogenic acfds, acct¡mulate in deflcient roots
(Perkins & Aronoff 1956, tJatariabe et aL. 1964, Dear & Aronoff 1965,
4
Dear 1968) and the oxl,daÈion of Ëhese by polyphenol oxldase
produces the broÌm colour (Shiroya et al. L955, H¿che L96?).
1.1.4 Lateral root gro¡¡th
tllthln 72 hr of trangferrfng tonato plants to
deficÍent nedfa, local sarellf.ags assocÍated slth the energence of
lateral roots are obsenred along the nain rooÈs (I{trfttington 1959,
Albert & I{Ílson 1961n Yth & Clark 1965). These appear at least
5 cm from the tlp Ln normal roots, but wfthÍa 0.5 cn in deficieut
roots. lfhittington (1957) suggested that this occurs because,
although root elongetÍon ceases ln boron-defLclent plaots,
dlfferentlatLon and developuent contÍnue; Èhus, eveD the root tLp
er¡entr¡ally reaches a stage of maturfty, which normal.ly oaly occurs
7-10 cn fron the tLp.
L.2 Some General es
The ablllty of borate to cornplex ¡rfth adJacent azle-hydroxyl
groups, and the failute to demonstrate a requLrement for boron fn
anl.nals, have lnfluenced the directlon of much r¡ork on boron
deflcl.ency. ltre conplexÍrrg with such substances as sugars and
polyphenols (Ztttle 1951, Torssell 1956, Neales 1967) has received
much attentlon, and formed the basis of nrany h¡rpotheses, but little
evidence to support these has been obtafned (stok 1957 " Dear 1968,
5.
flf.ldes & Neales 1969). Eowever, whate\¡er the role and sfte of
ectiotr of boron, such ¿ormplexes Eay ne11 be lnvolved.
Gauch & Dugger (1953) coasidered that boror prouotes sugar
translocatlon þ fonclng lonlzed sugar-borate conplexes ¡¡hfch move
more readlly through oembranes than non-ÍonLzed sugar nolecules.
Boron-deffci.eacy s¡rnptons, such as necrosLs of apical buds and root
tlps, were considered to be manlfesËatfons oi " "tg.r
deflciency at
these sites of actlve grorrth. Although widely accepted for several
years, thls hypothesLs has oftea been questloned (Neales 1959,
Neales 1960, Mcllrath 1960, ALbert & lfllson 1961, Yih e Clarke
1965). Carbohydrate netebolisn and starch-sugar balaoees (Dugger
et aL. 1957, Dngger & Iluryhreys 1960, Scott 1960, Dyar & lilebb 1961,
Zaleski L964, Berziua 1965, Lee et aL. 1966, Danl et aL. L97Ob),
and citric acid and glyo:ryllc acld eycles (Lee C Aronoff L967 " Lee
1969, llnashov 1970) have bee¡ suggested as sites of action for
boron.
the f.nvolvement of boron Ln ce11*a11 structure or synthesls
has also recef.ved much attentfon. Deficfency causes vasÊ changes
1n cell¡rall conponents of root tips, e.g. ll.gnfn, pectin, protefn
a¡rd cellulose (Odhnoff L957, !ühittLngton 1959, Odhnoff 1961, Slack
& t{t¡lttingtoa 1964, Dutta & Mcllrath 1964). Soæ of the phenolfc
conpor:ods ¡rhich accr¡mulate ln deflcfent tfssue (Sectfon 1.1.3) are
precursors of llgntn (ttccalla & Neish 1959). The fryortance of
these obserrratlons has been dl.scussed by Neales (1960), Odhnoff
(1961) and Slack & whlrtineton (1964). They agree rhar rhe vasr
changes ln ce11-¡a11 coupositÍoa are secondaty effects due to the
o
ÍnhibLtloa of ce1l eløgatfon and divisioa and the resuLtlng
lncreased maturlty of root-tfp cells. Slack & 
?.
5-6 day defLcient sunflower 1e¿ves (T{nas¡s\¡ L97O). After 2-4 days
of deffcfent condltlons, a decrease rùas recorded in the esterifL-
catlon of orthophosphate by ehloroplasts isolated from sunflo¡¡er
cotyledons, but not leaves (Tftnashov L967b), Changes Ín the
seoeitfvity of. boron-deficlent srmflower prfnary leaves end root
segrents (after 5-ó days 1n defÍcfent oedfun) to totrtbLtors of
reeplratLon and phosphorylatlon have been reported by Tluashov
(1970).
Decreased levels of AtP ca¡r be explafned by increased ATPase
acttvÍty (Sectlon !..3.4.2), wtrlch could also cootrlbute to the
lqrer apparent phosphate esterlflcatÍon values ln the above assays.
Tfnashov (L96U, also points out that effects on the nltochoadrfal
membtane could be important.
Effects orr nåny oxídatfve processes and enzyues have been
reported (MacVfcar & Burrís 1948, Nason et a,L. L952" Odtrnoff L957"
Mache 1963, Mathan 1965, Gupta L966, Mache L967, I{eiser & Blaney
L967, Tfmashov 19684).
Ttre nany effects of boron deficlency have been exterislvely
rerrLewed (Skok 1958, Hewitt 1963, Nasoa & McElroy L963, Shkolrnik
L967, Herrltt f971).
8.
1.3 Nuclelc Acfd Metebollsm
1.3.1 Nucleic acLd leveIs
Borou deficieuqy results J.a a reductfo¡r lD the anounts
of RNA ln grorrlng poLnts (shoots a¡rd roots) of suoflo¡'rer (Shkolrntk
& Maevskaya L96?a,, tloashov 1966, Jaweed & Scott L967, Tinashov
L967ù, tm,ato (Albert 1965" Johnsoo & Albert L967) and beans
(Wtrttttugton 1959, Berzl-na f965). Other plant parts are probably
affected to a lesser er.tent (Strkolenlk & Ìlaevskaya L962a, Bezzína
f965). DNA levels either do not chaage at all, or as much
(llhlttlngtou 1959, shkoltalk & Þlaevskaya 196?n, Albert 1965).
Except for the work of Albert (1965) and Jar,reed & Scott (1967),
these results were obtafned fron grossly deficient plants. Usfng
tloe-course studles, Albert demoastrated that root elongatloa
ceased before changes in the levels of RNA could be detected. Ttre
F,lilA content began to decrease at the same time as the tlps tu:íned
brown, This was also the stage at which deficLent roots lost thelr
abfllty to respond to added botoo. Jaweed & Scott observed the
sa¡ne sequence of changes Ín suufLq¡er roots and shoots.
Treatoent ¡¡'ith borÍc acLd lncreases the level of RNA f.n
gernfuating wfreat seedlLngs (Danl et aL. L970a), and reduces the
loss of Rl{A fn senescl.ng tonato-leaf dlscs (Paruar & Hamond 197f).
L.3.2 Incorporation of labelled precursors fnto nucleíc
acfds
lrro approaches, whlch glve dtfferent results, have
o
been used. cory et aL, (1966) and Cory 6l Flnch (1967) grew 2- to
3-day-old vì,sia, faha seedllngs Ln complete or boron-deff:lent
medfu¡m for tfme lntervals between I and 52 hr, a¡rd then træsferred
the plants to I yl,t-phosphate solution coatafoing 0.25-1.0 nCi of
[32pf ] for 50 r¡Ín. The specÍf;lc actfvfty of .¡¡¡l@ tlre
ffi RNA lncreased fn 0-2 m root È1Ps and the sectlon
5-10 m frou the tip after 12-15 hr in boron-deffcieat medlum, and
after only 3-4 hr fn Èhe secÈlon 2-5 mr¡ fron the tip. the
nagnltude of the specLffc actfvity fncreased wtth Ëhe tl¡ne of
growth 1n the liqufd mediurn, for both ao¡:mal and deflcÍent plants'
but the Íocrease was oore rapLd ln the deffcfeut roots. The
difference betlreen the specÍffc actlvlties of RNA fron nomal and
deflcieot roots was greatest fn the section 2-5 nm from the tlp.
Ttrus, these workers obsenred an early increase Ln lncorporatl'on of
label Íoto RNÀ, which becaoe Eore pronor¡r¡ced wlth continued gror^rth
1n deffclent mediuû. The section 2-5 urn from the típ, which was
affected firet, probably corresponds to the lm¡edlate post-
Beristematfc regÍon ln whfch Albert 6l l{{lson (1961) and Reed (L947)
obse¡n¡ed the ff.rst amato'nical eyuptons of deficieûcy. It nay also
eorrespond to the regLon of most rapid nutrient uptake.
Prellninary e:<perlmen.ts with lfAK chronatography (Cory & Flnch
Lg67> lndf.cated that 1o Ëhe second sectlon (2-5 nn from the tiP),
the specffic actlvlties of soluble RNA, DltA and partieularly
rfbosomal RNA were greater fn boron-deflcient tha¡r nomal tlssue.
These changes in the lncorporaËíon of [32pf] fnto RNA were
not due to ehanges Ío the speciff.c activity of the total nucleotlde
10.
pool -
a fect rrhlch also precludes the possÍbtllty that the
changes ¡lere due to a generalized lncrease fn the perneab!-lity of
defl-cient rsdLcles to radloisotope.
Strkolrnik & Kositsyn (L962), ShersÈnev & Kurllenok (1962) "
llmashov (L967a) and Rapota (1970) grelt pea a¡rd sr¡nfloner plants
showLng clear sfgos of boron deficlency (at least 6 days I'n
deffcfent ¡nediun), 1n a culture solutlon corntalning [lhC]adenine
or [32pt] for 2-4 days. A deficiency of the micronutrlent resulted
in a decrease 1n the specifÍc activfty of RNA from aplcal leaves
and roots, and a slfght increase fn cotyledoos and stems- The
incorporatLon of l3n]tny*fdine into DNA of pea roots nas elimf.nated
by deficlency (Rapota 1970). Usfng a 40 nin pulse of [32pt],
Sherstnev (L967) denonstrated the sane effect on incorporatLon
into RNA of pea roots. The TB-niA fractLoo fron lfAK chronatography
¡ras affected more than rlboeonal or soluble Riì[A, but less than a
DNA-bound RNA fractlon.
Ttre dlsagreement between these two sets of results may be
dr¡e to the different experimental conditlons and plant sPecfes
enployed. The Russlan workers used grossly deflcLent plants and
studied the lncorporaË1on into the total root and leaf systens,
whlle Cory et aL. (1966) and Cory & Finch (1967) nsed o'nly:r_o-gt
tþs af ter short exposure to deflcienÈ condltloos. Tlro explatrations
are possLble to accounË for these results. Elther Èhe lncrease
Ín the lncorporation of radiolsotope Lnto the RNA of deficlent
roots fs conffoed to the root tip and the reverse f.s true for the
remalsder of the root system' ot stfmulatlon of lncorporatÍon
11.
occurs only before visible synptoms aPpear and at later steges
l-ncorporation is lnhlbited. The flrsË explanatLon seems ncst
plausible, because the thÍrd root segnent (5-10 nrn from the tip)
in the experlments of Cory et aL. (1966) ¡¡as affected less thaa
the other segments, and the q,hanced fncorporatLoa ln the fLrst
tr,ro sections ltas still lacreaslng after 30 hr, the etage ¡¡hen root
elongatioa was severely lnhibited. The results rePorted fn this
thesie also support thls explanatl.on.
1.3.3 Nucle des
The only nucleotide whl.ch has been extensively
studied ln conuectlon with boron-deficiency r¡ork ts ATP. The
level of ÀTP decreases ln defLcient sunflower plants (Shkoltnfk c
Ìl,aevskaya L96?þ) but only after deflcíency s]'mptons have appeared
(8 daysr growth fn deficlent nedLr¡n). The aplca1 bud Ls affected
more than cotyledons or ste¡ns. ltrls was fnftfally attrlbuted to
:m uncoupllng of o:ddatfve phosphorylatlon; however, lt hae slnce
been sho¡m (l.faevskaya & Alekseeva 1964) that increased ATPase
actlvÍty precedes and probably causes the fall tn ATP content (see
Sections 1.2 a¡rd L"3.4.2). After defLclency e¡rnptoms appear, AÞlP
also decreases, but A.DP Lncreases 1n the apical shoots of sunflor¡er
(lLnashov 1968ø).
Introduction of RNA (0,2 glLltte) into boron-deficlent
nedir¡m prevents deflciency synpÈoos ln wtreat, flax and sr¡nflower
(Strkolrnlk & Troitskaya 1961" L962a and, I962b, Shkoltnik &
Maevskaya 1962a). At 15-200, elimlaatl-on of sJ¡nptoms ls complete,
72.
but et higher tenperatures, only a reducÈfon fn luteoslty occurs.
Norrnel eeed fornatLon takes place and the RNA content of <t"efLclent
plants receLving RNA ls slutlar to that of uornal plants.
Ttr¡ruldine, guaníne and cytoslne sfnllarly prevent deffciency
s)'qtoms and the loss of RNA I'n tomato root tlps, but uracLl,
orotic acld and adenfne are less effectfve (Johnson & Albert L967).
As it was assr¡med hat RNA is degraded to nucleotldes, nucleosides
or bases before enterlng the plants, both of Èhese observatlons
were Lnterpreted to lndicate that boron has some role fn the
Ðmthesls of oucleotldes. Selmaa et aL" (1954), however, have
pofnted out the danger of boron contamÍnaËlon ln e:çerlmenËs of
thts type, anrd as the speclfic actfwity of [32p1 lncorporated fnto
the total nucleotide pool ls not affected by deftciency (Sectlon
1.3.2), the interpretatLon of these erçerluents mrst be treated
wlth caution.
TÍmashov (1966) suggested that boton affects nucleotLdes
through l-ts fnteractlon wLth nagnesfum, rrhich affects a cofactor
of oucleotlde netabolLsm; thls role ¡tas supported by Pa¡mar &
Eamond (197f ).
1.3.4 Enzr¡mes involved ln nuclelc acld metabollsm
1.3.4.1 RNase
RNase actl.vl-ty Lncreases l-n the growing tips,
young leaves and rooÈs of grossly deficlent sr¡nflo¡rer and pea
plants (Sherstnev û Rasrmova 1965, Timashov I967a\. Incluslon of
73.
heparin, an RNase inhLbitor, ín the deflcÍent nutrient nedLum'
teûporerÍly elintnates deffeiency synptons, buÈ does not Frevent
necrosLs of grow-Ing polots. Itt yot¡r¡g leaves of sr¡nflo¡¡er, the
Rl[ase aetivlty rlses o'nly rhen the plants shon'- lncipl.eot boron-
deffclency syrptons (Abrol 196l>. The actfvlty can be decreased
1f the plants are returned to eomplete nedir.¡m after 4 but not 6
days of deflelency í¡¡tof f966), but does not retutïr to the aórnal
level (St¡kol'nik et aL. 1964r. The lacrease Ln actl"vl.ty ie large
(3- to 8-fold) and probably causes the reductfon Ln RNA cooteot
(Section 1. 3. I ) .
1.3.4.2 ATPase and other phospha
AIPase actfvlÈy fncreases Ln deflclent
sr¡nflower leaves, termlnal buds, cotyledons and roots, amd Vieia
faba root tlps (Haevskaya & Alekseeva L964, lllnde & Fiuch L966,
TLmashov L968b), when assayed at varíous pE values (5.1, 5.4r 5.7,
7.2, 8.2). Dlfferences c¿ur be detected after onLy 24 hr 1n
deffclent uedirn for Vieí.a faiba root tlps (ninae & FLnch 1966) aad
30 hr ln sr¡nflower roots (Timashov f968b). The dlfferences
contluue to lncrease 1f the planÈs remal-n ia deflcient medfun,
partlcularly after vlsfble deficiency e]rmptons appear. Al,fP, ADP
and GIP are s1¡nilarly degraded nore rapfdly ln deff.clent sunflor¡er
leaves, cotyledons arid roots (Tfnashov L968b). the Vieí.a faba
aeLd enzyme (pE 5.1) also hydrolyzes pheayl-phosphate and
pyrophosphate wtth sín1lar lncreases 1n activlty, but a¡r alkalLne
pyrophosphatase eo,zJlme (pH 7.2 arlld 8.2) decreases ln actlvlty
L4.
after 24-48 hr of boroa deficlencl¡ (Utnde & Ftndr 1966).
Phosphatase actlvlty 1s also enha¡rced fn sunflower leavee end
cotyledone after 4 daye fn deffclent nedfr¡n (Ttnastrov 1967b).
Binde & Ffnch (1966) obser¡¡ed thet the changes f¡ these
enzy¡e actlvitfes (ATPase, phoephatase' phenyl-phosphatase, and the
acld and alkal1ne pyrophosph¿tases) wfth lncreaslng distance from
the root tip were the same as those resultfng from boron deficl'eacy
- l.e. the enzyme actÍvfties in deflcient root sections were
slnilar to those of secËlons further from Èhe tlP in notmal roots'
Because of this, they suggested thaÈ these enz)me acËfrrlty changes
are due to
rf... gênêral physlologlcal and norphological changes
shlch ate far removed ln the chain of causatlon from
the otfglnal event that 1s suscePtlble to boron
deficiencyr'.
Ttrese lncreases 1n ptrosphatase actLvLty result fn hfgher
levels of Ínorganlc phosphate fn deficLent plants (need L947,
HLnde 6t Ffnch 1966, Timashov 1967b), ¿rnd 1or¡er organÍc phosphate
levels (nee¿ L947).
1.3.5 @nt
Introduction of 8-azeguaolne I'nto couplete nutrLent
medir¡n luhfbtts growth of sr¡nflower plants and produces
norphological changes slnllar to boron defÍcl.ency (Shkoltn|k et aL.
1965). lreatment with 2-thiouracil, $-azauracll or barbLtud.c
acld results fo slnllar oorphologfcal changes ln toûato' lacluding
L5.
the reductLon of root elongatlon. The RNA colntent 1s also reduced
(Johnsoo t Albert L967, Albert 1968). Guaniae aod uracLi' reverse
these effects,
Slnflar lnductlon of senescence by tr€:.tment nrlth base
analogues hae beeo reported by others (Heyes 1963' Key 1966, lleyes
t Vaughan 1967), aod Kulkarul. & Rege (1971) found that 8-azaguanfne
fnduces the productLou of differeat tyPes of RNA and RNase.
Shkolrnlk et aL. (1965) consLdered that wfth borø deffciency,
Jr:st as ln the presence of base analoguee, a synthesfs of altered
nuclelc acÍds, protelas and enz)tnes occurs, but Johneon & Albert
(1967) favoured a role |a the slmthesis or utLlLzatfon of certafn
altrogenous baees (Sectlon 1.3.3).
1.4
1.4.1 Protein levels
Boroa defLcfency decreases the level of protefn and
lncreases the an{no acfd and non-Proteln nitrogen content of nany
plant orgaos and specl.es (griggs 1943, Sherstnev & Kurllenok 1964'
Ylh e Cbrk 1965, flfnde et aL. L966, ZabaLotny & llfranyenka 1967 'Shlrallposr et dL. 1969). Total nÍtrogen has been reported to
increase (odhnoff 1957 , I{trfttingtoa 1959, Ilfnde et aL. 1966 'Shlralfpovt et aL. 1969) and decreaee (Briggs 1943' Sarlo &
76.
Sadgopal 1965), but the uee of grossly deflcient plaots and
calculatLons based on dlfferent paráneters (Per ce1l, dry weight
or fresh weight) may well result in these conÈradfctÍons.
1.4.2 ProteLn synthesls
I{hen plants are gronn in a nedfu¡m contafnfng labelled
amino aclds f.ot 20-48 hr, the lncorporatfou Ínto Protefns extracted
from pea rootsrand from roots, Yomg leaves and shoots of sunflower
is reduced by boron deflcfency (Sherstnev & Kurllenok 1963,
ÎÍmashov & Volkova L967, Rapota 1970), but only after the
appearance of visl.ble deffciency syuPtoms (Borshchenko & Sherstnev
L96Ur. Cotyledons, stems, and nltochondrial and rl.bosomal
fractlons of deficfent sunflolrer roots, however, f.ncorporate
fncreased amourits of radioactlvLty (Tlnashov & Volkova 1967),
which Èhe authors attribute to the faster rate of differentiation
of these defLclent tissues.
In v|trc lncorpotation of ItrC]tyrosfne into proteins by
rlbosomes Lsolated from the roots of pea plants showfng ttre first
slgns of deficÍency is reduced (tsorshchenko & Sherstnev 1968ó).
The fomatlon of polysomes appeers to be lnterrupted Ín plants
deprived of boron, possibly due to the fncreased RNase activLty
(Sectl.on 1.3.4. 1) . Ten-da-y defLcfeut stmf lower roots and ehoots
have a decreased rLbosome content (Ttraashov 1967a).
Although the fneorporatlon of labelled ¡nlno aclds lnto
protein ls reduced, the in tùw fncorporatloû Lnto s-RNA (fornatfon
77,
of amino acyl-s-Rl{A) 1s lncreased by borou deffcfency' eve¡ before
deffcf.eacy synpto¡s appear (Borshchenko & Sherstnev 1968ø). After
only 3 daye 1a deflclent medfun, the speciffc actfvity of the
an{no acyl-s-Rl{A ts doubled. ThLs suggests that boron deflciency
lnterrupts nomal proteLn synthesLs, but does noÈ interfere with
the foroatlon of anloo acyl-s-RNA. the rate of thf.s reactfon has
also been measured in titrc. Uslng a ParÈ1411y purifled enz¡rme
extract from pea roots, Borshchenko & Sherstnev (f9684) found no
effect of boron deflclency on the lncorporatfon of rhC-labelled
amlno acids (tyroslne, glyeine or threonine) lnto TCA-Lnsoluble
a¡nino acyl-s-BNA, before or after the appearance of deflclency
synptons. lllnde et aL, (1966) obtafned st-ûf.lar results when
roeesurÍng the amino acid-dependent AÏP-pyrophosphate exchange
actlvfty of a soluble extract fron fleld bean root tips. tltren 19
amLno aclds were added, however, Èhe actlrrlty decreased after the
plante had beeo grown for 48 hr, and fn some cases 24 hr, Ín
deflclent medfr¡m. Sfnce the activfty also decreased uP the root
(f.e. away fron the tip), Illnde et aL. (1966) suggested that Ëhe
decreesed actlvlÈy ¡¡as due to accelerated maturatÍon fn deffcient
root tlps (see dLscussfon fa Sectioo 1.3.4.2). these reeults
Buggest that the iacreased fncorporatl.on of lbc-labelled a¡¡fno
acÍds f¡to arnlno acyl-s-RNA, and its htgher speclfic actlvfty, are
due to a slorser rate of utillzatlon in proteln synthesf.s, and oot
to ar lncreased rate of aulno acyl-s-RNA synËhesfs.
18.
1.5 Plant llormones
1.5.1 Auxlns
1.5.l.I Auxln levelE
A relaÈionshlp between boron and IAA has
often been suggested. Early workers (Eaton 1940, Mol¡at L943,
MacVlcar & lottlnghan 1947, Torssell 1956, Odhnoff L957, Dyar &
Webb 1961, Shkolrnik & lfaevskeya L962a) equated boroa deflcfency
with IAA deflcfency, and suggested that boron nay be lnvolved 1n
I.AA synthesls. Most enperlneutal work, hor¡ever, has fafled to
substantl.ate thls Ldea (see Selnan et aL. f954).
Braodeuburg (1949) a¡d Neales (1960) suggested that boron
deficlency nay reeult ln the accumulatfon of hfgh levels of auxl.n.
Slack & Coke (f965), Jaweed & Scott (1967) and Coke & lùhlttfngton
(1968) have elaborated on this hypothesis. These authors realfzed
that rnrriy of the norphological sSmptoms and physlologfcal a¡rd
bfochemical changes lnduced by boron deficlency are sÍm1lar to
those assoclated Írlth hlgh concentratÍous of IAA and auxin-type
herbfcl.des. They have extensfvely dlscussed the sLnflaritÍes and
posslbl-e relatlonshlps. The results of Dyar & I{ebb (1961) in facr
conform to this hypothesfs - although they argued the converse.
NAA treaÈment of normal bean plants lnhfbtted the translocatl.on of
photosynthetlcally fncozporated lbCO2, Ðd produced norphologlcal
changes (reduced root grorùth a¡rd fnitiatlon of lateral root
prLnordla) sinLlar to boron defLciency. If Èhe nutrLent medfr¡m
L9.
corntains fO UH-IAA, the root Srowth of Ví.eia fúa Ls t-nbibited to
a slmllar extent to that resultiag from a deflciency of bc,ron (Coke
ô t{httttngton 1968). If both treetiments ere applled ei-uultaneously
(f.e. additlon of IAA and deletLon of boroa), the turhibitlon of
root grolrth fs even greaÈer.
Ar¡<fn levels ln deffcfe¡rt and oorual tissue have been
esrinated by Odhnoff (L957), Shkolrnl:k et aL. (1964), Jaweed &
Scott (L967) and Coke & llhittfagtoo (1968). Odhnoff's results
were rather varlable, but there ¡tas some evÍdence of slfghtly
hlgher ar¡xln levels fn boron-deflcfent bean shooËs. Shkolfnl.k eú
aL. for¡nd decreased amounts of free and fncreased anounts of bor¡nd
auxlns !n shoots, hypocotyls and roots of s¡¡nflower and maLze, but
the signifLcance of the separatlon lnto botmd and free forus ls
obscure (see BentLey L962). After 4 days ín deflcfent nedfr¡ur (the
earlLest tÍme examlned and 4 days before the aPPearanc,e of
deff.clency synptons), Jaweed & ScoÈt found thet the IAA conteats
(che¡nical e6tLmatfon) of sr¡rflgrter root a¡rd shoot apfces were 70-
802 hlgher than those of nornal plants. Thfs was maintatned untll
after sl¡Bptons appeared, wherr the levels decreased. Coke &
!ühttËlogtoa obtaf.ned evldence th¿t extracts fron deff.cÍent bean
root tlpe contaÍûed tltlce as much IAA as normal root tlps after
onLy 24 hr of gronÈh fn deflcient nedLr¡m. The dffference appeared
to be even greater after 42 }l;r.
