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Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)
¢3~em/J~ry~ u / P h ~ o / ~ r . 2 0 {1977) 141-1-55 ©.Elsevie~/North-HollandScientificPublishersLtd. |
CHEMICALCO~N ~D ULTRASFR~ OF THE E P I C ~ ~ W A X ~ FOUR MWrANTS O F PISUM SA T/VUM (L) P J , H O ~ W A Y ~ , Grace M. H ~ T ,
E.A. BAKER and M J X . MACEY *
Long Ashto~ :ReseUch Starion~ University o f BristoL Bristol BSI8 9/1F. UK and (M.J.K.M. )
School.of~ny,.Univer~ty of N e w South Wales,Kensington,N.$.W.: AuHralia 2033
Received h nuaty lOth, 1977,
accepted March 2nd, t977
The action of mutations affecting the epicuticular wax of t~sum sativum has been investigated at the chemicaland ultgastructural level. Upper and loweg surfaces of the leaves were found to differ markedly in both uluastructure and chemistry. Mutations affectoci prim~ily either the lower (wa. wb and wsp) or the upper surface (w/o). but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and .~.,abut the chemical effects of wb and w/o were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.
i. Introduction Mutations affecting the waxy surface of pea leaves have been well described and documented [1 |. The number of such mutations known is increasing but few have so far been ~ m i ~ y ~ r a c t e t i s e d . Of the latter, the chemistry of whole waxes from p e a ~ v e $ of mutants containing double recessives in vm. wb, wsp were analysed and compaged with ~ normal type by Macey and Barber [2]. Results of analysis for ~ ~ w a x w e r e e s s e n ~ y similar m those of Kotattukudy [3]. Analyses of aU f ~ o ~ ~ : t o tlw c o n c e r t that wa andwb showed signs of blocks to fatty agidelor4gation,~.~ ~ C a ~ s ,--~C:ts ~wb) and C3o -~ C32 (we) ~ranges whilst wsp showed s o m e : ~ ~ Of C ~ free a ~ and aldehyde, together with a reduced content oncerning certain mutants of I that the wax deposits on the ifferent ultrastructure [5,6 ] to reexamine the waxes visibly affected in different ous" on the upper surface ~t not on the lower. In the
141
I42
PJ. Holloway et al.; Epicuticular wax of Pimmmtivum
present work, a combination of cl~romato~aphic and mass spectrometric techniques was ttsed in order to re-examine the influence of mutations on the composition and morphology of pea wax.
H. Experimental All plants were grown from seed under identical conditions. The mutant lines of l~sum sativum (L) used were bred from lines supplied by Dr. l.C. Muffet (Botany Department, University of Tasmania). The mutations represented were wa, wlo, wb and wsp. The growth conditions and methods used for scanning electron microscopy (SEM) wax isolation and analysis were the same as those described previously for Brassica napus waxes [7]. All structural assignments were verified by mass spectrometry.
Ill. Results A. UItrastructure o f wax deposits
Scanning electron micrographs of the normal form and the 4 mutants are shown in figs. 1-6; mutant type surfaces are on~y included where they differ from ~he normal one. In accordance with visual appearance [2], a considerable reduction of crystallite form and alteration of ~tru,'~.ure occurred on the lower surfaeo ~i" wa, wb, wsp. but in wlo on;y the upper surface was affected. The completely different appearance of normal upper arid lower surfaces is illustrated. The upper surface (Fig. 1) cor~sists of a series of angular interlocking plates, raised from the susfaee but forming a dense cover over the cuticle. The lower surface (fig, 2)is composed m~nly of ribbom with ¢re_~uht¢ edge~ w~ch project fromthe surface forming a mect:anically looser but perhaps dense~ arrangement than the upper surface. The density and form cf the wax ~tructure~ is mue~ reduced and modified on the lower surfaces 9f ~a (fig. 3), wb (fig. 4) and wsp (fig, 5)° However, on wb ribbons are ~till present but in wa only short ribbons occur and they are:~parsety distributed. In wsp there ~.sa complete loss of cryst.atline ultrastracture and the wax appears as an amorphous crust on the surface of the cuticle. There is zho a &astic r~duction of crystalline deposits on the u p ~ r surface of wlo (fig. 6) the wax being mainly amorphous but w'th a few Flatelcts bein[~ ,islble. B. General wax cornpos~tkm
Table I shows ~he wax yields and composition of flte different [ines. There was a markc~ reduction in the quanUtie~ of wax pe~ unit area on tho~ ~,rfaees of the
P.J. ttolloway et aL, Epicuticutar wax o f Pisum sativum
Fig. |.
Fig. 2.
143
f~
~w
~b 4~
~J~ulL
Fig. 5,
Fig. 6. Figs, 1-6. Scanning electron micrographs of the epicuticular wax deposits of normal aad mutant leaves of Pisum ~ttvum. Fig- 1. Normal (Meteor) upper surface, × 5000. Fig. 2. Normal (Meteor) lower s~face, × 5000. Fig. 3. wa lower surface, × 5000. Fig. 4. wb lower surface, X 5000, Fig. 5. wsp lower surface, X 5000. Fig. 6. w/o upper sgriace, × 5000.
