Epicuticular waxes of eceriferum mutants of Arabidopsis thaliana

Phytochemzstry, Vol. 33, No. 4, pp. 851 855, 1993

003 l-9422/93

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EPICUTICULAR

WAXES OF ECERIFERUM ARABIDOPSIS THALIANA

MUTANTS OF

ABDELALIHANNOUFA,JOHN MCNEVIN and BERTRANDLEMIEUX* Department of Biology, York University, 4700 Keele Street, North-York, Ontario, Canada M3J-lP3 (Received in revised form 22 November

Key Word Index-Arabidopsis composition.

thaliana; Cruciferae;

1992)

eceriferum

mutants;

epicuticular

waxes;

_ Abstract-The principal surface lipids of Arabidopsis are n-nonacosane, 14- and 15-nonacosanol, 15-nonacosanone, C,,-C,, free fatty acids, C,,-C,, primary alcohols and C 26-C& aldehydes. We have analysed the chemical composition of the epicuticular wax of 10 Arabidopsis thaliana eceriferum (cer) mutants. One of the mutants (cer2) is blocked in the elongation of octacosanoic acid and accumulates large amounts of primary alcohols and fatty acids in its epicuticular wax. The surface lipid composition of another mutant (cer4) appears to be defective in the production of primary alcohols and accumulates elevated levels of epicuticular alkanes. We have also identified a mutant (cerl) which accumulates epicuticular aldehydes and is severely deficient in alkanes on its surface. Seven other mutants had only slightly different epicuticular wax compositions compared to those of wild type plants.

INTRODUCTION Mutant plants with altered wax chemical composition and/or ultrastructure have been found in many plant species such as Brassica oleracea [l-3], B. napus [4], Zea mays [S], Hordeum vulgare [6] and Pisum sativum [7]. However, the recent isolation of eceriferum (cer) mutants in Arabidopsis thaliana [S] opens new possibilities for the isolation of the genes which regulate wax biosynthesis. Indeed, the application of chromosome walking to the physical mapping of the Arabidopsis genome [9, lo] has already led to the map-based cloning of a gene which regulates the level of lipid unsaturation in membranes [ 111. Moreover, the proven effectiveness of T-DNA insertion mutagenesis in Arabidopsis [12] will make this technique the approach of choice to clone CER genes because the known Arabidopsis mutants with metabolic defects in wax biosynthesis are readily detectable by the naked eye [S]. Epicuticular waxes are composed of very long chain lipids [ 131 which are derived from the elongation of C,,-C,, fatty acids through sequential additions of two carbon units [6]. Some of the resulting long chain fatty acids are reduced to aldehydes which can be reduced to primary alcohols [14] which can be esterified to C,,-C2,, fatty acids [6]. Alkanes are believed to be derived by either the decarboxylation of fatty acids to alkanes [6] or the decarbonylation of fatty aldehydes to alkanes [15], whereas secondary alcohols and ketones may arise by the sequential oxidation of alkanes [6]. Twenty-one different loci are implicated in the formation of epicuticular wax in Arabidopsis (i.e. cerl-cer20 *Author to whom correspondence should be addressed.

and tt-5) [S]. The wax blooms on the surface of wild-type Arabidopsis plants exist predominantly in two shapes, plates and large tube structures [8]. Similar tube-like structures on the surface of Brassica sp. are believed to be caused by mixtures of symmetrical secondary alcohols and ketones [16]. Primary alcohols, on the other hand, tend to crystallize into plates while aldehydes lead to the production of filamentous wax types [17]. In this study, we present a chemical analysis of the epicuticular wax components of 10 cer mutants isolated from A. thaliana by Koornneef et al. [S]. RESULTSAND

DISCUSSION

Overall chemical composition

The chemical compositions of the epicuticular wax extracts obtained from 10 cer mutants of A. thaliana are shown in Table 1. In the wild-type plants, alkanes, secondary alcohols and ketones are more abundant than those classes derived from the elongation/reduction pathway. The epicuticular waxes of the cerl and cer2 mutants, however, are characterized by an abundance of compounds derived from the elongation/reduction pathway, namely, fatty acids, aldehydes and primary alcohols. At the other end of the spectrum of chemical phenotypes, the surface of the cer4 mutant was found to be almost completely lacking in primary alcohols. A more detailed analysis of the epicuticular wax composition of five of the cer mutants is given in Table 2. Although the chemical composition of the surface lipids isolated from the cer3 and cer15 mutants is not as markedly different from the wild-type as that of the aforementioned mutants, they 851