1.5.L.2 Aru<Ín degradatlon
Increases ln peroxf.dase actLvity Ín boro¡-
20.
defl.cl,ent bucknheet leaves (üadre 1963 a¡rd 1967, and beao root
tips (Odhnoff 1957), and an rtar¡<l¡ oxldase" fn buckwheat l-eaves
(Uache L967) have been reported. llowever, one lnterpretatlon of
the auxln theory ts that elevated levels of euxfn are due Ëo
lnhlbitlon of ar¡rl.n degradaticn (Slack & Coke 1965). Some evfdence
for a decreased rate of IAA degradatlon has been obtalned. At the
tlme when vlsible deficLency s]ttnPtons first aPPear' there ls a
decreased lAA-oxldase activity 1n sr¡aflcn¡er hypocotyls and growing
points (shoots and roots), æd the actl.vlty alnost disappears when
growth ceases (Shkolrntk et aL. 1964). Shoots a¡rd lateral roots
are most severely affected, Boron-deficient bean roots abeorb
thQ-labelled IAA more slowly than noroal roots, and the raÈe of
decarbo:rylatlon is sloner 1n the deficfent tissue (Coke &
lühlttlngton 1968). In ú,trc studies with horse-radlsh peroxidase
have sho¡¡n that the Presetrce of boroa stimulates the aerobic
oxldatlon of IAA by this enzyne (Parlsh 196E). Parish suggested
that boron nay functfon by facllltatlng Èhe foroatlon of hydrogen
peroxide" whlch 1s required for the reaction. This nay be related
to the obsetratlon by Shkol'nik & Steklova (L954) that dal1y
applfcations of hydrogen peroxfde can partly correct defLcfency
synptoms¡. InactLvation of lAA-oxfdase actfvfty could also result
from an accr¡mulatloa of phenoLlc compounds (Sectlon 1.1.3, Slack &
Coke 1965, Janeed & Scott L967, Coke & Wtrittfngton 1968, Henltt
1971) r¡hlch luhfblt rAA-oxldase (nauu û Klefn 1957, thina¡ra et aL.
Lg62r lomaszewskf 1963, Tomaszewski & Thinan¡r 1966, Psenak et a7..
1970, Rt¡nkova et aL. L972). Thls uay be because boron bLnds wfth
z1
and Lnactlvates phenolLc coryouods (slack & coke 1965, Coke &
WtrlËttagton 1968).
1.5.1.3 Relatlonshlp betneen boron. arurfng and
nucleic acld metabo!.fem
Shkollnük et aL. (1964) suggested that a
study of the relatfonshlp of boron to auxlu and nuclefc acid
netabollsm |s of great fnterest, but so far l1ttle work has been
done 1n thls field.
IAA ler¡els ere fncreased by boron deflcfency before decreases
1n RNA coûtent ca¡r be deËected (Jaweed & ScotË L967). Btgh
concentratlons of auxln enhaace RNase activlty (see Sectfon 4.4).
the decrease ln RNA content of boron-defÍcl.ent tfssue nay therefore
be lndlrectly due to a high IAA level. IAA treatnett of planÈs
stLnrrlates RNA synthesis (see nore detaLled dfscussLotr fn Sectfon
4.4), whLch nay be lelated to a slmllar effecË of boron deffciency
(Sectlon 1.3.2). It 1s known thåt 2-thlouracll, whlch disrupts
nucleLc acl.d netabolfem and produces morphologf.cal changes slnllar
to boron deficiency (Sectfon 1.3.5)r also lnhibits lAA-oxldase
(Gaspar et aL. i968). These obserr¡atl.ons ærely suggesË ll.nes for
further LnvesËlgation.
1.5.2 GibberellÍc aefd
Gibberelllc acld-Lnduced prollferatlon of debudded
tobacco plants, æd elongatlon of kldney bean and lettuce
22.
seedlLrigs, Ís reduced ln the abeence of boron (Skok 1958, Dar¡fels
& Struckneyer 1970). TlrLs effect nay nerely be due to ínhlbition
of grmth by boron deffcf.ency. Boron (1 uu) and GA3 (4.33 uM)'
together or alone, offset the loss of Rl.lA, DNA, protefn and
chlorophyll fron senescfng toï¿ato leaf dfscs (Pa¡mar & Hamond
f971) and the authors suggest that boÈh boron and GA3 are fnvolved
1n nucleotide metabolÍso.
t.6 Outll.ne of thls Investlgatlon
Wheu comparlng plaots gror¡ür fn couplete medir.m ¡¡1th those
gronn f.n boron-deflclent mediun, the earliest effects of the
deflcfency should provlde the best lnfottatlon abouË the prLnary
stte(s) of actl.on of the element. The stLmulatlon of [32pf]
locorporatLon Ínto the RNA of Vieia faba root ttpe (Sectfon L.3.2)
Ls the earliest effect of boron deffclency yet recorded. Since
nany other changes 1n pJ,ant growËh and develoPment also lnvolve
nuclelc acld netabolism, further lnvestfgatlons of thls aspect of
Èhe deffcfency r¡ere undertaken. Sone of the e:ßped.mental r¡ork has
been nodelled on approaches used to Lnvestfgate the effects of
pla:rÈ hormones oo suclefc acLd netabollsn. Io thls way,
comparl.sons could be nade between the effecte of boron defl.efency
and thoee of hormones. Thls has lnvolved the followlng lioes of
work: -
23
8. Establlshlng wtrether aû increased lncorporattou of
radloacÈ1ve precutsors Lnto RNA occurs fn the raet tlps
of boron-deffcient Pltaseolus aureust, æd determlning the
extent of thls fncrease before and during the onset of
boron deficfency. ÍhÍs has fncluded a study of some
factors ¡¡hfch nlght affect the uptake and lneorporetfon
of precursors fnto RNA.
b. Studfes of the propertfes of the labelled RNA, aLmed aË
deternlnlog whether it 1s a general lncrease 1n
synthesls, or whether particular (or nenr) species are
affected more than others.
c. Studies to deternl.ne whether changes ln nucleÍc acl.d
metabolism cao be related to changes Ín actlvity of some
of the enzJrmes lnvolr¡ed.
d. Extensfon of thfs work to the stage when the plants
extriblÈed gross norphologfcal deffcfency s¡4toms, 1n
an aÈtempt to e:rplaLn some of the effects of boron
defÍctency on nucleic acld and protein metabollsn
reported in such plants.
24
2. MATERIALS AT.ID I{ETflODS
2.L P1a¡rt Materlal
2.1.1 Chol.ce of plant r¿
Phoseohts Øæua (¡nr¡ng bean) was chosen as the
experÍoeotal plant for the following reasons:-
a. It Ls htghly susceptfble to boron defÍciency.
b. It ls a sul.table plant to grolt ln nutrlent
culture.
c. The radlcle and roots are easfly harvested.
d. Much bloehemlcal work, PartÍcular1y relatfng to
nuclelc acl.d netabolls¡¡ has been done wLth thfs
plant, 1n thÍs laboratory (Ong e Jackson 1969'
L970" L97L, L972a an.d b, Jackson & Ong 1972) and
elser¡here (e.g. Stockx & Vandendrleesche L96L,
Barber L962, Ioring et aL. 1966, Laskowskl 1967,
Ishll et aL. 1967).
e. Faclllttes were already avallable rrtthin this
laboratory for the cuLture of thls plant tnder
condftl.ons of borotr deficfenry.
Root tlpa were used because thls 1s where the flret vÍsible
syrptons (root elongatlon Sectloa 1.1.1) and phyalologl.cal (cell
enlargement end dJ.el.ntegratlon) and blochenLcal (lacreased
25,
lncorporatLou of [32pr] fnto RNA, SectLon 1.3.2) effects of boron
deflcLency have been recorded. Ttre firet a¡ratomlcal synp';oms of
deffclency occur in the lnmedlate post-ner1sÈenatfc reglon of
tonato a¡rd sr¡nflolrer root tlps (Reed L947, Albert & Wflson 196f)
and the earliest and greatest effect on [32pf] iacorporatfon fnto
PÌ{A 1s 1n the 2-5 m section of root tLps of Ví.cia faba eeedllngs
(Cory et aL. 1966). ltre equivalenË stages of root development 1n
Phaseohæ cal?eus are Lncluded Ln 5 m root tips. Maay prevLous
boroa-deflciency studles have used root tips, so that comparisons
ca¡r be o¿de ¡¡Lth the present work.
Feedlng radioactfve nucleic acl.d precutaors Ínto excised
root tfps is readlly carrLed out. Incubatlo¡ solutlon voh¡nes are
snaller (and therefore require less radloactlve ptecursor), æd
the condttfons of incubatlon (tenperature, aeratlon, shakfng ratet
etc.) ca¡r be ruore accurately controlled than when usÍng Lntact
plants. Entr:y of radl.oactlve precursors into the tiseue 1s also
enhanced by excislon (Ingle & Key f965). Neales (1964) has shown
that excfsed tomato rootg have sl.ullar boron requfreneûts to
lntact roots, and the Lacorporatfon of labelled precursors into
dlfferent RNA species (as separated by ÞfAK chromatography and
sucrosedenslty-gradle¡t centrtfr¡gatlon) fs slnllar fn most lntact
and excLsed plant tÍssues (Ctrerry et aL. 1965, Ingle &.Key 1965,
Chea 1971). Hovever, LoenLng (1965) and Rogers et aL. (1970) have
reported dfffereuces fn r-RNA netabolfsm 1n excl-sed root tl.ps, and
these r.r111 be dlscussed further 1a SectLon 3.5.2.
26.
2.L.2 Culture of Phaseohts cureuß
For all culture techniques aod nutrl.eut preparatlon,
double glass-diettlled lrater was further dlstflled fn a sllíca
apparatus¡ passed Èhrough a nflllpore fLlter (Celnen Iûstruûent
Coupany, Ann Arbor lffchigan, U.S.A. ) 0.2 uD pore slze) and stored
1n eealed plastlc contafners. All equipment was efther autoclaved
or surface-sterlllzed rrlth etha¡rol (or concentrated ttGl) aod
erçosed to u.v. llght for at least 30 mia.
PltdseoLus ø'treug seeds purehased from ândersonts Seeds Ltd.
(Brfsbane, Queensland, Australla) lsere surface-sterlllzed r¡1th
ethanol for 1 nln and wlÈh 0.2f @lv) soófi¡n hypochlorlte for
I hr, then rl.nsed three tlæs wlth nater and soaked ln water
overnight. The seeds were germÍnaÈed 1n the dark on molst blottlng
peper (prevfously lrradiated wltb u.v. lfght) at 30o for 72 hr 1n
covered contalaers.
Ttre seedlfngs were growri ln l0 lftre black plastfc contafnere
40 crn long x 25 cn ¡¡lde x 15 co deep. Iloles (120), 5 m f.n
diareter, were drilled through black Perspex sheet,s whfch were
plaeed on top of the open coûtafners. the radleles (3-5 cu long)
nere passed through these holes into the nutrfent solution belor¡.
The coryosl.tlon of the nedfrro was based on that of Eoagland &
Arnoc (f950) aod 1s sho¡¡n ln Table 1. Fe-EDTA lras prepared by the
method of Jacobson (1951). Boron (H3803) ¡sas omftted fron the
boron-deflcíe¡rt oedir¡n. FllÈered al.r ¡ras bubbled through four
sllica tubes 1n each contalner. The seedllngs were grown fu a
27.
TABLE I t Conpoeit¿.o?t of nutrùerú nedhnn
NUTRTENT
KNOs
Ca(No3)2 4H2o
ugSO¡ 7E2O
KE2POa
K2HPOa
B (as H3803)
Ma (as ÈhC12 4EzO)
Zn (as ZnSOç 7H2O)
Gu (as CuG12 2EzO)
Mo (as Na2MoO¡)
Fe (ae Fe-EIÍIA)
CO}ICENTRATION (ngllltre)
255
590
245
68
25
0.25
0.25
0.025
0.01
0.05
1.25
phytotron (Ellfs & Clark Ltd., Electrfcal- Engfneers, Adelafde,
South AustralÍa) using a day-length of 16 hr. Day teoperature was
25o and nfght tenperature 2Oo.
lhe nutrÍent ¡¡as changed e\rery 5-6 days. To do thfs, the
Persper sheets co¡taiufng the plants were reroyed and the roote
placed olr, and covered !rf.th, moíst, u.v.-irradÍated blottlng paper.
The nutrÍeût containers ¡vere rfneed three t1¡res, and reflIled ¡vLth
nutrleot æ,dLun. After tÞroroughly rlnsfng the roots rrlth rater,
the plants were retu¡:ned to the nutrl.ent medir¡¡¡. Norrnally,
batches of plants were gro!fl1 ln paLrs, and at the flrst nutrient
change (5 days after pla¡rttrrg) one contaÍner was refllled with
boron-deflcient medfr¡m.
28.
2.1.3 Root-elongatlon Deasurements
Root-tfp elongatfon was deterilLned by narkfag the
oaia roots 10 m fron the tlp lr-Ith a ulxture of ftnely grotmd
charcoal Ín laaolfne (Eydrous I{oo1 Fat, Drug llouses of AuetralÍa'
Adelaide, South AustralLa) anó neasuring the subsequent facrease
fn the dlstance fron the tfp to the oark. Measureænte to the
ûearest nÍn were made on L5-25 plants.
2.L,4 Harvestlng of-root tips
Root tips (5 om loag) were e:<cised from the naln a¡rd
lateral roots wlth a sterj.le scalpeI, and stored io a ch|lled
beaker 1n Íce rmtll the halvest was coopleted. Snall saruples
(2OO-500 root tÍps) requlred 30-60 mÍn and large samples (1000-
2O00 root tlps) 2-3 hr to conplete harvestlng.
2.t 5 Incorporation of labelled precursors lnto excfsed
root tlps
ExcLsed root tlps were Lncubated at 30o for 4 hr 1n
plastlc test-tubes (1.35 x l0 c'n) 1D t-2 El of nutrl.ent medLr¡ß
(Table I - wlth or wl.thout boron) contaÍnLng the tadloactive
precursor, 50 rrnlts/nl of PenÍcflll.n G a¡rd 27t (vlv) sucrose. the
nutrLent ædLr¡m was dl.luted 4-fo1d rvLth ¡vater for use l-n [32pf]
lncorporaÈ1on oçerlments to reduce the leve1 of r¡nlabelled
phosphate. The levels of radfoactl\¡e precursots used !Íere -a. l2-rhC]urldlne, I pCl/nl, 40 nCl/mol;
29.
b. l3n]urf¿fne (geaerally labelled), 2O vCtlmt, 5OO nnCi/nnol;
c. [32pr], 5OO ucf/nl, 3.0 mCl/unol.
After lncubation, the tips were ¡vashed sfx times wlth a 1et
of d1stllled water before honogenfzation, which, unlese oÈherr¡l-se
lndfcated, wes by 20 plunges ln a glass-glass homogenfzer (10 nl).
2.2 NucleLc Acfd Extractfon and DeterninatÍoa
2.2.L Extractlon
2.2.1.1 Total nucleic acLds
Nuclelc acfds were extracted and separated
Lnto acfd-soluble, RNA and DNA fractioas by the Schnldr &
Thatrahauser (1945) nethod as nodlffed by Bonner & zeevaart (Lg62).
In sooe e:çerfments, the acid-soluble fractÍon Ìras extracted urlth
0.2 M-Pc'Â, Lnstead of. 57 (w/v) TCA, and fn orhers rhe fnlrfalextractfon In 801l (v/v) ethanol at lOOo was onÍtted.
2.2.L.2 NucleotÍdes
Root tips were houogenized Ln le% (¡r/v) TCA
[1 nf/eSO ng (f.w.)] an¿ the nlxture shaken þ hand ar 20 for15 mfn. After centrlfugatlon for 20 mln at 20 and g00g, the
pellet waɡ re-extracred Flth 5z (w/v) TcA and the conblned
30.
superoatatrts ïere washed tea tlæs r¡1th ether to temove ÎCA. the
waehed supennatant lras used for chromatography on DEAE-Seþhadex or
Dowex-l.
Ttre acld-lnsoluble pellet renafnfng was hydroLyzed ln 0.3 l{-
KOË for 20 hr at 37o, theo neutrallzed rrlth 0.6 M-PCA. After
sÈandlug at 2o for 15 nla, followed by ceotrLfugatlon at 4009 for
5 nln, the eupemataot fractLon was uaed for chromatography on
Dovex-l.
2.2.L.3 RNA
Excised root tl.ps (100-400 ng) wereIhonogeulzed at 20 Ln 2 nl each of buffer [10 n]t-phosphate, 50 n]t-
KCl, l0 nll-lÍgCl2, L.5Z (¡¡/v) SoS, O.2Z (w/v) beatonite; pE 7.61
æd, 9OZ (v/v) phenol [cootainlag O.l"/, (w/v) 8-hydroryqulnollne].
The clxture was shakeu by hand at 20 for 20 nin and ceûtrffuged
for 20 nin at 20 aad 10009. the aqueous layer was re-extracted
with phenol and bentonlÈe, and the RNA preclpftated from the ftûal
aqueousr pbaee with 2.5 r¡oh¡oes of ethanol at -15o overnlght. Thl.s
precl.pitate was ¡yashed r¡lth 702 (v/v) ethanol, 952 (vlv) erhanol,
and twl.ce wfth eÈher, then dried in oac,uo. tdith larger a^mounts of
plant naterlal, I nl each of buffer aad phenol was used for each
200 ng of plant materLal.
RNA was extracted fron cotyledone and young shoots by the
same oethod, except that I nl eectr of phenol and buffer for each
gram of tfssue ¡sas used, and the phenol extractlon was repeated
õ7.
three tLnes.
For DNA-RNA þbrldLzatfon studies the Rl{[A was further
purÍfled by dÍssolvÍng the preclpitate 1n 10 nu-Trls buffer
(contalnfng 10 nlf-!rgCl2; ptr 7.4) and f.ncubatÍng n'tttr 25 Uglnl
Ilfase (RNase free) for 30 mia at 37o. After deproteÍaizatf.on wfth
phenol, as above" the aqueous phase was washed wlth ether end
dialyzed f,ot 24 hr agalnst three changes of 2xSSC (SSC - 0.15 lf-
NaCl, 0.015 M-sodfi¡m cltrate; pII 7.0).
2.2.L.4 DNA
DNA was prepared from 72 hr germl.nated
seedllng radLcles, grown as descrfbed fn SectÍon 2.L.2, by a
nethod based on that of Kado & Yfui (197f). The radLcles r¡e¡e
horcgenlzed for 2 nln at 650 aÈ ful1 speed Ln a Sorvall Omrluixer
¡vith an equal vofu¡oe of buffer (0.05 M-sodLr¡o borate, 0.01 M-EDTA;
pE 9.0). Soltd SDS a¡rd then NaCl were elowly added to fLoal
cooceûtratlons of 57( (wlv) ar¡d l5Z (w/v) respectLvely, and the
nfxture stÍrred gently for 15 uLn aÈ 650 after each addÍtfon.
After eoolfng to room temperaturer an equal volt¡oe of a nÍxture of
chloroform-lsoanyl alcohol (24¡L, v/v) was added a¡rd sr¡lrled
geatly for 15 mia at room temperature. After centrlfugiag for
10 nfn at 1010009, the agueoua phase lras renoved and re-extracted
lrl'th chlorofot'n-lsoauyl alcohol, a¡rd thea preclpLtated overafght
aÈ -15o 1n 2.5 vohmes of ethanol. Ttre preclpitate was dfssolved
ln 0.1XSSC and repreclpitated rrlth ethanol. After re-dissolving
ln O.txSSC, the ptecipitate was fncubated at 37o for 60 nln with
32,
0.1 ng/nl RNase (prevÍously lncubated at 90o for 5 mtn), follored
by 60 mÍn wlth 0.4 ng/nl dlastase, and 60 nln with 0.8 ng/r,l
pronase (self-dfgested for 30 uiu at 37o). The prouase dÍgestlon
was contfnued ovenÍght at rooD temtr'erature. Ítre solutloa was
deproteLuÍzed twice ¡¡fth an eq'.al voh¡ne of chloroforn-1soauyl
alcohol and dialyzed, f.ot 24 ht agaiost three changes of O.lxSSC.
After adJustLng to 0.3M-KOH and lncubatfng for 5 hr at 37o, the
solutlon wes neutralized with I I{-HCI a¡rd the DNA pelleted by
centrifuglng overnight at 4O,O00g in a Beckman ModeJ- L ultra-
ceritrffuge (Tltanfr¡n-5O head). The DNA pellet was dissolved fn
0.lxggg and stored at -15o.
DNA prepared by thfs nethod has a maximrn absorptLon at
258 nn (l'tgure 1), whlch fs sinilar to that reported for Dl{A
prepared from embryonfc axes of uung bean by Chen (1971).
2.2,2 DetennÍnatlons
2.2.2.1 Absorptl.on spectra
Ttre optical extLnctl.on of a neutralfzed
allquot was measured at 260 nn and the RNA or DNA conteot
calculated uslng extfnctfou coefffclerits of 6.7, 9.5 a¡rd 12.0 for
double-stranded, sfngle-straaded and hydroLyzed saryles respectf.vely.
2.2.2.2 Illboee
RNA-rfbose !úaa deternlned by the nethod of
MeJbaun (Schnelder 1957), usfng cuprfc chloride as the catalyst
0.6+,(^
(t,óroC'
+,CLo
1.0
0.8
0.4
0.?
0 210 230 2 274 29A 310
l,lavel ength : nm
FIGUR.E 1 : Absozpti.on spectrun of DNA. DNA was
prepared by the method descríbed in Section 2.2.1.4 and
the spectrum \¡ras determined using a Gilford Model 2400
recording spectrophoÈomeÈer.
93.
(r.ln c SetrJelde f969) anð AlfP as the reference etandard. Pyrlnf-
dlaes react very poorly fn thls assay (f,ln t SchJefde 1969). RNA
contents deternlned by rfboee a^esays of samples of hydrolyzed
pure RtlA (after ùfAK chrornatography) were oaly 55-60Z of the values
calculated from the abeorptlo'E at 260 nn. Ttrls 1s sfnl.lar to the
results of Lin e SchJeide (1969), ând all values have therefore
been corrected for a 6O7( estfnation of RNA conte¡rt by rLbose
detemfnatloa.
2.2.2.3 DeoxyrLbose
DNA ¡ras deternlned as deoxyrlbose fn an
alfquot of the acfd DNA fractl.on before neutralizatÍon (see Bonner
& Zeevaart 1962) wfth the Dische dfphenylarn{re reagent, usÍûg the
procedure adapted by Burton (1956). The dffference between
optfcal extfnctfon at 595 æd 650 nm rÍas neasured a¡rd these values
!üere coûpared r¡lth those of a d-Al[P standard. Pyrinidiues also
fall to react slgniflcantly ln thls assay (Burton 1956) and values
¡rere corrected as for the rfbose agsay.
2.2.2.4 Phosphate
An alfquot (0.1-0.2 ul) of the RNA fractlon
was dlgested by adding a drop of, IOZ (¡r/v) ltg(NOe)z 1n ethanol ar¡d
heating 1D a test-tube over e strong f1æe rntll all the bror¡n
fi¡nes had been oçelled. !ùtreu cool, 0.9 n1 of HCI vras added and
the solutlon ¡yas heated 1n a bo1llng nater bath for 15 nin. After
cooling, the solutlon was analyzed for phosphate by the nethod of
34
ûtea, et aL. (1956). The optfcal extlnctlon at 820 nm rtas neasured
and referencc standsrd;' of AlfP were developed at the sâue tlme.
2.3 Analysie of
2.3.T RNA
2.3.t.1 MAK chron¡toqraphv
Ìlethylated albrnín and cofumr preparatfon
were by the roethod of Ma¡¡dell & Eershey (1960), as modffied by
lfo'nfer et aL. (1962), but uslng celfte (H. Selby & Co., Adelalde)
Lnstead of Kfeselguhr. Colr¡urs (0.65 x 10.0 cn) were equlllbraÊed
ntth 0.1 l1-Nacl in 0.05 M-phosphate buffer (pII 6.7) and auclefc
acid samples (0.1 to 0.5 mg) were dlssolved 1n the sane buffer and
applied to the cohlur. The colr¡ur was l¡ashed wfth buffer rmtfl no
radioactl.ve or u.v.-absorbing naterlal could be detected and then
eluted wfth a llsear gradlent of NaCl (9:? l: _1._l- Y)_"r" 0.05 M-
phosphate buffer (pII 6.7). the RIIA tenaciously bound to the
colr¡nn (TB-RIIA) was eluted wlth 0.5"Å (wlv) SOS. Gradl.ents of
60-100 ml were used, a¡rd fractlons (1.5 nl) collected. The
absorbance at 260 nm was measured for eactr fractlon a¡rd the
radÍoactirrlty neasured by dryfng allquots onto glass-paper squares
(2 x 2 co) and countÍng as descrfbed 1o Sectloa 2.5.L.1 or, Ín the
case of [32p], by Cerenkov radlatlon tn l0 nl of 0.1 t[-phosphate
35.
as descrÍbed in Sectlon 2.5.1.3.
The u.v. absorptlon of proteln and other üaterLal removed
froo the columr by SDS prevents the estfnatlon of nuclefc aclds
by oeasuring the optleal exttocËfon et 260 n¡r. A rcdlfled rlbose
assay (Sectton 2.2.2.2) was used. I{hen the sæple and orcl.uol
reagent were mlxed, the black preclpftaÈe forned was renoved by
centilfu:1ng at 4OOg fot 10 nin and the clear supeErietant fractl.on
bolLed for 60 rofn. After ceatrífugfng agala, the absorptÍon of
the clear fractlon was rDeasured aË 660 nn. At 1o¡,r values, the RNA
content fs rmderestlnated by this method, as sholrri Ín Ffgure 2.
Eowever, lf the allquot used 1n the assay contains tnore than
20 nnol of RNA, the resulÈlng error fn the deternfnatLon is snall
and, by refetrlng to Flgure 2, can be easÍly corrected for.