•
•
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I
•
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.
~
I
~
~il ~
P~L tlolloway et aL, Epieutieutae wax o f Pitum satirum
147
~ble 2 Effect of mu!.ation on the wax deposits and components of apper and lower surfaces of Pisum ~ t i r u m leaves expressed as a percentage o f the llolmarl form.
Classes
w~
~Io
wb
wsp
Uppe~
Lower *
Upper * Low~
Upper-
Lower * Uppe.,
Lower
AJkane~ Esters Aldehydes Sec, a-loohols t~m. ak~hol$ Fatty acids
13.5 147.7 17.4 trace 135.6 160.0
12.8 1343.8 160.0 16.9 126.1 420,0
77.0 55.9 19.6 58,3 29.6 60.0
95.8 812,5 650.0 I66.2 58.0 140.0
10.3 85.3 67.4 trace 117.7 230.0
41.1 568.8 280.0 60.0 211.6 226,7
t27.3 1 lO.O
2.0 I93.8 420.0 4.6 43.5 346.7
Total wax deposits
114.9
42,4
38,6
111 ~2
98,0
52.0
94.1
14.4
32 24.7 21.7
* Surface showingulUastructmat differences f~om normal form under the SEM.
mutants which showed ultrastructural differences from the normal form. Alkanes predominate on the lower surface and primary alcohols on the upper; this is generally true of mutants as wetlas the normal form. The genette reduces alkanes on the lower surface to 13% of the normal form (table 2). There are corresponding increases in esters, primary alcohols and fatty acid but the overaU wax production is reduced. This mutant retains the normal $1auc~m appearance on the upper surface, in spite of some changes in wax chemt ~ y . At the level of analysis of tables I and 2, w b has a similar effect to wa,obut alkane reduction on the lower surface is less pronounced. The main effect of w/o is to reduce the ~r~ount: of wax on the upper leaf surface (table 2). Although the prin~ry aicohoh ate most affected, significant reductioM oc.~rred in all other components; At the same time there were large increases of a l d e h ~ and esters on the lower surface, and as found for wa the effo~ctof the 8erie is apparently not confined to onemfface. The other three genes examined, h o w ~ , ~ d rtOt xedu~ wax yields from the upper surface, but subsequent analysis tho~:~t~ effect ~of these pries could be detected m some of the components ~ . l i ~ n e ~ l e d u c e s alkanes on. the lower surface to a greater extent than°wa or ~ { ~ 2 ) ; ~ter~ ~ aldehyd~ are in~ased on the lower surface but d e - . . =~olX :~ ~ su~m:e. Primary alco~ls m.v~..tually unaffected. Plants w, th - . . . . . . ~ .... M ~ a n d w b phenotype. nusually high (29% of the ~ax fraction will be disattempt ~ be made to
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:148 "
r
_
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.
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-
summarise the .~ :
s y n t h e , s i s .
_
C, Alkane~
grea.sec[
.
.
.
.
.
.
.
.
.
.
-
.r:
shorterchainlcngth,:
. . . . . . --
.
:---
"
.....
'~~-
-
"
'
•
D. Secondaryalcoh"~
~
.
-~.... ~-: .:.-.. - : -
:-
.,.-:- ~::.~.- -,:-,:~.
-
"
~
....
'
.
. -
1.5
97.4
1.4
90.3
~
,
1.4
-
67.7
.
I. 3
-
16~0 1.2 80,2
0.4 0.9
.
.
.
.
L
,
wsp
5.0
10.0 I0.2 7.6 I1.3 2.5 30.7
22.7
Upper
w=p
2.7
3.6 0,8 ! .8 0.3 13.8 0.6 76,4
Lower
''
9 6 70 1140
118 10470
-
4 2 7 1
Upper
Upper
LOwer
wa
Normal
6 5 1175 5
Lower * 1t 8 2 9 723
Uppex *
wlo
: S u r f a c e showing ultrastjuctural differences from normal form under the SEid.
C2s C27 C29 C31
L
Homologues
103 9929
-
6
Lower
7 5 2 9
Upper
wb Lower
8 4 29 160
Upper 9 4 4 13
Lower *
*
6 2 707 3544
4~
t~
f
JL
0.6 0.8
trace 0.5
0.6
96.4
1.0
74,5
4.2 4,5 1.4 15.2
5.6
.
Lower
.
Table 3B Amounts of some a!kane homologues produced by normal and mutant pea leaves (#g/gl. cm leaf surface).
•
trace
trace
85.2 0.9 4.0
41.6 2.0
5.4
0.9
2.0
0.6
trace trace trace 1.0
12.2
0.5 1.8 trace 9.5
lower
Upper
Upper
wb
!
0.9
1A
OA 5.6
4.5 0.9 3~6
23.8 3,4 9.5
Lower **
.w/o
-~*~Contaim about 12% of lowe~ homologues to Cts. ** Contains traces of ihot,t ~ chain homotogues to C IS-
C~3
c~
C30 C3t
cim
CT/
C=6
0.? .~$ O~
Upper *
w
the upper and lower surfaces of normal and mutant lines of I~sum sativum leaves.
