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J.':ETABOLISM AND TRANSLOCATION OF LINM.�ARIN IN CASSAVA (MANIHOT ESCULENTA CRANTZ)
A Thesis Presented in Partial Fulfilment of the Requirements
for the Degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand.
Badu Michael Kofi Bediako November 1977
ABSTRACT
The met ab o l i sm o f linamarin in c assava ( Manihot es culent a C rant z ) has been investi gated. I n fo rmati o n
on the b i o synthet i c pathw ay , s ynthet ic sites , t rans l o c at i on and turnover o f t he c yanoglucoside has been ob tained b y precurs or adm in i s t rati ons to various part s o f cass ava plants grown under parti al l y cont ro lled conditions in the gl asshouse .
i i
Three vo lat i le 14c - l abel led precursors o f l inamarin i sobutyron it ri le , is obut yral doxime and 2-hydroxyi s obut y
ronitri le were prep ared , purified and administered t o c as s ava leaves by a new techn ique in which t he leaves were allowed to t ake up precurso r vapour in an en c losed glass chamber . The inc orporat ion o f t hese precurs ors ,
and o f L-valine a dm inis tered by so lut i on upt ake , was c ons istent with a pattern o f linamarin biosynthes is i n c ass ava invo lving the react i on sequen ce through val ine , i s obut yraldoxime , i s obut yronitrile and 2-hydroxyis obut y
ronitri le· est ab�ished for other plant s.
The s o luti on administ rati on of L- [u-14c J valine t o vari ous o rgans o f the pl ant indic ated that the leaves and the shoot apex s ynthes ised linamarin more e fficientl y than the w o ody stem and the roots and tubers . More
det ai led i nves t i gati ons o f leaf biosynthes i s showed much higher incorporat ion of 14c -val ine ·int o linam ari n b y the pet i o les and m i dribs ( 4S-62% 14c inco rpo ration b y pet i o les and 20% by midrib s ) than the leaf b l ades ( 2%) . There was no d irect rel at i onship between endo genous
l inamarin content ( which was higher in the bl ades than the pet i o les ) and the apparent abi lity to s ynthes ise
i i i
l inamarin from exo genous valine . Howeve r , the low ab i lity of t he b l ade t i s sue to inc orporat e valine into l in am arin could be due t o more act ive compet ing pathw a ys removing the exo ge nousl y adm in is t e red val ine . I n
furthe r inve st igat i ons w it h tub e r pe e l s and the edi b l e c o re s , sim i l ar compet ing pathw ays have been imp l i cat e d fo r an app arently low b io s yntheti c e ffi c i e ncy o f
linamarin .
The t rans lo c at i on o f l inrunarin w as dem ons t ra t e d b y sp e c i fic al l y l abe l l ing 1 4c - l inamarin in att ached l e ave s w i t h 2-hydroxy [ 1 -1 4c J is obut yron it ri le vapour and
fo l lowing t he change in lab e l led l inam arin content i n t h e l e af and the dist ribut i on o f l inamarin to othe r p arts o f the p l ant . I n both non-tuberous and t ub e rous p lant s there w as a rapid l o s s o f 1 4c - l inamar in due t o t r ans l o c at ion from the ful ly exp anded le ave s u p t o 69 hours aft e r s ynthe s i s . How eve r a res idual compon ent o f the 1 4c- linam arin (25-37% of t h at initi al l y s ynthe s i se d) rem ained in t he l e ave s . A c o mpartment at i on o f
s ynthe s ised linamari n i n cass ava l e a f tis sue s into a re adi ly mob i le and p art i al l y immob i le fracti on woul d a c c ount for these ob s e rvat ions . In s ene s ci ng l e ave s a cont inuous l o s s o f b o th 1 4c - l ab e l le d and e ndo genous l i n am arin o ccurred l e aving almos t no re s idua l c omponent although this was at tributed to bo th t rans l o c at i on and t urno ve r .