2.3.1.2 Poll'acrylamide-gel electrophoresLs
Polyacrylæ1de gels cross-l1nked r¡ith bis-
actyla¡r{dè were prepared as descrLbed by LoenLng (1967). The
buffer used contalned 36 nlf-Trle, 30 nM-NaH2PO4 ard I ül.f-EDtrA
(disodfr¡n ealt), pE 7.8. The developlng buffer also contained
O.2Z (wlv) S0S. Electrophoresis was at roon temperature t¡ 2.4f
(¡¡/v) gels in 0.6 x 9.0 cn glass tubeE. If the tubes ¡¡ere cleaned
in alcohollc KOH and lretted rflth O.2f fulv) SUS before castLng,
the gels could be easlly removed fron the Èube by lnJecting a
snall volr¡ne of !üeter ¡rith a syrfnge between the ge1 a¡rd the glass
surface. Ttre gels were pre-run at 5 na/gel for I hr before
appllcatlon of the sample, at 2 malgeL for 10 nin after loadl.ng
80
60
L840L
lJJ
ès.
20
050
FIGURE 2 : Ez,r.or ín z"Lbose assaA for RNA content ofTB-RNA fraction. Identical samples of TB-RNA
containing varíous knom amounts of r-RNA were assayedfor ribose as descrtbed in Section 2.2.2.2 arrd theerror in the determinatíon of added r-RNA calculated..
0 l0 20 30
RNA : nmol
40
36
the sample, and then at 5 na/gel and 15 volts/cm for the tines
lûdlcated in the resulÈs (Sectio¡r 3-5-2). Brmopheaol blue ¡ras
run as a narker. Samples (less than 100 ul) dtssolved 1n
developfng buffer cofitainLng 5% (wlv) sucrose were layered over
the gels.
After separatlon of the saqle, the ge13 ltere removed from
the tubes and soaked in 0.1 M-phosphate buffer (pll 7.0) for 30 rrln,
then transferred to a quartz cell (0.6 x 12.0 x 1.0 cu) and
scanued 1n a Joyce-Loebl Ctrromoscan, fftted ltfth a 265 nn
Lnterference f1Iter. Ttre gels rùere then frozen at -15o aad cut
lato 1.5 m elices. the sllcing aPParatr¡s consisted of 7O razor
bladee mounted sfde by sÍde wlth equal spaclng. The frozen gels
were laLd ooto several sheets of parafilm on a Perspex sheet and
the raao¡ blades pressed ffuo-ly dorm through the gel' The s1lces
remafaed bet¡¡een the blades and could be easlly reuoved wlth a
spatula. these sllces were dried on fllter-Paper squares (1.3 x
1.3 cn) a¡rd cor¡oted {n toluene-based sclntillatlon fluld as
descrl.bed in Sectlon 2.5.1.1.
2.3.L.3 Base compositLon
Yeast RNA (f rng/nl) was added to fractlons
from l{AK chromaÊography and the RNA precfpitated wl.th 2.5 volu¡res
of ethanol. After washíng r¡Ith ether and drylng ì.n vaato, the
preclpÍtate w¿rs dLssolved lû water and reprecipftated wlth a ftna-l
concentratlon of 0.2 M-PCA and thfs precÍpltate hydrolyzed f.n
0.3 M-KOE at 370 for 20 hr. Tl¡e solution was neutralízed wLth
37
1.2 U-PC'A and, after ceûtrlfuglag at 40ogr for l0 aln, the
Supernatant was evaporated tO dryneSg undef a gtreârir of e.eol,
ffltered a1r. The resldue wag then dlssolved 1n 0.1 nl water.
Paper electrophoresls ltes used to sepa-rate the four
oonophosphaÈes. Samples (up to 40 Ul) or authentlc rnarkers
(0.1 Unol of eactr) ltere spotted onto the orl.gfn points of l{tratnan
3 MM paper. The papers were wet wÍth buffer (0.1 M-citrate' pE
3.6) a¡rd excesa tüas¡ reno\ted wlth absorbent PaPer. Electrophoresls
lfas carrled out for I20 nin at 26.5 volts/cmr wlth the Paper
1ærsed ln a ta¡rk of carbou tetrachlorLde as coolant, Nucleotfdes
noved towards the anode and çrere located wrder short-wavê Û.V.
radfation a¡rd a cofiËact photogrePhLc prfnt produced with the same
radlation source. The electrophoretogr€us !úere then cut loto 5 nn
sections aod radfoassayed wLth a Beckman Lowbeta II gas-flow
planchet counter (Section 2.5.2) to deternlne the ratlo of counts
lncorporated into the four uucleosfde nonophosphates.
2.3.L.4 DNA-RNA hvbrLdizatlon
The technlque of Gl1lespie & Spiegelnan
(1965) was¡ used. DNA eamples (Sectlon 2.2.1.4) were dlluted to
50 ug/nl 1n 0"lxSSC and denatured by adJustlng to pll 12.5 wlth
0.1 ll-KOII a¡rd stlrring gently for 15 oln. Ttre solution was then
neutrallzed with 0.1 Þî-UCl and dfluted to 5 ug/nl !,riËh 2XSSC.
Filters (SartorLus Membtane Fflters, Göttlngen, l{est
Gernany¡ MF 50, 50 m dl.ameter) were pre-sôaked for aÈ least t hr
38.
1n 2XSSC and r¡ashed r¡1th 100 nl of the same buffer. the deaatured
DNA solutlon was passed slorly through the fÍltersr whÍch -vere
thea ¡¡ashed r¡fth 50 nl of 2xSSC. After drylng cvernfghtr the
filters were further drled at 80o in taauo for 4 hr. Blaok fllters
frere treated sln1larly except chat 2XSSC was used lnstead of DNA
solution. DLscs, 5 n¡n Ín dlameter, rtere cut from the fllters with
a sterillzed cork borer, and some checked for uoiform DNA
distributfon by deo:cyrlbose asaay (Sectfon 2.2.2.3). Before use,
the ftlters¡ were pre-lncubated for I hr Ln 2xSSC at 70o. Any DNA
not tlghtly bormd to the fllter was lost durLng this pre-iucubatLon
and further losses !üere very sna1l (see Figure 33 l-n Sectlon 3.6-2).
Ilybrlds were formed 1n tightly stoppered 0.8 x 7.0 cm test-
ttùes by in'nersl.ng three DNÀ ftlter discs (0.4-f .5 ue/disc) and
three blank filter dlscs 1n 0.2 nl of 2xSSC containing the amouûts
of RNA Lndicated Ln the results (Sectlon 3.6,2). The tubes and
solutlons Ìüere pre-heated to 70o before addftl.on of the fllter
dlscs. Incubatfon ¡vas for 6-8 hr at 70o. The Rl{A solutlon r¡as
then renoved and the flLter dfscs further fncubated fn 2xSSC for
5 mfn to nLnfnlze non-speclfic hybrfdizatÍon (Blshop 1970,
Benedich & McCarthy 1970). Ttre dÍscs vere washed t¡dce fn 2XSSC
(standing ln lce l0 nin each wash), then incubated for t hr at 30o
tn 1 nl of 2xSSC containl.ng 50 Ug of RNase (pre-fncubated 10 nfn
at 90o). After washlng three Èl.oee hrfth 2XSSC, the filters ¡rere
drled and radioassayed as described for samples on paper Ín
Sectfon 2.5.1.1. The bla¡rk values were deducted fron the
e:<perimental values to glve the amor:nt of hybrid fomed. Uader
39
these condltÍons, a hlgh degree of speclficlËy ls achieved (Church
& MeGarrhy 1968, Thonpson & Clela¡rd lg7Lbr. Several addftlonal
ÐNA fflters from the same batch ¡yete carrÍed through the entlre
procedure ¿md then assayed for DNA conteot either by the deoxy-
rl.bose ¿rssay or by lncubating ln I nl 1 M-ItCl at l00o for 15 nln
a¡rd measurlng the absorptioa at 26O nn (Section 2-2.2.Lt.
Coqetftlon rùas achieved uslng the sinuLtârìeous lncubatLon
technlque. Fllter dlscs were incubated wtth const¿urt amounts of
labelled RNA (approaehiag the prevlously deterulned saturatlon
level) and lncreasing amowrÈs of r¡olabelled' conpeting RNA as
lndLcated in the results (Section 3.6.2r.
2.3.2 Nucleotfdes
2.3.2.1 DEAE-Sephadex
The nucleotides in the acld-soluble fractfon
(Sectlon 2.2.L.2) were separated by chronatography on DEAE-
Sephadex.
DEAE-Sephadex (A-25) was hydrated overnLght 1n water and
then washed by decantatlou, flve tlmes wfth 0.5 I'l-NaOlI, four times
¡vfth ¡¡ater, five tlnes rífth 0.5 M-sodir¡n blcarbonate and ffve
tloee wl.ttr water (Ca1&¡e11 1969). A slurry of Èhe Loo-exchanger
was poured fnto coluurs 0.9 x 20 cn a¡rd ¡¡ashed !úfth at least
200 û1 water. Ttre aeld-soluble fractfou (dlluted 5-fo1d wLth cold
water to ensure a lorr salt concentratl.on) was then loaded onto the
coltm at a flo¡r rete of 40-50 rnUhr. Unlabelled marker
40.
nucleotldes (0.25 Unol each) were loaded wfth the saryle. l{tren
loadiag was completed, the colur¡n was lr:rshed r.rlth water un:1.1 no
u.v.-ebsorblng Eaterlal was present I'n the effluent.
The nucleotldes were eluted wfth a llncar gradLeat of
trlethylamonlr.rm blcarboûate 1n wtrich the nfxlng vessel lnitlally
coataLned 400 nl of water and the reserrrolr 400 nI of the buffer
(1.0 U, pII 8.0). Ttre flo¡y rate ¡¡as 40-50 nl/hr. Ttre effluent !ûas
monitored auto,matically at 254 nn with an LKB Uvicord II ultra-
vfolet absorptloneter, and fractl.one of 4 n1 were collected and
radfoassayed. for [lhC] aod ¡3n1, allquots of the fractlons r¡ere
6potted onto glass-paper squares and radfoassayed as descrfbed 1n
Sectlon 2.5.L.1, and 32P-costainiug panples túere assayed by
Cerenkov radiation (SectLou 2.5. 1. 3) .
2.3.2.2 Dowex-1
Nucleotides ln the acld-soluble and
hydrolyzed acid-lnsoluble fractfons were algo separeted by
chronatography on Dor¡ex-l resÍn.
Dowex-l (Blo-Rad, AG l-x8; 200-400 nesh) !ùas converted from
the chlorfde to the for¡rate forn by soaklng fn 0.2 M-NaOE for
60 nÍn, washing wfth water untÍl approaching neutrallty, then
washÍng twÍce wlth sodit¡m formate and once l¡1th 5 M-fornf.c acid
(Hurlbert et aL. L954). After agall washfng wl.th water untll the
pB approached oeutralfty, the resin !ùas stored. at 2o r¡nder lteter.
Columrs (L.2 x 10.0 cn) of the resln were prepared and
47.
washed with 2-3 bed voft¡nes of 882 (v/v) formlc acld and then r¡lth
rùeter r¡otll ttre pll approached neutralfËy. The sanple lras then
loeded end washed wfth ¡rater r¡nt1l no u.v.-€bsorbing material l¡as
present 1n the effluent.
Elutfon ¡¡as achleved wfth a te¡o-stage llnear gradÍent system.
A 0-4 M gradieat of fornLc acÍd (100 nl) was followed by gradfents
of either O-0.5 M (for seperetfon of oononucleotides) or 0-1.0 l'f
(total aucleotl.des) .amoniun form¿te Ln 4 M fornfc acid (100 n1).
Ttre efflt¡ent was monltored a¡rd fractions collected and radloassayed
as 1n Sectfon 2.3.2.L.
2.4 Enzvme Actlvítv DeterElnatlons
Only crude plant extracts ¡¡ere used for enzyne detettlnatlons.
The rate of actfvity lras detemlned fron the fnltlal llnear part
of the tfme cun¡e Lf the actlvlty was ûot llnear for the full
reactÍon tine. Zero-tLne and boLled-extract controls were
included wlth all ¿rs¡6ays.
2.4.1 RNase
Root tfps were honogenlzed fn 8 n1/100 ng
acetate buffer (0.1 tl; pII 5.4), a¡rd the degradatloo of
acLd-soluble nucleotldes and olLgonucleotfdes r¡as used
for Rl{ase actLvlty (tlalters & Loriug f966). The assay
(f .w. ) of
RNA to
aa an essay
mfxture
42.
Ln a total vol¡¡me of 2 nl of acetate buffer (0.05 M; pÉ 5.4)
corÈafned 4 o.D. units of, dLabyzed yeast RNA a¡rd 0.1 nl of plant
eatract. the reactlon was lnitiated by addlng plant extract, and
termLnated lrtth 2 ul of 3 oÌ.[-uranyl acetate fa 0.2 M-HCI.
Incubatlon was at 37o and sançles were taken et verlous Petlods
between 2 æð 30 nln. After precfpltatlng ln Lce and centrlfuglng
for 10 min at 20 and 8009, the optical extfnctlon of the super-
natent solutl0n was measured at 260 r¡n a¡rd the rate of RNA
degradatlon calculate¿.
2.4.2 UrLdlne kfnaqe
Root tips were houogenfzed at 2o In 2 ø'Llg (f 'w') of
50 nlf-lris (pB g.0) cootainlng I nll-glutathlone, and then passed
through a French Pregeure CeIl at 21500 p.s.i. The couversion of
lfhC]urf¿loe to IIhC]Uæ vas used as a measure of uridlne kinase
actirrtty. The assay nlxture ln a total volt¡rne of 0.4 ml contafned:
50 pnol Îr1s (pE 8.1), 5 ttmol I'fgcl2, 1.5 l¡nol nercaptoethanol'
15 ¡r'ol ATp, 5.6 nnol [Z-1rC]uridfne (4.e x 10b c.p.n./nnol) and
O.O5-0.1 nl of plant extïact. Ttre reactlon was fnltfeted by addfng
the plant extracË and, after lncubatf.ng at 30o for varior¡s perf'ods
between 30 and 120 nLu, was terninated by acifllficatLon ¡¡Lth
1.25 Dl lgt (¡¡/y) TCA. After etafidlog at 2o the supernataût lras
re¡noved and extracted tlrice rvlth ether, a¡rd then 0.04 n1 of each
saryle (to whtch was added 0.1 umol each of uracLl, urldfne and
IIMP) was sPotted onto l{hatma¡r 3 MM chroaatography paper and
developed by ascendlng chronatography 1n 0.25 M-fomlc acÍd. The
43.
marker substances !ùere located under u.v. radlatiou, a¡d a contact
photographíc print ltas prePared wlth Èhe same light source. Tte
ctrromatogrârn wEùE¡ cut lûto 5 @ strfPs and radLoassayed (Section
2.5.L 1) to detemf.ne the coraverslon of IlbC]urldfne to [fa6Jmæ.
2.4.3 RNA polFerase
Plant extract ltas PrePaïed aa in Sectlon 2'4'2, and
Èhe fncorporatlon of labelled nucleoslde trlphosphate lnto lCA-
insoluble ¡raterlal was used as a measure of RNA synthesLs. The
assay uixture 1n a total vofu¡ne of 0.15 n1 contained: 1 Umol
!hC12, 20 Unol (!¡E¡+)ZSO¡+, 0.1 Umol heat-denatured calf th¡rnus DNA'
20 unol trfs (pE 8.0), 0.4 unol glutaÈhf'one,0.4 unol CTP'
0.4 uu¡l GTP, 0.8 unol ATP' I nnol [r+c]uÎP (105 c.P.n.) a¡rd
o.o1-0.05 nl of plant extract. Ttre reaction was fnitlated by
addlng plant extracÈ a¡rd fncubatfon r,ras at 37o for various periods
bet¡reen 15 and 60 nfn. Ttre reactlon was terninated by placlng the
¿rssay tubes ln lce a¡rd addÍng O.2 r¡1 of 0.5 Èf-PPi and 0.05 nl of
10 mg/nl yeast RNA follor¡ed by 2 nl of 102 (r¡/v) TCA. lhe mixture
rùas cenËrlfuged a¡rd the precipitate ¡¡ashed four tlmes wlth l0Z
(w/v) TCA and finally dlssolved overnl.ght ln 1.5 ¡n1 of 2N-NH4O1I.
Then 1.0 nl was dried onto a planchet and radloassayed as in
Sectlon 2.5.2.
44.
2.5 Radloisotope Deternloatloos
2.5.L Lfquld sclnttllatlo'¡t
2.5.L.1 Geoeral
lfost saoples containing [3g] ana [lac] ana
sone contalning [32pJ were radioassayed on paPer, efther as areås
cuË from electrophoretograms and chronatograms or as alf.quots of
sauples dried onto squeres of glaes-flbre PaPer (2 x 2 cn; T{hatman
GF-S3). Unless ottrerwfse indicated, sËandard glass vfals (Packerd
Instrument co,, chícago, IlllnoLs, U.S.A.) were used nrlth 2 ul of
sclntlllatlonr fluld cootainÍng (1n glLl:tre of A.R. toluene):
2,5-dlphenyloxazole (PPo), 3.0, a¡rd 1,4-bIs-2-(51heny1oxazo1yl)-
benzene (PQPOP), 0.2. Radloassays rûere usually done 1n a Packard
lrfcarb model 3375 lfquld sclntillation sPectrometer, but sone
[1+C] sauples were assayed fn a Unfh¡x bench-top rcdel 6851
apectrooeter. Sanples contalnlng perchlorlc acld (PCA) were firet
made alkallne wtth 2 M-K0H and cooled in lce to allow KCIO4 Èo
precLpitate, since the presence of perchloric acid results in
severe aod variable quenchlng. Channels-ratLo quench-correctlon
curves calculated usfng Packard Instrument Co. standards have been
used to deternfne cotmting efffefency and absolute dlslntegratlon
raÈes where indlcated.
2.5.1.2 Dual-label analysLs
For experfnents fn whlch [3n] aua [r+c] were
4Ð.
used sinultaneously, eaqrles were spotted ooto glass-flbre Paper
aquates and radfoaseayed tn 2 nl scfntillatlon fluld (SectLon
2.5.L.1) tJr nyLon scfntillatlon vialE (Nuclear Ctrlcago Cor7., Des
Plalnes, IllÍoois¡ U.S.A.). Saqles were assayed lrfth the three
dÍscrlÊtnator char¡nels of the Peckard ltquid sclntlllatlon
spectromeÈer, and count retes for both [3nJ ana [lhC] *.r"
corrected for quench aud converted lnto estimatee of absolute
dÍslntegration rates by meæs of a series of standard quench
curves. These curyes were derived froo data obtafned with a set
each of [3tt] aud [14C] queûch sÈandards prepared Ln the s¿rme forn
as actuel sanples, as Lndfcated fn Elgure 3. The numerical
calculatfons, made with a desk-top calculator (SharP Cornpet-32
fitted with a lfenorizer-60 Autoo¿tic Programer) lúere essenttally
sl-n|Iar to those described by lletenyL & Relmolds (1967) ' excePt
that channels-ratlo values (derlved froo the [IaC] present fn each
saryle) ¡¡ere used for quench deternloatloû instead of an extetnel
standard channels-ratlo value. the [3II] sta¡rdards nere corrected
for progressive decay.
2.5:L:3 Cermkov radiatlon
Aqueous [32PJ sry1es were usually
radÍoassayed by Gerenkor¡ radlatlon. Sauples ltere nade rp to
constant vol¡¡oes (fO-14 nl) wtth 0.1 M-phosphate buffer (plt 7-0)
and aesayed using the pre-set trltLun cha¡nel of the Packard
liqul.d scintlllatlon spectr@eter. Quenching was always el.nllar
withf.n each set of saoples, so correctlons for quenchlng were aot
nade.
EIGIRE 3 : Øpnnh-eow,ctí.on eoLibmtiott cun)es for l3ul + [1+C]
ùtøL-1.abeT eqterirænte. 1\¿o sets of chemlcally quenched
lsotope standards were prepared:- Seyiee b) Sfx sanples of¡2-lr+c]urfdlne (911000 d.p.n.) drled onto 2 x 2 csr squares ofglase-fibre peper were sealed 1n nylon scintlllatLon vl.ale
sontaioing 2.O nl of scintlllation fluid plus gtaded amor¡nts
(0-20 Ul) of carbon tetrachloride as quenchfng agent. Setiee
ft) As for lal uslng [5-3E]urfdfne (5531000 d.p.m,) fn place
of [z-1\g]urÍdÍne.
The standards were prepared ag 'natched pal.rs (one each
of [lhC] and [38] containing ldentical a¡or¡ots of queachlng
egent), so that ueasured [3n] efffciencies could be related tothe channels-ratfo values obtafned rftth the correspondfng [thC]series.
Standards and experlnental sanples were radLoaesayed lnthe Packard sclntlllatlon spectrometer rrÍth the three channels
calibrated as follq¡s:
ChøræL Diec*iní*øtor LiniteRED 20 - 500GREEN 175 - 1000BLUE 325 - 1000
Ilhc] d.p.m. =
[ 3tt] d.p.n. -
Ieotopee deþected[3n] ana [14c]
I rrcJI Ihc]
tuin (7)100
II
For each sauple the clran¡reLs-ratÍo value deternfned fot[14C] (BLITE cormÈ: GREEN cor.nr;1.e. B:G) was used for corÍtt-efffcfency estiuatfon by meaos of the stardard quench-correctloucuti\¡es obtalned with the standards (tt¡ts fig¡¡re). Ttrus, theamourits of the Ísotopes (as d"p.n.) 1n each sample were calcu-lated as follo¡¡s:-
GREEN c.D.m.
REÐ c.p.n. - (tlhcl¿.p.r. x [lhc] co,nt-efffcfeacv tn RED)[ úE] cornt-efflcfeucy io RED
Ttre standard quench-correctfon cuÌves of thfs fLgure aÌè!-A; [lbc] count efflcfency fu GREBiI v. [1+c] B:G ratloB : [3n] co,rot effLciency 1n RED v. llhcJ B¡G ratloC : [tac] count efficiency La RED v. [l4cJ B:G rario
:.* 40
(Jc(U
(J
.E 30
(u
C')cP520o(JoCLo+,?, l0
H
50
0.1 0.2 0.3 0.4
Channels ratio : Blue:Green
0.5 0.6
FIGURE 3 : Quench-corryection ealibtwtíon curDes for l3A) +
[ 14c] duaL-LabeL erper'tments.
00
B
A
46,
2.5.2 Gas-flow analysls
Low-activity samples !üere often radl'oassayed 1n a
Becloan Ioqrbeta II gas-flow planchet courter (background 3 c.p.u.).
Aqueous saqles were drfed ooto rlaged plane.hete (5 cn diaoeter)
and electrophoretogran sectlous l*ere attached to the planchets
rrlth adhesLve tape.
2.6 Other lechniques
2.6.1 Protefn deteminatlons
Proteln was deteruloed with reference Èo a sta¡rdard
solutfon of bowl.ne serum albr¡nLn by the Folin procedure of Lowry
et aL. (f951) after precipitatÍng the protein Ln 57. (w/v) tCl.
The optlcal extinctloa l¡as measured at 725 nm.
2.6.2 CeI-l cor¡nts
Ttrfrty 5 m root tfps were lncubated in I nl of 107"
(w/v) chromic acld for 24 ht at 30o (grolrr & R:lckless 1949).
llaceration was conpleted by presslng any lumps agalnst the side of
the test-tube with a glass rod aad fotclng the suspensfon through
the flne hole of a pipette. Cell counts were nade 1n dupllcate oD
each suspensÍon usfng a haenacytometet.
47.
2.7 Materials
2.7.L Buffers
Buffers and other aqueous solutfons were made up ln
elÈher glass-dlstllled vater or water further distllled ln a
efllca apparatus (Sectloa 2.1.2). Stock eolutioos (1 tt) ot
phosphate, Trls (slgna ctræ1cal Co., St. Louls, MÍssourl, u.s.A.),
citrate and acetate buffers rtere prepared as described by Gonori
(f955) aad stored frozen at -15o. Èfeasurements of pH were oade
ax 2O-25o on a Beckman pH netet sta¡rdardlzed r¡ith 0.05 M-potassiuû
hydrogen phthalate buffer (pE 4.0) or staudard conceatrated buffer
(pH 7.0) produced by Becknan Instrumeats Iûc. (Fullertoo,
Callforaiâ¡ U.S.À.).
2.7.2 Bl.ological p¿.terLals
Bovfne serun albtmin was purchased from the
Co om¡ealth Sen¡n LaboratorÍes (AustralLa) and yeasÈ RNA fron
Schwartz BloResearch Inc. (Orangeburg, New York, U.S.A.). Calf
thSrugs DNA was prepared by Dr. J.F. Jacft-son as descrfbed by
Jackson et aL. (1968).
DN¿se (elecÈrophoretfcally purified, Rlilase free) a¡rd RNase
rrere obtalned fron tlorthington Biocheml-cale CorporatÍon (Freehold,
New Jersey, U.S.A.), pronase from CalbÍochem (Los Angeles,
CalifornLa, U.S.A.) and diastase from AJa:c Chemlcals (Auburn, Netr
South Ï{ales, Australfa).
48.
2.7.3 Nuclefc acfd
all nuclelc acid compoûeDte were purchased f¡om stgna
GhemÍcal Co. (St. Iouts, ÈllssourL, U.S.A.).
Stock solutlons lrere prepared fn distl.led rtater aad titrated
to pE 7.O-7.5 with 0.3 M-HCI or 0.3 Dl-NaOH. Concentratlons ¡¡ere
caleulated on the basl.s of nolar extlnctlou coeffÍctents and
optical- extlnctÍon readl.ngs at the wavelength of maxlmr¡o abeorption
measured oa a Shfnadzu spectrophot@eter. Molar extlnction
coefffcients were obtained fron publfshed sources (e.g. Sch!üaltz
BloResearch Inc., Radioeheul'cal Catalogue, 1969).