852

A. HANNOUFAet ul.

Table 1. Amounts (in pgg-‘)

Lines Landsberg cerl cer2 cer3 cer4 cerl5 cerl6 cerl7

cerl8 cerl9 cer20

of epicuticular wax components on the surface of Arahidopsis wild-type and on 10 eeerzferum (cer) mutants

Fatty acids 63.3 + 3.1 47.5 +2.4 161.3+10.6 17.51*0.75 44.55 + 4.03 168.4 + 5.4 46.6 + 3.4 64.5 + 2.8 26.3 + 2.8 81.5*6.5 57.7 * 2.4

Primary alcohols

Aldehydes

23Ok 8.9 67.9 + 2.0 313.6+ 14.8 112.4+4.1 10.27+0.74 31.8k2.1 82.lk4.6 73.6k2.2 104.7*7.5 125.126.8 125.6f 4.4

103.4+ 3.2 79.5 * 3.0 040~0.01 1.50+0.07 131&8 47.8 f 2.2 45.2 & 1.9 57.9f 1.1 32.9k2.6 60.3 + 3.2 51.4* 1.0

Alkanes

Secondary alcohols

Ketones

374+ 13.1 9.2kO.3 25.3i 1.1 26.4 +0.8 421+21 164.6k5.2 165t5 194+4 1552 10 80.5+1.5 3047f54

81&l 2.5,O.l 8.5 + 0.9 5.7 +0.2 143+6 61.8 t3.1 185&4 75.4 + 1.7 130,4 33.3 kO.9 33.3kO.5

281+11 15.0_+0.5 2.5 & 0.3 36.8 + 1.3 301 f4 127.7k6.3 39g+5 19624 322&7 96.5 + 3.2 101*3

Values are the mean ? s.d. (n = 5). Table 2. Amount of epicuticular wax compounds (wt %) on the surface of five Arabidopsis cer mutants Compound Tetradecanoic acid Pentadecanoic acid Hexadecenoic acid Hexadecanoic acid Octadecenoic acid Octadecanoic acid Icosanoic acid n-Pentacosane Docosanoic acid n-Heptacosane I-Tetracosanol Tetracosanoic acid 13- and l~Heptacosano1 13-Heptacosanone n-Nonacosane Hexacosanal 1-Hexacosanol Octacosanal Hexacosanoic acid 14- and ~5-Nona~~nol 15-Nonacosanone n-Hentriacontane I-Octacosanol Octacosanoic acid Triacontanal I-Triacontanol ~-Amy~n Triacontanoic acid

Landsberg 0.13

tr 0.12 0.42 0.18 0.18 tr nd 0.11 0.27 0.29 0.5 nd nd 32.74 0.27 2.80 0.30 1.55 7.15 24.80 tr 10.68 0.55 8.56 6.53 0.04 1.87

cerl

tr* tr tr 0.50 0.32 0.32 tr nd 0.27

nd 0.72 0.77 nd nd 4.14 0.23 5.72 nd 3.02 1.13 6.76 nd 6.31 2.25 35.59 0.18 tr 13.96

cer2

cer3

cer 4

cerl.5

tr tr tr 0.27 0.45 0.18 tr 0.41 0.06 3.57 0.35 6.93 1.66 0.49 0.96 0.08 38.28 nd 18.75 tr tr nd 21.88 4.57 tr 0.74 tr 0.27

tr tr tr 0.59 0.84 0.45 tr nd 0.15 0.20 0.20 0.50 nd nd 12.87 tr 3.17 nd 0.74 2.82 18.22 tr 9.36 3.7t 0.74 42.92 tr

tr tr 0.05 0.29 0.14 0.14 0.11 0.18 tr 0.35 nd 0.25 nd nd 39.01 0.27 0.13 0.50 I.24 13.61 28.64 0.47 nd tr 11.70 0.85 0.15 2.03

tr tr tr 1.19 2.57 3.83 12.87 nd tr nd 0.22

i 68

1.66 nd nd 25.14 tr 0.88 tr tr 10.25 21.18 2.16 2.59 tr 7.93

1.59 tr 5.80

*tr, Less than 0.2 pgg- r. nd, Not detected by mass spectroscopy.