Trans l o c at ed l in amarin w a s di s t ribut ed t o a ll
p a rt s of the p l ant but the gene ral pat tern o f t rans l o c at e f l ow differed b e t w e en non-tube rous and tuberous p lants . An api cal dire c t i on o f l inam ari n d is t ribut i o n exi s t e d in
the n on -tuberous pl an ts w hil e tuber-directed l in amarin transl ocation prevail ed in the tuberous pl an ts. Leaf
senescence apparen tl y en hances l in amarin tran sl ocation to the tubers.
There w as l ittl e turnover of freshl y syn thesised 14c-l inamarin in detached l eaves and tuber tissu es over
a period of 1 to 3 days. How ever the l ow recoveries
of 14c-l in amarin in the w hol e pl an t tran sl ocation
experimen ts su ggest that active turnover may be oc currin g durin g translocation or in certain sin k tissues.
\
(.
iv
ACKNOWLEDGERffiNTS
I wish t o thank m y Supe rvi s o r , Dr B.A. Tap p e r (A pp l ied B i o che m i s t ry D i vis ion , DSI R) fo r h i s he l p fu l advice , sugg e s t i ons , en c oura gem ent s and assist anc e throughout t he course o f this s tudy . I would l ike t o expre ss my grat itude t o D r G.G . Prit chard , m y un ive rs ity supe rvis o r , fo r he l p fu l comm ent s and cri t i c isms dur i n g the preparat ion o f t h i s the s i s .
I am a l s o gra t e ful to :
Miss Y a ri l yn M acdonald fo r t e chni c al a s s i s t an c e i n the gas chromat o graph ic as s ay o f c y anogen ic gluc os ides .
Dr J . C . H awke for t emporary supe rvi s i o n in the abs ence of Dr Prit chard .
Dr R . W . B a i l e y fo r the o pportun it y to us e the
faci l it i e s at the App l i e d Bi o che m i stry Divi s ion of DSI R .
In addit ion , I wish to acknowl edge wit h th anks the financ i al support fo r thi s wo rk by the Unive rs i t y Gran ts Comm it t e e , New Ze aland , grant ed und e r the
Commonwe alth Scho larshi p S ch em e .
0
V
CONTENTS
ABSTRACT
P age numb e r i i
ACKNOWLEDGEMENTS V
LIST OF CONTENTS LIST OF FIGURES LIST OF TABLES
SECTI ON 1 - INTRODUCTION
vi ix x i
1 . 1 Hydro lysis o f C yano gen ic glyc o s ide s 1 1 . 2 Toxi c i t y as s o c i at e d with cyanogenic
glycos ides 4
1 . 3 B i o synthe s is o f cyano gen i c gl yc os ides 5 1 . 4 C yano glycos ide s o f c a s s ava and th e ir
import ance 9
1 . 4 . 1 C as s ava t ox i c i t y 1 0
1 . 5 Aims o f the pre sent study 1 2
SECT I ON 2 - N:ATERIALS AND METHODS 2 . 1 Source of p l ant mat e ri a l s 2 . 2 P l ant culture
14
2 . 2 . 1 Propagat ion 1 4
2.2 . 1 . 1 Propagat ion f rom s e eds 2 . 2 . 1 . 2 Prop agat ion from shoot and
s t e m cutt ings 1 4
2 . 2 . 2 Growth o f cass ava i n s o lut ion
culture 1 6
2 . 2 . 3 Growth o f c as s ava i n grave l culture 18 2 . 2 . 4 Growth and maint enance of c as s ava
p l ant s in the glasshous e 1 8
2 . 3 Chemi c a ls and so lvent s 21
2 . 4 Sourc e and prep arat i on o f 1 4c -l ab e l le d
compounds 22