2.7 .4 R¿dloisotopes
Labelled nuclelc acld eoryonents were obtalned from
the Radlocheaical Gentre (Amersham, England) and [32pf] (fu dllute
ECl) fro'n the Australia¡r Atonic EnergT GomrnLssfoo' Isotope
DtvLslon (Lucas Helghts, Sydney, New South lJales, AusÈralLa)'
2.7.5 Other materials
Solutes for lLquld scÍntillatfon flulds were from
Packard Instn¡nent Co. (Ctricago, Ill1nols, U.S'A.).
Bentorilte ¡¡as obtaLned as technical powder from Brit{sh Drug
Eouges (Poole, Eugland).
DEAE-Sephadex was srpplled by PharnacLa Elne chenicals
(Uppsala, Sweden) a¡rd Dor¿ex-l by Bfo-Rad LaboraËorÍes (Rlchmond,
CaLffornÍa, U.S.A.).
49.
Atl other ctrenlcals ¡vere of analytleal reagent grade,
obtalaed fron Ajax Ctrenlcal Conpany (Auburn, Ne¡v South Wa1-es,
Ar¡stralta), ÌÁay & Baker (Dagentran, Englaad), Unlvar (Auburn, Ne¡v
South lfales, Atrstralla) and Brltigh Ilmg Houses (Poole, Eogland).
50.
3. REST]LTS AND DISCUSSION
3.1 Morphologl.cal Sy¡mtons of Boron Deficfency
Before enbarklng g(r bloche¡rLcal studles, some of the vfsible
sruto4¡ aod general phystologlcal effects of boron deff.cLency
¡¡ere studied. Thls was done for the followlng re¿rsons:-
a. To detemine whether Phaseolus al?eua erhlblts sLuilar
deflciency spËons to those reported fn other plant
specl.es.
b. To establtsh a tlme sequence for the laitfatÍon of
visible s)tnPto¡ns and physlologfcal effects of the
deficÍeucy ln Plnseolus auteus.
c. To determlne the lnteractions betr¡een these factors, so
that the changes Ln biochenical paraneters could be
correlated with the early gro!ùth and physl-ologlcal
effects of the defl.ciency.
3.1. I Root tLon
Boron deficlency has a rapf.d effect on root
elongation, as shown in Figure 4. Inhfbltfon of root elongatfoo
was usually observed after 24 hr æd complete cessatlon occurred
after 120 hr growth tn deficleot nedl.um. Ttrls ls somewhat Longet
thari the cessation of root elongatf.ou after 24 hr reported by
Neales (1960), AlberË & lt1lsoo (1961) and Yth & Clark (1965).
Differences betrreen specLes (Neales 1960) or stages of plant
00I
E
coP(õc')co(¡J
PooÉ.
ls0
50
00 100 200
Tirne after transfer : hr
300
FIGURE 4 : Effect of boz'on defiei-encg on root elonga-
tion. Plants \¡rere gror,üTì in normal and defícíent
medium as described ín Section 2.I.2. Roots were
marked at the time of transfer to defícient medium and
Ëhe subsequent elongation measured as described inSection 2.1.3. The Ëíme after the transfer of plants
to boron-deficienË medíum is plotÈed on the abscissa.
-B
+B
57.
gronth (l{aclnnes 6¡ Albert L969r, or the preseDce of traces of
boron Lo the nutrlent nedir¡n nay contrlbute to this slcnrer
reactlon. Soæ variatlon between plafit batches occurred, but only
plants ln which lnÌrlbttl-on and cessation of elongatfoû took place
wlthl.n 48 hr arld 150 hr reepectlvely r¡ere used for the açerlmental
work reported in thls thesl.s.
3.L.2 thlckening of roots
ThÍs eras usually obsen¡ed Just prlor to the cessation
of root elongation. Figure 5 lndicates that ¡¡hen measured as ac
lncrease !n fresh wefght, this ffrst occurs after 50-100 hr 1n
defLeienÈ nedir¡m. Yfh e Clark (1965) observed simllar changes
after 72 trr. the dlfference bet¡¡een deficient and notmal rooÊg
lncreases w"ith the tlme that the plants are grown Ln deficient
medium; this appears to be mainly due to cell ealargement, and
not to an fucreased number of cells (Table 2). The method used
for the estiuatlon of cell nt¡mber does aot pernit accurate
deterninaÈlqrs in merísteûatlc reglons containfng large nr¡mbers
of snall and lnconpletely separated cells, æd falls to
differentlate betl¡eea cell types. Horever, Ëhe results indlcate
tbat the deficiency has lltt1e if any effect oû the nunber of
cells per 5 nnn root tLp. The dfffereûce fn wefght Pel root tlP
is due largely to increased cell slze. The suspenslon of boron-
deficlent root-tip cells contaLned Eany very large cells' several
tLrnes the size of the largest observed Ln suspensfoas obtalned
from norual root tips.
=Its
tt)E
ô+,{Joof-
'-(uo-+i!g)(l,3
0.8
0.6
0.4
0.2
100 200
Time after transfer hr
FIGIIRE 5 z Effect of bonon deficiencA on auer.q.ge
ueíght per root típ. Plants \^rere gro\¡rn in normal and
deficient medium (Section 2.L.2) and at various time
int.ervals 200-300 rooË tlps were excised, weighed, and
the average weight per root típ ca1culated.. The boron-
deflcient roots ceased el-ongating after 150 hr indefícient medíum.
03000
.B
o
+B
û2.
TABI,E 2 : Effeet of borcn defieiencg on t7,Ê nt¡rtbet øtd avetageueight of cells ì.n 5 ¡¡m rcat tiPs
5 nm root tips were exclsed, welghed and
mace¡ated as descrlbed fn Sectlon 2.6.2- Each set of
results ¡¡as obtalned from plants grotün for 200-300 hr
ín deflcfent medium, and values are the me€ms of two
deterol.nations on each samPle.
cel1s/rip cells/ng (f .w. ) Anerage weight(ue)/cell
+B -B +B
25,3OO 32,800
22,600 27,80A 58,600 25,000 0.018 0.040
19,100 20,2OO 50,800 18,100 0.020 0.055
3.1.3 Bror¡ning of root tÍPs
The colour of boron-deffcient root tlps began to
change from white Èo bror¡n after approxlmately 100 hr 1n deflcient
medirrn (i.e. Just prior to the cessatlon of root elongatlon). The
colour becane darker with tlne and eventually affected the entire
root systen.
3.1.4 Lateral roots
.B +B -B
Lateral root grotrth was lnhfblted, anrd eventually
5'ó.
stopped at appïoxÍnately the same time as the n¡{a root (Figure 4).
Ètaay of the lateral roots ceased grwth when only 3-5 m long.
As the deffcleacy PlogÌessed (250-300 hr), lateral root' laLtfals
(1-2 m long) appeared at 2-5 ñn lntentals rlght to the root tlP.
Ttrls then contrlbr¡ted to the íncreased welght of the root tlps'
and nakes ¿my coryartson betrreea oomal and deficlent tlssue ûore
difflcult at Èhls stage.
3.1.5 Sr¡rnptons 1n ae,rial p1aût parEq
Synptons of boron deficLency sln{lar to those
reporÈed fn other plants were obse:nred, buË Ëheee only occurred
several days after root elongatioa ceased' the stem and leaf
petloles beca:¡e thlcker and more brittle and the leaves a darker
greea colour tha¡r ln normal plants. After 250-300 hr the terulnal
shoot dÍed and fel1 awaY.
3.2 Physlological Effects of Boroa lÞff-ciencv
3.2.t Protefn
I{tren the proteLr coûtent Per root tip 1s plotted
agaiust ttne (ptgure 6a), the changes are sirrllar to those
obqewed for fresh weight (Ffgure 5). As plants grow aod the
root system becomes more extensfve, notmal root tiPs becoae
thLnner and contain less protelo, eventually reaching a constant
b
+B
-B
I
a
.B
+B
o-+J
poor-C',
=
g
(u+Jo!ô-
8
6
4
2
0
Bdlsu)g)c
10cc,+JoLÀ
50 50 100 150 200
Time after transfer : hr
250
FIGURE 6 : Effect of boron deficíeney on tLte proteincontent of 5 nrn noot típs. 1000-2000 roor ripswere excised and homogenized as described in SectÍ.on
2.4.2. Protein r¡/as determined as d.escribed in Section2.6.L
a Protein (Ug) per root tÍpProtein (mg) per grarn (f .w.) of root tipsb
54.
value lOO-120 hr after the first nutrleot change. DefLcfent root
tlps folloç¡ a simllar '¡rattern for the firsÈ 48 hr of deflciêncY'
but then, as the tlps becone thl.cker, the protefo content lncreases
to a level much higher than that of normal root tÍPs. oo a freeh-
!üef.ght basls (rtgure 6b) the :rotef-n conteat of normal tlps
decreased slfghtly, r¡ntil lOO-120 hr after the fLrst ûutrient
change, while ln <lefl-cient tfps it declined steadlly throughout
the study, reaching a sllghtly lower level than the nomal root
tfps aftex 22O hr growth 1n deficieot medirn'
SÍnce protein levels closely folIo¡¡ fresh-weight changes'
some riet synthesfs must occur at all stages of deflclency (up to
200 hr -
Ffgure 6). Thls 1s, however, conslstent l¡ith a decreased
rate of proteiû synthesis (Sectlon L"4.2) because the raËe of
Lncrease ln fresh weÍght of non-elongatiag boron-defícient roots
is lesE tha¡r that of elongatfng normal roots (Odhnoff 1957), so
that a lower tate of Protein sJrnthesfs 1s required to mal'ntain the
proteln content showa 1a Flgure 6.
These results demonstrate the stgnfficance of ttre choice of
parameter on whlch comparfsofx; are made. Based on the content per
root tl-p, Èhere 1s an lncrease fn protelo followfng the lrtthdrar¡al
of boron from the mediun, whíle on a fresh'treight basl's a sllght
decrease 1s obserrred. From the results 1n Table 2, an lncrease 1n
the proteln content Per ce1l would also ï:e erçected. These
dÍfferences help explain the contradictory results dlscussed 1n
Section 1.4.1.
55.
3.2.2 RNA
Boron deficiency results ln a decrease ln th: RNA
level (RLgure 7) on elther basÍs shown (fresh-r¡elght or rooË-tl.P),
but the dfffereoce ís greater on a fresh-wei.c.ht basfs. RNA
deternfnations by Ëhe absorptLon at 260 nm and by the rfbose and
phosphate assays gave coryarable result,s. InclusLon of the
lipid-extraction steps recormended by Mr¡¡rro 6] Fleck (1966, p.125)
or omLssion of the luitial extractlon fn 802 (v/v) ethanol had
lfttle effect on these values. The effect of boron defl'ciency on
RNA content is first obsen¡ed Just prLor to the cessatios of root
elongation. Thls is sinflar to the observatl'ons of Albert (1965)
and Jaweed & Scott (L967, ¡rhere changes 1o RNA leve1 were not
detected r¡nËÍl Just after root elongation had ceased. The results
in Table 2 suggest that a elight decrease fn RNA per cell would
also be e:gected.
3.2.3 pNA
Changes in Dli[.,A cotrteut also depend on the vay in
which the results are expreased. Boron deficLeacy lncreases the
DNA coutent per root tLp, but has I1tt1e effect on a fresh-weight
basl.s (F1eure 8). TtrÍs increase ln DNA per root tlp (a¡¡d therefore
probably per cell) suggests that DNA synthesfs contLnues after
ce1l dlvislon stops, produclog an approxlnately doubled DNA
content per cell. Thus, although the RNA content per cell falls'
the DNA coûtent is doubled under boron-defLcient condftlons.
I
o-+,+,oof-
oE
z.d.
Bq-
O)E
oEc
z.æ.
6
4
2
5
0
0
0I
0 100 200
Tire after transfer : hr300
FIGURE_7 : Effect of boron deficiencg on RNA eontentof 5 rwn root tips. RNA \nras extracted from 250-300 root tips (Section 2.2.L.1) and detennined bV (i)absorption aË 260 nm, (ii) ritose merhod and (iii)phosphate method (SectÍon 2.2.2). Mean values fromthe three meÈhods are plotted.
a. RNA (nrnol) per root tipb. RNA (nnol) per mC (f .w.) of roor rips
.B
b
+B
.B
a
+B
ì
2.0
5
.0
0.5
b
a -B
I I
a
.B
I +B
I
cl+,+Jootoc
=o
0
2.0=(F
U)E
oFc
=o
t.0
0100 200
Time after transfer : hr
300
FIGURE 8 : Effect of boz,on defieieney on DNA contentof 5 nrn noot tips. DNA was exËracted from 250-3OO
root típs (Seetion 2.2.I.1) and derermined by thedeoxyríbose assay (Section 2.2.2.3).
a. DNA (nmol) per root tipb. DNA (nnrol) per urg (f .w.) of roor ríps
0
56.
3.2.4 Acfd-soluble fractlon
The acLd-soluble [O.e u-pcL ot 5I (wlv) TCA] fractioo
extracted by the SchnidÈ & Thanahauser method (Sectf.on 2.2.1.1),
even with the fncluslon of ltpid-extractfon steps (Munro & Fleck
1966) before acid extraction, contains large quåf¡tltles of
non-nucleotlde naterlal r¡hfch laterferes ¡rlth the various
nucleotide deterninatÍons (e.g. absorptlon at 26O nrn, ribose and
phosphate). Ttrus, the estLoatlon of nucleotf.des Ís not feasLble
on such a crude fractl-on, but ehanges Ín the levels of subst¿mces
reacting wlth the above assays may be of interesË. Flgure 9
LndicaÈes that large lncreases take place Ln these three grouPs
of substances (i.e. substances absorblng at 260 nn or reactLng 1n
rÍbose or phosphaÈe assays) after 50-100 hr 1n deficient medlum
(at the tfæ !,then root elongation ts inhíblted) ' No changes can
be detected at earlier stages. If the lfpid-extractioû stePa are
fncluded before the acfd extraction, the values are reduced by
50-707" but the general effects of the defLcLency are unchanged.
It 1s Lnteresting Èo note that the RNA coîtent of deffcLent
tissue decreases at the same Ëine as these substances fn the
acLd-soluble fractfon lncreaee. Thfs may suggest that with the
onset of deficlency s)'EPtoms, eLther synthesís of some macro-
molecular coupounds ceasesr or degradatl'on rates lncrease,
resultlng 1n lor¡er Ievels of the high oolecular-¡¡eight compormds
and accr¡mulatlon of precursors or breakdown of products.
The ¡neasurenent of nucleotfde levels fs dlscussed in
Section 3.4.
FlcttRE 9 z Effeet of boron defieí.enq on the aeid-soLub|.e
ft.actíon of 5 tnn rcot tips. the aci&soluble fractLott was
extracted fron 250-300 root tfps (Sectlon 2.2.1.1) atrd
determiaed by abaorption aX 260 nm and the rfbose and phosphate
asÊays (Sectfon 2.2.2). Ttre values plotted are the amourits of
^ål{P requÍred to produce an equlvalent optlcal abaotption or
colour fornatl.on 1a the assay.
a. & c. Phosphate of *B (¡) and -B (o) root tLPs
b. & d. trÈl.bose of iB (A) a¡r¿ -B (Ð root tfPs
b. & d. Absorptioo et 26O nn of +B (o) and -B (o) root tlPs
o
o
c
o
a
0I
o+,Poot.
oeg
20
l5
5
30
0
3,d 20
q)E
ocË10
00 t00 200 300 0 t00
Tirne after transfer : hr
200 300
FIGUIü_9 : Effeet of boron deficienq on the aeid-soLubLe fraetionof 5 rnn root típs.
d
b
57.
3.3 Uptake and IncorDoratlon of Labelled Precurso¡s
of NucLeLc Acídsi
3.3,1 Effects on total upÈake and incorpqrêltou
3.3.1.1 Uridlne concen 1n Èhe LscubatÍon
medfum
FLgure 10 shows the effect of the concentra-
tfon of urldfne 1n the fncubation medlum on both its total- uptake
and its lncorporatfon lnto RNA. For subsequenË [raC]urídine
experlments, 25 nmoUn-l r¿as chosen as a suÍtable coupromÍse
bet¡'reen the follo¡¡1ng factors: -
a. Economical use of both labelled a¡rd unlabelled utidLne.
b. Incorporation of sufficient of the radfoactlve materfal
(at the specLfic activLty of the [IaC]urf¿1ne available)
to give accurate results.
c. ReproducÍble partltlonfng of radloactLwity betçveen
acfd-soltùle a¡rd RNA fractlons.
[3g]urldtne !üas morc readLly avallable and the concentration was
lncreased to 40 nnoUnl.
3. 3. 1.2 IncubatLon time
the lnflueoce of lacubaÈ1on tlme on the
incorporaÈioo of IlaCJurfdine fnto acLd-soluble and RNA fractlons
Ls shor¡n ín Figure 11. Incorporation Ínto the acLd-soluble
EIGTIRE IO : InfLuønæ of widine ænænffiíon in the ¿nqûat'ían
ne.diz,on on. trytalce øtd inæzgowtíon of uridiræ. 50 root tipsfrom plaats grorñi fn conplete autrleot uedft¡n fox 7 daye were
lncr.rbated 1û 1 nl of nutrlent nedir¡u as descrlbed fn Sectlon
2.1.5, and extracted and separated lnto acfd-solt¡ble and INA
fractÍons (Sectloq 2.2.L.Lr.
êo lotal urfdfne uptahe loto root tLps
b. Z of total uptake facorporated lnto lNA
2
I
oEg
(l,l¿G'+,o-5(uc.lJ
LÐ
I
2
0
20
l0
0
30z.É.g
o
!L
òq
0 50 100 t50
Uridine concn. : nmol/m1
200
FrcuRE 10 : rnfLuence of uz+&tne coneentration in theineubatí.on mediu¡n on uptake ørd. ineorpotation of w.idine.
EIGTIRE 11 .: Effect of inctbatí.o¡t þhe øú borcn ¿Iefieienqon tqtal<e ød ítæozporuþípn of l,r+Cluridine. lo0-200
root tipe were lncråated La [rhc]urrdine as deecrlbed 1n
Sectfon 2.L.5 end extracted end eeparated í¡to ecld-eoIuble
and RNA fractlous (Section 2.2.L.Lr.
+B (A) aad -B (A) RNA
+B (¡) arrd -B (o) lctê-solr¡ble
anCL
+,+Joof-oO
EooX
jfIo
co+,G'!ooLooE
'úcrõ
(L,:G,+,o-5(ucEL=(J
d
0
4
4
2
2
2
0
4
0I 3
Incubation time : hr
FIGURE lL : Effect of íncttbation tine and boron
deficieney on uptake qnd íncozporation of lr+c)widtne.
20
144 hr
24 hr
+B
.B
+B
-B
48 hr-B
+B
-B
+B
-B
5A
fractíon fs rapfd over the flrst 2 þ¡t, then slows dor¡n |n young
root tlps (24 and 48 iir of deficfency) or reaches a plate¡ru fn the
older roots (144 hr). Radioaettwtty is locor?orated fu.to the RNA
fraction at an approxlmately llnear rete throughout the 4 hr
fncubation.
FÍgure 11 also demonstrates the effect of boron deflciency
on these factors. Increased fncorlporatfon lnto the RNA fractLon
occurs et all stages, and at a steady rate throughout the
lncubation períod. During the early stages of deficLency there
is Iittle effect on the acld-soluble fractlon, but after 144 hr
the incorporation Lnto deflcfent root tips is much hÍgher than
that lnto normal tfPs.
If these results ate etcpressed on a fresh-weight basls, the
24 and 48 hr resulËs ate uncha¡rged, but after 144 hr of deficLency
the lncorporatLon lnto RNA and acld-soluble fractÍons 1s not
affected by boron deficiency.
The total uptake of [32pf] follolus a sl.nilar pattern, exeept
thaË the plateau fs reached more rapldly (Flgure 12).
. 3.3.1.3 Boron deficfency
Durfng the first 48 hr of growth in boron-
deflcient medftn, deficLency lnereases the lncorporation of
llhC]urf¿1ne into RNA ¡¡lthout lnfluencing that fnto the acld-
soluble fractfon (Fígure 13). Beyond Ëhis sÈage, Lucorporation
1nÈo both fractions ls lncreased bv defl-cíency.
tnÈ+,+too!oo(\l
CL
(J
xÚ)lo
(u-s¿G'+,CLJ
èN(oLI
5
4
I
0
3
2
3
Incubation time: hr
FIGURE 12 z Effect of ineubatíon time on totaLtqtake of lszp¿1. 2OO root rips fron planÈsgror¡rn for 7 days in complete medium were incubatedin [32pti as descríbed in Sectlon 2.I.5. The uprake
- j-tfãs=calculated from the rate of removal of [32pi]from the incubation medium, by radioassaying (Sec-tion 2.5.1.3) aliquots at the indicared rímeintervals.
40 I 2
4
a^a+t+)oo'-oo
Eq()x
+Io
go+¡€toÈ!o(Jg
€ct(uJrõ+,o.5(u
Et-
t-f(J+Ht-¡
3
I
0
2
6
4
2
00 100 200
Tine after transfer : hr
300
FIGURE 13 z Effect of boron defieieney on trytakeætd ineorporatíon of lt4Clwidine. lO0-200 roortÍps were incubated in IIaC]uridíne (Seerion 2.1.5)and extracted and separated into acid-soluble and
RNA fracti.ons (Section 2.2.L.L).
b. Acid-soìuble
+B
-B
3.3. i.4 dl-s f 1þc fn RNA
[r+C]Urf¿lne taken uP by the planÈ can be
lncorporated Lnto RNA as IIMP or Cl{P as shown below:-
cTP ----ôTAUridine
-+ In'fP UDP
Ttrls 1s illustrated ín Table 3.
Î1me aftertransfer to
boron-deficlentnedir¡n (hr)
48
Ð9"
% of total radioacttvítylncorporated fnto RlüA
CMP
TABLE 3 : Effect of borcn defieiency on the base dietrùbutionof ltaCi ín RNA
Root tips were Lncubaùetl i¡r llqCJurt¿toe (SectLon
2.L.5) and the acid-insoluble (RNA) fraetLon extracted
and hydroLyzeð. Lo 0.3 ¡f-KOË as described in SectÍon
2.2.L.2. GIP and UlfP were separated by chromatography
on Do¡¡ex-1 (Sectlon 2.3.2.2).
-
Treatment
+B
-B
+B
-B
+B
-B
24
r44
39.7
42.O
4L.4
39.8
38.6
37.7
T'}fP
60.3
s8.0
58.6
60.2
6L.4
62.3
60
ApproxtnaxeLy 4O7. of the [laC] enters RNA as QfP' afid thfs
proportlon is not slgnf-flcantly Ínfluenced by boron deficlency.
No radLoactl-vlty ¡sas detected in Al'fP or GMP.
3.3"1.5 other trace element deflcieacles
It was lryortant to determlne ç¡hether the
lncreased lncorporatLon of radloactfve precursors Íato RNA fs a
specfffc effect of boron deficiency. Ttrus, the effects of several
other trace elements were fnvestfgated. The results gfven in
Table 4 sho¡s thât, durfng the flrst 48 hr, mangaûese and copper
deficlencies result in lncreased uptake of ll+C]uridine lnto the
acid-soluble fraction, but without siguificantly LncreasLng
Íncorporation Lnto RNA. At later stages of the deffcl'eocÍes,
lncotlporatlon lnto both fractlons 1s Íncreased. Iron deficLency
does not affect the uptake of lf'+C]urfdine lnto the acLd-soluble
fractlon ufitLl ûore than 144 hr of the defLcLency, but results in
an earLy lnhibitfon of incorporatfon lnto RNA,. The effects of
deflcfencies of, these trace elements on lfaC]tridine uptake and
incorporatlon are very different from those of boron defielency.
Ttrfs Ls particularly interestlng in the case of Lron, because the
effects of lrou deficlency on growth, cell divisLon and DNA and
RNA conËents of pea Eoot tlps are síoilar to those of boron
deficleacy (lbbott L972).
61..
TABLE 4 .. Effeets of truæ eLet¡ent defiei,encieí on Ll\ClwyidLne
tptake øtd ínætponatì'on
Deflcleat and nomal root tips (150 of each) were
fncubared tn llhC]uridLne fot 4 hr (Sectfou 2.1.5) a¡rd the acid-
soluble and RNA fractlons extracted and separated as descríbed fn
Sectlorr 2.2.L.I. Results are e:<Pressed as the ratlo:-
cotm lnto 150counta incorporated into root tlps
All sauples fncorporated, at least 10h c.p.m. into each fractfon.
-_
Deflcfentelement
Iroo
Marrgatrese
Copper
Boron
Iroa
Manganese
Copper
Boron
Tine after transfer to deficient nedir¡n (hr)
T2 24 48 L44 336
1.00
1.00
Aeid-soluble
l.2L
t.34
1.08
L.O2
0.97
L.42
L.27
I .08
0.90
1. 58
L.77
L.57
o.62
1.30
L.32
t.52
1.85
1. 10
1.33
2.62
1.06
1.56
1.08
3.23
0.75
1. 07
RNA
0.55
0.98
0.88
r.24
0.60
o.92
1. 10
I .50
62"
3.3.2 Effects of boron deflcl.encv on the specific actLvlty
of labelLed precursor lncorporation
3.3.2.1 RNA
Ffgure !.4 shows that there Ls an early and
very substa¡rtial increase ln the specific actfvity of the RNA
fraction following [l4C]urr¿fne Lncorporation. After 150 hr of
deficl-ency, the reduced RNA content of boron-deficient root tl.ps
(Ffgure 7) contrtbutes to the fncreased specific actLvfty.
Sful.lar resulËs ¡¡ere obtafned ¡vith Ëhe lncorporatlon of [32p1¡
lnto RNA (Table 5).