~Ihb~ 4
.++
+
. . . .
.
: + _
.
.
.
.
.
°J 2~ol
~.+P++gt+em
+
2+
++++
;51 ++
3+] 2°0 OJ
J+O +
~
.
.
M+~+
Nomml
C o m ~ + i g i o a (%) o f + c o n ~ f y .
.
+
| +2+
.
+ +
+.
•
+
+
+
+
+
+
m++
mat~
+
3+4 ++
2+!
47 +! 40++
6+6"
L@%~ ++
f r a c t u r e s f r o m the u p ~ r a n d ~ w ~ .
m=m
+
.
+
0.7 +
26+2 1+4
4 3 +0
|+l +
++2 0+~
++
0~$ 0+2
_ _ + .
. .
.
+
I+9+ +++ ++
~++
~++
+
-
:+
2+g
_
.
.
+
.
.
.-
. . . .
+++ +
of
.
4+6
.
.
m+ 2+s
.
wb
~ ++~ m m ~ m t ~
. . .
+
of
0+++ 0+8 0o3
m++
mfams
.
.
++,
+6+ + 2+~
S,2
.
.
+
+mm+,
+
+~,++,
+
+
+
~p
+ ~ m
61 +7
~t i~ ~ ~ave~ ~ ler~gth o~ ~ e upon: saAaceo (726 primary alcohot ~ted fo~ ~ - - 7 {u 0 a~d 40--5 { e~) of the to{a.i p~mar3 a~cohots m~ mu t ~.s y reP~cts the decre~ed elo~~gation capacity f~ ~ ~a w~de~ uiated m~ the basis of alka~e distributions.
|1
t~
of
is si ae ~o that of the primary A fo~m ~ m w~p [oweL which co~:}~ains of t r i o I~ at| o~ f~rm~ .C~ C ~ p~ed r~ateusualy in approxima t, ,H r, in wb u p ~ r and Iower a~d wsp upper the amoum of tea m Ca~ g ~ . U~|ike the p~ ~y d s a~d aikaaes, the aver° a~ of ~ gu~ a~ yde~ are m~{ sgnificanty different er~ ~ e m w~p IoWe~o
fatty acid
o~ ~ de becau~ of i ~ N:~tm~tial impor{ar~ce m reflect,mga of fatty a~d~ a p ~ a r ~ a~ byop~:~uct~ ef the d e n ~ R m system. ~a ty of ~ e |c~e~ surface, a~ ~eflected by average c ~gth0g a ~ t~ (Caa--Cn) (table 5). ~oth tl sl of the ~m~ ald comail 20--3R, of C 31: which i~ c o l .~ m ~ ~e.|aged m ~ b e ~ u~t of ~N Nedommam a~kaee of the wax {Ca~}. ~ ~ t . a ~ { wb r o~ of Ca~ ~d m Ca~ a~d tN~ result ~a~ ~ePe i= ff~e aid ~, al ~o~ i~ ~he p~ y a~cohdso The c~ose co~i~ f~ee fatty a~d ),des N pa~ticula~y dea~ with r e ~ c t to C~ i~ ~ p ; ~ t h ~ aed wb ~o de~ec~fale C 3~ acid.
~s
~uf
f~ wb@d g
:a a
i
~ of gee m ~
s g a ominanRy even ~rie~ of n-homo° C~a~ C4~ ~nd C46. ~ i n g the ~ * n t i { u e ~ (~o~ °d ~. :c effects at° t of g4a{ l 3 ~ 2 ~ ) tRe ~or real a=d A~ ~a~ ~ e e . ~ Y [21, the es~eAfied |e t t ; f t~e flee fatty Cae¢~fe
=g f~ee
primay a o~ are ge~all:y ~-a e×cep{ i~ wgp lowe~ g ~
~ C ~ ~ e h ~ ~de
,.
::U~
::..:. : : , , . : :~,~
,-
:i:~i -
. "
:/:::~;::::i
..
-:
.:~:.::::.
;,
.......
m ,s
~
:
i"
:
-
_
,
.
.
.
-3.6
7,~0 4~! 2'3
Uppe¢ *
.
9,,8 trace 15.7 2:3
30~
.
24.2
.
.
-3,4.8
.
.
23.5
2.5
1 . 2
14,1
24.7
Lowez *
...,
~om normal fogm trader the ~ M .
-
,~8
14.s
6.0
~8,6 17;9
Upper
.. .
12.0 :4.1
-
3.4
3.4
18,7
40A
$$.0 34.2 . . . -4.0
Uppm"
t4.1 6.4 1"5 2'8
Lowe¢
_ . .
•
.
.
. . . . .
-
20,9
"27.9
--
34.9 lr6~3 "
Lowm" * -
..
,v'sp
10.4
19.1
45.I 4.3 6"8 -!4.3
t
Tab~$ . Com~sition:(%) o f fatty acid_gractions!from the upper and lower surfaces of no~.al and mutant Linesof P/sum ~n'tqamleaves.
