Planta

Planta (1991)185:233-245

9 Springer-Verlag1991

A comparative study into the chemical constitution of cutins and suberins from Picea abies (L.) Karst., Quercus robur L., and Fagus sylvatica L. K. M a t z k e and M. Riederer*

Institut •r Botanik und Mikrobiologie, Technische Universit~it Mfinchen,Arcisstrasse 21, W-8000 Mfinchen2, Federal Republic of Germany Received 1 March; accepted 3 May 1991

Abstract. The compositions of BFa/CH3OH depolymerisates of cutins and suberins from leaf and periderm samples from Picea abies [L.] Karst., Quercus robur L., and Fagus sylvatica L., respectively, were determined by quantitative capillary gas chromatography/mass spectroscopy. Long-chain monobasic, co-hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhydroxymonobasic alkanoic acids constituted the major aliphatic monomers of leaf cutins. The total amounts of cutin monomers ranged from 629 mg- m-2 (Fagus) to 1350 m g - m -2 (Quercus). Cutin composition and amounts did not significantly differ between current year and three-year-old needles of Picea. Trans-esterification of periderm samples yielded a much greater variety of aliphatic monomers than obtained from cutins. In addition to the substance classes found with cutins, suberin depolymerisates also contained ~,co-dibasic acids while dihydroxymonobasic acids were lacking. Depolymerisates from periderms taken from different locations on a Picea tree did not differ significantly in their relative composition. The results are discussed in terms of the distinctive characteristics of the aliphatic portions of cutins and suberins, respectively. Discriminant analysis is applied for formulating a quantitative and inarbitrary classification rule for cutins and suberins. The precision, statistical significance and robustness of this classification rule are tested by employing it to a large set of compositional data (70 plant species) from the literature. The relevance of data obtained by depolymerization methods for elucidating the physical structure of cutins and suberins in situ is evaluated. Key words: Cutin (composition) - Discriminant analysis - Fagus (cuticle, periderm) - Picea (cuticle, periderm) Quercus (cuticle, periderm) - Suberin (composition)

* To whom correspondence should be addressed

Introduction

The biopolymers cutin and suberin are characteristic components of the aerial surfaces of higher plants. They represent the matrices of the transport-limiting barriers of plant cuticles and periderms. Cutin has also been identified as the main compartment for the sorption of lipophilic xenobiotics in leaf and fruit surfaces (Sch6nherr and Riederer 1989). During litterfall, cutin and suberin are transferred to the soil. Their role as a source of an aliphatic fraction of the organic matter of soils has recently been pointed out (K6gel-Knabner et al. 1989). Chemically, both polymers are closely related and, therefore, they are usually distinguished from one another by their morphological origin. Even though the lipid polymers of numerous plant species have been examined during the past twenty years, there is still no general concept unambiguously to classify both polymers. Although some qualitative criteria have been established (Kolattukudy and Agrawal 1974; Holloway 1983 ; Kolattukudy 1984), a simple quantitative and inarbitrary classification rule with general applicability is lacking. The need for such a rule becomes obvious when polymer samples of unknown or potentially mixed origin are to be classified. This may be the case when the actual nature of the lipid material found for example in the Casparian strip of roots, in certain Kranz-type vascular-bundle sheaths or in the protective layers of seeds and fruits is to be investigated by analytical rather than histochemical means. A simple and clear distinction between cutin- and suberin-derived aliphatic material is also a prerequisite for the analysis of lipid biopolymers contained in plant litter or humus. As a first step to establishing such a classification rule, we have comparatively studied the constitution of the cutins and suberins of three common European tree species (Picea abies, Faous sylvatica and Quercus robur) using nested samples. Subsequently, multivariate statistical methods have been used to obtain the appropriate

234

K. Matzke and M. Riederer: Comparative study into cutins and suberins

classification criteria and a quantitative classification rule. The validity and robustness of this scheme has been extensively tested using a set of 122 literature data on the composition of the cutins and suberins from 70 plant species.

Materialsandmethods Plant materials. Needle and bark samples of Norway spruce (Picea abies (L.) Karst.) were obtained from an approximately 70-year-old tree growing in the Fichtelgebirge (N.E. Bavaria, FRG). Plant material was taken in September 1989 from (i) twigs harvested at a height of 2-3 m, (ii) the stem at a height of 1.5 m and (iii) roots with a diameter of 7-10 ram. Outer bark of the stem was cut off using a knife. The twigs were separated according to age classes (one, two and three years old) and the needles were mechanically removed after submersing the twigs in liquid nitrogen. The periderm of the twigs and roots (thoroughly washed in deionized water) together with the cortex tissues was carefully separated from the vascular cylinder. Microscopic examination of this material confirmed that only tissues outside the cambium had been isolated. Leaves and outer bark from the stem of oak (Quercus robur L.) and leaves and periderm from about three-year-old twigs of beech (Fagus sylvatica L.) were obtained in June 1990 (S. Bavaria, FRG). The samples were dried at 90 ~ C for 2 d and afterwards homogenized in liquid nitrogen using a mortar. The resulting material was subjected to two exhaustive (checked by thin-layer chromatography and gas chromatography) extractions with boiling CHC13 and acetone, respectively, in a Soxhlet-type apparatus to remove free lipids. Each extraction step lasted for at least 24 h. The yield of this procedure was 295, 239, 174 and 117 g dry extractive-free corky substance per m 2 surface area of twigs (ageclasses 1987 to 1989) and roots of Picea abies, respectively. The surface areas were assessed by assuming a cylindrical geometry of twigs and roots. The yield of extractive-free dry needle tissue was 33 g per m 2 needle surface for each age-class. Nineteen g and 36 g were obtained per m 2 leaf surface from Faffus sylvatica and Quercus robur, respectively. Needle surfaces were estimated from the length of 150 needles (Riederer et al. 1988). The leaf surface areas of the samples taken from Fagus and Quercus were determined using a leaf-area meter (LI 3000; Licor, Lincoln, Neb., USA). Gravimetric measurements were made using an electronic microbalance ( i 0.1 mg; Sartorius, G6ttingen, FRG). Transesterification ofcutin and suberin. The samples were treated for 30 min at 25 ~ C with a freshly prepared 0.2 M solution of HC1 in 1,4-dioxane (Merck, Darmstadt, FRG) to transform epoxy groups into their stable chlorohydrin derivatives (Riederer and Sch6nherr 1986). Afterwards the HCl-dioxane was removed by evaporation on a rotary evaporator and repeated washes with CHC13. A 5- to 10-mg sample of the ground material was weighed into 1-ml screw-capped boron-silicate glass vials with PTFE seals (Wheaton, Millville, N J, USA). An aliquot of 200 ~tl BF3/CH3OH (16%, Merck) was added together with an appropriate amount of n-eicosane as internal standard and the samples were heated to 70 ~ C for 24 h. This procedure releases cutin and suberin monomers cross-linked by ester bonds and simultaneously transforms acids into their corresponding methyl esters (Riederer and Sch6nherr 1986). Preliminary experiments with bark of five-year-old Picea abies trees had shown that longer treatment with BF3/CH3OH neither increases the depolymerization yield (Fig. 1a) nor changes the qualitative composition (Fig. lb) of the depolymerisates. The further work-up of the monomer mixtures resulting from the transesterification reaction followed the methods described earlier (Riederer and Sch6nherr 1986, 1988b). Identification and quantification of monomers. The depolymerization products were analyzed using a Carlo Erba (Milan, Italy) 4200 gas