ÎABLE 5 : Effect of botwn defieí.encg on the speeific actùvityof RIIA follor,ring L32Píl íneorponctíon
400-800 root tips were fncubated fn [32pf](Section 2.f.5) and RNA rüas extracted by the rnethod
described fn Sectfon 2.2.L.3. Radloactlvfty was
assayed by Cerenkov radiatlon (SectIon 2.5.1.3) and
RNA by optical absorptlon at 260 nn (SectÍon 2.2.2.L).Each sanple Lncorporated at least 105 c.p.n.
Tlne after tra¡rsfer toboron-deflclent nedir¡n (hr)
24
48
L44
3L2
Increase 1n speciflcactivfty (Z)
53
69
100
202
c
Or.o(\¡
ooEèC'
x(Ð
Io
+,
+J(Jrlú
(J
l+-
(J(uq
ar'l
5
4
3
2
I
0
100 200 300 400
Time after transfer : hr
500
FIGURI 14 : Effect of boz,on d.eficieneg on the speeíficactivLtg of RIVA foLLouing lt4Clw¿*tne ineozpoyation.400-600 root tips were incubated itr l14c]uridine (section2.L.5) and RNA was extracted by the method described inSection 2.2.L.3.
0
+B
-B
63,
3.3.2.2 Acid-solt¡ble fractlon
In thfs serLes of experLænts, nucLeotldes
rûere uot separated fron the acl.d-soluble fraction for further
analysis. Àccurate speciffc actf.vlties could not therefore be
determlned, but values calculated on the basfs of the anount of
naterLal reactfng w"ith the rlbose assay nay be useful. SÍnce uany
other substa¡rces give a posftive result r¡ith thls aseay (Munro &
Fleck 1966), the results oust be treated wl.th cautÍon. Oa thls
basls, as shown 1n Flgure 15, Ít is ooly after at least 150 hr fn
deficlent medium Èhat the speciflc-act!-vity value 1s lnfluenced
by boron deflclency. Ccry & Fiuch (1967, obtafned siullar results
wLth accurate specl-fic-actlvfty neasurenents of the total
nucleotide pool (see Sectlon 1.3.2).
3.4 Nucl-eotfdes
. In tnl-tlal experÍments, wtren the acid-soluble extract
(Sectlon 2.2.L.2) of 300-500 ¡ng (f.w.) of root tlps was
ehromatographed on Do¡¡ex-l or DEAE-Sephadex, no nucleotldes or
nucleosfdes could be detected. If the root tfps were Lncubated
fn [1þc]uridl.ae, E5-957" of the [Ì4C] eluted wtth added urLdÍne and
otrly snal1 amor¡ots rùere assocÍated with nucleoÈide fractl.ons
(Ffgure 16). Three erçlanatl.oûs ere possfble to account for these
results: -
4
3
(uano-ot
oc
o-ox
NIo
2
I
00 100 200
Time after transfer : hr
300
FIGIIRE 15 z Effeet of boncn deficieney on thespeeLfie aetiuity of lL4Cl in the aei.d-soLubLe
fraction. 100-200 root tlps r¿ere incubared ín[laC]uri¿íne as described Ín Section 2.!.5 and theacid-soluble fraction extracted (Sectíon 2.2.I.Ð .
Ribose r¡ras assayed as described ln Sectíon 2.2.2.2.
.B
+B
c
oîtx
ô¡Io
2
4
25 50 75 100
Fraction number
125
FIGURE 16 : Ineozpoz,atton of l|aclurid.t ne into nueLeosidesar¿d nucLeotides of z,oot típs eæeised fz,om pLants groum 6 oy Z
døys in nutr'Lent medimu 900 root tips were incubated inllac]uridine (section 2.1.5) and nucleotides extïacted as
described i.n sectíon 2.2.1.2 and chromatographed on DEAE-
sephadex (section 2.3.2.r). The positi-ons of authenticmarkers are shor^m. rn Ëhís systeur uDpG elutes jusË prior toI]MP.
0
6
6
4
2
00 0
b. 7 days
=I
L=
èô- cL o- ô-== o ô FO= (J
= (J
ilt t t
a. 6 days
CLo=
I
o-F(J
I
CLF
I
I=
I
o-ac)o- ô-==c)=
tt
64
a. l{ucleotldes are lncornpletely extracted from the tl-ssue.
b" Nucleotlde¡r are degraded during extraction.
c. Ttre yormg root tÍps contain lorr amounts of nucleotides-
Ttre fl.rst expLanatfon fs unllkely, because other extractlon
procedures (e.g. 0.2 M-PCA) dld not $(tract hlgher levels of
nucleotÍdes, and nuch hlgher a,mouûts could be extracted fron
mâture root tLssue by the saÐe methods. Ttrus, approxf'oately
100 nrnol of AlP (with a maxfmum optical absorptlon of 0.20 per
fr¿ctlon eluted from a Dowex-l coltmr) rÍas extracted from one gr¿rm
of mature roots.
[l+c]urp facluded ln the extraction medlum is only sllghtly
degraded during extrectioo (lable 6). Only 9.3 pml of UTP (10.82
of added uIç) ls degraded. Even less I'DP (r.8 pmo13 7.57( of that
added) and no IIMP, both of whfch $rere coritanr{uants in the ¡t+glil["
sanple, ltere degraded. Ttris suggests that degradatÍon of uucleo-
ÈLdes fs uot an lluportant factor.
Ttrus, 1t is concluded that yotrng Phaseolua atæus root tÍPs
contaiD very low levels of nucleotfdes, whfch precludes the
deterul.nation of t.be speclffc activl.ty of the labe1 lncoraorated
into lndivldual nucleotides. Uaequivocal ldentlflcatÍon of
nucleotLdes Ís also Lupossible, and thelr fdentfffcatlon has only
been achleved by co-chromatography w-ith auther¡tic sta¡rdards (both
Iac-l"belled and unlabelled). Thus, although peaks are named
according to the sta¡rd¿rd w'lth whfch they correspond, oËher mfnor
nucleotfdes nay contrÍbute to the radfoactLvity withio the peak
65.
TAB1E 6 : Ðegrudatùon of l'* C-LobeLLed nucleotídee ãæingnueleotide esttaetion
0.05 nl of a IlhC]um staodard (44 d.p.n./pnol), also
containine [I+C]uDp, [r'+c]næ a¡rd [14C]urldtrle, was added to the
buffer before extraction of nucleotides frc.: 1000 root tips. the
acid-soluble extract (Sectlor 2.2.1.2) wae chronatographed on
Do¡¡ex-l (SectLon 2.3.2.2) -
?est eolu¡vts. A sample (0.05 nl) ofthe I rhC]Urp standard rnras also chronatographed directly on Dowex-I
- Cor¿trcL øLuwts.
Nucleotlde orNueleoside
Control Test
d.p.n. prnol % totaL d.p.n. prnol Z total
UrfdLne
UUP
UDP
UTP
62
zcr9
1060
3780
L.4
6.1
24.L
85.9
1.2
5.2
20.5
73.L
63
347
1330
3373
L.4
7.9
31. 0
76.6
L.2
6.8
26.0
66.0
Total 5171 117.5 100.0 5113 116.9 100.0
(grwn L962, Caldwell L969). Ttrls appJ.les particularly to [32pf ]
incorporatlon oçerLments.
3.4.1 [ rrc]uridtEi tncor.Ðoratlon
At eatly stages of deficiency (24 aud 48 hr) the
amouût of [lbC]uridtne incorporated lnto uucleotl.des fs very
snall. Ttre only sigoifÍcanÈ Lncoraoration of radloactfvfty was
oo.
futo a peak whtctr appears to be assoclated r¡fth [¡DPG. ltrl.s peak
elutes in slnllar positLons to those reported for UDPG fron both
Do¡rex-l and DEAE-Sephadex (Eurlbert et aL. L954, Cald¡¡ell L969,
Bror¡n a¡¡d Caesells 1971), but has not been posftively Ldeatl"fied.
Deficfency dld appear to sllghtly fncrease the l-ncorporation of
the tracer lnto thfs peak, but the vah¡es nere Ëoo low to permft
8n eccurate assesament of the effect. Ttrfs posalble lncrease may
be reLated to the fncreased ÍncotAoraÊ1on of lraC]glucose Lnto
pectÍc substaricee ktùa UDPG) by boron-deflclent ffeld bean root
tlps (Stactc & I,thittlngton 1964).
After 144 hr of the deficÍency (wheu the plants are 4 days
older) sfgnlficant æouûts of IllC]urfdfne are fncorporated Lato
the nucleotldes of both normal and deflcient plarits (Ftgure 17).
Fron the aaalysfs ln Table 7 ft can be seen that the defl.cfency
causes only snalI changes 1n the dLstrÍbutfon of radloactLvity,
the most sigofflcant being that a decreased proportlon of the
total lneotaoratÍon fs lnto trÍphosphates (40.8"Á of aucleotLdes 1n
aormal and 35.02 ln deflcleot tlssue) and an fncreased proportion
fs lnto the IIDPG and IIMP peak. At thÍs late stage of deficieney
there also appears to be an increese ln lncorporatfon Ínto alluridine nucleotldes and a sllght reductl.oa of lncorporatlon lnto
cytldfne nucleotldes. Ihe deffcieaey does not, honever, change
the ratÍo of the correspondÍng bases 1n RNA (table 3 Íu Sectl.on
3.3.1.4), suggestl.ng that the effect Ís due to ctrangee 1n size
rather than speciffc actlwlty of the urldfne a¡rd cyrLdine
nucleotLde poo1s.
0
6
4
o-<tX
ôlIo
2
25 50 75 t00
Fraction number
FIGURE 17 : Ineorpoyatíon of lt+Cluyidine ínto nueLeosides
ø¿d nueleotides after L44 hr gnouth in defi.eient me&iwn.
1000 root tips were incubated in Ilag]urídine (Secrion 2.1.5)and nucl-eotides extracted as descrlbed in section 2.2.r.2 anð.
chromatographed on DEAn-Sephadex (Section 2.3.2.L). The
positions of authentie markers are shown. In this systenUDPG elutes just prior to IIMP.
6
4
2
0
'-
I I I
oô-==(J=
b. -B
èô-¡-Fc)=o-èOA(-)=
lr
f-=
cL O-
==ct=
I
a. +B
ècLooc)=o- ô-Fl-(J=
67.
TABLE 7 :
lhe data l¡ere obtalned from the same set of resultE
used in Flgure 17.
Radfoactfvlty(c.p.m. )
Z of Total Z of Nucleotldes
+B -B +B -B +B -B
ùiet?Lbution of [lþC] ìn nuc'Leoeidee øtd Tcucleotidesof 744 hn defí.eient rcot tipe follourtng l,raCJurùdineinøtpowl;ion
Nucleotfde orNucleosfde
Nucl.eosides
Ct-lP
TIMP + UDPG
CDP
UDP
c1
UTP
7,838
95
L,279
64
293
237
884
8,398
57
1,620
50
383
188
947
74.0
0.9
11.1
0.6
2.8
2.2
8.4
72.L
0.5
14.0
0.4
3.3
[.6
8.1
3.5
42.8
2.3
10.6
8.6
32"2
L.7
50.0
1.5
11.8
5.8
29.2
3.4,2 [ 32Ptl tncoEroration
Using [32pf], the fncorporatfon of radioactivity lnto
nucleotfdes is much greater (Ffgures 18 a¡rd 19) but, as wlth
[Ihc]urrdfne, the lncorporatlon increases with plant age. [32pr]
Ís incorporated Ínto all oucleotfdes, but wlth the slnple
chrornatographÍe nethod used, coqlete separatloû qras not achieved.
Monouucleotides are very poorly separated, slnce Cl'{P, IIllP, Al'fP,
IIDPG a¡rd Pf are all eluted together. Based on the separatlon
¿o-(J
x
IIII
r.c)
Iof-o
I
(Y)
Io
l5
t0
0
5
00 25 50 75 100
Fraction number
125 150
FIGURE 18 : Ineorporation of l32Pi) into nucLeot'ides of 48 hr
deficíent root tips. 500 rooË tips were incubated in [3zpt1(Section 2.L.5) and nucleoËídes extracted as described inSection 2.2.I.2 and chromatographed on DEAE-Sephadex (Section
2.3.2.I). The positions of authentic markers are shown. In
thís system UDPG elutes with IIMP.
5
t5
0I
ilrltl
¡tsl¡tl
CL
=CL
==è=q)
I
t
Itt¡l¡l
!itl
È=(Jô-o(JI
ô-oô-oJ
I
ô-l-
I
ô-(.Ð
I
b. -B
ô-
=¡
ÈÈÞ-â(J (5il
ô-=(5Àô(JI
o-F(5
I
a. +B
ô-F-
I
ô- ô-oo=<ll
ô-À o-FO F(J(5
=ill
3
2
Ëo-(J
x
IIII
(oao
Lo
I
,o
I
0
3
2
l
025 50 75 100 125 ì50
Fraction number
FIGURE 19 z Ineozpoz,ation o¡ l3zeil into nucLeotides of 1_44 hz,
defietent root tips. 500 roor tips were incubated in [32pi](Sectlon 2.L.5) and nucleotides extracted as descríbed ínsection 2.2.1.2 a¡d chrornatographed on DEAE-sephadex (section2.3.2.L). The positions of authentíc markers are shown. Inthís system UDPG elutes with UMP.
0
t 9,
Ilt,¡,r
o-==ô-=ô-=(J
II
ô-=CL
=(J
^
ô-(5ô-Õ(-)
ô-F(5
b. -B
CLèoa=<tt
o- ô-l-oc) (5
ll
o-t-(5Iil lr
o- o- o- o-o= oQ(JCt =<
a. +B
CL ô-Fâ(-) (5
tt
68.
achÍer¡ed wlth d1- and trl-phosphates, boron deficlency appears to
have lLttIe or no effect on the dlstrlbutlon of radÍoaetfrity
between the nucleotides. this ls demonstrated for adenoslne end
uridLae nucleotfdes fn Table 8. After 48 hr of deficiency' the
ratio of UDP + ADP to UTP + ATP 1s not affected, whLle after L44
hr there fs a sllght lncrease (of doubtful signiflcance) 1n the
triphosphates relative to dlphosphates. The sllght varLance
TABI.E 8 : DisþrLbution of [3zp] beù'teen aåettosine ø¿d uridtnenueleoþíclee
It¡e data were obtaíned from the same sets of results
used ln Ffgures 18 and 19.
Time after Treatment ADP + UDP ATP + UTP
transfer toboron-deficient
nedfr:n (hr) c,p.u. Z total c.p.m. 7" total.
48
t44
+B
-B
+B
-B
39,633
33,807
7L,068
93,327
38.2
38.3
26.5
23.9
63,99L
54,516
lg7 ,996
297,604
61.8
6L.7
73.5
76.L
behreen this result a¡rd the effect of the deficieacy on llhC]uridfne
incotAoratlon (Table 7) nay be due to the different e:(Perimental
condLtioas or plant batches enployed.
Ttre total amount or [32pt] incoqporated Ínto these nucleo-
69.
tides (table 8) ts altered by boron deflcfency. After 48 hr of
grqrth ln deflcleot uedirn, the sltgþtly reduced locorporatlon nay
be due to increaeed rates of utlllzatfon of nucleotides ln Bl{À
synthesls, thus resultlng 1n lower levels of nucleotldes. Both
changee observed after 144 hr of deflclency can be attrfbuted to
the lncreased raturtty of defLcfent root tips. Total fncorporatlon
lnto nucleotides and the ratlo of lncorpotatlon Lnto trlphosphates:
dfphosphates lncrease ¡rith both boron deff.cl.eacy and plant age-
these tesults are conslstedt ¡d.th the obeen¡atÍon of ShkolrnLk &
Haevskaya (L96?b - Sectíon 1.3.3) that the AÎP leveI does not
fall uotll after the appearance of deffclency sJrytoms.
Incotporatlon of radloactlvity fnto the totel acfd-soluble
fraction of norn¿l root tlps decreases with plaut age (Figure 13
in Sectf,on 3.3.1.3), but the lncorporatfon lnto nucleotides
lncreases (Ffgures 16, L7,18 and 19; Table 8). Ttris Ls because
in yorrng root tLps oost of the radloactlvÍty taken up by the root
tips fs assocfated nrtth nucleosldes, bases a¡rd non-nucleotLde
oaterlaJ-, but as the plant ages much oore radl.oactlvity Ls
assoc ated rrtth the aucleotide fracÈf.ons. thls nay be due to
changes 1n growth (Flgure 4 La Sectfon 3.1.1) aoa dlfferentiatfon
rates. Ttre change with plant age is much greaËer than any effect
of boron defiefency.
70
3.5 Fractloaatlon of RNA
3.5. I l{AK chrooatography
3.5.1.f Appraleal of the technique
To investl.gate futther the eerly effect of
boron deflcfency on the fncorporatlon of precursors luto RNA,
lfAK chtonatography (Sectlon 2.3.L.1) ¡ras enployed. Ttre naJor
peaks separated O" ;ÞnfchnLque
have been r¡ell characterÍzed
(ttonler et aL. I962r¡sherldan Lg64, Ctrerry tg64, Y'ey et aL. Lg65,
Cherry et aL. 1965, Grroboczek & Ctrerry 1966, E1lem L967). Iloder
ldeal couditlons,- Èhe fractLons sho¡sn ia Ffgure 20 caa be
separated, and the maJor conponents of each are as follows:-
(a) Nueleotides a¡rd olfgonucleotfdes (E\rtng & Cherry L967);
(b) 4s(t-RNA) and Ss(r-RNA), wtrlch are sometlmes separated,
and are usually refer-red to as s-RNA;
(c) DNÀ and DNA-RNA hybrlds;
(d) Light rlbosonal nNe (1I-RNA) ¡
(e) Heavy rfbosonal Rl{A (hr-RNA);
(f) I'fesseoger RNA (n-RNA);
(g) RNA which fs tfghtly bornd to the colum, and eluted
wlth SDS or alkal1 (TB-B-¡¿\) '
lfuch work bas been done to characterLze fractlons (d), (e)
and (f), partlcul,arly (f). Ingle et aL. (1965), uslng dffferential
extractl.on techniques, shorred that Fractlon (f) contalns RNA whlch
1
+,
+t(J.l'o
!rotLoco+)CL5-oanEfõ
j
Fracti on nunùer -*
I'IGqRE 20 : Fz,aetionation of RNA by I'AAK chyoma-
tographg. Ideal separation by I4AK chromaÈography
from Cherry et aL. (1965), Ewing & Cherry (1968),
Ingle & Key (1968) and Johri & Varner (1970).
a
I
b
I
c
I
II
defttl
77.
1s different from the hr-RNA peak 1n base eouposition, rate of
synthesis and half-llfe. Because the base eoqositiofl !ta-< sfmllar
to that of DNA aad the half-I1fe approximately 2 hr, they
suggested that ft may be messeoger I0!lA. 0n sucroee gradLent
centrifugatlon this fractloa Ls heterogeneous (see also Johrl &
Varner f970) and not colncideat ¡rl.th the optical densfty patteras.
Others have conflroed the hlgh AllP content (Key & Ingle L964,
Chroboczek & Cherry 1966) aod sholttr that thts fractfon hybridlzes
nore readlly wtth homologous DNA than ariy other fractfoo
(Van nuystee 63 Gherry 1966), ls most sensitl've to Actlaomycin D
(Ctrroboczek & Ctrerry 1966), Ís associated nost ¡rLth and 1s most
actÍve ln the fo¡natioa of polysones (1,1n et aL. L966, Jach.Snczyk
& Cherry 1968' Johrl & varaer 1970) and is most capable of
supportLag protein synthesfs in titzp (Jachymczyk & Ctrerry 1968).
$rLs fractLon therefore posseases oany of the characterlstics
e:ßpected of messeuger RNA and has usually beea referred to as
u-RNA. Ttre poor separatloa of peaks (d), (e) and (f) 1ed Ingle &
Key (1968) to fractionate them further using polyacrylarnide-gel
electrophoresis. Aggregatlon of ribosomal and other RNA specl.es
results in each peak belng a conplex m{xture of RNA species.
after a 4 hr l32pr]-ratelllng period, much of the radioactlvlty of
peak (f) w¿ra associated ç¡tth 18s and 25s r-RNA. Wlth the
sinpltfied chromatographfc technlque used ln the work reported f.a
thls thesls, the aggregatLon Ls elten rore severe, resultLng 1n the
elutfon of one r-RNA * n-RNA fractfon (referred to as r-RNA).
TB-RI{A lpeak (e) ] fs knor¡n to conslst of several subf,ractÍons
72.
(nrsfng & Gherry 1968, l4lassod et aL. 1970). At least one of these
ls sfmLlar to m-ltlA lpeak (f)] and rechrouatography and base-
coryosltf.on analysis lndfcate that r-RNA (or a rLbosoual precursor)
Ís also a ajor conponent (Key 1967, Johrl & Varner 1970). A
rapldly labelled AlfP-rLch RNA fractfoa separated from total RNA by
counter-currerlt dlstrlbutloa ls fractlonated almost entlrely fnto
n-RNA and ÎB-RNA by lfÀK chromatography (hrfng & Cherry 1968).
Johrl & Vatner (1970) have suggested that these tlto rapldly
labelled fraetions have an open structure a¡rd are seperated
because of thelr dlfferent base eompositlons. Those specfes wlth
a high adeoLne conten.t (low guaoÍne and cytoslne) have llttle
secondary structure and foteract strongly wlth the nethylated
albun{n a¡rd are therefore eluted as TB-RNA, whÍ1e those epecf.es
¡d.th a low adenfne coDtent lnteract rflth r-RlilA and elute as n-RNA.
If the lnablllty Èo chase label fron TB-RNA fs due to the
accu¡oulatLon of label by other subfractlons (e.g. rlbosmal RNA -Ingle et dL. f965), lt 1s then possfble that the raptdly labelled
components of both n-RNA and TB-RNA represent planË nessenger-RNA
dffferiag only in base coqosition.
The fractlons vrfll be referred to by the above Damea, but Lt
must be eryhasl.sed that they are only radioactive and u.v.-
absorbLng naterial eluting ln this part of the elutlon pattern and
fn sme cases, especlally 1n grossly deficlent tissue (Flgures 22
aod 28), the ldentffication of the RNA epecfes mriy not be strlctly
correct.
73.
3.5.1.2 [lçc]urfAlne Lncotporatl.on
Élgure 21 shorys the elutLon patterns for
notral and defl.cieat saryles of Fù{A fron root tl-ps after 24 hr
grolrth ln deff.cLent medfum. Ttre incorporatfoa Ls fncreased lnto
all Rl{A specles, but particul¿rly ÎB-RÌ{A (fractLons 80-90) a¡rd
r-RNA (fractfoos 40-60). This pattêEi changee progreseÍvely, and
after 512 hr of boron deficÍeucy the sltuatLoa depicted fn Flgure
22 Ls react¡ed. Ttrere fs alnoet a coqlete breakdown of r-RNA
(shora by absorbance at 260 nn) la deficf.ent tlseue. Sone of thfs
appears to elute fn the t-RNA region, but the total RlilA level fs
considerably lorer than that of the nornel root tips (see Flgure 7
ln Sectl.on 3.2.2). There Ls also a vaat dlfference La the
distributlon of label from [IhC]uridine lncorporatlon studles.
TB-RI{A stlll incorporates approxinately tnrice as much radloactirrlty,
but the ratfos between fncotporatl.on lnto r-RNA and s-RNA are
reversed.
The incorporatÍon into the s-RNA, r-RNA aod ÎB-RNA fractioas
during the development of deflciency is shdrri ln Flgure 23. Durfng
the first 50 hr the effects shor¡n ln Ffgure 2l preval.l - f-. e.
iocotporatlon Ínto all fractlons 1s lncreased, but TB-RNA to a
much greater extent than r-RNA, ¡¡hich fn turn 1s sllghtly greater
than s-RNA. Ihrrfug the per{.od 50-120 hr, changes occur, the most
luportant belng that a¡r even hlgher fncorporation lnto the s-RNA
and TB-FùilA fractlons 1s obser¡¡ed compared to that Ln nomal
tlssues. Ttre effect on r-RNA becomes less notlceable, eventually
revertfog alnost to the same level of Lncor?oratÍon es that of
tttt
b
,
+B
,,.lq,
l¡lttl¡l
rtrlItltl¡tl¡ltt
'itl
IIIII
I
I
I
I
I
I
I
III ^
II
Ia. -B
I\\
IIII
I/i
I
IIII
I
\\\\I
II
t
425c. p.m
0. 15
0. l0
0. 05 I
3
2
I
0
ço(oc\t
0 0=o-
0. t5 3()x
NIo
0.10 2
0. 05
020 80
FIGURE 21 2 IAK chromatographA of RNA eætyaeted fnom 24 hrdefieøent Toot ti,ps. 4OO root Ëips hrere incubated in[IaC]uridine (Section 2.1.5), and RNA exÈracted (Secrion
2.2.L.3) and fraeËionated by lufAK chromatography (Section2.3.1.1).
oo
0 40 60
Fraction nunùer
0.05
0.15
0.10
0.15
0.10
0. 05
IIIt
o-C'
xc\¡Io
0
I
g
O(o(\I
oc;
0t00
FIGURE 22 . MAK ehrornatogrqha of RNA eætracted fnom5L2 hv' defic't ent root tips. 400 root tips were incubateditt [14C]uridine (Sectíon 2.L.5) and RNA exrraered (Secrion
2.2.L.3) and fracÈionated by MAK chromatography (Secrion2.3.1.1).
0 20 40 60
Fracti on nunùer
80
,t
It
n,l
b. +B
IIII
It
It
,,It
II¡II¡I
f¡
a
^
I
I
860
tl
tItItI,
.B
IIII¡tIIII¡
c.p.m
II
,l,,,
It,
co+(Fo
ès
500
400
1000 100 200 300 400
Tine after transfer : hr
500
FIGURE 23 : Effeet of boron d.efíaieney on ltaClutrLdt ne
ineorpozu,tion into RIVA fraetíons sepatated bg ILAK
ehromatogrøphg. 300-500 root típs were incubated inIIaC]uri¿íne, and RNA extracted and fracËionated by ]4AK
chromatography as for Figures 2l and 22. The Íncorporationinto deficient root tips is expressed as a percenÈage ofthe incorporation inËo normal root Èíps on the ordinate.