?-
19,4
14,8 8.6 e,:o
.~
t
sj
P.J. Hotloway e t a , t~picuttcular wax o[ Pisum sativum
153
I:V, Discussion Tt~ results presented generally support the concept that the chemical constitution r.of a WaX is in|imately related to its crystallite structure. In peas this is seen bothlin the differences whiehoccur between the upper and lower surfaces, and in the diffexen~sobsetved in mutants. 1! would appear that the closely interlocking plates which o ~ u r on the upper surface of pea leaves are composed mainly of primary+alcohols, Whereas Ihe rii:~bon4ike structures of the lower surface (which resemble structures found in Brassica oleracea) are mainly alkanes. The results:also provide a chemical basis for the observed differences in the emerald mutants of peas. Thus, wa and wb are described as glaucous on the upper surface but emerald on the lower. This can be explained by assuming that both genes affect the synthesis of alkane which is a predominant component of the lower, but not of the upper surface. The action of wa, as previously described [2] and confirmed here, involves an dongation block between C 3o "+ Cz:~. Thus the C32 acid is absent from the lower surface of the mutant, but present in the normal form, and the latter is correlated with maximalamounts of C3j alkane which is only a minor component in wa. Furthermore, there is significantly more C30 aldehyde in wa than in the norreal form, This probably results from part of the Cso add or acyl compound (result. ing from the block)being diverted to aldehyde formation, in addition to C29 alkane. E'~en though wa only appears to affect the wax on the lower surface minor chemical effects ~ n he detected in the wax from the upper surface. The gene does not affect the ultrasttueture of the upper surface because the latter is composed predominantiy of primary alcohols rather than alkanes, so that the chain length of these components (mainly C~6 and C2s) is out of range of the genetic block, The effect ofwb is le,~sclear; in previous work [2] it had been tentatively coneluded that this mutalion caused a partial inhibition of the elongation step C26 "" C2s. The tne~nt results show equivalent results for aldehydes and free acids on the lower surface but the effe~ is not seen in the primary alcohols. At the same time, alkane synthesis" is reduced a p ~ t l y at the C3t level, but closer analysis of the resull~ shows,,that C29 s y n ~ is increased in wb on the lower surface but decreased on ~ u p p e t surface (table 3B). Thus, there ale complex interactions influe~the final pauttre of the end lnoducts of wax synthesis which obscure the ~teq)fetation. One ~ explanation f ~ the-mutant is a .generally weakened e l o n g a ~ ~ m . b e y o n d . Cie; competition for .limited substrates could then perhal~ aeca)~t,fo~ differences in the chemi(ml phenotype of upper and lower wax. ~~effeet of w/o is a redue.tion of wax.on the upper surface (table 1). Thus, w ~ Netm:to affect Synthesis of most c~mponents on the upper surface, and no ~S~¢ ~ ~ ~ r ~ ~ ~~ from ~mi~a~ analysis. This rather sugp s t s i ~ t t ~ m u l ~ t i o n re!aim ettlm to wax +release otto an earlier step in fatty ~fd-qna~whi~ tsnot ~eflected in thee n a t ~ e of the surface wax. - alkanes are reduced in quan, ~ , ~ F n e )t~'),like-,)m, lent one :~finite effect..Whilst till , ~ ; l m ) d ~ t oae )elmira C3:v In oontrast to wa there is no evidence of any
minent in C tt
tween
C~. aldehvd .
.
.
_
..
-
. -
s~,rface where a l ~ e ~ s ~ the occurrence of the Cs~ acid lower surface, but not on tl~up clear-cut effects, the results sho,~ this simple, way. For i n s ~ i t h surface, and at the same time a3 are- ItkoIv to be secondmyeffe~ ca~:.~ of ~ihe complex nahum,oft 'Vn¢ present work shows that alcohols is still a dausible hypot maximum on the ~ntral carbon stitution in C2s and C2,11a l ~ C27-13-ol. Such a result~couldb' possible maximal substitution ra carbon atoms from,one e M of t~
~=in
r.~
a
variation This in u ing leavesfor a solWlpt S]
in P,'sum sat clear, but di plied:. Since that the flu, !•
~J. HoUo~,y et aL, Eplcu~'ularwslxof Pisum sativum
155
!term [I]:IL Lampz~ht,. l~nd~kzom~ Trycke~i A.B. (1961). ~ : ~ the Plant Br~medinl~l~tituUon. Weibullsholm Landsktona) ~70) s ~70) 13 [6] LA.Bager and P.LH ;loway, Mi~on 2 (1971) 364 [7] G,M~Hunt:, E,A, Baker and PJ. Holloway, Rep. Agric. Hort. Res. Stn. Univ. Bzistol t 974 :(1975) 84 [81 P.J. HoUoway, G.A. Brown, E.A. Baker and MJ.K. Macey, Chem. Phys. Lipids, in press [9] P.E. K o l a t t ~ y , .t.S. Buckner and T.-Y. Lieu, Arch. Biochem. Biophys. I56 (I973) 633
CHEMICALCO~N ~D ULTRASFR~ OF THE E P I C ~ ~ W A X ~ FOUR MWrANTS O F PISUM SA T/VUM (L) P J , H O ~ W A Y ~ , Grace M. H ~ T ,
E.A. BAKER and M J X . MACEY *
Long Ashto~ :ReseUch Starion~ University o f BristoL Bristol BSI8 9/1F. UK and (M.J.K.M. )
School.of~ny,.Univer~ty of N e w South Wales,Kensington,N.$.W.: AuHralia 2033
Received h nuaty lOth, 1977,
accepted March 2nd, t977
The action of mutations affecting the epicuticular wax of t~sum sativum has been investigated at the chemicaland ultgastructural level. Upper and loweg surfaces of the leaves were found to differ markedly in both uluastructure and chemistry. Mutations affectoci prim~ily either the lower (wa. wb and wsp) or the upper surface (w/o). but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and .~.,abut the chemical effects of wb and w/o were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.