I

E

g

10

' I ' I ' [ ' I/ ,1 , I -

o

9

~,

~.-

8

N

o ~6 -K)

2

-~

100

~

6o

~

4o

s A7i

g 2o

~

00

I

10

"1"

20

30

i"

I/ / 40 60 70

BF3/CHsOH treatment [h] Fig. IA, B. Effect of the duration of treatment with BF3/CH3OH at 70 ~ C on the yield of monomers (A) and the qualitative composition of bark depolymerisates (B) from five-years-old Picea abies trees

chromatograph (GC) equipped with flame-ionization detector, an on-column injector and a WCOT fused-silica capillary column (20 m long, 0.32 mm i.d.) coated with CP-Sil 5 CB (df=0.13 ~tm) from Chrompack (Middelburg, The Netherlands). The GC system operated with hydrogen carrier gas at a pressure of 40 kPa, a detector temperature of 320 ~ C and the following temperature program: injection at 50 ~ C, 2 min at 50~ C, 40 K - m i n -1 up to 150~ C, 6 K 9min- 1 up to 300 ~ C, 13 min at 300 ~ C. Peak areas were recorded by electronic integration (C-R3A; Shimadzu, Kyoto, Japan). Specific correction factors accounting for losses during sample work-up and differential response were applied to the peak areas of 18-hydroxy-9-octadecenoic acid, dihydroxyhexadecanoic acid, trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid (Riederer and Sch6nherr 1986). A factor of 1.10 was assigned to all other compounds. The identified monomers comprised between 80% and 94% of the total amount of alkanoic monomers detectable by gas chromatography. In each case, all dominating constituents were identified.

K. Matzke and M. Riederer: Comparative study into cutins and suberins

235

Cutin and suberin monomers were identified by electron-impact mass spectroscopy (70 eV; Finnigan-MAT 12S, San Jos6, Calif., USA) using reference compounds and published spectra (Kolattukudy and Agrawal 1974; Holloway 1982b). Mass-spectroscopic identification proved to be essential since some of the compounds had identical retention times under the given chromatographic conditions. Capillary gas chromatography-mass spectrometry was carried out under chromatographic conditions as detailed above but using helium instead of hydrogen as carrier gas.

Results

Statistics. Transformation and analysis of the raw data as well as discriminant analysis were performed using SPSS/PC + statistical

Composition of leaf depolymerisates. The total amounts o f d e p o l y m e r i s a t e s r e l e a s e d b y t r e a t i n g t h o r o u g h l y ex-

Table 1. Composition of BF3/CH3OH depolymerisates from leaves of Fagus sylvatica and Quercus robur. -, not detected; tr, traces

software (SPSS Inc., Chicago, ILL., USA). The values quoted in Tables 1 to 4 and Figs. 1 and 2 are the means of five samples in each case. The experimental error was determined by five injections of identical samples representing needles, leaves, twigs, stem and roots. Coefficients of variation were smaller than 5% for all compounds.

Compound"

Fagus m g ' m -2

Quercus %

mg'm

2

%

Monobasic alkanoic acids Hexadecanoic Octadecanoic Octadecenoic Octadecadienoic Octadecatrienoic Docosanoic

62.4 5.2 224 b 4.5

9.9 0.8 35.7 b 0.7

Total

296

47.1

Dibasic alkanoic acids

-

44.8 6.4 15.5 79.6 c

3.3 0.5 1.2 5.9 ~

tr

tr

146

10.8

-

~o-Hydroxymonobasic alkanoic acids

16-OH-hexadecanoic 18-OH-octadecenoic

8.9 19.8

1.4 3.2

12.7 133

Total

28.7

4.6

146

1.9 8.0 5.5

0.3 1.3 0.9

15.4

2.5

0.9 9.9 10.8

2-Hydroxymonobasic alkanoic acids 2-OH-hexadecanoic 2-OH-docosanoic 2-OH-tetracosanoic Total

m

Dihydroxymonobasic alkanoic acids DiOH-hexadecanoic d

110

17.4

361

26.8

Trihydroxymonobasic alkanoic acids' 9,10,18-TriOH-octadecanoic

12.7

2.0

77.2

12.3

Heptadecanol Octadecanol Eicosanol Octacosanol

3.3 tr tr

0.5 tr tr

Total

3.3

0.5

18.5

1.4

Epoxyhydroxymonobasic alkanoic acids 9,10-Epoxy- 18-OH-octadecanoic

411

30.5

1-Alkanols E

m

m

m

m

m

Alkanediols Unidentified Total monomers

85

13.6

265

19.7

629

100.0

1350

100.0

" Where appropriate, determined as the corresponding methyl ester and trimethylsilyl ether derivatives. Absolute values are rounded to three significant digits b Sum of octadecenoic, octadecadienoic and octadecatrienoic acids (individual compounds not sufficiently separated under the chromatographic conditions used) c Sum of octadecadienoic and octadecatrienoic acids (individual compounds not sufficiently separated under the chromatographic conditions used) d 10,16-Dihydroxyhexadecanoic acid with Quercus, 8,16- and 9,16-dihydroxyhexadecanoic acids with Fagus (positional isomers not separated)

236

K. Matzke and M. Riederer: Comparative study into cutins and suberins

Table 2. Composition of BFa/CHaOH depolymerisates from needles of Picea abies from different age classes. -, not detected Compounda

1987 mg

1988

9 m -2

%

mg

1989

- m -2

%

mg

9 m -2

%

Monobasic alkanoic acids

Hexadecanoic

29.8

Dibasic alkanoic a c i d s

.

3.5 .

35.8 .

.

4.3

30.5

3.5

2.1 8.1 10.9

10.3 45.1 55.5

1.2 5.3 6.5

.

~o-Hydroxymonobasic alkanoic acids

12-OH-dodecanoic 14-OH-tetradecanoic 16-OH-hexadecanoic

18.8 64.8 107

2.2 7.6 12.6

Total

191

22.4

2-Hydroxymonobasic alkanoic acids

17.2 67.2 90.7 175

.

21.1

.

.

.

111

12.9

382

44.5

.

Dihydroxymonobasic alkanoic acids

9,16-DiOH-hexadecanoic

300

35.2

307

36.9

Trihydroxymonobasic alkanoic acids

9,10,18-TriOH-octadecanoic

50.1

5.8

22.8

2.7

96.2

11.3

97.2

11.7

19.8

2.3

Epoxyhydroxymonobasic alkanoic acids

9,10-Epoxy- 18-OH-octadecanoic 1-Alkanols

.

.

.

.

.

139

16.1

.