^ s-RNA
O r-RNA
O TB-RNA
300
200
74"
norûal root tfps. It Ís fuportant to note that this fs the tl.me
(50-100 hr) vhen fntribftlon aod thea cessatlon of elongsf,ton
occur, and the relevanee of changes 1n RNase actlvlty w111 be
dÍscr¡ssed ln Sectfon 3.7.1.
DurÍng the ffrst 100 hr fn deflcient nedLtn the effects of
the deficiency on the speclflc actfvÍty of these fractLons (¡'lggre
24) ts the sane as that on total counts' but beyond Ëhls stage
different effects of boron deftclency on the RNA content of each
fraction (Figure 25) change the specific-actlvtty pattenr.
Because of the greatly reduced level of r-RNA but contÍnued
lncorporaÈion of radl.oactÍvlty, the specffLe actlvlty of this
fractlon coatLnues to lncre¿¡se, eventually (Sfe hr) reachLng a
value alnost 16 ttnes thet of the aormal root-tip r-RNA. Changes
1n s-Rl{A and TB-RNA are slmllar to total counËs, becauEe the Rl'lA
cooteût of these fractlons does not change as uuch.
3.5. 1.3 Dual-1abe1$U.Jtudfes
IÞgradatlon of rRNA fn deflcÍent root tipg
(Ffgures 22 and 25) fs probably due to lncreaeed RNase actfvLty
(Sectfoa L.3.4.1) but degtadatíon could occur elther in vùø
during the Íncubatlon perLod (f.e, soon after label lncorporatlon)
or durfag the extractf.on procedrrre. 1o deternLne wtrether
artlfacts of the extractlon procedure were affectLng the
dl.fferences betweea defLcient and no¡mal tissue, nixf.ng
ex¡:erÍneuts enrFloyfng dual-Labe11ed samples were r¡sed.
FIGURE 24 : Effect of borcn defùcíeney on the epeaifie aetiuity of RNA f,ractiona sepanted
by IIAK olwomatogtaphy. 300-5OO root tfps were fncubated fn lraC]urf¿fne and RNA
extracted and fractfonated by MAK chromatography as for Figures 2l and 22. The specfflcactLvtty of deflcfent root tips fs expressed ae a percentage of the speciffc actlvfty ofnor¡ral root tÍps on the ordLnate.
ê. 0 Èo 170 hr 1n boron-defLcient medir¡m
b. 170 to 500 hr Ín boron-deficient medLr¡n
s-RNA
r-RNA
lB.RNA
A
o
a
a
e+
oÈc
400
300
200
.l00
2000
I 500
I 000
500
0150 200 ]00
Time after transfer : hr
300 400 5000 50 100 200
FIGURE 24 = Effect of boron deficieney on the specí,fíe actíui.ty of RNA fz,aetions separated. byMAK chnomatogn;ryhy.
co+
120
t00
80
60hoèq
40
20
00 t00 200 300 400
Tirn after transfer : hr
500
FIGURX 25 z Effeet of boron defieieney ort RIVA eontent offractíons sepatated by XaAK chromatognophU. 300-500
root Èips were incubated in [1a6]uridine, and RNA was
extracted and fractionated by MAK chromatography as forFigures 2L and 22. The RNA content of deficient Èips isexpressed as a percentage of the conËent of norrnal rootËips on Ëhe ordj.naËe.
A s-RNA
O r-RNA
O TB-RNA
75.
Tuo sanples of nomal root tips (300-400 tlps each) ¡¡ere
fncr¡bated fn l3H]uridlne as descrÍbed 1n SectLoa 2.1.5 and t¡,ro
saqles of deflcfeut tips treated sinllarly tn llhC]urfdfne.
After lncubatfoß, one saryle frm each was cosbfned æd the Rt{A
extrected (Sectlon 2.2.L.3) fron the coubfned deficfent lrhg-
labelled) and not¡¡al (3u-taUetled) saryle. After extrectLoû, the
RNA was applied to a llAK cohm and eluted. Analysfs of the dual-
labelled fractlons was perforned as descrlbed io Sectlon 2.5.L.2
to sepetate the radloactlvLty lnto that derlved from deffcfent
(1bc-1abelLed) a¡rd nornat (3n-utelled) RNA. Ttre remainlng
saryles of aor¡nal and defLcfent root tlps were extracted
separately, tben applfed- to a lfAK cohur a¡rd eluted and analysed
as above. The procedure Ís outlLned fn Figure 26. If degradetfon
or any other artLfact of the extractlon ûethod is affectlng
deficient aod aorual tlsst¡e differently, the samp1ee nlxed durfng
RNA extractlon would produce labellfng patterns differeut fron
those ¡shich were mlxed after extractÍou. If degradatl.on of RNA
w¿ls occurrÍng durlng extractlon of deflclent but not noroal tÍssue,
both should be degraded in the nixed experlment.
Identical patter¡s sere obtained ç-rith uixed and unnixed
sæples, ÍndÍcating that changes 1n chrouatographfc elutLon
patterns of RNA extracted frou deffcLeot root tips are due to
ehanges ín vivo and not artffacts of the extractlon procedure.
Only the nfxed samples are shoqTn 1n FLgures 27 arð,28.
Dffferencee between colums are also ell.nfnated by this
procedure. This pemits the detectlon of mrch smaller changes ln
+B Incorporati
Harvest of+B
root tips+
Incubatìon ([3H])
P¡IXED EXTRACTION
Itlix+
RNA Extraction+
l4AK Chromatography+
Anaìysi s
UNMIXED EXTRACTION
Harvest of-B
root tips+
Incubation ([14c])
I-B Incorporation ([1
Harvest of-B
root tips+
Incubation ([tac])+
RNA Extraction
)
Harvest of+B
root tips+
Incubatìon ([3H])+
RNA Extraction
l{ix*
t'lAK Chromatography
I
+
Analysi s
+B Incorporation (t3Hl) -B IncoruoratÍon (f 14Cl)
FIGTJRE 26 : Proeedtre fon &aL-LabeL eæperítrcnts
24 hr
ì
a
t,
?,d. p. m.
b
48 hr
,t
ì6
12
I
0
2
l0
Fraction nunùer
FIGURX 27 z I4AK chnornøtographA of funL-LaheLled RIVA sønpLes
after, 24 øtd 48 hy, of defíe\eneA. Root típs were íncubaredÍn 14C- or 3H-1abe11ed uridine, and the RNA extracted and
chromatographed as described in the text (Sectíon 3.5.I.3) and
Figure 26.
8
6
IIII
ó+
I(Ðl-¡
ÈEx
NIo
4
12
4
2
0
4
I
@I
(-)ii-
IJ
åEX
NIo
8
4
000
,,
a
,I
tIÍltlll¡tlll¡l¡l
,r
b
l¡
t
I
288 hr
,It,
I
ú
I
,t
a
ll
t
144 hr
tt
t,
,tItI
I
l6
12
4 I
2
12 12
l0 20 30
Fracti on nunùer
40
FIGURE 28 2 MAK chromatographa of duaL-LahelLed satnpLes after144 øtd 2BB hy of deficienq. Root Ëips were incubated in14c- or 3tt-1abel-1ed uridine and the.RNA extracted and. chroma-
tographed as described in the text (Section 3.5.1.3) and
Figure 26.
I
6
IIII
æ+
!ttt-¡
F
o-g
xN¡o
4
00
I
coI
(-)
t-J
Eo!x
c.lIo
I
4
0
8
4
0
500
76
the laeorporatÍo¡ patterns, and coaflrn¡ the df.fferences found fn
ttre prevlous¡ section (SectLon 3.5. 1.2). After 24 hr of the
defl.cfeucy (Ffgure 27) lncorporatlon of label lnto TB-RNA and
r-RlilA fs locreased nore than that fnto s-RNA, but after 48 hr
s-RNA and r-FùilA are equally affected. After longer perlods of
defl.cLency (ftgure 28) the effect on s-RNA becoues greater. Thtg
fs more cLearly deoonstrated fn Figure 29. The incorPoratLoû fnto
IB-RNA Ls lucreased most by deflclenc? untLl almost 250 hr, but
although Íncotporatlon Ínto r-RNA ls lncreased more than fnto
s-RNA ac early stages, there Ís a closs-over at epProxinately
44 hr. Thus, the effect of boron deficiency oa Precutsor
Lacorporatlon Ln the first 44 hx fs dLstfnctly dtffereat fron that
at later stages. Tt¡is cha¡rge fn the e-RNA a¡rd r-RNA curves began
ax 24 hr, the tlne r¡hen a restrlctlon of elongation was ffrst
obse¡xted.
Instead of usfng the reversed-label method to elfolnete
dÍfferences due to differeatlal locorporatlon of the t¡so radfo-
feotopes (ElLen 1967), the dLfferences rùere determfned dÍtect1y,
using two sanples from plants gro!ùn ¡rith sufficíent boron. These
are plotted Ín Flgure 29 es 0 hr of deflciency.
' 3.5.1.4 [32P1] incorporatlon
IncotlporaÈ1ou of [32pr] lnto RNA gave
sinllar results for the s-RNA and r-RNA fractlons (Ftgsre 30).
Ëowever, ¡þs rn{lfal fncrease 1n fneoraoratf.ou fnto the TB-RNA
fractiou was not as great as that for labelled urldine (Figure 29)'
@+ó
,
o+JG'É.
t.4
1.2
1.0
0.8
0.60 100 200
Tinæ after transfer : hr300
FIGURE 29 z Effeet of boron defi,eieney on p?eeu.?soy
íncorporation into &nL-LabeLLed aul fractions sepaz,ated
by XAAK eVtz,omatogrqphA. Root tips were incubated in14c- and 3n-1ab"11ed uridine and. the RNA exËracted. and
chromatographed as described in the text (Section3.5.1.3) and Figure 26. The values plotred on theordinaËe are:-
7" lota]- -B radioactivi ty"Á
for each RNA species.
s-RNA
r-RNA
TB-RNA
Total tB radíoactivity
^oo
ó+o
I
o+)ct&.
1.6
t.4
1.2
0
0.8
0.6
0 t00 200
Time after transfer : hr
300
FIGURE 30 : Effect of boron deficieney on l32p¿l incorpor-ation into RNA fraeti,ons separated by IIAK ehnomatography.
1000 root tips were incubared in [3zpf] (Section 2.L.5) and
RNA extracted (Section 2.2,L.3) and fractionated by MAK
chromatography (section 2.3.1.1). Aliquots of the col-lecredfracÈions were radioassayed by cerenkov radíation (section2.5.L.3). The values plotted on Èhe ordinate are:-
"/. Total -B radioactivity"/" TotaI tB radioactivíËy
for each RNA species.
^ s-RNA
O r-RNA
O TB-RNA
I
77
and after 350 hr the lncreaeed fnco4oratlon L¡¡to TB-RNA was stll1
greater tha¡r for elther s-RNA or r-RNA.
3.5.2 Polvacrylamide-gel electrophoresls
Ttie superlor separation of RNA specÍes by
polyacrylarn{{s'gsf electrophotesis was used to foIlqr the
tncorporatLon of labelled precursors into r-RNA epeeies and to
dete¡mine the apparent molecular nelght of the labelled specfes.
Iü1th the extractLon ueËhod rrsed, root-tÍp RNA preparatlons did not
readlly enter the gels, end only with snall âmor¡nts (2-5 ug) of
RNA sanple lras couplete entry achleved. At hlgher loadl.ngs, only
sLlght1y more RNA entered the gels, and lc¡¡ molecular-welght
species appeared to enter more readtly than those lrith hlgh values.
ltrÍs lras lnsuffÍcieût RNA for a reliable densitometer trece. the
entry of RNA prepared from cotyledons and young leaves of
PhaseoLus alæeus by a slnflar nethod (Sectlo¡ 2.2.1.3) was not
fahfbited and the de¡rsftoneter traces for thls prep¿ratÍon are
shorrn Ín Flgure 31" The reason for Èhe dffference betweeu these
t¡ro RNA preparaÈlons has not been deternfned. It nay be due to
sllght dlfferences f.n the extractfon technfque (1.e. ratio of
buffer to plant materlel during homogenLzatlon, and nnnber of
phenol extractioas), or to the presence of a htgher concentration
of some fnterfering substance fn root tlps (e.g. polSrphenols).
Electrophoreels of the RNA prepared from cotyledons and
leaves rer¡ealed a nr:¡nber of htgh molecular-ryelght species,
correspondlag to those reported for greeu plant tissue by Loenlng
I
l6s
IIIt
2 3sb
t¡lt
IÍ
25s l8s
.B
aatl
,t
a
+B
I
Êcol.OC\I
+,fitgo+,CLLoa^4l
5
l5
l0
0
0
0
5
a
III
È(J
xN¡o
5
0 l0 20 30 40 50
Distance moved : rm
60 70
FIGURE 31 : PoLyacrylønLde-geL eLectnophoresis of 32p-
LaþeLLed RNA eætraeted from 24 hr deficient noot tips.400 root tips were incubared in [32pi] (Section 2.1.5) and
RNA extracted as described ín SectÍo¡ 2.2.1.3. 5 pg wereloaded onto Èhe gels, together r^¡ith 40 Ug of RNA extractedfrom cotyledons and young leaves (Section 2.2.L.3).Electrophoresís (Sectíon 2.3.I.2) was for 115 min.
78.
& Ingle (1967) and Ingle (f968). In FLgure 31, 5 sg of 32P-
labelled root-tip RNA was nrn with 40 Ug of RNA extracted fron
eotyledons and yor.rng leaves. Ttre denslt@€ter Èrace fs therefore
prlnarLly that of green-tlssue RlilA, and the radloactfvÍty cunre Ls
due to the [32p1] incorporated lnto the RNA of oormal and boron-
deflcfent root tfps. Ttre deffciency facreases the incorporatlon
of [32pf] fnto all specÍes of RNA, but particularly fnto r-RNA'
affecting the tlto r-RNA specles equaIly.
Ttre radloactivfty traces for l¿ter etages of deffclency are
shorm 1n Figure 32. The incteases Ln [32pf] Íncorporatlon are
elrnllar to those observed wlËlr lfAK chronatography and the t¡ro
r-RNA specLes are agatn equally affected.
Loenfng (1965) and Rogers et d7,. (1970) have shorm that root
tfps excfsed from 2- to 3-day-old pea seedll.ngs aad cultured La 2%
(w/v) sucrose, syntheslze 18s aad 25s r-RNA slolly, resultlng fn
¿u¡ accuaulatlor¡ of rlbosonal precursors. ThÍs situation ltas not
obserrred ia the present work. Holrever, Chen (1971), uslog excised
PhASeoLrc Cilreus root tl.ps lacubated Ín a co4lete nineral medfum
l¡ addition to sucrose, also obtalned labelled 18s and 25s r-RNA
after lncubatÍng for 18 hr tn l3u]urldfne.
Ilnlike the 2- to 3-day-old pea seedlÍags getmLuated on
veruiculate noisteoed wÍ'i:h !ûater used by Loenfng (f965) and Rogers
et a7,. (1970), the root tLps used ln thfs rrork wete exclsed frou
mature plants (at least 14 days after germfnatl.on) gro(tliug 1n a
conplete mlneral medfu¡m. In thLs sËudyr the root tips were longer
Eo-(J
xNIo
0
5I
t5
10
5
l0
02040 60020Distance moved : mm
40 60
FIGURE 32 : PoLyacrgLann de-geL eLectnophoresis of 32p-
LabeLLed RNA eætyaeted from 48 b e b) md i.g8 k e il hrdefieLent root típs. 400 root típs r¿ere incubated in[32pi] (Section 2.I.5) and RNA exrîacred as described inSectÍon 2.2.L.3. 2.5 Ug were loaded onto the gels and
elecËrophoresis (Section 2.3.1.2) was for 105 min.
5
0
tlf'
I
c
188 hr+B
inI l¡I lt
I iri ¡¡t l¡
a
tlIt
Il¡,ll¡ a
It¡l
48 hr+B
IIIIIII
tt
6ll
d
ll
l̂tll¡l
t,t
f,,
ttlt
188 hr
-B
IIII
tt------- ----'
I
n
b
48 hr.B
nt¡l¡
79.
tha¡r those reported by the above auËhors, a¡¡d locubation was fn
nutrleaÈ nedir¡m of einílar or ldentlcal compoeltion to that 1n
wtrich the root tfps had been growtag (a¡rd sLnflar to that used by
Ctren 1971). The root tfps are therefore not subJected to sr¡ch a
severe step-down fn nutrltlonal coadftfons as those of Loening
(1965) and Rogers et aL. (1970). In addltlon, the lncubatfon
perlod 1s longer thafr that euployed by these authors, and loenfng
(f965) has lndlcated that locger f-ncr¡bation perlods result in the
fomatlon of more riboeonal RNA (as obserrred by Ctren 1971).
Repeated re-extractlon of the fnterPhase layer in the phenol
nethod (Loening et aL, f969) Ls aecessary teo'obËaÍn a good yl.eld
of the labfIe (Rogers et aL. 1970) r-RNA Precursors' Thus, wlth
the present extraction techníque, much of thls RNA fraction ¡¡ould
renaln in the r:nextracted lnterphase layer, and any f.u the aqueous
layer would probably be degraded. Consequently, fa the system
used here, sooe synthesLs of l$s and 25s r-RNA could be oçected
a¡rd evea !f there |s an accunulatlon of rfbosomal precursora,
these lrould Dot be e:(tracted ¡rf.thout befng degraded. It must be
eryhasized, however, that the lates and types of RNA eynthesls {n
the excised root tlssue nay Dot accurately reflect the sltuåtLon
1n the Íntact tiesue. Ttris does not decreasè the l.ryortaoce of
the differencea obsenred betr¡een nornal srd boroo-deflclent ÈÍesue.
80.
3.6 Propertles of RNA
Ttre fncreased lucot¡poretloa of precursors Lnto RNA nay be
due to a general Íacreased rate of synthesisr or s)¡ûthesls of
partlcular (or nan) RNA baée sequences. If Lhe increase ls not
general, but 1s restrlcted to particular base sequencesr tbis nay
lnvolve changes Ln the properties of the newly synthesized RNA.
1o lavestlgate this, base cooposttion (of Lncorporated radíoactfve
precurr¡ors) and DNA-RNA hybrldization studiee ltere rurdertaken.
3.6.1 Base tion
lhe effect of boron deficl.ency on the oucleotide
dl.strÍbutl.on of [32p] 1n the Rl{A species separated by UAK chroma-
tography ls shown ln Table 9. tùtr11e there are differeûces between
Rt{A specles, the base coupositioa of todlvldual specfes f.s not
eÍgnificantly altered by grorth 1a deficl.ent nedirm fox 24 or 48
hr. After 360 hr there are sore changes, the most fuportant beÍng
that the s-RNA fractlon becomes more LLke the r-RNA fractlon.
Ttris Ls consistent nlth the suggestlon that ia grossly deffcient
tfssue much of the radfoactivfty elutlng as s-nNA is due to
degraded r-RNA (Sectlon 3.5.1.2). TB-RùIA ls,not affected at any
stage of deflclency.
Effects of boron deffciency on the base dlstrlbutl.on of
label 1a total FUA were studied by alkaliae hydrolyeis of the 32P-
labelled acid-insoluble (RNA) fractloa (Sectlon 2.2.t.2) followed
by chronatographic separatfon of the mononucleotldes on Dowoc-l.
¿7
TABLE 9 : Dietributíon of Í32p1 Øþr?g eonponent nueLeotídee ofENA speeies eepævted bA I,IAK ehrcnatogrqhU
3OO-400 root tLps were lncubated fn [32pf] (Sectl'on
2.1.5), NA was extracted (sectLo¡ 2.2.1.3) and the RNA specles
were aeparated by lfAK chromatography (Sectlon 2.3.1.1). After Rl{A
wss recovered and hydrolysed, the nucleotfde distrlbutfon of [32p]
was analysed by peper electrophoresis as described ln Sectl.on
2.3.1.3. Values are exptessed as Èhe percentage of the radfo-
acttwlty associated rfith each nucleotLde.
Tirre aftertra¡rsfer to
boroa-defÍcientnediun (hr)
lreatment CMP A}fP GMP m{P
95
2930
+B
-B
+B-B
+B
-B+B-B
+J-B
+B
-B+B-B
24
48
360
24
48
360
24
48
360
s-RìÍA fractfon+B 2I.4-B 20.3
28.828.4
25.725.6
28.729.6
28.728.L
29.328.2
29.027.7
28.028.5
31.230.4
28.927.O
31.328.4
27.231. I
29.430.5
30.429.7
31.729.6
2s.926,2
25,326.0
25.725.2
2L.g24.3
22.224.5
22.02L.3
23.L22.0
22.322.L
22.822.3
+B-B
20,82I.522.L18.0
r-RNA frectl.on16.6t6.217.5r8.215. 219.9
TB-RNA fon
n.a23.3
2525
2223
2422
32
I9
II
2L.22t.52L.622.O
82.
This also Índicated that boron deficiency does not affecÈ the
nucleotLde dl-strlbutLoo of [32p] after 48 hr growth f.n dafÍcienË
mediun, but AllP and GMP increase s]|ehtly after 144 hr (Table 10).
TASLE 10 : Disþrí.bution of [szp] úØLg eonpotænt nucLentiiles oftotal RNA
900-1000 root tlPs were Lncubated fn [32pr] (Sectlon
2.I"5), and the acld-insolubLe fracÈ1on rùas extracted as descrfbed
in secrlon 2.2.t 1 and hydrolysed tn 0.3 u-KoE at 37o for 16 hr.
Ttre mononucleotides !üere separated on Dowex-l (SectÍon 2.3.2.2).
Values are e:rpressed as Èhe percentage of the total radioactlvity
associated wfth each RllA fractio¡r.
TLue after TreatûenÈ CtfP AMP GMP ttMP
tra¡rsfer toboron-deflcLent
nedlr¡n (hr)
48
L44
+B 23.3
-B 23.5
23.O
2L.9
25.3
25.4
25.2
26.5
26.7
26.6
28.1
28.6
24.6
24.5
23.7
23.O
+B
-B
Thus, differences Ín base Cfstrlbutlon of [eZp] uithfû total
or llAK-fractionated RNA can be detected only after long periods of
growth in boron-deficLent medfi¡n. However, the sensÍtlvity of
these oethods is only sufficlent to detect large changes fn
corrposition, and changes in RlfA base sequences need not neeessarlly
83.
l-nvolve changes i¡ base coupositf'on'
3.6-2 DNA-RNA hybridlzatfon
Wtth the conplex RNA Preparatlîns obtalned from
hlgher organLsms" ft f.s diffícult to obtain sufffcfent locus
speciflclty tn DNA-BNA hybridlzatlon reactl-ons to eoable sinple
lnterpretation of the results (Church 6] McCarthy 1968, Paul 63
Gilmour 1968, Blshop !969, Blshop et aL. 1969, ln[ccarthy & church
1970). Studles involving saturatfon deternfnations are
parrlcuLarly dffftcult to interpret (ltccarthy 6l church 1970) '
Although 6tf11 subJected to the same crfticlsms' competltlve
hybrtdization exPerlnents can r¡Lth the use of discrfniaatotly
anneallng coadítÍons (Church ûr McCarthy 1968) provLde useful
f-nfornation. Conrpetitíon betr¡eea iclentical RNA preparatfons I's
always greater thas that betr¡een preParatlons from different
orgaûs or organlsms (see Paul & Gllmour 1966, Ctrurch 6l McGarthy
f968). It 1s oaly the sensLtlvLty of the technLque ¡vhich can be
questloned. l{ithin an RNA populatf.on, fndirridual base sequences
anneal !fitth DNA at vastly dLffereut rates (due nafnly to
differences l-n concentration) and some RNA base sequeoces
synthesised from rmÍque DNA base sequen'ces faLl to react' Ttrese
factors result ln an underestlmatio¡r of the extent of homolog1r
betr¡een the RNA and DNA sanples, a¡rd thus l¡111 lead to under-
estimation of differences beËlreen RNA populatlons. the technfque
cannot be used to ptoue l-tlentity, since this nay be readlly
confused qrith lack of resolution. Qualitative differences are
84
not easlly dlstingulshed from quåfrtitaÈLve dlstortlors 1n the
popularlon distrlbutlon (McCarthy 6' Gtrurch 1970). Ttronpsan &
Cleland (L97Lb) have recently dfscussed and lmproved the
applicatLon of the technlque to plant nucleÍc acids.
Ia spite of these lÍnltatLons' conPetftlve DNA-RNA
hybrtdfzatlon Ls the most seasltfve technlque aval.lable for
coryarfng RNA sa4les. So long as it 1s realLzed that any
measured dLfferences wÍll be ninlnal estLnates of the real extent
of dlssrntlarfty, the tecturique can be applied to conplex RNA
saryIes.
Bfstrop et aL. (f969) have pofnted out that straLght-fonrard
lnterpretatlon of competÍtlve e:çerÍnents is dependent on the
folloving condítfons: (1) Use of a eoocentratlon of labelled RNA
sufficl.ent to saturate the DNA, and (2) use of a concentratlon of
rr¡labelleal RNA suffLcient to dilute out ldentl.cal labelled R,tilA
sequences eoopletely. In practlce, however, these condftf.ons
cannot be met, and only approrlnatlons are possible.
PrelLnLnary experinents wfth the technique demonstrated the
follorÍng facts:-
3. Some DNA (up to 2O7") ls Ìost from the filters durlng
incubatfon, but thls only occr¡rs durlng the ffrst hour"
so that after the t hr preincubatlon (see SectÍon
2.3.L.4) further losses are snall (ffgure 33).
b. Ttre amount of hybrid foroed reaches a maxLmum after
10-12 hr of annealing (FÍgure 34), a tÍoe course
z.ôtFovlanoJès
20
l5
l0
0
5
012345 15 16 17 18
Incubation tìme : hr
FIGURE_33 : Rate of DNA Loss from nitroceLluLose
ftLtez.-dises. 5 discs each containÍng 1.0 ¡rg ofDNA were incubaÈed in 0.2 rol of 2xSSC at 70o for thetimes indicated and the DNA remaining on the filterswas deterurined by the deoxyribose assay (Section2.2.2 .3) .