nevertheless appear to have altered chain length distributions compared to those found on the surface of wild-type plants (Table 2). The detailed chemical composition of five other mutants (cerl6-cer20) is listed in Table 3. These mutants display much less variation in the partitioning of carbon between the two pathways of wax biosynthesis; however, all of these mutants have reduced amounts of epicuticular wax when compared to wild-type plants.

The chemical composition of the wax of the cerl mutant (Table 1) indicates that there is a substantial accumulation of aldehydes, acids and primary alcohols which could be consistent with either a partial block in the decarbonylation of aldehydes to alkanes or the decarboxylation of fatty acids to aikanes. The chemical composition of the surface wax of the Arahidopsis cerl mutant

Epicuticular waxes of Arabidopsis thaliana mutants

853

Table 3. Amounts of epicuticular wax compounds (wt %) on the surface of five Arabidopsis cm mutants Compound

Landsberg

cerl6

cerl7

cerl8

cerl9

cer20

Tetradecanoic acid Pentadecanoic acid Hexadecenoic acid Hexadecanoic acid Octadecenoic acid Octadecanoic acid Icosanoic acid Docosanoic acid n-Heptacosane l-Tetracosanol Tetracosanoic acid n-Nonacosane Hexacosanal I-Hexacosanol Octacosanal Hexacosanoic acid 14- and 15-Nonacosanol 15-Nonacosanone 1-Octacosanol Octacosanoic acid Triacontanal 1-Triacontanol

0.13 tr* 0.12 0.42 0.18 0.18 tr 0.11 0.27 0.29 0.50 32.74 0.27 2.80 0.30 1.55 7.15 24.80 10.68 0.55 8.56 6.53 0.04 1.87

0.10 tr tr 0.14 0.62 0.10 1.59 tr 0.10 0.10 0.33 17.81 0.08 1.13 0.76 0.54 20.09 43.21 4.42 0.65 4.06 3.27 tr 0.99

0.17 tr tr 0.17 0.97 0.14 2.62 tr tr 0.14 0.41 29.35 0.15 1.12 1.32 0.77 11.41 29.65 4.36 1.89 7.29 5.52 tr 2.63

0.04

0.60

tr tr 0.12 0.05 0.06 0.06 tr 0.26 0.23 0.35 19.84 0.12 2.52 0.32 0.92 16.86 41.76 7.12 1.10 3.83 3.71 tr 0.71

tr tr 0.50 3.10 0.47 tr 0.77 1.42 1.40 0.65 15.89 3.08 8.99 4.58 6.17 7.16 20.75 13.72 2.37 5.31 2.80 tr 2.90

0.16 tr 0.13 0.33 0.25 0.10 tr tr 1.44 0.46 1.44 43.77 0.33 3.93 0.31 4.58 4.94 14.99 10.61 0.76 6.99 3.64 tr 0.82

/I-Amyrin Triacontanoic acid *tr, Less than 0.2 pgg-‘.

is similar to that of the gi5 mutant of maize, which is believed to be the result of a block in the reduction of fatty acids [18]. The Rigo mutant of B. nupus [4] and the wsp mutant of pea [ 191 both accumulate elevated amounts of aldehydes on their surfaces. Unlike maize surface waxes, alkanes are a major component of the epicuticular wax of Arabidopsis, thus, cerl appears to have more influence on the production of epicuticular alkanes than does g/5. The pronounced reduction in nonacosane in cerl (Table 2) and the absence of all other alkanes indicates that this locus may encode an aldehyde decarbonylase which is responsible for the production of epicuticular hydrocarbons [14]. Fatty aldehyde decarbonylase activity has been detected in particulate preparations from the cell wall of Pisum sativum [14]. Labelling studies with cell wall particulate fractions of the cerl mutant should display a reduced decarbonylase activity in this line when compared to the activity obtained with extracts from wild-type plants. Recent results obtained with Botryococcus braunii microsomal preparations may lead to the purification of an aldehyde decarbonylase and the cloning of the gene which encodes this cobalt-porphyrin enzyme [20]. Mutant with reduced levels of primary alcohols The wax of the cer4 mutant is rich in triacontanoic acid when compared to that of the Landsberg line from which the mutant was derived (Table 2). The very sharp reduction in primary alcohols (Table 1) in the wax of this