Alkanediols

1,12-Dodecanediol 1,14-Tetradecanediol

35.0 18.5

4.1 2.2

30.9 15.9

3.7 1.9

19.7 6.4

2.3 0.7

Total

53.5

6.3

46.8

5.6

26.1

3.1

Unidentified

132

15.5

148

17.7

151

17.6

Total monomers

853

100.0

833

100.0

859

100.0

a Where appropriate, determined as the corresponding methyl ester and trimethylsilyl ether derivatives. Absolute values are rounded to three significant digits

tracted leaf material with BF3/CH3OH differed significantly a m o n g the three species studied. The lowest coverage was found with Fagus (629 m g . m -z) while from leaves of Quercus 1350 mg of m o n o m e r s were released per m z (Table 1). Intermediate values were found for needles of Picea abies which, depending on needle age, had coverages ranging from 833 to 859 mg 9m -2. N o significant difference in total coverage was observed between Picea needles of different age classes (Table 2). Long-chain monobasic, co-hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhyd r o x y m o n o b a s i c alkanoic acids were found in the leaf depolymerisates of all three species (Tables 1, 2). Fagus leaf depolymerisates were further characterized by the occurrence o f three homologues of a series of 2-hydroxymonobasic acids and traces of 1-alkanols. In Picea needle depolymerisates, two n-alkanediols were present which were missing with the two deciduous species. The carbon chain-lengths of all m o n o m e r s ranged from C12 to Cls in Picea (Table 2) and from C~6 to C22 (traces of 1-octacosanol disregarded) in Fagus and Quercus (Table 1). Most constituents were saturated, the only exceptions being octadecenoic, octadecadienoic, octadecatrienoic, and 18-hydroxyoctadecenoic acids which were found in the depolymerisates f r o m the two broad-leaved

species. In addition to aliphatic material, ferulic and cumaric acid as w e l l as fl-sitosterol were identified in depolymerisates from needles and leaves. Dihydroxymonobasic acids constituted the most abundant c o m p o u n d class in the depolymerisates of Picea needles ( 3 5 4 5 % of total) and also considerable amounts were found in Quercus leaves (27%). In Fagus, the contribution of this class (17 %) was exceeded only by that of unsubstituted monobasic acids (see Discussion). O f the total mass of the constituents of the depolymerisates of Picea needles 16 to 21% consisted ofco-hydroxymonobasic acids (Table 2) while this class contributed only 11% and 5 % in Quercus and Fagus depolymerisates, respectively (Table 1). 9,10-Epoxy-18-hydroxyoctadecanoic acid was also an important constituent making up approx. 31% in Quercus and between 11 and 16% in Picea and Fagus. 9,10,18-Trihydroxyoctadecanoic acid was more prominent in Picea than in the two broad-leaved species but did not exceed 6% in any case. Unsubstituted monobasic acids were released in much higher amounts from Fagus and Quercus leaf material than from extracted Picea needles. With Picea, needles from three different age-classes had been subjected to depolymerization. As a whole, the qualitative and quantitative composition of depoly-

K. M a t z k e a n d M. R i e d e r e r : C o m p a r a t i v e s t u d y i n t o c u t i n s a n d s u b e r i n s Table 3. C o m p o s i t i o n o f B F a / C H a O H d e p o l y m e r i s a t e s f r o m stem p e r i d e r m s ( b a r k ) ofPicea abies a n d Quercus robur a n d f r o m b r a n c h p e r i d e r m o f Fagus sylvatica. -, n o t d e t e c t e d Compound a

Picea

Faous

Quercus

%

%

%

Monobasic alkanoic acids Octadecanoic Octadecenoic Octadecadienoic Eicosanoic Docosanoic Tetracosanoic Hexacosanoic

Total

0.4 2.8 5.5 7.0 1.2

0.4 0.4 0.1 3.6 5.1 1.1

0.3 0.5 0.8 2.5 -

16.9

10.7

4.1

6.1 1.4 9.3 4.4 2.3 1.2

6.5 1.8 0.4 2.3 0.8

0.9 2.4 2.9 -

24.7

11.8

6.2

Dibasic alkanoic acids Hexadecanedioic Octadecanedioic Octadecendioic Eicosanedioic Docosanedioic Tetracosanedioic

Total

merisates differed only slightly between one-, two- and three-year-old needles (Table 2). The most conspicuous change was the doubling of the amount of 9,10,18-trihydroxyoctadecanoic acid from the first to the third year albeit the contribution of this constituent to the total remained fairly low in any age class. The amounts of 9,16-dihydroxyhexadecanoic and 9,10-epoxy- 18-hydroxyoctadecanoic acids were lower by factors of 1.3 and 1.4, respectively, in the oldest needles than in those from the current year.

Composition of periderm depolymerisates. Transesterification of periderm samples from Picea, Fagus and Quercus yielded depolymerisates which were composed of a much greater variety of constituents than the corresponding leaf depolymerisates. Depolymerisates from periderms of the three species also contained monoba60

20-OH-eicosanoic 22-OH-docosanoic 24-OH-tetracosanoic 26-OH-hexacosanoic

8.7 2.9 23.8 8.0 3.2 -

2.3 2.4 2.0 0.8 1.9 7.2 3.9 0.6

1.1 1.1 13.8 2.1 10.3 12.2 4.7

Total

46.6

21.1

45.3

-

-

18-OH-octadecenoic 18-OH-octadecadienoic

2-Hydroxymonobasic alkanoic acids 2-OH-tetracosanoic

1.4

Dihydroxymonobasic alkanoic acids

A Fogus sylvatico

50 q)

8

~o-Hydroxymonobasic alkanoic acids 16-OH-hexadecanoic 18-OH-octadecanoic

237

{3

40

-

30

-

20

-

10

-

0

12

10

14

23.8

13.7

-

17.6

7.6

18

20

J 22

24

26

28

B

2 9

50 -Quercus robur 40 -

O_

.30 -

[3

20 -

Trihydroxymonobasic alkanoic acids -

16

leaf cutin stem suberin

60 !

i~~

leaf cutin stem suberin

[

10 9,10,18-triOH-octadecanoic

[

~ ~

0 10

12

14

16

18

20

22

24

26

28

~ ~

needle cutin twig suberin stem suberin root suberin _

Epoxyhydroxymonobasic alkanoic acids 9,10-Epoxy- 1 8 - O H - o c t a d e c a n o i c

60 C Pice(] abies

50

1-Alkanols Heptadecanol Octadecanol Eicosanol Docosanol Tetracosanol Hexacosanol

0.5 1.0 0.8 -

4.2 4.8 0.9

1.2 1.4

8

1.0 -

0-

50

60 60 0

20

Total

2.3

9.9

3.6

40

10 9

0 Alkanediols

-

-

-

Unidentified

9.5

5.1

18.1

100.0

100.0

100.0

10

I

I

t

I

12

14

16

18

I

I

I

I

20

22

24

26

28

Carbon number Total monomers

a W h e r e a p p r o p r i a t e , d e t e r m i n e d as the c o r r e s p o n d i n g m e t h y l ester a n d t r i m e t h y l s i l y l ether d e r i v a t i v e s

2 A - C . C h a i n - l e n g t h d i s t r i b u t i o n o f the a l i p h a t i c m o n o m e r s o f c ut i n a n d s ube ri n f r o m different a n a t o m i c a l l o c a t i o n s o f Fagus sylvatica (A), Quercus robur (B), a n d Picea abies (C) o b t a i n e d af ter d e p o l y m e r i z a t i o n w i t h B F 3 / C H 3 O H a t 70 ~ C for 24 h Fig.