30
+?zoct
-o(uN
!l-oèloz,É.
025
FIGURE 34 : Rate of DNA-RIVA hgbrLd forrnation.4 discs each contalning 1.0 Ug of DNA were incubatedín 0.2 mI of 2xSSC conÈaining 10 ug of 32p-1abe11ed
RNA at 70o for the Èimes indicaÈed. The amount of
50 l0 t5
Incubation time : hr
20
ao
85
which ls siuLlar to that rePorted for nucleic acids from
higher organÍsns under sLsrilar annealfng coadíÈj.ons
(uetu & Bishop L969, chen 1971)-
c. TreatuenË of DNA-containl.ng dlscs ¡uith DNase I before
annealÍog almost ellmÍaated the fomatlon of hybrid.
Under the condf.tfons used [100 ug DNase/nl tn 10 nM-
MgCl2 and 10 ott-Trls (pE 7.4) anal incubated for 60 nin
at 37o] 1t ¡¡as found that DNase I removes oost of the
Dl{A fron the fLlter dfscs.
d. DNA-loaded fllter dLscs have negligtble RNase acËivlty
(Table l1). Ttre loportaÍrce of thls has beea dlscussed
by Gillespie & SpLegelman (1965).
TABLE 11 : Degrudntíþn of RNA &rLng øtnealing
FLlter discs containing 1.5 Ug of DNA r¡ere
lncubated lu 0.2 nl of 2xSSC contalnLng 37 yg of
yeast RNA for 12 hr at 70o. RNA degradatlon vas
deter:nlned by Ëhe nethod descrlbed Ln Sectiot 2.4.L.
_-_
Fllter dLscs RNA degraded(ue)
0.80
0.48
0.96
1.68
RNA degraded(z)
2.2
1.3
2.6
4.5
2
2
86
In atteuptfrg to fulffl the first couditlon of Bishop et al.
(1969) (t.e. the use of a concentratlon of labelled RNA sufffcfent
to aaturête the DNA), saturatLoû cuEves r¡ere obÈaloed fcr each RNA
preparatfon (Flgures 35, 37 and 39). Because of the small a¡ror.ltts
of RNA avaflable, conplete saturatfon could not be achieved, and
ooly cunres approachlag saturatlon were posstble. In each case,
the curve for RNA froo boron-defl.cfent root tlps is hlgher than
that frou nomal tlssue becauee of the higher specffic actfvlty of
the forner (e.g. ttre 144 hr samples had specifÍc actfvlties of
1816 and 1040 c.p.m.lvg for RNA frm defl.cLeot a¡rd nornal plants
respectfvely). The quantl.ties of Rl{A avallable requÍred that
values far from the saturatlon level be used fn courpetitfve
hybrldlzatlon studies, and restricted dilutlon ¡vfth r¡nlabelIed R!ûA
to only 10- to I5-fold (FÍgures 36, 38 and 40).
After 24 and 48 hr of grotrth tn deftclent medÍum, no
dlfferences 1n the conpetLtÍon curves could be detected (Flgures
36 aod 38). thus, at these early stages of deficLency, w-lthLo the
llntts of sensiÊivity discussed earller lo thLs sectLon, the tno
RNA preparatf.ons (fron aormal and deff.clent plants) do not dlffer
in their base sequence coryositÍon.
After 144 hr of boron-defLcÍent growth, r¡nlabelled RÌ{A frorn
both normal and deflcient tissues conpetes equally ¡¡ell wlth
labelled RNA fron noroal plants (l'tgure 40a) but not rrith labelled
RNA fron deftclent plants (ftgure 40b). Rl{A fron no:mal tfssue
does not coßpete as successfully wlth labelled RNA fron deffcLent
tisst¡e as does the hooologous RNA, ladicating thaË there nay be
00
Eo(J
!G'N€f-
4
z,É.
200
150
50
00 l0 30 4020
RNA : ¡rg
FIGURI 35 ¿ Saturation cwues for DNA-RIVA hybnd fornationaftez' 24 hr of deficient gnoath. 3 discs each conraining0.4 Ug of DNA r^rere annealed (Sectíon 2.3.1.4) ¡¿íth theamounts of 32p-1abe11ed RNA indicated and the hybrid fonna-tiop.delermined as described in SecÈion 2.3.I.4.lúüf ,,1- 4 -B ÂNÊ ,À .l-/"rilø þ4J /r' +"9 ú,Nn( ou- ö"2' ,r) .
35
30
20
lroo(J
;(u
IO!
å60
z,É,
40
20
50 100
Unlabelled RNA : pg
150
FIGURE 36 : Competitiue DNA-RNA hybrLdization after24 hr of defieLent grot'tth. 3 díscs each contain-
íng 0.4 ug DNA were annealed with 11 pg of 321-
labelled *B (a) or -B (b) RNA in the presence ofvarious amor¡nts of competi.ni +n (o) or -B (o)
unlabelled RNA. Hybríd fornation was determined as
described in Secti.on 2.3.I.4.
00
b
oO
a
oa
o-()
E80(uNT'L¡
z.d.
120
0
0 l0 20 30 40
RNA u9
FIGURE 37 : Satt¿yati,on ezæues foz, DNA-RNA hgbrLd
forrnation after 48 hz, of defieient gnouth. 3 discseach containing 0.7 Ug of DNA r^reïe annealed (Sectíon
2.3.L.4) wiÈh the amounts of 32p-1abe11ed RNA indicated
+B
.B
b
I I
a
40
60
20
0
0
60
40
Eo-u
!oNE
'--oszÉ.
20
0 50 100
Unlabelled RNA : pg
t50
FIGURI 38 : Conrpetítíue DNA-RNA hgbrídization after48 hn of defieient grouth. 3 discs eaeh conraín-ing 0.75 uC of DNA ürere annealed wíth 15 Ug of 32p-
labelled +B (a) or -B (b) RNA in the presence ofvarious amounts of competing *B (O) or -B (O)
unlabelled RNA. Hybrid formation was determined as
described in Sectíon 2.3.L.4.
oC)
80-q(lJNo!-o-c
=É.
120
l0 20
RNA : ug
30
FIGURE 39 : Saturation eurues for DNA-RNA
hybrl d formation aftez, L44 hr of defi.eientgrouth. 3 discs each containíng 0.5 pg ofDNA were annealed (Section 2.3.L.4) r¿ith the
amor:nts of 32P-Labe11ed RNA índicated and the
hybríd formation deÈernined as described infhr "1"+¿ ,(^l /)
00
Section 2.3.I.4.we"d ørU,^ l8l0
lw tu¡*.,ta-
d,
^^^"1 l0l(¿t
+B
.B
30
=o(J
E(uNEs-â
z.É.
20
0
40
0
0 t0 20 30 40
Unlabelled RNA : ug
50
FIGURI 40 : Saturation ezæues for DNA-RNA hybrid forrnationaften 1-44 hr of defieLent gz,outh. 3 discs each conrain-ing 0.5 uC of DNA Trere annealed with 5 pg of 32p-1abe11"d
+B (a) or -B (b) RNA in the presence of varíous "¡1er¡¡¡r,.s
ofcourpeting *B (O) or -B (O) unlabelled RNA. Hybrid forma-tíon was deÈeruined as described in Section 2.3.L.4.
I 0
60
20
b
II I
a
87
sone RNA base sequences ln the saryle from deflcLent tlssue !'rLth
¡vhfch RNA fron norn¿rl tissue does not comPete. The possfbflfÈy
that degradatlon of r-Rl{A fnto lolrer rclecular-r,reLght materlal at
thls stage of deficlency gemtts nore rapid anneallng and produces
the dlffere¡ces obsenred (tr'lgire 40b) ts un1l.ke1y, becar¡se such
degradatlon should produce a slmflar effect rrlth labelled nomal
RNA (Figure 40a). the presence of any 32p-tabelled contamfnanË ffr
the Rl{A preparatLon should also effect coryetitl-on w1Èh both
normal and deficient labelIed RNA. Ttre dtfferef¡ces could be due
to changes in the concentratlon of partfcular base sequences
within the RNA populatiofle or to the synthesls of addftfonal RNA
types Ín deficient root tips. These dÍfferences lndfcate that Ëhe
technlque Ls capable of detecting at least some chdrges ulthin the
RNA preparatlons.
I{tthLn the linits of seosLtivity of the technl'que, these
results sr¡ggest that early stages of boron deflcÍency (up to
48 hr) do not result in any changes ln the type or distrLbution of
newly synthesLzed, hybrfdfzable RNA. After 144 hr of deffcfency'
some base seguences are produced in defÍcient toot tips whlch
eLther are not present or are present fn dLfferent concefitrations
1n normal root tlps.
Atteqts to carry out coryetftfve hybrfdlzatÍon studies with
the RNA specLes separated by llAK chronatography ltere r¡nsuccessful
because of the ltntted quantfÈ1es of RNA avallable fron the
systell.
d8.
3.7 Enz¡rme Actlvity
Eoz¡rme actlvl.ties of crude extracts rrere determined as
described ía Sectlo¡ 2.4. RNase actlvLty has been sho¡¡¡¡ to
increase 1n grossly deffclent tissue (Secticr 1.3.4.1), but Lt 1s
of interest to detetnfne ¡¡hether thfs lncrease can be correlated
with the decreased BNA levels observed 1n this and other studies
(Sectloas 1.3.1 and 3.2.2).
It ls also loportant to deÈerûd.ae whether lncreased
lncorporatlou of labe11ed ptecursors fnto RNA can be orplained by
Íncreases 1n the actl.vÍty of enzynes lnvolved. Urldine klnase and
RNÀ polynerase were chosen for study because they uay represent
the first and last eazynes fn the pathlrey of lncorporatfon of
urLdlne fnto RNA.
3.7.f RNase
RNase acttwLty was detemined as descrl.bed fn SectLon
2.4.L, a¡rd the reeults are sho¡¡n 1n Ffgure 41. On efther basÍs
(per ng of proteln or per root tlp), boroa deficiency results Ín
a sharp lncrease 1n actlvfty, but only after at least 100 hr of
gronth 1n deffcleot nedLum. thfs ls the stage at whÍch the RNA
level begfns toldecliae (FLgure 7 ln Section 3.2.2) ard sllghtly
precedes the cessatÍon of root eloogation (trfgure 4 fo Section
3.1.1). The fall ln RNA conteat Índuced by boron defLcl.eocy may
therefore be due to lncreased RNase actLvÍty. Ttre decrease ln
Rllase actfvlty per root tip in both nomal and deffcÍent plæts
0.5
0
0.2
0
o+)
+)oo!
oEc
,lJo!rõt-U)o!
zÉ.
Éo.PoLo-(')c
F
oF
=
!(uErõLÞ)(lJ!
=É.
2.0
.l.5
1,0
0.3
0.1
50 100 150
Time after transfer : hr
200
FIGURE 41 : Effect of boron defieiency on RMase
actiuity. 100 root típs r¿ere harvested (Sectíon
2.I.4) and the protein content (Section 2.6.I) and.
RNase activity (Section 2.4.I) determíned.
0
.B
+B
b
+B
-B
89
o\rer the flrst 50 hr (flgure 41a) ls largely dr'¡e to the decreese
ln freghweight of these tiPs (Flgure 5 Ln SectLon 3.1.2), but the
decrease per mg of protein (ftgure 41b) has not been e:<plained.
It Eåy be due to soûe Detabolfc change tn seedllng growth'
partlcularly ln lateral root growth, whlch Ls rapfd at thfs stage.
3.7.2 Uridine kfnase
Table 12 sho¡re that oaly when calculated on e Per
root-tlp basis does boron defLcieocy affect the uridiae kfnase
ectlvlty. Even on thlE basis, Ít 1s only after 91 hr gronth Ln
deficfent medÍuo that a naJor effect 1s obsenred, suggestÍog that
the fncreased fncorporatlon of precursors fnto RNA ls ûot due to
Íncreased rates of precursor syothesls. Ttrl-s was also lndlcated
in Section 3.4. At all stages of deficleDcy, a large proPortlon
of [lhC] *.lthitr rhe acid-soluble fractfoo 1s stíll 1n the form of
lthC]uri¿ine (or uracfl -
Êl.gures 16 and 17 and lable 7 fn
Sectioa 3.4), possibly because thÍs 1s a rate-limLtÍog step in the
pathway of nucleotide synthesls. After 48 hr of growth ln
defLclent mediuu, the rates of lncorporation of exogenous urLdfne
(fron therlûcr¡batlon uedirn) lnto RNA of defLcient and norual root
tl.ps are 0.8 and 0.4 pnol per mg of root ttps (f.w.) per hr
respectlvely. ltrl.s is less than Èhe urldfne kfnase actlvlty
me¿rs¡ured ìn ví'þzø (Table 12), but as eodogenous uridlne Ís also
fncotporated ioto Rl{A, the total rate of urLdine utilizatlon nay
be as great as the ín Viþzp rate of IIMP slmthesis, lfLthout
detemLning the specifl.c actlvlty of [lhC] fn specf.fic nucleotLde
Btg 12 z Effect of borcn defLaiencg on reidí,ne kitøse aabLví'fu
2000 root tlps were hanrested (sectÍon 2.L.4ì and the
protein co1,Èent (Sectlo¡ 2.6.1, and urfdlne kinase actfvltfes(Sectlon 2.4.2, detetmlned.
90.
pnol UMP produced/hr
/ng proteln l¡e(
/root tÍptissuef.w.)
T1æ aftertransfer to
boron-defl.clentoedlun (hr)
49
l"eatDent
+B
.B
+B
-B
943
1040
1455
L292
14.3
L6,2
19. 9
20.4
5. 18
6.24
5.8291
8.02
pools, the total rete of utfllzatlon cenûot be calculated' lhe
accr¡nr¡latÍon of [f[CJurt¿1ne could also occur because urldl'oe 1e
takea up lnto parts of the cell or tl.esue lacking urÍdÍne kl.nase
actlvity.
3.7.3 RNA polynerage
lllth the crude extract ueed, Ít r¡as luposslble to
obtafn en eccurete estlnatlon of RNA polynerase actfvfty. Flgures
42, 43 æd 44 show that the reaction rates ere tot lÍnear wl'th
efther lncubatfon tfne ot amorlrrt of plant extract. ThlE fs
probably because hlgh lenele of auclease and phosphatese enzyoes
30
.= to(u
oIoO)E
o
:Es30lt'LoCLLo(Jg'- 20o-
20
l0
00 5 l0 15
Incubation tire : min
20
FIGURE /+2 z Effeet of ineubation time on RIIA
poLymerase actiuity aften 49 (a) cnd. 97 (b) hr ofgz,outh in defieLent medíwn. 2OO0 roor rips wereharvested (Section 2.I.4) and the protein contentdete:mined (Section 2.6.1). RNA polymerase acrivírywas determined (Section 2.4.3) usíng 0.02 rnl ofplanË extract. The effects of Actinomycin D (10 Ug/nl) and RNase (150 ug/urL) íncluded ín rhe incubarionmixture are shor¿n in (b).
b 9l hr
.B
oo
+B o
RNase o
ActD .
49 hra
+B
.B
30
20
=(l,+Johlog)E
oo-
0T'(lJ+Jro!o?zao(Jg
èF= l0
050 l0 t5 20
Incubation time : min
25 30
FIGURI 43 : Effect of ineubation time on RNA polymezase
actiuity after 21,6 hz, of grouth in defieient meùLum.
2000 root Ëips were harvested (section 2.r.4) and the proteincontent determined (section 2.6.1). RNA polymerase activitywas determíned (Section 2.4.3) using 0.01 m1 (o), 0.02 ml (^)or 0.05 rnl (O) of plant extracË.
.Bb
a +B
40
60
20
b +B
.Bo
o
I I
a
+B
-B
0
0 23Prote'in : mg
FIGURE 44 : Effect of uazging the amotatt of eætz,aet
used in tLte RNA polymez,ase a.ssaA. 2OO0 roor ripswere harvested (Section 2.I.4) after 216 hr of growthin deficient medium, and the proteín content deter-mined (Section 2.6.L). RNA polymerase activity r^ras
determined (SecÈion 2.4.3) after íncubating for lO
min (a) ot 20 rnin (b)
gzo:T(¡,+,rõ
oUo5-o(Jç
È40=
54I
97.
ln the extract (especlally that from grossly deficÍent tlssue)
Lnterfere wl.th the essay. Eowever, under boron-deflclent
conditl.ons, the actf.wl,ties of many phosphatase and nuclease
enzynes are fncreased (Sectlons 1.3.4.1, 1.3.4.2 aod 3.7.1) so
that the reductLon of the measuted RNA pol¡rnerase activlty would
be greatest in the assay of deficlent tfssue. Itr vfers of this,
the results suggest that there nay be a¡r lncreased' in r¿uo RNA
polynerase actfirlty tn deficlent root tfps. If the results ere
calculated on a pet toot-tip basls, the actlvlty ln defLcient
tissr¡e 1s equal to, or greater than, that of nonnal tlssue at all
stages of deflcfeucy.
Ioclusiqn of Actinonycin D (10 uglnl) or RNase (150 uglnl)
in the reactLon nÍxture reduced the lncorporatlou of UTP by
5O-7O7" (figure 42b), lndicating that the produet fo:med fs
predonLoantly RNA.
92.
4. GENERAL DISCUSSION
4.L Morphologlcal and Physfologl.cal Effects of Borou DefLcfency
the norphologlcal and physlological lesioos of boron
deflcleney reporÈed here agree closely wÍth prewlous reports ia
the líterature (see Sectton 1.1). The sequence of development of
these synptoms ls also sfmilar. It is clear that after 80-100 hr,
the deficiency influences lr¡rDy oorphologícal a¡rd physlologlcal
characterfstlcs. Some of these are paraneÈers on whtch blochenical
caleulatLons are based, e.g. protein, fresh wel.ght, cell number
and size. Becar¡se boroa defLcLency affects Ëhese paraneters Ln
dlfferent ways and to a different extent, calculations based on
different parameters nay l¡rdicate different effects of the
deffciency. Ttris {s demonstrated uany times in Section 3, when
results are expressed Ln different ways (e.g. Flgures 6, 7, 8, 9
as¡d 41, and Table l2). This must be taken Lnto accor¡nt Ln
evaluating nany reported effects of boron defLclency. During the
flrst 50-70 hr of deficieoey, these parameters are aot affected
and the basis used to e:cpress results Ls therefore less iryorÈant.
Most of the results fn thfs thesfe are expressed on a root-tip
basls. Tlrls was chosen because it approxlmaÈes to a per ceil
basls and Ís the most convenl.ent paraneter to assess. Ilonever,
by reference to Figutes 5 or 6, or Table 2, the results may
readlly be converted to other foms.
93.
4.2 RNA llegradaÈfon
l1re decrease f.8, the level of Rt{A reported by prevloue
r¡orkers (Sectlon 1.3.I) was coaffimed, br¡t tlæ-courne gtudfes
fadlcated tirat thls ouly occutred at or Jtrst before the eÈage rhen
root elongatlo'n eeased. Ttre ßllase actfvlty lncreaeee et aPProxl-
oately the eaue stage of the deflciency' auggestlng that the løer
level of RNA is due to a higher rate of degradatlon by Rlìlase. The
fractlonatlon of RNA by llAK chrouatograpþ prowldes fi¡rther
evldence for thle. At advaneed etagee of the defLcl.encry, the
level of r-RNA is reduced aad the amrnt of RNA 1o the e-Rl{A
regloa focreases s11ght1y. Ttre base coryosltion of 32P-labe[ed
RNA ln the s-Rl¡A regLon suggests thaË thfs fs dr¡e to degradatloa
of r-RNA lnto lol¡er mlecular-neight paterfal, sone of ¡shfch rrfll
fractfonate wlth s-80[A on üAK colums. TtrÍs degradatf.on of r-RNA
also explafue tbe fahfbltlo'n of protela synthesis dlscr¡ssed fn
Sectlon L.4.2. Interruptlon of polysooe fo¡matfon and decreased
rÍboEoue coûtelt lvere teported, but ooly after the agpearance of
vLsfble deficleacy slqltous¡ f.ê. at tbe stage when a decrease 1n
the r-RNA codtent was obsenred ln the present sork. Ttrue,
fncreased RNase actlvfty reEults ln degradation of RNA (Partlcularly
r-RllA), leadlng to an lnternrptlon of protefn synthesls. The
Íncreased RNaee actlvlty eould therefore be a key factor 1n
fnduclag the late syqtons of boron deftcl.ency.
Ttrese ehanges ln RNA cootent and RtIase actfvÍty are sinllar
to those occurring 1n uaturing atrd seneselag tleeue. Severel
94.
dfffereat t¡¡pes of ENese €nz)¡me6 harÉog dffferent l¡tracell,ular
dfetributlon have been reported (Keeeler & Eagelberg 1962, I{Ílson
L963r^ and Þ, hgle & llagenan 1965, SrÍvastava f968). In geoeral,
Lt appears thst total RNase activlty' and partlcularly the soluble
cytoplaeafc eûz¡oes lncrease l"lth the rnaturatloa of plant tfseue
(Kessler & Eagelberg 1962, Shannon E Baneon L962, Ingle E Bagenan
1965, McHale E Dove 1968, Phlllfps & Fletcher 1969, ltflllkan &
Ghosh f971). At the sane tlme the RNA level decreaees. Detached
leaves and leaf diece also show loss of RNA aod lncreased total'
cytoplasnfc a¡rd chroaatla-bornd Pû{ase aetLrrlty durlng the oneet
of se¡resceoce (Chertly 1963, Ibve 1967, llcËale & Dove 1968'
Srlvastava 1968, Eckh¡nd t Moore 1969, Atkf¡ E Srfvastava 1969).
IÞcreaged RNA levels of attached aod excl.sed senescing leaves are
prLnarlly due to degradatfoa of r-RllA. Srlvastava & Atlcfn (1968)t
uslng aucroae deaeity gradient ceutrffugatfon, aod Ctrerry et aL.
(1965), rrsing l{AK chronatography, deuonstrated a rapl.d declÍne fo
r-RNA, r¡fth a snaller decrease (Srfvastava & Atkin) or slfght
fncrease (Gherry et aL,) ln g-RNA. Srfvastava & Atktn fotmd no
change 1n the base conpositfon of 4 hr 32P-1abe11ed r-RNA, but the
base co,ryosltlon of s-RNA becaoe uore like r-RNA durfng the
advanced stages of senescence. Both groupâ suggested that the
e-RNA peek f.s coóÊa-{trated wlth degradatl.on products of r-nNA and
n-RNA. Ttrese changes are efmllar to thoee fnduced by boroo
deflcl.ency (see Ffgure 25). IÞgradation of rfbosones and poly-
sooes fn seneeclng tiesue hes been reported by Oota (1964) and
SrLvastave & Arglebe (L967), RNaee activity also fncreases, and
95
RNA conteot dec¡easee wÍth lncreasfng dlstance-from the tlp (i.e.
increaeLng uaturlty) cf broad bean (Robineon & Carürrlght 1958)
egrd. lene (ptlet & Brar¡n 1970) roots.
Elongatfoo ceeses Ín botm-deffcíent rootar but naturatl.on
contlaues, so that the tlssue wfthfn the 5 @ root tips eventually
reaches a stege of aaturity usually ooly fonnd much oore dl'stant
fron the tfp. Later, seDeace[ce takes place' The boron-deflcl'ent
root tip therefore ¡eeenbles the tåturlng and senesclng tlssue
dl.ecwsed above, and the changes 1o RNA content a¡rd RNaee actLvlty
are sloflar. Thus, the locreased nNase actlvfty aod resultfng
degradatloa of RNA and laterruptfon of protefn synthesfs can be
explafned by the accelerated Daturatlon and senesceuce of
deficlent root tips.
The Ínductlon of boroa-deflclency s)¡qtoms by base analogue
treataeat (Section 1.3.5) could be due to the lnductíorr of
senegcence by these coûpormds whÍch disrupt RNA and Protefn
synthesis (SugÍura 1963).
4.3 RNA SvnthesLs
Itrrrlng the errly etagec of boro'a def{clÊncy, there Ls as
lncreaeed rate of fncorporetfoo of radfoactl.ve Precuraors fnto
RNA sithout any appafent effect on the total Precursor pools. Ttre
total level of radloactivlty 1n the acld-soluble fractlon and lts
OA
dlstrlbutLoo betræen the dffferent nucleotLdes and nucleosfdes is
not sfgnLfl.cantly char.ged r¡ntl1 at least 48 hr after tra¡1"'ferrfng
plants to deffclent ûediuo. The ebsence of any slgnlficant effect
on urldlae kinase actfvity or the dÍstrlbutlon of [thC] (lntroduced
as lrhC]urldloe) bet¡¡een IIMP r"nd CltP lrtthfn the oewly syntheslzed
RNA also suggests th¿t deffcÍency does noÈ affect the total
aucleotide pool. Thus, lacreased íncotToratfon of radloactlwlty
lnto RNA must be due either to chenges fn the propertíes of snall
speclfic nucleoÈlde pools or to an lncreased rate of RlÍA syntheafs.
DIAK chrouatography lndfcated that at tlrese early stages of
deflcfeney, the ÍocotporatLon of precursors f.nto TB-RNA 1s
st1il¡lated more thao the lncotToratlon fnto other RNA fractf'ons.
As the rapl.dly labelled coúponents of ÎB-RNA have proPertles (e'g.
baee coryositfon and half-l1fe) slnllar to those orpected for
æRNA (lester & Dure L967>, these results oay be due to an lnftlal
stLnulatlon of æRNA syntheal.s. If rRNA ls the oaJor labelled
couponent of the RNA fractfon, thfs could occur !ùlthout causing a
detectable ehange 1n base coqosltlou. After a 4 hr labelllng
perlod, rRNA would probably be a ml.nor coupotreat of the r-RNA
peak (Ingle & Key 196E), ¿md a slfghtly greater lncrease Ía the
ÍocotporatÍon lnto thfs fractfou thaa r-RNA would not sfgniflcantly
change the base coqosftlon of the peak. Thue, the fact that the
lncorporation of precursors fnto TB-RNA ls Lncreased rcre than
r-BNA or s-RNA oay reflect a greater effect of deflcfeney on rn-RNA
syathesl.s at these early stages of deflcleacy.