i. Introduction Mutations affecting the waxy surface of pea leaves have been well described and documented [1 |. The number of such mutations known is increasing but few have so far been ~ m i ~ y ~ r a c t e t i s e d . Of the latter, the chemistry of whole waxes from p e a ~ v e $ of mutants containing double recessives in vm. wb, wsp were analysed and compaged with ~ normal type by Macey and Barber [2]. Results of analysis for ~ ~ w a x w e r e e s s e n ~ y similar m those of Kotattukudy [3]. Analyses of aU f ~ o ~ ~ : t o tlw c o n c e r t that wa andwb showed signs of blocks to fatty agidelor4gation,~.~ ~ C a ~ s ,--~C:ts ~wb) and C3o -~ C32 (we) ~ranges whilst wsp showed s o m e : ~ ~ Of C ~ free a ~ and aldehyde, together with a reduced content oncerning certain mutants of I that the wax deposits on the ifferent ultrastructure [5,6 ] to reexamine the waxes visibly affected in different ous" on the upper surface ~t not on the lower. In the
141
I42
PJ. Holloway et al.; Epicuticular wax of Pimmmtivum
present work, a combination of cl~romato~aphic and mass spectrometric techniques was ttsed in order to re-examine the influence of mutations on the composition and morphology of pea wax.
H. Experimental All plants were grown from seed under identical conditions. The mutant lines of l~sum sativum (L) used were bred from lines supplied by Dr. l.C. Muffet (Botany Department, University of Tasmania). The mutations represented were wa, wlo, wb and wsp. The growth conditions and methods used for scanning electron microscopy (SEM) wax isolation and analysis were the same as those described previously for Brassica napus waxes [7]. All structural assignments were verified by mass spectrometry.
Ill. Results A. UItrastructure o f wax deposits
Scanning electron micrographs of the normal form and the 4 mutants are shown in figs. 1-6; mutant type surfaces are on~y included where they differ from ~he normal one. In accordance with visual appearance [2], a considerable reduction of crystallite form and alteration of ~tru,'~.ure occurred on the lower surfaeo ~i" wa, wb, wsp. but in wlo on;y the upper surface was affected. The completely different appearance of normal upper arid lower surfaces is illustrated. The upper surface (Fig. 1) cor~sists of a series of angular interlocking plates, raised from the susfaee but forming a dense cover over the cuticle. The lower surface (fig, 2)is composed m~nly of ribbom with ¢re_~uht¢ edge~ w~ch project fromthe surface forming a mect:anically looser but perhaps dense~ arrangement than the upper surface. The density and form cf the wax ~tructure~ is mue~ reduced and modified on the lower surfaces 9f ~a (fig. 3), wb (fig. 4) and wsp (fig, 5)° However, on wb ribbons are ~till present but in wa only short ribbons occur and they are:~parsety distributed. In wsp there ~.sa complete loss of cryst.atline ultrastracture and the wax appears as an amorphous crust on the surface of the cuticle. There is zho a &astic r~duction of crystalline deposits on the u p ~ r surface of wlo (fig. 6) the wax being mainly amorphous but w'th a few Flatelcts bein[~ ,islble. B. General wax cornpos~tkm
Table I shows ~he wax yields and composition of flte different [ines. There was a markc~ reduction in the quanUtie~ of wax pe~ unit area on tho~ ~,rfaees of the
P.J. ttolloway et aL, Epicuticutar wax o f Pisum sativum
Fig. |.
Fig. 2.
143
f~
~w
~b 4~
~J~ulL
Fig. 5,
Fig. 6. Figs, 1-6. Scanning electron micrographs of the epicuticular wax deposits of normal aad mutant leaves of Pisum ~ttvum. Fig- 1. Normal (Meteor) upper surface, × 5000. Fig. 2. Normal (Meteor) lower s~face, × 5000. Fig. 3. wa lower surface, × 5000. Fig. 4. wb lower surface, X 5000, Fig. 5. wsp lower surface, X 5000. Fig. 6. w/o upper sgriace, × 5000.
•
•
~L
I
•
r
.