238

K. Matzke and M. Riederer: Comparative study into cutins and suberins

Table 4. Composition of BF3/CH 3OH depolymerisates from periderms obtained from twigs (three age-classes) and roots of Picea abies. -, not detected Compound a

Twigs

Roots

1987

1988

m g - m -2

%

1989

mg.m 2

%

mg.m 2

%

mg.m-Z

%

Monobasic alkanoic acids

Octadecanoic Eicosanoic Docosanoic Tetracosanoic Hexacosanoic Total

67.2 825 743 639 110

0.3 3.3 3.0 2.5 0.4

48.6 540 542 441 100

0.2 2.6 2.7 2.2 0.5

40.9 384 409 240 62.7

0.3 2.5 2.6 1.5 0.4

26.9 12.0 -

4.5 2.1

6.6

2 380

9.5

1 670

8.2

1 140

7.3

38.9

Hexadecanedioic Octadecanedioic Octadecendioic Octadecadiendioic Eicosanedioic Docosanedioic

1 180 283 2 450 418 979 451

4.7 1.1 9.8 1.7 3.9 1.8

941 283 1 860 291 735 477

4.6 1.4 9.1 1.4 3.6 2.3

589 127 1 240 124 310 361

3.8 0.8 8.0 0.8 2.0 2.3

46.9 28.0 20.8

Total

5 770

22.9

4 580

22.4

2 750

17.7

95.7

16.1

1 940 2 630 7 830 2 000 368 328

7.7 10.4 31.2 8.0 1.5 1.3

1 670 3 230 5 550 1 640 294 296

8.2 15.8 27.1 8.0 1.4 1.5

1 210 4 070 3 800 1 050 166 159

7.8 26.2 24.4 6.8 1.1 1.0

17.9 7.5 218 48.8 34.9 -

3.0 1.3 36.7 8.2 5.9

15 100

60.1

12 700

62.0

10 500

67.3

327

55.1

2-OH-tetracosanoic

.

.

.

.

Dihydroxymonobasic alkanoic acids

.

Trihydroxymonobasic alkanoic acids"

-

Epoxyhydroxymonobasic alkanoic acids

.

39.3 200

0.2 1.0 .

21.4 121 .

1.2

142

Dibasic alkanoic acids

7.9 4.7 3.5

~o-Hydroxymonobasic alkanoic acids

16-OH-hexadecanoic 18-OH-octadecanoic 18-OH-octadecenoic 20-OH-eicosanoic 22-OH-docosanoic 24-OH-tetracosanoic Total 2-Hydroxymonobasic alkanoic acids

.

.

.

.

.

10.6

1.8

.

1-Alkanols

Heptadecanol Octadecanol Tetracosanol

71.6 262 .

Total

334

0.3 1.0 .

. 1.3

239

Alkanediols Unidentified Total monomers

.

-

0.1 0.8

-

-

0.9

-

-

.

.

-

1 560

6.2

1 270

6.2

1 060

6.8

121

20.4

25 200

100.0

20 400

100.0

15 500

100.0

593

100.0

a Where appropriate, determined as the corresponding methyl ester and trimethylsilyl ether derivatives. Absolute values are rounded to three significant digits sic, o ) - h y d r o x y m o n o b a s i c , 2 - h y d r o x y m o n o b a s i c (only present in Q u e r c u s stem a n d P i c e a r o o t periderms), t r i h y d r o x y m o n o b a s i c (except P i c e a ) a n d e p o x y h y d r o x y m o n o b a s i c (except P i c e a ) a l k a n o i c acids a n d 1-alkanols ( T a b l e 3). H o w e v e r , they differed f r o m l e a f d ep o l y me r i s at es as they a d d i t i o n a l l y c o n t a i n e d ~,co-dibasic a l k a n o i c acids while b o t h d i h y d r o x y m o n o b a s i c acids a n d a l k a n e d i o l s were missing. T h e n u m b e r o f h o m o l o g u e s within the different s u b s t a n c e classes was m u c h h i g h e r than o b s e r v e d with l e a f d e p o l y m e r i s a t e s . In a d d i t i o n to the c h a i n - l e n g t h s o f C16 a n d C18 w h i c h d o m i n a t e d l e a f

d e p o l y m e r i s a t e s , higher h o m o l o g u e s u p to C26 were present in samples d er i v ed f r o m p e r i d e r m s (Fig. 2). 1 8 - H y d r o x y o c t a d e c e n o i c acid was the m o s t a b u n d a n t u n s a t u r a t e d c o m p o n e n t in the p e r i d e r m d e p o l y m e r i s a t e s o f the three species (Table 3). In b o t h P i c e a an d Q u e r c u s , ~ o - h y d r o x y m o n o b a s i c acids were the m o s t a b u n d a n t class o f c o n s t i t u e n t s (47 a nd 45%, respectively), while p e r i d e r m samples f r o m F a g u s were d o m i n a t e d by 9 , 1 0 , 1 8 - t r i h y d r o x y o c t a d e c a n o i c acid. This single c o m p o u n d c o n t r i b u t e d m o r e to the total m a s s o f p e r i d e r m c o n s t i t u e n t s t h a n the different h o m o l o g u e s

K. Matzke and M. Riederer: Comparative study into cutins and suberins ofco-hydroxymonobasic acids together. Dibasic alkanoic acids (C~6 to C24) made up 25% of the monomers in Picea depolymerisates as well as 12 and 6% of Fagus and Quercus depolymerisates, respectively. Depolymerisates from periderms taken from different locations on a Picea abies tree did not differ significantly in their relative composition (Tables 3, 4). In depolymerisates from twigs (one to three years old), stem and roots, co-hydroxymonobasic acids were the most abundant substance class followed by dibasic and monobasic acids. Since intact periderms could be isolated only from young twigs and roots, total coverages of the monomers in the depolymerisates could be determined only for these organs (Table 4). No coverage data are available for stem barks. Total coverages were much higher in twig periderms than in the root periderm studied. Owing to the continuing formation of bark during the dilatation of the twigs, the total amounts of depolymerisates increased from 15.5 g 9m 2 in one-year-old to 25.1 g 9m -2 in three-year-old twigs of Picea abies. Discussion

It was one of the objectives of the present work to study comparatively the qualitative and quantitative composition of the lipid biopolymers making up the aerial interfaces of three major species of forest trees in Europe. Unfortunately, pure samples of these polymers could not be obtained because of (i) the impossibility of enzymatically isolating cuticular membranes from mature leaves of these species (data not shown) and (ii) the very nature ofperiderms which are complex tissues. Thus, at best cutin- or suberin-enriched samples could be investigated. Nevertheless, a careful treatment and work-up of the samples enabled us to obtain depolymerisates from leaves which contained all the typical constituents of cutin known from previous studies with isolated cuticles (Kolattukudy 1981, 1984; Holloway 1982b, 1984; Riederer and Sch6nherr 1986). Depolymerisates from periderm samples as well contained a spectrum of constituents characteristic of suberin (Holloway 1983; Kolattukudy 1984). Thus, we assume that the qualitative compositions of the depolymerisates provide representative pictures of the actual compositions of the cutins and suberins of the three species investigated. There is one compound class of aliphatic depolymerisates, however, which in this context should be treated with caution. In leaf and periderm depolymerisates from all three species considerable amounts of monobasic alkanoic acids have been found (Tables 1-4). This compound class has only one functional group and is therefore unable to participate in any cross-linking reactions within a polymer. Single-substituted monomers can form only endpoints in a polymeric network while double- and triplesubstituted monomers may be incorporated in linear and three-dimensional polymers, respectively. For this reason, it is doubtful whether the monobasic acids found in leaf and periderm depolymerisates indeed originated from cutin and suberin, or whether they were cleaved from other parts of the mate-