2 . 4 . 1 Some l abe l led compounds 22 2 . 4 . 2 [ 1 -1 4c J i s obut yro nit rile 22 2 . 4 . 3 2-hydroxy [ 1 -1 4c J i s obut yronit r i l e 25 2 . 4 . 4 [u-1 4c ] i s obut yraldoxime 26
2 . 4 . 5 [ 1 -1 4c J Linamarin 26
2 . 5 Pre paration o f l in amaras e 27 2 . 6 Ext rac t i on o f p l ant m at e rial- 28
vi
Page numb e r
2 . 7 Ana l yt ic al me thods 28
2 . 7 . 1 Linamarin e s t i mat i on 28 2 . 7 . 1 . 1 Enzymi c hydro lys i s method 29 2 . 7 . 1 . 2 Gas chrom ato gr aphic m ethod 29
2 . 7 . 2 Pap e r chromato graphy 30
2 . 7 . 3 Thin l aye r e l e ct ro phore s is 31 2 . 7 . 4 D e t e ct ion of cyano gen ic gluco s ide s 31 2.7 . 5 M e a surem ent o f radi o ac t ivi t y 3 2
vii
SECTION 3 - ADMINISTRATION OF LINAMARIN PRECURSORS T O CASSAVA
LEAVES 34
3 . 1 Adm in i s t rat i on o f 1 4c pre curs ors 3 5 3 . 1 . 1 Val ine adminis t rat i o n t o le ave s 3 5 3 . 1 . 2 Adm inis t rat ion o f 1 4c vo lat i le
pre curso rs t o le ave s 36
3 .2 R e s u l t s 39
3 .2 . 1 As s im i l a t i o n of L- [u-1 4c J val ine 39 3 . 2 .2 As s imilati on o f 2-hydroxy [ 1 -1 4c ]
is obut yroni t ri l e 41
3 . 2 . 3 As s imi l at i on of [ 1 -1 4c J
i s obut yronitri l e 41
3 . 2 . 4 Ass imi lat i on o f [ u-1 4c J
i s obut yra ldoxime 45
SECT I ON 4 - SITES OF LINM.IfARIN SYNTHESIS IN CASSAVA
4 . 1 Linam arin cont ent o f c a s s ava t is sues 48 4 . 2 B i o synthe s is o f l inarnarin from val ine
b y det ached o rgans o f c a s s ava 53 4 . 2 . 1 Methods for valine administrati on
t o the di f fe re nt t is sue s 53
4 . 2.2 Re sults 55
4 . 3 Linam arin s ynthe s is b y d e t a ched le ave s 61
4 . 4
4 . 3 . 1 I nt roduct i on 6 1
4 . 3 .2 Experime nt a l pro c e dure 62 4 . 3 .2 . 1 Pre l im inary e xpe riment to
as s e s s e f fect o f c arri e r
additi on 62
4 . 3 . 2 .2 Linam arin synthes is in d e t ached le ave s ha rve st e d at di ffe rent t ime s a ft e r
pre curso r addit ion 6 5 Linam arin s ynth e s i s in c a s s ava tubers 72 4 . 4 . 1 Upt ake o f
co res 4 . 4 . 2 Linamarin 4 .4 . 3 L inamarin
l abe l le d val ine b y
s ynthe s i s b y - tuber s ynthe s i s b y tub e r
tub e r cores pe e ls
7 3 7 5 79
Pag e numbe r SECTION 5 - TRANSLOCATION OF LINAMARIN IN CASSAVA
5 . 1 I nt ro duc t i on 8 3
5 . 2 Pre l im inary e xpe rime nts on t rans locat i on 84 5 . 2 . 1 Trans loc ati on fo l lowing adm inis t rat i on
o f pre curs o r t o the matu re l e a f 84 5 . 2 . 2 T r ans loc at i on fo l lowi ng adm inis t ra t i o n
o f pre cursor t o the s hoot apex 88 5 . 3 Linam arin t rans lo c at i on in imm ature
non-tubero us c as s ava p lants 90
5 . 3 . 1 Linam arin turnove r i n the source
le a f 91
5 . 3 . 2 Trans loc at i on o f l inam ar in from
l e ave s 93
-
5 . 4 Linam arin t rans l o c at ion in mature
tube rous c ass ava p l ant s 1 00
5 . 4 . 1 Pre l imin ary expe riment to me asure
incorporat i on aft e r 4 hours 1 02 5 . 4 . 2 Tran s l oc at i on fro m a mature gre e n