~
I
~
~il ~
P~L tlolloway et aL, Epieutieutae wax o f Pitum satirum
147
~ble 2 Effect of mu!.ation on the wax deposits and components of apper and lower surfaces of Pisum ~ t i r u m leaves expressed as a percentage o f the llolmarl form.
Classes
w~
~Io
wb
wsp
Uppe~
Lower *
Upper * Low~
Upper-
Lower * Uppe.,
Lower
AJkane~ Esters Aldehydes Sec, a-loohols t~m. ak~hol$ Fatty acids
13.5 147.7 17.4 trace 135.6 160.0
12.8 1343.8 160.0 16.9 126.1 420,0
77.0 55.9 19.6 58,3 29.6 60.0
95.8 812,5 650.0 I66.2 58.0 140.0
10.3 85.3 67.4 trace 117.7 230.0
41.1 568.8 280.0 60.0 211.6 226,7
t27.3 1 lO.O
2.0 I93.8 420.0 4.6 43.5 346.7
Total wax deposits
114.9
42,4
38,6
111 ~2
98,0
52.0
94.1
14.4
32 24.7 21.7
* Surface showingulUastructmat differences f~om normal form under the SEM.
mutants which showed ultrastructural differences from the normal form. Alkanes predominate on the lower surface and primary alcohols on the upper; this is generally true of mutants as wetlas the normal form. The genette reduces alkanes on the lower surface to 13% of the normal form (table 2). There are corresponding increases in esters, primary alcohols and fatty acid but the overaU wax production is reduced. This mutant retains the normal $1auc~m appearance on the upper surface, in spite of some changes in wax chemt ~ y . At the level of analysis of tables I and 2, w b has a similar effect to wa,obut alkane reduction on the lower surface is less pronounced. The main effect of w/o is to reduce the ~r~ount: of wax on the upper leaf surface (table 2). Although the prin~ry aicohoh ate most affected, significant reductioM oc.~rred in all other components; At the same time there were large increases of a l d e h ~ and esters on the lower surface, and as found for wa the effo~ctof the 8erie is apparently not confined to onemfface. The other three genes examined, h o w ~ , ~ d rtOt xedu~ wax yields from the upper surface, but subsequent analysis tho~:~t~ effect ~of these pries could be detected m some of the components ~ . l i ~ n e ~ l e d u c e s alkanes on. the lower surface to a greater extent than°wa or ~ { ~ 2 ) ; ~ter~ ~ aldehyd~ are in~ased on the lower surface but d e - . . =~olX :~ ~ su~m:e. Primary alco~ls m.v~..tually unaffected. Plants w, th - . . . . . . ~ .... M ~ a n d w b phenotype. nusually high (29% of the ~ax fraction will be disattempt ~ be made to
•
•
.
.
.
-
.:.
.:
~
.
:
-.
~'.',
•
.
.
.
~
.
.
:148 "
r
_
'
"
.
"..
-
summarise the .~ :
s y n t h e , s i s .
_
C, Alkane~
grea.sec[
.
.
.
.
.
.
.
.
.
.
-
.r:
shorterchainlcngth,:
. . . . . . --
.
:---
"
.....
'~~-
-
"
'
•
D. Secondaryalcoh"~
~
.
-~.... ~-: .:.-.. - : -
:-
.,.-:- ~::.~.- -,:-,:~.
-
"
~
....
'
.
. -
1.5
97.4
1.4
90.3
~
,
1.4
-
67.7
.
I. 3
-
16~0 1.2 80,2
0.4 0.9
.
.
.
.
L
,
wsp
5.0
10.0 I0.2 7.6 I1.3 2.5 30.7
22.7
Upper
w=p
2.7
3.6 0,8 ! .8 0.3 13.8 0.6 76,4
Lower
''
9 6 70 1140
118 10470
-
4 2 7 1
Upper
Upper
LOwer
wa
Normal
6 5 1175 5
Lower * 1t 8 2 9 723
Uppex *
wlo
: S u r f a c e showing ultrastjuctural differences from normal form under the SEid.
C2s C27 C29 C31
L
Homologues
103 9929
-
6
Lower
7 5 2 9
Upper
wb Lower
8 4 29 160
Upper 9 4 4 13
Lower *
*
6 2 707 3544
4~
t~
f
JL
0.6 0.8
trace 0.5
0.6
96.4
1.0
74,5
4.2 4,5 1.4 15.2
5.6
.
Lower
.
Table 3B Amounts of some a!kane homologues produced by normal and mutant pea leaves (#g/gl. cm leaf surface).
•
trace
trace
85.2 0.9 4.0
41.6 2.0
5.4
0.9
2.0
0.6
trace trace trace 1.0
12.2
0.5 1.8 trace 9.5
lower
Upper
Upper
wb
!
0.9
1A
OA 5.6
4.5 0.9 3~6
23.8 3,4 9.5
Lower **
.w/o
-~*~Contaim about 12% of lowe~ homologues to Cts. ** Contains traces of ihot,t ~ chain homotogues to C IS-
C~3
c~
C30 C3t
cim
CT/
C=6
0.? .~$ O~
Upper *
w
the upper and lower surfaces of normal and mutant lines of I~sum sativum leaves.