239

rial subjected to transesterification (e.g. cell walls). The same argument may hold for the presence of phenolic compounds in leaf and periderm depolymerisates. In many cases it is simple to tell whether the monomers found after transesterification of a material are derived from cutin or from suberin. The distinction is clear as long as it is definitively known that the material is derived from primary or secondary plant surfaces. In some cases, however, either the exact origin of the material is unknown (e.g. when investigating aliphatic residues in soil or sediments) or when tissues formed by primary and secondary growth cannot be separated (e.g. in the outer layers of the caryopses of Triticum; Matzke and Riederer 1990). Then, precise criteria are needed for distinguishing monomers of cutin from those of suberin. The data produced in this study are perfectly suited for deducing such criteria since cutin and suberin samples were taken from the same trees (nested samples) and identical protocols were followed during the analysis of both types of polymers. Comparing the data obtained for Picea, Quercus and Fagus (Tables 1-4, Fig. 2) revealed the following differences between cutin and suberin: (i) all cutins examined were characterized by substantial amounts of dihydroxyhexadecanoic acids. (ii) Suberins from all three species contained considerable amounts of ~,co-dibasic and long-chain (>C18) monomers which were lacking in cutin depolymerisates (Fig. 2). It is questionable, however, whether these qualitative rules are sufficient for classifying cutins and suberins reliably and with a high statistical significance. In order to test these criteria and to establish inarbitrary and more stringent quantitative criteria, the data on the composition of cutins and suberins of Picea, Quercus and Fagus were subjected to a discriminant analysis. This is a multivariate tool for classifying cases which are characterized by a set of variables. The aim of discriminant analysis is to find the best linear combination of independent variables to classify cases. For this purpose, a set of cases with known group membership is used as a training set in order to select the best discriminating variables and to calculate the coefficients Bo...B, of the equation D = Bo+B1 9XI+B2 9X 2 + . . . + B , " X, .

Eq. (1)

From this equation, the discriminant score D can be computed for each case, with xl...x, representing the values of the variables used for classification. Based on the value of D, cases whose group membership is unknown may be classified into one of the groups and the significance of the classification may be calculated (James 1985; Norugis 1986). In order to obtain such a classification rule for cutins and suberins, we formed a set of eight variables which were suggested by the qualitative properties outlined above. Variables were the mass percentages of all homologues of the following compound classes: 1-alkanols (variable symbol OL), monobasic (OIC), a, co-dibasic (DOIC), co-hydroxymonobasic (coOH), dihydroxymonobasic (DOH), trihydroxymonobasic (TOH) and epoxyhydroxymonobasic (EPO) alkanoic acids. Additionally, the sum of the relative amounts of monomers

240

K. M a t z k e a n d M, Riederer: C o m p a r a t i v e s t u d y into cutins a n d suberins

Table 5. Relative c o m p o s i t i o n a (in m a s s percents o f total) a n d d i s c r i m i n a n t scores o f cutins a n d suberins. - , n o t detected No. Species b

Material

OIC

DOIC

e)OH

branch root stem twig stem

10.8 6.6 17.0 8.3 4.1

11.8 16.1 24.7 21.0 6.2

21.1 55.1 46.5 63.1 45.2

leaf needles leaf

47.1 3.8 10.8

-

4.6 18.8 10.8

cork layer cork layer crystal idioblast root cork layer fresh b a r k industrial b a r k root root c o r k layer bark i n n e r seed coat cork layer root cork layer cork layer cork layer green fibres green fibres seed coat tuber cork layer root p e r i d e r m s t e m b a r k (1 yr) s t e m b a r k (2 yrs) s t e m b a r k (3 yrs) stem b a r k (15 yrs) s t e m b a r k (15 yrs) root bark bark cork layer cork layer bark cork layer rhytidome sapwood cork layer cork layer cork layer cork layer cork layer cork layer cork layer cork layer cork layer cork layer tuber tuber bundle sheath

10.7 2.8 7.8 18.0 4.0 8.0 1.1 6.4 9.4 14.0 6.2 4.4 7.9 3.4 1.1 2.9 9.0 1.5 3.7 8.1 9.3 10.1 8.8 10.4 14.0 15.6 16.6 6.2 6.0 2.1 2.0 1.6 1.3 1.3 0.3 0.2 0.5 2.3 0.3 2.9 4.0 6.7 8,2 11.1 8,0 3~8

25.9 20.5 34.8 16.0 8.0 9.7 11.1 22.0 22.0 9.9 43.0 42.9 31.6 24.0 24.0 11.9 40.1 15.3 25.3 3.8 21.0 21.8 13.3 21.3 17.8 16.1 13.1 14.4 27.0 31.3 27.7 27.8 10.5 17.5 15.1 14.5 17.2 32.7 35.9 31.6 29.2 26.9 30.6 23.9 20.2 24.6 34.2 32.0 24.0 7.2

39.6 45.3 32.0 29.0 21.6 28.8 28.0 36.0 43.0 13.4 47.2 45.9 51.2 31.0 61.2 18.7 24.3 78.7 71.9 75.7 36.0 48.6 25.0 17.8 18.8 19.3 16.8 13.9 30.0 50.6 53.7 54.5 23.9 21.3 21.3 34.9 35.2 40.7 50.1 51.3 52.7 49.7 50.8 51.6 49.7 50.5 31.7 35.3 16.0 23.1

DOH

TOH

EPO

C20

OL

Dr

Ref. d

23.8 -

17.6

36.5 30.7 36.4 23.6 39.4

2.42** 1.81" 3.21"* 2.35** 1.41"

29 29 29 29 29

Initial training set Suberins

1 Fagus sylvatica 2 Picea abies 3 Picea abies 4 Picea abies 5 Quercus robur

13.7

7.6

9.9 2.3 1.1 3.7

17.4 38.8 26.8

2.0 3.6 1.4

12.3 13.0 30.5

0.5 -

2.9 -

-2.43** - 2.57** -2.63"*

29 29 29

0.3 2.8 1.0

10.9 3.5 2.7

4.2 3.7 3.1

29.1 9.4 7.8

2.0 2.1 0.6 15.9 36.0 37.9

2.0 4.7 4.0 6.0 5.0 3.0 3.9 3.2 3.6 3.0 1.5 6.8 9.5 1.6 1.2 15.5 4.0 1.5 8.9 9.5 12.5 12.9 12.5 9.5 4.0 2.6 1.8 2.3 5.6 2.4 2.3 5.1 2.3 2.3 0.7 1.2 1.9 6.6 1.2 5.7 6.2 6.1 11.1 12.5 6.0 22.7

49.8 33.9 21.5 23.4 30.1 23.2 23.7 13.2 12.2 16.8 21.8 37.2 29.9 20.2 51.0 26.0 29.4 97.3 97.0 95.0 10.8 14.5 21.6 26.5 31.7 34.2 32.6 31.4 17.9 35.3 39.6 21.4 28.5 16.2 16.8 24.8 20.4 37.1 17.1 23.3 31.5 27.6 26.5 40.7 40.2 34.8 28.4 24.6 14.3 22.6

4.31'* 2.72** 3.96** 1.35" 1.43" 0.60 0.74 1.82" 1.80" 1.32' 5.21"* 5.72** 4.01"* 2.24* 3.96** 1.95"* 5.22** 4.88** 6.14"* 3.68**

