SUPPLEMENTARY MATERIAL
Methodology notes
Complete Depolymerization: To determine the time required for complete cleavage of
ester bonds in the polyesters of extracted Arabidopsis leaf and stem residues,
hydrogenolysis was carried out for 24, 48 and 72 h at 80 C. After 48 h, no further
changes in the yield or composition of extractable monomers were detected by GC and
therefore this period was used for the analysis. The yield of diethyl ether-soluble
products was typically less than 1 % of the initial mass of dry residue. In addition, the
solvent-extracted tissue residues were treated with cellulase (5 g/l) (Sigma) and
pectolyase (1 g/l) (Sigma) in 0.05 M acetate buffer (pH: 4.0) at room temperature (20°25°C) for 14 h. After filtration, the residue was washed thoroughly with water, reextracted with 2:1 (v:v) chloroform:methanol and air-dried for at least two days.
Digestion of leaf and stem residues with cellulase and pectolyase prior to
hydrogenolysis, a treatment that removed over 90% of the dry weight of the residue, did
not produce substantial changes in the content or distribution of hydroxylated aliphatic
products.
Comparison of hydrogenolysis and methanolysis: The relative distribution of
dicarboxylic and ω-hydroxy-fatty acids obtained from methanolysis was in good
agreement with the distribution obtained after LiAlH4/LiAlD4 treatments (Table S1).
However, palmitate, oleate and linoleate in the methanolysate represented a larger
fraction of total fatty acid components (30% in methanolysate versus 5% in
1
hydrogenolysate). These differences could be the result of the transmethylation
producing fatty acid methyl esters (FAME) from fatty amides present in the crude
residue. By contrast, LiAlH4 will reduce amides to alkyl amines, which will not be
recovered. A second difference between methanolysis and hydrogenolysis was that
epoxy fatty acids were not recovered in the methanolysate. As epoxy fatty acids
constitute less than 10% of the total polyester monomers this loss does not alter bulk
compositions substantially. Overall, the hydroxy-fatty acid and dicarboxylic acid
monomer load was slightly underestimated by the NaOCH3 method relative to the
hydrogenolysis method. Hydrogenolysis / deuteriolysis produced very consistent
compositional data. It also produced consistent monomer content data, whereas
methanolysis gave more variable monomer contents, with values up to 50% less than
hydrogenolysis. In addition there were fewer unidentified and overlapping peaks when
compared to methanolysis. Thus, we used hydrogenolysis / deuteriolysis of dry,
delipidated tissue residues to determine the polyester monomer composition and
content of Arabidopsis tissues.
Polyester composition of leaves and fruits from different plant species:
To
validate the methodology used for the analysis of Arabidopsis tissues, the polyester
composition of fruit skins from apple (Malus pumila) and tomato (Lycopersicon
esculentum) were analyzed. The results of depolymerization by hydrogenolysis (Figure
S1c) gave polyester monomer compositions similar to those of previous studies
(Eglinton and Hunneman, 1968; Walton and Kolattukudy, 1972b). Importantly, the fact
that high levels of tri- and tetra-hydroxy-aliphatics were detected at similar proportions
2
to previous reports demonstrates that the method used in this study does not
discriminate against polyhydroxylated fatty acids during extraction, derivatization or
chromatography to give inefficient quantification of these components.
Analysis of double bond position and geometric isomers in dicarboxylic acids.
Results are presented in Figure S2
Identification of the major ω-hydroxy-fatty acid monomers
Here we briefly report on the characterization of the major ω–hydroxy fatty acid
monomers in Arabidopsis leaf and stem tissue. Mass spectra of the TMS ethers of the
C16 and C18 family of cutin monomers obtained by hydrogenolysis were similar to those
previously observed by Walton and Kolattukudy (1972b). Likewise, the mass spectra of
monomers obtained by methanolysis were similar to those reported earlier (Eglington
and Hunneman, 1968; Croteau and Fagerson, 1972). In addition to mass spectrometry,
as described immediately below, the GC retention times of known major cutin
monomers from control tissues aided identification.
The mass spectrum of the TMS ether of hexadecanetriol showed strong ions at
m/z 275 and 317 and weaker ions at m/z 289 and 303 (Figure S3a of supplementary
material). These indicated that the predominant isomer is hexadecane-1,7,16-triol but
that hexadecane-1,8,16-triol was present at lower amounts (ranging from 10-30% of the
3
major isomer). Deuterolysis increased the most abundant molecular ion by 2 amu,
indicating the presence of only one reducible group (ester) in the intact molecule and it
shifted the m/z 317 peak to 319. Thus the substrate must be 10,16-dihydroxypalmitate.