l e a f ove r a 7 d ay peri o d 1 04 5 . 4 . 3 Trans l o c at i on fro m a s en e s c ing le a f
o ve r a 7 day peri od 1 06
5 . 4 . 4 Chang e s in linamarin cont ent in
le ave s during s e ne s c ence 1 1 1
SECTION 6 - DISCUSSION 1 1 4
6 . 1 General me thod of appro ach us e d in the
study 1 1 5
6 . 2 P athw ay o f linamarin b i o s ynthe s i s in
c as s ava 1 1 7
6 . 3 S it e s o f l inam a rin s ynthe s is in c ass ava 1 21 6 . 3 . 1 B i o synthes is in the pet io late
l e a f system · 1 24
6 . 3 . 2 B i o s ynthe s is b y tub e r t i s sues 1 27 6 . 4 Trans l o c at i on o f l inam arin in cass ava 1 31
6 . 4 . 1 Kine t ics o f l inamari n trans l o c at i o n
from le ave s 1 32
6 . 4 . 2 D i s t ribut ion o f t rans lo c ated
l inam ari n in c a s s ava 1 36 6 . 4 . 2 . 1 D i s t ribut ion o f trans loc at ed
linarnarin in non-tuberous
p l ant s 1 37
6 . 4 . 2 . 2 D i s t ribut ion o f t rans loc at ed linamarin in tub e rous
c as s ava p l ant s 1 38 6 . 5 Turnove r o f l inam arin in c as s ava 1 41
1 46 REFERENCES
vi i i
F igure N o .
1 . 1 1 . 2 1 . 4 . '1
1 .4 . 2 2 . 2 . 2 2 . 4 . 2 3 . 1 3
4 4 . 1 . 2 4 . 2 . 1
4 . 3 . 2 . 2 4 . 4 . 3
5 . 2 . 1
LIST OF FI GURES
Tit l e Nuin"ber �
H yd ro l ys i s o f l inamarin in p l ants 2 Pat hways for the b i o s ynthe s i s o f
c y ano gen i c glyc o s i d e s 8
A m at ure cas sava p l ant growing in pe at
culture 9b
Cas s ava tub e rs 9b
I mmature cas s ava p lant s gro wing in
s o lut i on culture 1 9
Preparat ion o f [ 1 - 1 4c J i s obutyronit ri le 24 Adm i n i s t rat i on o f 1 4c vo lat i le
pre cursors to attache d cas sava lea f 37 I n c o rporat i on o f radi oact i vi t y from
2-h ydroAy- [ 1 -14 c] i s obut yronit ri le , [ 1 -1 4c ] is obutyron i t ri l e and [ U-1 4C ]
i s obutyra ldoxime in cas sava l eave s 4 2 Di agram o f i mmature tub e ro us c ass ava
p l ant 49
Linamarin e s t imat ion b y gas
chromato gra phic t e c hn i que 51 I n c o rpo rat i o n o f radi oact ivity from
L- [u-1 4c] val ine int o 1 4c-linamarin
by c assava t is sue s 57
Bio s ynthe s i s of linamarin b y
p e t i o l at e l eaf 68
Rad i o act ivi t y re cove red from tuber p e e ls 4 hours a ft e r uptake of 1 4c -
val ine 81
Rad i oact ivit y r e cove re d from shoot apex , lowe r s t em and s ource l eaf o f
immat ure p lant s 7 d ay s a ft e r uptake o f 2-hydroxy[ 1 -1 4c ] - i s obut yronitri l e
b y the source leaf 86
eh anges 1n . 14 c- 1naru ar1 n an 1. . d
endo genous l inamari n in de tached
l e aves o f vari e t y man i oke 92 K 1ne 1cs o · t · f 1 4c - 1namar1n rans-1· · t ·
l o c at i on out of leave s of immatur e c as sava p lants . 14c - l in amarin
l eve ls in l eaf di s c s 9 5
. Kine t ic s o f 14c - linamarin trans
l o cat ion out of leave s of imm ature cas sava p l ant s . Time -cours e o f spe c i fic act ivity o f l inamari n in
le a f di s c s 96
ix
Figure No .