17 17 27 19 17 6 6 19 19 17 12 10 17 19 17 17 17 24 26 24 19 17 16 16 16 16 16 16 19 7 7 17 17 21 17 21 21 17 13 13 13 13 13 13 17 17 17 3 18 9

m

-

m

Cutins

6 Fagus sylvatica 7 Picea abies 8 Quercus robur Test set Suberins

9 Acer griseum 10 Acer pseudoplatanus

11 Agave americana 12 Beta vulgaris 13 Betula pendula 14 Betula verrucosa 15 Betula verrucosa 16 Brassica napobrassica 17 Brassica rapa 18 Castanea sativa 19 Cedrus libani 20 Citrus paradisi 21 Cupressus laylandii 22 Daucus carota 23 Euonymus alatus 24 Fagus sylvatica 25 Fraxinus excelsior 26 Gossypium hirsutum 27 Gossypium hirsutum 28 Gossypium hirsutum 29 Ipomoea batatas 30 Laburnum anagyroides 31 Malus pumila 32 Maluspumila 33 Malus pumila 34 Malus pumila 35 Maluspumila 36 Maluspumila 37 Pastinaca sativa 38 Picea abies 39 Pinus silvestris 40 Populus tremula 41 Quercus ilex 42 Quercus robur 43 Quercus robur 44 Quercus robur 45 Quercus robur 46 Quercus suber 47 Ribes americanum 48 Ribes davidii 49 Ribesfuturum 50 Ribes grossularia 51 Ribes houghtonianum 52 Ribes nigrum 53 Ribes niyrum 54 Sambucus nigra 55 Solanum tuberosum 56 Solanum tuberosum 57 Solanum tuberosum 58 Zea mays

-

-

-

2.2 0.4 0.4 0.8 0.8

-

31.9 0.6 1.0 1.6 26.4 4.5 -

0.5

-

-

9.4 4.6 3.8 2.1 2.4 1.0 0.1

1.2 14.4 19,2 22.1 24.8 23.6 38.8 -

1.9 0.7 3.7 3.7

0.7 0.5 0.6 4.0 0.6 3.8 2.9 0.6 0.8

1.1 16.6 25.3 25.3 24.0 3.6 5.4 1.3 1.3 1.3 0.6 1.1 0.9 1.6 0.8 0.6 -

19.6

12.8 10.2 0.1 1.0 28.7 2.9 -

0.3 25.9 16.0 15.4 11.5 13.6 2.8 0.5 31.4 22.7 22.7 10.6 17.1 15.3 1.2 0.3 0.5 -

6.0

4.6

1

.

5

4

*

1.82" 1.54" 3.00** 3.02** 3.02** 2.45** 3.12"* 2.59** 4.15"* 3.84** 3.15"* 1.45" 2.09* 1.78" 2.32** 1.50" 4.51"* 4.00** 3.70** 3.75** 3.33** 3.67** 3.47** 2.97** 3.36** 4.32** 3.98** 1.90" 0.67

K. Matzke

"Fable 5.

and M. Riederer:

Comparative

study into cutins and

suberins

241

Continued

No. Species b

Material

OIC

DOIC

coOH

DOH

TOH

EPO

C20

leaf leaf leaf leaf leaf fruit leaf leaf leaf fruit leaf fruit leaf fruit peel inner seed coat juice-sac leaf fruit leaf fruit leaf leaf leaf leaf fruit leaf leaf leaf white fibres leaf seed coat fruit leaf

2.0 9.6 0.9 59.6 2.4 6.3 1.0 9.0 2.0 9.0 0.8 1.1 0.4 1.0 10.0 1.0 10.0 24.3 5.0 2.7 0.6 2.0 1.5 16.0 1.1 0.9 1.6 1.5

1.1 7.9 3.0 -

2.1 3.9 1.9 3.9 2.0 63.0 10.1 63.0 8.0 2.3 10.9 1.2 1.4 34.0 9.0 47.0 7.0 6.9 2.7 1.0 2.3 10.5 5.4 12.8 1.5 3.3

10.5 15.3 20.7 13.8 24.8 5.9 19.8 69.3 34.0 79.0 30.0 82.0 31.9 2.0 30.3 89.5 62.0 79.0 51.0 81.0 30.2 33.4 66.2 23.9 24.2 18.0 19.3 23.1 20.1 16.2 11.8 25.5

22.1 19.3 37.5 13.5 10.4 10.9 . 12.7 . . . . . 34.0 12.3 2.2 . . . . 7.5 1.2 1.0 17.4 14.4 16.9 26.3 10.6 9.6 8.6 6.3

. . 50.2 18.5 52.0 . . . . . . . . . . . . . 43.1 22.6 . . . . . . 49.6 . . . . 54.5 . . 33.8 30.2 . . . .

fruit leaf leaf leaf

3.3 5.7 59.6 3.5

-

4.5 8.0

9.2 31.4 4.2 26.9

28.0 . . 11.6

. .

. . 24.2

. . . 12.8

seed coat leaf fruit leaf fruit fruit fruit fruit

0.9 6.9 1.2 38.6 2.4 1.4 1.2 1.7

1.1 1.4

17.4 5.2 4.3 3.0 31.0 20.1

8.1 36.8 71.1 5.5 30.0 18.9 38.0 23.8

10.5 . 2.6 . 11.3 6.5 27.0 15.1

21.5 . . . . . . . 34.8

13.1 . . . . . -

leaf leaf fruit leaf stem stem leaf seed fruit leaf leaf leaf leaf leaf leaf fruit leaf inner leaf outer fruit

1.6 3.8 0.7 5,3 16.6 12.5 2.6 3.4 1.1 11.0 1.1 16.6 26.9 0.9 5.8 13.1 2.2 26.5 37.7 3.3

32.9 20.8 7.5 9.4 0.1 -

5.1 6.5 6.1 27.4 19.3 3.8 22.3 1.9 5.1 1.2 4.7 3.2 38.2 3.3 17.3 -

39.5 36.8 20.5 38.2 20.2 40.0 37.2 59.1 65.3 49.5 10.8 10.9 51.0 2.8 2.9 21.9 18.4 5.0 21.5 9.8 17.9

7.6 7.0 26.5 5.4 0.1 0.2 3.7 6.5 . 34.9 3.9 4.9 12,1 . 13.4 20.8 5.9 3.6 14.0

. . . . 3.1 . . . . . . . 64.3 . . 49.7 36,7 31,9 .

OL

Dr

Ref. d

1.64"* - 1.80"* - 1.03" -2.04** 2.35** 2.65** 2.57** 2.16"* 3.40** 2.04'* 3.39** 1.99"* 3.46** -2.63** 0.69 -2.23** - 3.31"* 2.82** 3.40** 2.49** 3.46** 2.55** -2.20"* -3.16"* 2.02** 2.13"* - 1.82"* 1.57* - 1.26' - 1.62"* - 1.48" 2.24** 2.50**

1 25 25 25 1 1 1 1 1 2 2 2 2 10 10 10 10 2 2 2 2 1 23 28 1 1 22 1 26 8 8 1 1

1.36* 2.84** 2.55** - 1.67"*

1 l 1 8

- 1.37" 2.98** 3.24'* 2.57** 2.32** 2.41"* - 1.18" -0.99*

8 1 1 1 1 1 5 14

2.60** 2.57** 1.58" 2.67** 2.96 0.48 - 1.32" -2.46** 3.32** 1.5l* 2.46'* 2.40** -1.17" 2.53** -0.57* 1.85'* 2.47** - 1.71"* - 1.79"* -2.38"* 2.10'*