~Ihb~ 4
.++
+
. . . .
.
: + _
.
.
.
.
.
°J 2~ol
~.+P++gt+em
+
2+
++++
;51 ++
3+] 2°0 OJ
J+O +
~
.
.
M+~+
Nomml
C o m ~ + i g i o a (%) o f + c o n ~ f y .
.
+
| +2+
.
+ +
+.
•
+
+
+
+
+
+
m++
mat~
+
3+4 ++
2+!
47 +! 40++
6+6"
L@%~ ++
f r a c t u r e s f r o m the u p ~ r a n d ~ w ~ .
m=m
+
.
+
0.7 +
26+2 1+4
4 3 +0
|+l +
++2 0+~
++
0~$ 0+2
_ _ + .
. .
.
+
I+9+ +++ ++
~++
~++
+
-
:+
2+g
_
.
.
+
.
.
.-
. . . .
+++ +
of
.
4+6
.
.
m+ 2+s
.
wb
~ ++~ m m ~ m t ~
. . .
+
of
0+++ 0+8 0o3
m++
mfams
.
.
++,
+6+ + 2+~
S,2
.
.
+
+mm+,
+
+~,++,
+
+
+
~p
+ ~ m
61 +7
~t i~ ~ ~ave~ ~ ler~gth o~ ~ e upon: saAaceo (726 primary alcohot ~ted fo~ ~ - - 7 {u 0 a~d 40--5 { e~) of the to{a.i p~mar3 a~cohots m~ mu t ~.s y reP~cts the decre~ed elo~~gation capacity f~ ~ ~a w~de~ uiated m~ the basis of alka~e distributions.
|1
t~
of
is si ae ~o that of the primary A fo~m ~ m w~p [oweL which co~:}~ains of t r i o I~ at| o~ f~rm~ .C~ C ~ p~ed r~ateusualy in approxima t, ,H r, in wb u p ~ r and Iower a~d wsp upper the amoum of tea m Ca~ g ~ . U~|ike the p~ ~y d s a~d aikaaes, the aver° a~ of ~ gu~ a~ yde~ are m~{ sgnificanty different er~ ~ e m w~p IoWe~o
fatty acid
o~ ~ de becau~ of i ~ N:~tm~tial impor{ar~ce m reflect,mga of fatty a~d~ a p ~ a r ~ a~ byop~:~uct~ ef the d e n ~ R m system. ~a ty of ~ e |c~e~ surface, a~ ~eflected by average c ~gth0g a ~ t~ (Caa--Cn) (table 5). ~oth tl sl of the ~m~ ald comail 20--3R, of C 31: which i~ c o l .~ m ~ ~e.|aged m ~ b e ~ u~t of ~N Nedommam a~kaee of the wax {Ca~}. ~ ~ t . a ~ { wb r o~ of Ca~ ~d m Ca~ a~d tN~ result ~a~ ~ePe i= ff~e aid ~, al ~o~ i~ ~he p~ y a~cohdso The c~ose co~i~ f~ee fatty a~d ),des N pa~ticula~y dea~ with r e ~ c t to C~ i~ ~ p ; ~ t h ~ aed wb ~o de~ec~fale C 3~ acid.
~s
~uf
f~ wb@d g
:a a
i
~ of gee m ~
s g a ominanRy even ~rie~ of n-homo° C~a~ C4~ ~nd C46. ~ i n g the ~ * n t i { u e ~ (~o~ °d ~. :c effects at° t of g4a{ l 3 ~ 2 ~ ) tRe ~or real a=d A~ ~a~ ~ e e . ~ Y [21, the es~eAfied |e t t ; f t~e flee fatty Cae¢~fe
=g f~ee
primay a o~ are ge~all:y ~-a e×cep{ i~ wgp lowe~ g ~
~ C ~ ~ e h ~ ~de
,.
::U~
::..:. : : , , . : :~,~
,-
:i:~i -
. "
:/:::~;::::i
..
-:
.:~:.::::.
;,
.......
m ,s
~
:
i"
:
-
_
,
.
.
.
-3.6
7,~0 4~! 2'3
Uppe¢ *
.
9,,8 trace 15.7 2:3
30~
.
24.2
.
.
-3,4.8
.
.
23.5
2.5
1 . 2
14,1
24.7
Lowez *
...,
~om normal fogm trader the ~ M .
-
,~8
14.s
6.0
~8,6 17;9
Upper
.. .
12.0 :4.1
-
3.4
3.4
18,7
40A
$$.0 34.2 . . . -4.0
Uppm"
t4.1 6.4 1"5 2'8
Lowe¢
_ . .
•
.
.
. . . . .
-
20,9
"27.9
--
34.9 lr6~3 "
Lowm" * -
..
,v'sp
10.4
19.1
45.I 4.3 6"8 -!4.3
t
Tab~$ . Com~sition:(%) o f fatty acid_gractions!from the upper and lower surfaces of no~.al and mutant Linesof P/sum ~n'tqamleaves.