At later stages of deftclency (aftet 144 hr or nore), the
97.
increased speclflc actl.vftJ of lucorporatloa of precursore into
RNA uay be due to the lncreaeed level of radfoactfrrtty wlthlo the
precursor pools. If, however, nucleotide atd nucleoeide levels
ere lncreesed by the deffciency, aE are nany other polyner
precuraors [e.g. auino acids arrd alnnonia (Sectfon 1.(.1),
fnorgaric phosphate (Sectlons 1.3.4.2 a¡rd 3.2.4) aud componente of
the actd-soluble fractlon (Sectlon 3.2.4, ], the lacreaeed aoouût
of radfoactlvlty wlthfu thfs fractÍon rnay be due to an Íacreased
level a¡d not an Lncreased epeclfic actÍvtty of nucleoeidee and
nucleotÍdes (eee Flgure 15). The dfffeÍent rates of Rt{A tutilover
(dr¡e to lncreased degradatl.on by RNase) and the ¡åny other
physlologlcal and blocheofcal changes fnduced by boroa deficfency
nake any coqarLsoo of Rl{A synthesLs lates durlng these late
stages of deflcleucy dlfffcult to lnterpret.
Ihrrlng these later stages ln the development of boron
deflcfency, the fncorporatlon of Precursofs Ínto rfbosonal Rl{A
(pRNA peak aod degraded I-RNA fn the s-RNA peak) Ls iucreased
more than TB-BNA. Ttre tlvo subfractlons of r-RNA separated by
polyacrrylamide-gel electrophoresls are equally affected at all
stages of deffclency.
I{lthout further characterizatlon of the labelled Rl{A withtn
eEcl¡ fractfon, !t 1e not possfble to state unequfvocally rñtch
RNA speeles are effected by borm deffciency. the tesults are
consfstent Irlth the hypothesls that l-oftfally çRNA synthesÍs fs
fncreased, but after 50-100 hr of grdrth fn deflcient uedir¡m' the
s)lnthesls of r-RNA fs stlaulated uost. It fe certaln, however,
98.
that dfffe¡ent ßNA fractfons are affected at dlfferent stages Ín
the developEent of thr, deflcf.errcy. If, as suggesÈed by Ingle &
Key (1965), these RNA specf.es are sFrtheslsed from dlfferent
precuraor pools, the dLstrlbutLon of 1abe1led precursors between
speclftc pooLs, rather than theLr LncorPotatlon lato RNA' nay be
lafluenced by the deflcÍency to produce the differeDt labellt¡g
patterns. Ttre slullar effecte of the deffcieocy on [32Ptl,
lrbC]urfafne end [3n]urtdt¡e lncorporation provlde Ladirect
evLdence against thl.a. If dtfferent pools are used, they ottst
have sluÍlar equilibtir¡n ProPerties.
Thus, the effects of boron deficfeacy on the lncorporatl'on
of precursors lnto RNA can be dlvlded fnto two dÍstfnct steges.
I{lthln the fLrgt 48 hr, the fncorporation ls stlnulated wlthout
eny epparent effect on the total uucleotf.de and nucleosfde pooI.
The lacrease Ls rnostly lnto Its-RNA a¡rd r-RNA (posElbl'y both due to
çRNA). Beyond thls stage the lucorporatlon into nucleotldes and
nucleosides also lncreases and that Ínto r-RNA Ls stinglated nore
thaa lnto IB-R¡¡A. TtrÍs change occl¡Ìs between 40-50 hr after
transferrlng plante to borordeflcient nedir¡n (FLgutee l3r 29 and
30).
Like changes Ln RNA content and RNase actlvlty, the later
effects of boroa defielency on RNA pf'ecutsor Íncotporatioa (eecond
stage descrfbed above) nay be due to the acceleEated rate of
ngtufatlon and Eeneecence fn deficlent root tiPs. Seseecence of
attached or exciged leavee results fn Lncreased Lncorporatlon of
[32pr] Íûro RNA (Gherry et a7.. 1965, Srlvaetave 6l Atkln 1968'
oo
Ecklr¡od E Moore 1969) and changes 1û the dlstrlbutLon of 1abel
bet¡reen RNA fractiong are ræry efnllar to Èhoee obeenred Ln boron-
defieient root tÍps af'ter the æsedtiort of rcot elpngatiott (Cherry
et aL. 1965). Ttlus, after 80-100 hr gro¡th fs boron-defl'cl.ent
nedlum, the Íncreaeed Lncorpoxatfon of precursors fato RNA' the
changes Ín RNase actfvfty, RNA conteat and Protefn synthesle and
posslbly oaûy other s]tryto@s of the deffclency appear to be
assoclated ûdth naturatlon and senesoence of the deff.cfent tfssue'
and are therefore far remved from tbe fnitfal efte of actÍqr of
boroo defÍciency.
Wtthln the flrst 48 hr, the chaoges 1n RNA s¡¡utheefs could
be due to a dlrect effect of boro'n on the synthesLs of partlcular
RNA species, but Èhe abseace of any detectable changes Ín the
properrfes of the newly synthesfsed RNA (DNA-RNA hybrfdlzatlon
and base cæposftlon) do Dot aupport this ldea. An lncreased RNA
polyrerase actlvlty could produce the lncreased precursor
fueoraoratlon but ney not accouût for the dlffere¡t effects on
dfffereot RNA spee{es. In the abeence of any evldence suPPortiog
a prlnary effect of boron on the control of RNA synthesl.s' ft
aeems oost lÍke1y that although thLs Ís an early effect of the
deflclency, ft 1e et1ll a eecoadary effect. Sever¿l poeslble
e:planatlons for thle ate dfscusaed fn this and the followiag
sectÍons.
Ttre lnitlation of adventitLoua roots of Cì,eer æientínwt
lnvo1r¡es an lncrease 1n the Íncor¡poratLoa of precursors fnto Bi¡lA
(Jalouzot 1971). In pea roots, a lower proportíon of t-nwA fe
700.
eesocfated ¡rlth rapfdly dividfng cells (Vanderhoef & Key 1971)¡
wheu cells chaoge frour e dividtng to a noo-dtvldlag atate, the
aootmt of 4s t-nNA expreseed aB e percetrtage of total RNA lncreaees
frm 5.67 to 11.12. Bro¡En û llangat (f970) have also showa that
wlth the lnltiatlon of rootfng 1o petl.olee of Flwaeohts tnþaria,
there 1s a 3.5-fo1d lncreaee 1n r-RNA conceûtratlon' whlle g-RNA
and DNA are rot affected. Ttre ler¡el of triphoaphatee and
pertlcularfy,UDPQ lacreased Just prlor to the rootlng of thls
tlssue. Thrls, an luereased íncorpoËetÍon of precursors lnto RNAt
affectlug partlcularly r-Rl{A aad ÎB-RlfA, and the sllghtly fncreased
incorporatlon of precursors into nucleotidee (Tables 7 and 8)
could be assocLated rrlth the actl\tely divtdtng celle of
adventftLous root laftlatlou. It 1s not kûowr when these are
ialttated fn deflcteût root tipe, but Íf lnltlatLon ¡rere lnduced
by deflclency duting the fl.rst few hours, this nay well explein
the fncreaeed RNA precursor lncotaoratfon. tÎre absence of any
effect otr the total nucleotlde pool at 48 hr aay ho¡ver¡er suggest
that lateral-root lultlaË1on ls not responslble for dlfferencee Ln
precutsor lncotporatl.on at thÍs early atage. Ttrfs posslble
intetpretatloa e4hasfzes Ëhe dlfficultl.es aesocl.aËed stth
etudylng ehanges 1n cqlex tissues. The changee uay be due to
only a enaIl gtor¡p of specLalf.zed celle r¡fthfa the tfegue alrd uay
enen be naskÍng fnportant changee la other groups of celle. If
the Íncreased precurÍror fncorporatÍon fnto INA were due to the
fnftlatlon of adventftlous roota, lt nay explafn the different
reEults obtafned wlth the tlro systens dlecussed iu Sectlon 1.3.2.
\I,/AITE Ii'iS'i¡TUTE
LIBRARY
Io ¡rhole root or plant systema, adveotl'Èloue roots
relatLvely tolqortant a¡rd ân olterell reductÍon ln
iocoraoratl.on eould be recorded Ln apÍte of the lncrease 1n tfesue
where laitietÍoa occurs.
4.4 Plant llottotres
ltre slnflarlties between arrxfn treatmer¡t of plant tissue arid
borou defLctency have been extenelvely rewlewed by Jaweed & Scott
(1967) and Coke & llhlttlagton (1968). Some addftloual pofnte are
relevant to the root-tLp system. Ttre lnhfbitfoû of ceII dfvlslon
and elongatl.on but eontfnu-,d radfal enlargement of 2,4-D treated
apLcal soybean hypocotyls observed by Key et aL. (f966) and the
swelllng of chl.coty root tlssue (Flood et aL. 1970) are slmller to
the effects of boron deff.cfency. Follor¡1ng the auxln-Índuced
swelllng of root tlssue, root prloordia are inltlated 1n the
pericycle, leadlng to the aPPearance of adventl'tLous roots
(C,oldacre L959, Fan & Mcl,achlEn 1967, Jackson & Earney 1970' Grat¡t
& Fuller 1971).
IAA apptted to pea or bea¡r shoots ol stena 1s rapfdly
transported r.rnchanged to root tips and laterel-loot primordfat
where lt ls degraded Ëo lnactlve foms at such a tate as to
naintaÍn e const¿mt level of IAA fn the root syoten (MorrLs et 4,L.
1969, Iversen et aL. 1971). Thus, any ehaoge Ín arsÍn oetabolfsm
would have an early effect oû root-tÍp grwth. Inhibltfon of
7.
r,¿í
702.
auxiD degradatlm would reptd.ly teeulÈ ln ar¡rLn toxlcl.ty tn root
tips as dlecuseed 1n Sectfon 1.5.1.2.
Follodng the ¡rork reported 1o thls theels' further
corqarÍso'ns ca'r be 'n¡de. Ar¡xlns (2r4-D a¡rd IAA) lncrease the
total (strannon et aL.'f964) and microsmal (Birnfnghan Ê Mcl,achlan
L972, Slase activÍty but reduce tåe solr¡ble cytoplasufc enzynÊ
actfvlty (ptlet L970, Pflet & Brâr¡n f970). In maturing tlssue,
honever, the lncrease f.n the activlry of the latter eozyoe
contlnr¡es Lodepeodently of hormme treatuent (Bfimlnghan &
Uel.echlsn L972, so that ln lAA-treated, maturlng and eeneecing
tfssue the actirrlty of both rlbonuclease eîzynes lñcreases.
Ar¡xfn treatDent (IAA, 2t4-D, NAA) lncrea8ea RNA 1evele fn
nost 1ûtact plant EysËeo3 and reducea or prevents the logs of RNA
fn nost exclsed dystens (see Key 1969). RiboeonEl RNA fncteaees
nore than s-RNA (T'ey et aL. L966, F1Èes et aL. f969). Incot¡rora-
tfon of nuclelc acfd precursors ([llC]nucleosldes or [32pt]) tnto
BNA fs also enhaneed by auxfn treetûent fn both tlpes of systeûs
(fe.¡ g Shannon 1964, Sacher 1967, Tresavas 196fu, Key 1969'
Hatestg 1971). Most auxÍn-responslve tfsEues shor an enhanced
precursor f.ncorporatÍon after a 1ag perlod of 5-120 nin (Clfck &
Eackett 1964, llasude & Kanfseka 1969; Key 1969). þtake of
precursore luto the total nucleoÈlde pool fs enl¡anced 1o eome
tlseues (Xooden 1968), but the levels of incor?oratÍon lnto Rl{A
are such that thfs c¿rüiot account for the effect of ar¡x1n orn the
apparent XûIA elmtheals (Îr€stevas 19684 ad þ) c '
1.03,
The applfcaÈfon of atxlns to fi¡tact tissue Pronotes or
lutrlbits gronrth, depeadfng upon the ar¡xin concenËratlon, but RNase
actLvity and proteln and uuclelc acfd levels fncrease ltlth applled
anrxfn, even lnto the herblcldal cosceotretion range. llerbicLdal
actlon appear6 to be dr¡e to ar- o\¡er-Productfon of RNA and protein'
leadiag to nasslve tlssue prolfferatlon, dfsorganlzed growth and
eventually death of the plant (Sbannon et aL, 1964, Rey et aL.
1966, Key 1969). thls 1s eÍollar to the el.tuatloo occurTÍog r¡nder
boron deffclency. Grol¡th fs fnhlbfted, but cell e¡paosfon
cottinues, wtrlle RNase actlvlty and RNA synthesis fncrease, and
the Èissue eveutually dÍes. At late stages of boron defl.clency'
RNA levels fa1L, but thfs 1s probably due to the lncreaeed
cytoplasnic RNase activfty resulting frm the lncreased rate of
maturatlon and seneecence of deficfeut root Ëlps.
Further characterlzatlqr of the newly synthesfzed (labelled)
RNA by sucroae density gradfeat seParatlon (Banflton et aL. 1965)'
after fncubatürg sub-apleaL Aae¡ta coleoptfle segnents 1n
l3H]uridtne a¡rd IAA for I hr, lndlcated that all classes of RNA
lrere equ€rlly affected. Ilowever, after ehorter treatuent perlods
and separatlon by lfAK chromatograPhy, changes slnilar to those
faduced by borcr deffclency have been noted. InftÍally'
Íncorporatfotr lnto rRl.IA fs stlnulated by ar¡xia treetnent of
eloogatlng æd dfwidlng tissue (soybean hypocotylr Peê lntertrode
and oat coleo,ptlle) but later the lncotaoretfon lnto r-RNA 1s
stfnr¡lated nost (Xey g Ingle L964, Ingle et aL. 1965, Jactr¡rnczyk
& Cherry 1968). At early sÈagea' the lncotaoretioû fnto TB-RNA
704.
1s etloulated more than any other fractLoo (Tester E llr¡re L967,
Obrlen et aL. 1968, Tanl¡oto & lfasuda f969). lhe base conposftÍon
(of lncorporated [32p]) 1s also sllghtly altered, the AMP:II]íP
raËlo decreasf,ng. The sinllarity between the changes wlth tioe
after ar¡xfn treetmeût and bor-,n deflcÍency f.s shor¡n by conparing
the plot of Taninoto & Masudars (1969) results (¡'tgure 45 of thfs
thesfs) wtth Figure 24. Dlfferent specl.es lrfthfn the TB-RNA
fractlon are affected at dlffereot stages after ar¡rin treatEeût
(Key 1969, Ìflassod et aL. 1970). If, as has beea suggested by
Tester & Ih¡re (1967), the rapld"ly labelled TB-RNA Ís mostly pRNA
(see also Sectfou 3.5.1.1), aru<Ln treatment appears to tnittally
stfnulate e[NA s]mthesfs. Ttrfs hypothesis ls supported by the
obeenratlon that ar¡xln treatment rapidly Índuces polysome
foruatlon (Trewavas L96&, Fltes et aL. 1969).
Ttrus, fron the avallable data, the folloulng suggestÍon by
Eltes et aL. (f969) could apply equally well to eLther ar¡xin
treatnent or boroa deprlvatloa:-
"In the tnltfal hours after treåtment there Ís an
apparent burst of messengerRNA s)/nthesl.s such that
nonorlbosomes¡ are converted to polyrlbogomes.
Preernably this response fs to traûsftory Iov ar¡xia
concentratloas. Follorvfng closely onto thls perLod
there fs an lncrease La rfboso,De productloo ¡¡hlch
eppeere fndepeodent of DNA syathesls and ceII dlvlslon.t'
00
E(u+,rõ(uL+,
hoèe
200
150
00 50 100 150
Treatnpnt tirne : min
200
FIGURE_45 z Effeet of 2r4-D treatrnent on the specifieaetiuíty of [32P] dneoz,porated i,nto different RILA
fractions. Values r¡rere calculated from Tabl-e I ofTanimoto & Masuda (1969). Fractions are those separa-ted by IUIAK chromatography. The specifÍc activity of2,4-D treated pea stems J.s expressed as a percentageof the specific activlÈy of normal tissue on theordinate.
TB-RNA
r-RNA
s-RNA
50
ooÀ
705.
Elongatlon-stinr¡latlng concetrtrattons of au¡fo do not
produee æy detectable changes 1n the cqrPetftive DNA-RNA
þbrlillzatlon curnes of total RNA exttacted fro excl,ted Pee-stem
tlse¡¡e. Htgher concentretÍoaa of 2r4-D result 1¡ dÍffereocee
betr¡een hybrldt zable RNA spec:lee slnllat to Ëhose ¡tror¡n here to be
fnduced by boron deflelency (Îtrøpcon & Clels¡d l97lå).
Thus, the early effecta of boron defleÍency on Rilaee actirrlty'
the r.ptake and fncotporatlon of precuraors loto RNA, and Ëhe
propertles of the nenly elmtheelzed RNA are siaLler to the effects
of ar¡¡cfa treaÈEeat. All of theee deficfeocl¡-lnduced chaogee could
be e:cplalned by au accr¡oulatLoû of auxfn, pasefng through grøth-
proootfng to lnhibftfng to herbicl.dcl coÍccstratlons. the eane
effect cor¡ld be achfeved, however, if boron and a¡¡rt¡e were
operatlng lndependently ot together through the same efte of
actÍon or bloctrentcal process.
Tt¡e æde of ectf@ of ar¡xine ls stlll not clear. Dlrect
effects on Bt{A sy¡thesls have been suggested, aûd dfrect-bfadfng
wlth chroostt¡ (Fellenberg 1969 art 1970) a¡rd g-BNA (Kobayashl. &
Ysnakf L972, reported. Reduced thero¡l Etebfllty of reconetituted
nuclco'pæotelns (fealoUerg 1 9 7 I ) and lncrerscd elrtoneÈl¡tFalaoc Lated
RNA polynerese rrlthout lncreaaed teqleCe eva{laþÍlfty (ObrLe¡ et
aL. 1968) have also been derootttated. Although coûtl.oued nNA
syatberfe 1e eaEer¡tfal fot euñtn-tûduced græth ln a vatf.ety of
eysteÉ (eee Key f969), the 1çorcæce of thls aa eû fnlÈ1el slte
of actÍoc 1s r¡Bcertaln (Nelsoa & Ilsr 1969r lfer¡umn & Palner 1971).
Reccnt reÞott! fndfcate that othcr factots uay be lryortmt ln
706.
nedfatl.ng the atrxln effect (Hardfn et al. 1970). -Protefns
(ltatthysse & Phflltps 1969, Venis f971), otner ho¡mones (Thourpson
& Cleland L97lb> and c-AlfP (Salooon & Mascareahas 1971) have been
suggeated. IAA stLmulates the lncorporatlon of IrfC]aaenlne fnto
c-AllP (Azhar & Krlshna MurtL '!.971, Salomon & Mascarenhae 1971),
reeultlag ln a 5- to lO-fold increase fa the basal leve1 of c-AlfP
wlthfn l-2 nfn after ar¡xÍn treatoent of At¡end coleoptlle sectfoûs
(Salouon & Mascarerhas 1972b). Ttre ar¡x1o (2,4-D) faduced cell
ercpanslon Ln Jerueale¡n Artfchoke tuber tlssue fs synergfstically
entranced by c-AI'IP (fadsatca û l.[aeuda f970), and the presence of
c-Æ{P and IAA durlng chronatln extraction also results 1n an
lncreased rate of Rl{A synthesls by the f.solated chro¡natfn (Salouon
& l,lascarenhae 1972a). Boro¡ could lnf luence nuclefc acld
sÊtebollan ví.a some effect o'n c-AlfP, but the regulatory role of
c-AlfP ín tiUo has aot yet been rrnequfvocally establlshed 1n plants
(Ockerse & l,fi¡oford 1972).
Treatnent of plant tfssue wtth gibberellfc acld (Chandra &
Varner 1965, Fletcher & Osborne 1966, Johrl & Varner 1968,
Broughton L969, Poulson & Beevers 1970) and cytoklnlns (Osborne
1962, Oota 1964, Carpenter & Cherry L966, Burdett & I{arelag 1968,
Kulaeva et aL. f97f) algo lncre¡raes tt¡e lacorporation of radlo-
actfve precuraors Íato RNA of both exclsed and latact plant tissue.
CytokfnLns affect all Rl{A fractlons (separated by l{AK chronatog-
raphy) equally, but gfbberellfns àtmr¡late the lncorporarloû of
precursors futo r-RNA and ÎB-RNA Eore thrm s-RNA. RNA polynerase
actlvlty aseoclated wlth lsolated chronatfn (ttcConb et aL. 1970)
707.
and BNase actfvity ln fsolated barley aleurone layers (ChrLspeels
& Yarner L967ù are also lncreaeed by gfbberellln treatnent. GA¡
treatueût does not Lnduce any dlfferêncee fn readlly hybrtdLzable
Ft[A specles in epicotyle of dwarf pea seedll¡ge (Itroûpsoû & Clelaod
;971a,r, but rnllke boron deffcleacy, GA and kfnetLn lnduce chæges
1n nucleotl.de patterne (Brcm É Cassells 1971, Coll|ne et aL.
L9721. c-Æ{P may also be lnvoll¡ed ln the sctlon of gibberelllos
(Catsty & Llpplneott 1969, Pollard f970). Because of the sfnLlar
effects of boroa and GA, ln bre¿lcfng dotoancy 1n Theneda triødna
Beeds, Cres$yell & Nelsot (L972, have recently suggested that boron
nay be lrylLcated L¡ the synthesfs of GAr.
l.fany lnteractfons beüweeu plant ho¡:nones have been teported
(e.g. Van Overbeek et a7'. 1967, Ctrriepeels & Vamer L96Tb,
vanderhoef 6l Key 1968, srivaetava 1968, Ktran & Belt 1969, Pllet
1970) a¡rd 1t fs posslble that boroa nay fnfluence these. Inter-
actlons betneen kfnetlo a¡rd ar¡¡Ína (Cioldacre 1959, Key et aL.
1966, Blrntnghan & l{cl¿chlan L972), md partlcularly the kfoetln
tntrlbitlon of auxin-lnduced [32pf] fncorporatl'o'tr 1ûto RNA
(Vanderhoef 6l Key 1968), could be lnfluenced by boron defÍclency
to produce the changee observed l¡ auclelc acLd netabollsrn.
4.5 Concluslons
Boron defl.ciency affects several dLstl¡rct aspects of RNA
rnetabollsm. The lncreased fncorporatLoo of radl.oactlve Precursors'
708.
wtrlch 1s obeerr¡ed fn plant Eoots after only 6 hr grolrth 1n
defl.cient nutrlent, 1s the earlleet effect of the defl'cieocy yet
recorded. Thls occurs wl.thout any slgnffLcant changes ln the
total precuraor pool. Much later 1B the development of the
deffcfency, lncreased rates of RNA degradatfoa result fn lower
levels of RNA and oay aleo contrfbute to the changee 1n the total
nucleotfde pool which are obeen¡ed at this stage. These effects
oa RNA netabollsn, whlch are not Índuced by deffclencfes of lron'
u¡lng¿¡rrese or copper, aPpear to be speclffc to boron.
Many eyrytoos and effects of boron deffciency glve the
appear¡mce of belng assocl.ated ¡rtth changes ln grotth rates and
the state of natur:Lty of defl.clent tissues. Only changes which
precede effects on grorth and develcrpnent can be attrÍbuted to a
more direct effect of the deffclency. the lncreased lncorpotatloo
of precursors lnto RNA, as ehotfir 1n the Preseot work, |s one of
the fe¡¡ changee ¡¡hich have been denoustrated during thts eerly
stage of borou-deffclent growth. So far, ao evldence 1s avaflable
to suggest that the prlnary role(s) of boron Ínvolves soûe aspect
of nucleic acld uetabollsm. However, correlatfoas betneen the
effects on nuclel.c acld metabollsm of boron deficfency afid other
fectors (e.g. hormone ectfoa or other tJæes of dlsturbences to the
no¡:¡¡al blochenical evente taktng place during grütth) nay provlde
cfu¡es for the prfuary sfte of boroa actfon. For thls reesoot
further work on BNA polynerase for exanple rnay be useful, but fn
thl.s case sooe form of purlffcatfoo ¡¡ou1d be oecessery Prl'or to
eDzyEe aetfrrlty measurerents to obtain neanfngful results.
709.
h¡rther lnvestlgatfoo of a poseibls favolveæat of plant
hor:uones Ln the expreesfon of boton deficlency synptons fs aleo
requfred, but rntll oore lnfornatfon Ls avafleble about tf¡e
bLocheofstry of ho¡none actfm, lnter?retation of the results
would be difflcult.
Ìlost work on borou deffcLency has e4loyed r¡trole plant or
conplex tÍesue Bystems. I{1th such naterial, the control of
experfnental condltlons and the fntetpretetlon of results fs
dlfftcult. Further progress w111 probably require the derreloPneût
of eone siqllfied system. Ttre elongatlon of pollen tubes
(Wtrlttfngton 1959, Ban'ef Ê Ra¡rdhasa 1967) and the groltth of
pollen-derfved tlssuc cultures (1,Lh et aL. f966) eould be useful
nodel systeor¡. Ttrus, the reported effecte of plant hormones oo
ttre grape pollen syeten (Banzal & Rar¡dhæra f967) provide
prellnlnaty íafors¡atfon for the study of posalble honnone-boron
lnteractfons fn thle Daterfal. Ttre denmstratlorn of boron
requlrenents for sone algae (Eyster L952, Mcllrath & Skok 1958'
Lesln 1966, Getloff 1968, Boyd 1970), partlcularly r¡nf.cellular
epeciee, a¡ð, Azotobaeter eltnoøcø (Gerreteen & Ðe Eoo'p f954)
suggests thaÈ these could also be t¡sed for bloctreoical etudlee.
710.
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