5.4 . 2 . 1
6.2 6
Tit le
D iagram i l lustrating the d i st ribution o f 1 4C-linamarin from low e r to upp e r l e ave s wi th respe ct t o phyl lot act i c c onfigurati on
L ina� arin t rans loc at i o n in mature c as sava
L i n amnrin t ran s l oc at i o n in mature c as s ava . Time -cours e o f ch anges in l i n am a rin in s en e s c ing source
Pa
t
eNum e r
1 01 1 05
l e a f d i s c s on a tub e rous p l ant 1 1 0 Ef fect o f s enesc e n c e on linam arin
c on t e nt o f at t ached and d e t ached
l e ave s o f c ass ava 1 12
P athway o f l inamarin b iosynthe s is
in c a s s ava 1 1 9
Transve rs e s e ct i on o f a young sto rage
c as s ava root 1 2 9
0
X
Tab le No . 2 . 2 . 1
2 . 2 . 2 3 . 2 . 1 . 1 . 3 . 2 . 1.2 3 . 2 . 2 . 1 3 . 2 . 2 . 2
3 . 2 . 4 .1
3 . 2 . 4 . 2 4 . 1 4 . 2 . 1
4 . 4 . 1 4 . 4 . 2
5 . 2 . 1 5 . 2 . 2
LIST OF TABLES
Tit le P a
�
eNum e r Compo s i t i o n o f s t o ck nut rient
s o lut ion fo r c as s ava p lants growing in pe at b e ds
Compos it i o n of nut r ient so lut ion for s o lut i on and grave l cultures
I n c orpo rat i o n of val ine into l inam arin I n c o rpo r at i on of val ine into l inam a ri n
i n other s pecies
Incorpo rat i on of 2-hydroxyis obutyro
n it r i l e int o l inam a ri n
Inc o rporat ion of 2-hydroxyisobutyro
n i t ri le int o l inam arin in o the r
1 5 1 7 40 40
43
s p e c i e s 43
I n c o rpor at i on o f i s obu t yroni t ri le int o
l i n amarin 44
Inco rporat i on o f is obut yro n it ri le i nt o
l inam arin i n othe r s peci e s 44 I n c o rpo r at i on o f i s obut yraldoxime into
l inamarin 46
I nco rpo rat i o n o f i s obut yr a l doxime into
l in am a ri n in othe r s p e c i e s 46 Linamarin distribut i on in c a s s ava 52 Incorporat i on of L- [ u-1 4c J val ine into
l in am arin by c as s av a t is su e s 56 Effe ct o f c arri er valine on 1 4c
d i s t ribut ion and inc orpor at ion int o
l inam a rin b y de t ached l e a f. 64 D i st ribut i on of 1 4c in L- [u -1 4c ]
v aline fed lea f 6 9
I nco rporat i o n o f 1 4c -v a l ine into
l in amarin b y le af t i s sue s 70 Upt ake of l abel l ed va l ine b y tuber c o r e s 76 L- [u -1 4c J va line adm in i s t r at i on to
e d ib l e tub e r cores . 77
L- [u -1 4c J va line adm i n is t r at i on to
p e e l s 8 0
D i s t ribut i o n o f re s idu a l linamarin
s ynthes i s e d in c as s ava le ave s 87 D i st ributi on of l i n am ar in s ynthes i s e d
i n sho ot apex �9
D i st ribut i on of 1 4c - l inam arin in non
t uberous p l ant s 7 days a ft e r synthe s is
in uppe r s ource le ave s · 98
x i
Tab le No .
5.4.2.2
6 . 2 6.4.2.2
T i t l e Page
· t · b t · f 14c 1 · · ·
D 1 s r1 u 10n o - 1namar1n 1n non- tube rous p lants 7 days aft e r
Number
synthe s i s in l owe r l e aves 99 Di stribut ion of l in amarin act ivit y
in tube rous plant 4 hours aft er
s ynthesis 1 03
Dist ribut i o n o f l inamarin act ivit y in tube rous p l ant 7 days aft e r
s ynth e s i s i n leaf 1 07
Linam arin translocat ion in tuberous p l ants - Distribut ion during lea f sene s cence
Summary o f L-Cu1
�
4cJ valine incorporation into C-linamarin b y c as s ava t i ssue s
Dist ribut ion of t r ans located l i namarin in tube rous plant s
1 09
1 22
140
0
x i i