?-
19,4
14,8 8.6 e,:o
.~
t
sj
P.J. Hotloway e t a , t~picuttcular wax o[ Pisum sativum
153
I:V, Discussion Tt~ results presented generally support the concept that the chemical constitution r.of a WaX is in|imately related to its crystallite structure. In peas this is seen bothlin the differences whiehoccur between the upper and lower surfaces, and in the diffexen~sobsetved in mutants. 1! would appear that the closely interlocking plates which o ~ u r on the upper surface of pea leaves are composed mainly of primary+alcohols, Whereas Ihe rii:~bon4ike structures of the lower surface (which resemble structures found in Brassica oleracea) are mainly alkanes. The results:also provide a chemical basis for the observed differences in the emerald mutants of peas. Thus, wa and wb are described as glaucous on the upper surface but emerald on the lower. This can be explained by assuming that both genes affect the synthesis of alkane which is a predominant component of the lower, but not of the upper surface. The action of wa, as previously described [2] and confirmed here, involves an dongation block between C 3o "+ Cz:~. Thus the C32 acid is absent from the lower surface of the mutant, but present in the normal form, and the latter is correlated with maximalamounts of C3j alkane which is only a minor component in wa. Furthermore, there is significantly more C30 aldehyde in wa than in the norreal form, This probably results from part of the Cso add or acyl compound (result. ing from the block)being diverted to aldehyde formation, in addition to C29 alkane. E'~en though wa only appears to affect the wax on the lower surface minor chemical effects ~ n he detected in the wax from the upper surface. The gene does not affect the ultrasttueture of the upper surface because the latter is composed predominantiy of primary alcohols rather than alkanes, so that the chain length of these components (mainly C~6 and C2s) is out of range of the genetic block, The effect ofwb is le,~sclear; in previous work [2] it had been tentatively coneluded that this mutalion caused a partial inhibition of the elongation step C26 "" C2s. The tne~nt results show equivalent results for aldehydes and free acids on the lower surface but the effe~ is not seen in the primary alcohols. At the same time, alkane synthesis" is reduced a p ~ t l y at the C3t level, but closer analysis of the resull~ shows,,that C29 s y n ~ is increased in wb on the lower surface but decreased on ~ u p p e t surface (table 3B). Thus, there ale complex interactions influe~the final pauttre of the end lnoducts of wax synthesis which obscure the ~teq)fetation. One ~ explanation f ~ the-mutant is a .generally weakened e l o n g a ~ ~ m . b e y o n d . Cie; competition for .limited substrates could then perhal~ aeca)~t,fo~ differences in the chemi(ml phenotype of upper and lower wax. ~~effeet of w/o is a redue.tion of wax.on the upper surface (table 1). Thus, w ~ Netm:to affect Synthesis of most c~mponents on the upper surface, and no ~S~¢ ~ ~ ~ r ~ ~ ~~ from ~mi~a~ analysis. This rather sugp s t s i ~ t t ~ m u l ~ t i o n re!aim ettlm to wax +release otto an earlier step in fatty ~fd-qna~whi~ tsnot ~eflected in thee n a t ~ e of the surface wax. - alkanes are reduced in quan, ~ , ~ F n e )t~'),like-,)m, lent one :~finite effect..Whilst till , ~ ; l m ) d ~ t oae )elmira C3:v In oontrast to wa there is no evidence of any
minent in C tt
tween
C~. aldehvd .
.
.
_
..
-
. -
s~,rface where a l ~ e ~ s ~ the occurrence of the Cs~ acid lower surface, but not on tl~up clear-cut effects, the results sho,~ this simple, way. For i n s ~ i t h surface, and at the same time a3 are- ItkoIv to be secondmyeffe~ ca~:.~ of ~ihe complex nahum,oft 'Vn¢ present work shows that alcohols is still a dausible hypot maximum on the ~ntral carbon stitution in C2s and C2,11a l ~ C27-13-ol. Such a result~couldb' possible maximal substitution ra carbon atoms from,one e M of t~
~=in
r.~
a
variation This in u ing leavesfor a solWlpt S]
in P,'sum sat clear, but di plied:. Since that the flu, !•
~J. HoUo~,y et aL, Eplcu~'ularwslxof Pisum sativum
155
!term [I]:IL Lampz~ht,. l~nd~kzom~ Trycke~i A.B. (1961). ~ : ~ the Plant Br~medinl~l~tituUon. Weibullsholm Landsktona) ~70) s ~70) 13 [6] LA.Bager and P.LH ;loway, Mi~on 2 (1971) 364 [7] G,M~Hunt:, E,A, Baker and PJ. Holloway, Rep. Agric. Hort. Res. Stn. Univ. Bzistol t 974 :(1975) 84 [81 P.J. HoUoway, G.A. Brown, E.A. Baker and MJ.K. Macey, Chem. Phys. Lipids, in press [9] P.E. K o l a t t ~ y , .t.S. Buckner and T.-Y. Lieu, Arch. Biochem. Biophys. I56 (I973) 633