1 1 1 1 11 11 8 8 1 4 1 1 3 1 15 1 1 20 20 20 1

Cutins

59 Agave americana Agave americana Agave americana Agave americana Allium cepa 64 Bryonia dioiea 6 5 Bryonia dioica 6 6 Chlorophytum elatum 6 7 Citrus aurantifolia 6 8 Citrus deliciosa 6 9 Citrus deliciosa 7 0 Citrus limon 71 Citrus limon 7 2 Citrusparadisi 7 3 Citrus paradisi 7 4 Citrusparadisi 7 5 Citrus paradisi 7 6 Citrus reticulata 7 7 Citrus reticulata 7 8 Citrus sinensis 7 9 Citrus sinensis 8 0 Clematis vitalba 81 Clivia miniata 8 2 Coffea arabica 83 Euonymus europaeus 8 4 Euonymus europaeus 8 5 Ficus elastica 8 6 Gasteria planifolia 8 7 Gossypium hirsutum 8 8 Hordeum vulgare 8 9 Hordeum vulgare 9 0 llex aquifolium 91 Ilex aquifolium 9 2 Irisfoetidissima 9 3 Irisfoetidissima 9 4 Lactuca sativa 9 5 Lactuca sativa 9 6 Lactuca sativa 9 7 Lamium album 9 8 Lycopersicon esculentum 9 9 Lycopersicon esculentum 1 0 0 Malus pumila 101 Maluspumila 1 0 2 Malus pumila 103 Maluspumila 1 0 4 Malus pumila 105 Malus pumila 1 0 6 Malus zumi 1 0 7 Malus zumi 108 Pinus radiata 109 Pinus radiata 1 1 0 Pisum sativum 111 Pisum sativum 1 1 2 Rosa canina 113 Rosmarinus officinalis 114 Salix scouleriana 115 Sanseveria trifasciata 1 1 6 Solanum tuberosum 1 1 7 Spinacia oleracea 118 Spinacia oleracea 119 Tamus communis 120 Tamus comrnunis 121 Triticum aestivum 1 2 2 Triticum aestivum 123 Triticum aestivum 1 2 4 Vitis vinifera 60 61 62 63

(summer) (winter) coat

bran bran

53.2

. . . . . .

. . . .

.

.

. . .

. . . .

. . .

. .

.

.

.

-

.

.

. .

.

. .

. . . .

. . . .

. .

. .

2.4 1.3 2.5 6.2 . . . .

9.2 . . . .

. 0.6 . . . 26.5 13.3 15.5 . .

. 3.9 2.3 . . . 12.1 1.7 0.3 . . . . . 1.1 . . . . . 0.2 15.8

. . . . 35.6 4.2 . . . . 2.9 . . . .

. . . . 18.0 7.7 1.5 0.9 . . . . 5.5 . 0.4 . . 3.2 22.5 .

242 Table

K. Matzke and M. Riederer: Comparative study into cutins and suberins 5. Continued

No. Species b

Material

OIC

DOIC

coOH

DOH

TOH

125 Vitis vinifera 126 Vitis vinifera 127 Vitis vinifera 128 Zea mays 129 Zea mays 130 Zea mays

fruit leaf leaf leaf leaf seed coat

2.2 5.5 35.4 3.0 1.7 2.8

1.0 -

4.4 32.4 32.4 56.9

16.7 21.1 15.4 13.7 13.7 20.0

7.5 3.6 3.4 2.7 7.3

EPO . . . . 18.5 18.5 2.6

C20 .

OL . -

. 8.0 7.8 1.4

. 1.0 3.9 2.7

Dc 2.38** -2.55** 2.54** - 1.74"* - 1.61"* - 1.45"

Ref. d 1 1 1 8 9 8

" OIC,

n-alkanoic acids; DOIC, c~r acids; o)OH, o)-hydroxyalkanoic acids; DOH, dihydroxyalkanoic acids; TOH, trihydroxyalkanoic acids; EPO, epoxyhydroxyalkanoic acids; OL, 1-alkanols; C20, compounds with chain-length > C2o b Systematic names as given by original authors c Discriminant scores calculated from the mass percents by using Eq. 3). Posterior probabilities of group membership are indicated by *, 0.9900 C2o was calculated as a further variable (C20). The initial training set for discriminating cutins and suberins consisted o f eight cases (three cutins, five suberins) which were taken f r o m the results in Tables 1 4 (Table 5, cases 1-8). A stepwise variable selection p r o c e d u r e based on minimizing Wilks' l a m b d a (maxim u m tolerance level: 0.001, m i n i m u m F to enter: 1.00, m i n i m u m F to r e m o v e : 1.00) was applied to choose those variables which significantly c o n t r i b u t e d to the characterization o f cutins and suberins (Norugis 1986). F o u r o u t o f eight variables were selected by this procedure and were subsequently used to calculate the following canonical discriminant function:

showed that a relatively simple equation (Eq. 2) based on a fairly small training set rather effectively distinguishes between cutins and suberins f r o m very diverse sources. F o r the purpose o f expanding the data base the canonical discriminant function was calculated from, all data (initial training and test sets) were merged and another run o f the procedures for variable selection (same criteria as above) and estimating the coefficients o f the discriminant function was performed. N o w , two additional variables ( D O I C and T O H ) were selected. The new canonical discriminant function correctly classified all but three o f the 130 cases in the merged set. The three misclassified cases were "cutin" f r o m oneyear-old stems o f Pinus radiata, g r o w n u n d e r s u m m e r (case n u m b e r 108) and winter (case 109) conditions (Franich and V o l k m a n 1982), respectively, and "cutin" f r o m the non-chalazal region o f the inner seed coat o f grapefruit (case 73; Espelie et al. 1980). F r o m the data (Table 5) it is very likely that the material o f Pinus radiata consisted o f m u c h m o r e suberin than cutin, p r o b a b l y owing to the initiation o f periderm f o r m a t i o n below a superficial cover o f a cuticle. The b i o p o l y m e r f r o m the seed coat o f grapefruit was considered by the authors to be cutin because o f its high content o f polar acids. H o w ever, additional significant a m o u n t s o f long-chain and dicarboxylic acids imply that this b i o p o l y m e r was possibly a mixture o f cutin and suberin. In conclusion, all three outliers must be assumed to be derived f r o m m o r e than a single b i o p o l y m e r source. Thus, these three cases were excluded f r o m further computations. A n o t h e r discriminant e q u a t i o n was fitted to the remaining 127 cases:

D = - 17.58+ 1.10 9 O L + 0 . 3 4 - e 0 O H + + 0 . 2 7 9 C 2 0 - 0.19 9 D O H

Eq. (2)

The discriminant scores calculated f r o m Eq. 2 using the g r o u p means o f each o f the four variables (group centroids) are 11.12 for suberin and - 1 8 . 5 4 for cutin. A p p l y i n g this e q u a t i o n individually to the eight cases included into the analysis gives values o f discriminant scores ranging f r o m 9.71 to 12.77 and f r o m - 18.01 to - 19.02 for suberins and cutins, respectively. All cases o f the training set were classified correctly when Eq. 2 was applied. This discriminant function having been established on a rather n a r r o w d a t a base, it seemed desirable to check its performance. Therefore, a large test set o f data on the c o m p o s i t i o n o f the two biopolymers in question was compiled f r o m literature d a t a on 72 cutins and 50 suberins (Table 5). E q u a t i o n 2 was applied to the test set a n d the discriminant scores were calculated. The m o s t p r o b a b l e g r o u p m e m b e r s h i p o f each case was determined f r o m the discriminant score (for further i n f o r m a t i o n see Norugis 1986). 90.2% o f all cases in the test set were classified correctly (94.0 % o f all suberins and 87.5 % o f all cutins in the set). This result was encouraging because it

D = -2.49+0.14. DOIC+0.045 -TOH+0.042 - C20+ +0.039 9 O L + 0 . 0 1 4 . a ) O H - 0 . 0 1 3 9 D O H Eq. (3) The canonical correlation (in the t w o - g r o u p situation o f discriminant analysis equivalent to the usual Pearson correlation coefficient) is 0.9318 which indicates that