In agreement with these results, the mass spectrum of the TMS ether of methyl
dihydroxy-hexadecanoate showed a strong ion at m/z 273 and weak ions at m/z 259
and 289, indicating that 10,16-hydroxy-hexadecanoate is the most abundant isomer
with lesser amounts of the 9,16 isomers.
The deuterolysis derivatives of octadecane- and octadecene-triols showed an
increase by 3 amu in their most abundant molecular ions indicating the presence of
either a carbonyl or an epoxy group in addition to the carboxylate and the ω–hydroxy
groups. The mass spectrum of the TMS ether of octadecanetriol derived from
hydrogenolysis showed intense ions of similar magnitude at m/z 303 and 317 indicating
the octadecane-1,9,18-triol isomer (Figure S3b). These data do not currently distinguish
between a 9(10)-oxo and 9,10-epoxy functional groups present in the substrate. By
contrast, the fragmentation pattern of octadecenetriol derived from hydrogenolysis gave
a strong fragment ion only at m/z 317 (Figure S3c). A similar mass spectrum was
reported for the hydrogenolysis product of 9,10-epoxy-18-hydroxyoctadec-12-enoate
(Walton and Kolattukudy, 1972b), so we infer that the same substrate is present in
Arabidopsis tissues. Unfortunately, the low levels of these components obtained after
methanolysis made the mass spectrometric detection of an epoxy group in these
monomers difficult.
4
30
10
120
100
80
60
40
20
0
m/z
+
428
432
426
Abundance
430
30
10
4
120
100
80
60
40
20
0
199
323
147
130000
110000
90000
115
95
149
80000
70000
60000
50000
40000
30000
60
100
140
180
220
260
300
340
380
420
0
10000
20000
432
428
Abundance
430
m/z
426
103
73
387
371
312
297
186
55
75
18:1
1000
2000
3000
4000
5000
6000
7000
8000
9000
95
18:2
16:0
60
100
140
180
220
260
300
0
10000
20000
30000
40000
50000
43
115
129
Abundance
-15)
(M
325
75
299
103
55
89
LiAlH
LiAlD
m/z
16:0
Abundance
18:1
18:2
1,18-hydroxy-C
1,16-hydroxy-C
309
151
276
109
81
9000
8000
7000
6000
5000
4000
3000
2000
1000
323
428
199
75
60000
426
147
95
73
130000
110000
90000
70000
420
380
340
300
260
220
180
140
100
60
274
307
149
107
135
121
94
41
55
79
67
Abundance
50000
40000
30000
20000
10000
0
103
280
240
200
160
120
80
40
m/z
1-hydroxy-hydroxy
1,18
(TMS)
C
dicarboxyli
acid
esther
100
80
60
26.00
24.00
22.00
20.00
Abundanc
Time
45
40
35
30
25
10
5
pA
22
21
20
19
18
17
16
15
14
1
7
8
0
3
(x10,000)
20.67
25.45
25.90
16.
18.
19.
a
b
c
d
e
A
C
B
cC
methyl
e
(min)
Table S1. Aliphatic composition of leaf polyester from wildtype Arabidopsis by two different methods (mol%)
LiAlH4
NaOCH3
C16 DERIVATVES
hexadecane-1-ol
2.5
hexadecane-1,16-diol
10a
*hexadecane-1,7,16-triol
14
Hexadecanoate
6
16-OH-hexadecanoate
1
Hexadecane-1,16-dioate
9
10,16-OH-hexadecanoate
6
C18 DERIVATIVES
octadecane-1-ol
<1
Octadecanoate
1.5
octadecene-1-ol
1.5
Octadecenoate
17
octadecadiene-1-ol
1
Octadecadienoate
10
octadecane-1,18-diol
2
<1
1,18-OH-octadecenol
octadecene-1,18-diol
b
10
18-OH-octadenenoate
Octadecene-1,18-dioate
5
1,18-OH-octadecadienol
Octadecadiene-1,18-diol
52c
octadecane-1,9,18-triol
2
octadecene-1,9,18-triol
4
mg / g dry residue
1.8
18-OH-octadecadienoate
7
Octadecadiene-1,18-dioate
38
9,18-OH-octadecanoate
nd
18-O-9,10-epoxy
octadecanoate
nd
9,18-OH-octadecenoate
nd
18-OH 9,10-epoxy
octadecenoate
nd
1.9
The data shown are the average of at least three replicates.
The standard errors are not shown and correspond in all
cases to less than 5% of the average. nd: not detected.
*: 1,8,10 isomer also present in low amounts (see text);
Based on deuteriolysis: a: approximately 50% dicarboxylic
acid and 50% -fatty acid; b: approximately 70% dicarboxylic
acid, 28% -fatty acid and 2% diol; c: approximately 80%
dicarboxylic acid, 10% -fatty acid and 10% diol.
5