K. Matzke and M. Riederer: Comparative study into cutins and suberins

0.16 n

Cutin

r----] Suberin

0.12

>k 0 cG)

Group centroid.~

0.08

Oq)

m 0.04

0.00

b

I

~-2

'

|

I

II

HHIIH( H

0

Discriminant

i

2

4

Scores

Fig. 3. Frequency distribution of the discriminant scores and group centroids calculated from the discriminant equation (Eq. 3) for the merged data set (Table 5, except cases 73, 108 and 109). Negative scores are indicative of cutins, positive ones of suberins

83.5% of the total variance of discriminant scores are attributable to the differences among the groups. The discriminant scores from Eq. 3 calculated for all 127 cases form two clearly distinct frequency distributions (Fig. 3). All discriminant scores for cutin samples are negative (D at the centroid of the cutin group: -2.23) while those for suberins are positive (D at suberin centroid: 2.91). Since all variables included in Eq. 3 were measured in identical units (mass percent), the effect of the different variables on the discriminant scores and consequently on group membership can be evaluated directly from the coefficients of this equation. All variables with a positive coefficient (DOIC, TOH, C20, OL, o)OH) are variables indicative of suberin while a negative sign marks the variable indicating cutin (DOH). The large negative constant Bo ensures that minor amounts of dibasic, trihydroxy-, o)-hydroxymonobasic or long-chain alkanoic acids or of 1-alkanols sometimes occurring also in cutin depolymerisates will not result in a misclassification of these samples. Applying Eq. 3 to the total data base (cases 73, 108 and 109 excluded) results in a 100% correct classification of the 127 cases. Success in classifying cases, however, is only one criterion for the goodness of a classification rule. Another one, which is equally important for the practical usefulness of the rule is the statistical reliability of group assignments. The probability that a case with a discriminant score of D belongs to one of the two groups can be estimated by Bayes' rule (Norugis 1986). When group membership is unknown, what is needed is an estimate of how likely membership in one of the two groups is, given the available information. This likeliness is called the posterior probability. When applying Eq. 3

243

to the total data base, the posterior probabilities of 97.6% of the cases were > 0.990 (Table 5). Only three cases were classified with posterior probabilities below this limit. Thus, Eq. 3 is both a very precise and reliable classification rule. This is surprising and reassuring in view of the diversity of the materials included in this study and the differences in the analytical methods used to produce the data. Robustness and bias, i.e. the impact single cases have on the classification rule, are further criteria which must be considered. Both were evaluated by a Tukey's jackknife-like procedure: using the variables included in Eq. 3, 127 different discriminant equations were calculated from the merged data set with each of the cases left out once in turn. Subsequently, the discriminant score was calculated for the case which had been left out when estimating the coefficients of the discriminant function. All left-out cases were classified correctly which indicated (i) that none of the cases exerted an undue influence on the discriminant function, and (ii) that the classification rule based on the variables chosen by a stepwise selection procedure using Wilks' lambda was fairly robust. Thus, Eq. 3 seems to be suitable for the inarbitrary classification of cutins and suberins according to quantitative data on the monomeric composition of depolymerisates of lipid biopolymers. The classification rule expressed by Eq. 3 generally confirms the qualitative rules derived from more limited data sets (Kolattukudy and Agrawal 1974; Holloway 1983; Kolattukudy 1984). It had been proposed that suberins were sufficiently characterized by the presence of significant proportions of dicarboxylic and very longchain (C2o to C3o) acids. Holloway (1983) considered the proportion of monomers with chain-lengths > C18 as the most important single criterion indicative of suberin. In fact, 95.3 % of all cases in Table 5 were classified correctly when C20 was used as the only variable. When DOIC was also included, 98.4% of the 127 cases were assigned to their actual group. The reliability of a simplified classification rule based only on C20, however, is significantly reduced when compared to that of Eq. 3. Using only a single variable, the posterior probabilities for classification are generally much lower. Only 41% of all cases are assigned to one of the two groups with a probability > 0.990 (69 % > 0.950) which contrasts sharply with the excellent significance of the classification based on Eq. 3 (see above). Moreover, discriminant analysis also revealed some deficiencies of formerly proposed classification criteria. Large amounts of polar compounds including epoxy-, dihydroxy-, and trihydroxyalkanoic acids had been frequently suggested as distinctive characteristics for distinguishing between both types of biopolymers (Kolattukudy and Agrawal 1974; Espelie et al. 1980; Kolattukudy 1984). However, our results clearly show that only the presence of dihydroxyhexadecanoic acids in depolymerisates is a useful single indicator for cutin. Additionally, the results obtained in our comparative study suggest that the differences between the monomeric composition of the aliphatic portions of cutin and suberin are much less clear cut than previously assumed.

244

K. Matzke and M. Riederer: Comparative study into cutins and suberins

A reliable classification of depolymerisates from the two lipid polymers is only possible when based on a multivariate rule taking into account several qualitative and quantitative properties. As a consequence of the principal similarity of the aliphatic portions ofcutin and suberin a critical reevaluation of the methods used up to now for investigating the structures of both biopolymers seems to be appropriate. Unless radically new procedures will be developed in the future, the current methods relying on rather crude depolymerization procedures appear to have reached their limitations. They indeed are valuable tools for analyzing the qualitative and quantitative composition of the aliphatic portions o f these polymers but are unable to allow deeper insights into the modes of cross-linking and the physical structures of cutin and suberin in situ. A major drawback of these methods is that they cannot provide any information on the interrelationships between aliphatic monomers and the non-lipid components of cuticles and periderms like polysaccharides or amino and phenolic acids. Schreiber and Sch6nherr (1990) recently have reported that the courses of the thermal expansion of extracted cuticular membranes (matrix membranes) and of pure cutin preparations were markedly different. They concluded that the cuticular matrix is a composite material whose physical properties are influenced in a non-additive way by the components present. Special importance has been attributed to the effects of cutin/ polysaccharide interactions. Therefore, the results from this study unfortunately cannot be used to resolve the continuing debate on the validity o f the structural formulas proposed for cutin and suberin (Kolattukudy 1980a, 1980b, 1981, 1984). Those proposals should be considered rather as illustrations of cross-linking reactions being possible within the polymers than as representations of their actual physical structures. The same is true for the discussion concerning the role of phenolic constituents in both suberin and cutin. This question cannot be answered as long as destructive methods of investigation are used. All depolymerization procedures will inevitably and indiscriminately release considerable amounts of phenolics from periderms. Owing to the heterogeneity of this material there is no way of deciding on the actual origin of these phenolics. Besides suberin, cell walls and lignified parts must also be considered as possible sources of covalently b o u n d phenolics. Progress in elucidating the physical structures of cutins and suberins seems only to be achievable if nondestructive and more direct methods of investigation will supplement the current knowledge on the qualitative and quantitative composition of these biopolymers. Initial promising results have been obtained by applying advanced techniques of solid-state nuclear-magneticresonance spectroscopy to cutin samples (Zlotnik-Mazori and Stark 1988; G a r b o w and Stark 1990). The authors are indebted to Drs. J. Winkler and H. Krause (Laboratorium ffir Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universitfit Mfinchen, Garching, FRG) for performing capillary gas chromatography-mass

spectrometry and their valuable help in the identification of cutin and suberin constituents. The work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bayerisches Staatsministerium ffir Unterricht, Kultus, Wissenschaft und Kunst.

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