mutant indicates that this gene may encode a fatty aldehyde reductase. Fatty aldehydes and fatty acid reductase activities have been resolved by protein fractionation and are thus likely to be encoded by separate genes in plants [15]. The block in the synthesis of primary alcohols in this mutant resulted in an increase in fatty aldehydes which could then be decarbonylated to alkanes. Thus, the cer4 mutant has higher amounts of surface lipids derived from alkanes (i.e. secondary alcohols and ketones) than does the wild-type (Table 1). Mutant with shorter carbon chain leitgths

The elevated content of fatty acids and primary alcohols (Table 1) occurring on the cer2 mutant is best explained by a complete block in the elongation of octacosanoic acid to triacontanoic acid (Table 2). The chemical composition of cer2 is similar to that observed in the B. oleracea glossy1 [2] in that the chain length distribution of secondary alcohols is shifted towards 13and 1Cheptacosanol in the epicuticular wax of both mutants. Similar mutants have also been characterized in pea (wa) [7J in 2. nrays [Zl] and H. vulgare [6]. Moreover, the partial purification of an epidermal fatty acid elongation complex from leek, would support these genetic results [22]. The small amount of n-nonacosane in the wax of the cer2 mutant would suggest that the elongation-decarbonylation pathway has a separate fatty acid elongation system as has been suggested by other authors [6,23]. Alternatively, it is possible that the

A. HANNOLJPAet al.

854

mutant allele of the cer2 locus is leaky or that the elongase is encoded by a gene family. Mutants with reductions in overall wax biosynthesis

The chemical composition of seven of the mutants examined (cer3, cerlS-cer20) could not be explained in terms of simple blocks in known biosynthetic steps. Several glossy mutants in maize also have a similar defect in the production of the epicuticular wax layer rather than in the production of specific epicuticular wax compounds [24]. The accumulation of shorter chain length fatty acids on the surface of the cerl5 mutant suggests that it is partially defective in the early stages of fatty acid elongation. The cerl6, cerl7 and cerl9 mutants are similarly affected, although the accumulation is far less dramatic. The ratio of secondary alcohol to ketone of the surface of the cerl6 and cerl8 mutants also differs from that found on the surface of the wild-type (Table 1).

plants in 4 ml of CHCI, for 30 sec. The extracts were evapd under a N, stream and the dried wax residues prepd for GC by silylation with bis-(trimethylsilyl)trifluoroacetamide (BSTFA) at 120” for 10 min. Excess reagent was evapd under a N, stream. Samples were redissolved in CHCl, for FID-GC analysis on a 530 pm o.d. DB-5 column. This instrument was operated with a N, flow rate of 4 mlmin- ’ at an initial temp. of 170” followed by a temp. prog. of 5” min- ’ to 3 lo” for 20 min. This temp. prog. separates all the wax components of Arabidopsis except for wax esters. Quantitations were based on an int. standard of nonadecane. Acknowledgements-This

work was supported by NSERC grant OGPOO46649 to B.L. We thank Karl Espelie (University of Georgia) for assistance with the identification of epicuticular wax compounds and Louisa Mak for technical assistance. REFERENCES

CONCLUSION

Although our results show that Arabidopsis cer mutants are similar to wax biosynthesis mutants isolated from other plants, the fact that the map positions of 10 of the Arabidopsis cer loci are already known [25] offers a unique opportunity to isolate the genes at these loci by map-based cloning. Moreover, we have recently isolated a collection of 13 T-DNA tagged cer mutants from 13 000 Arabidopsis transformants generated by the seed cocultivation method [12]. Our genetic characterization of these mutant lines indicates that five different CER genes have been tagged by T-DNA insertions; these correspond to the genes at the cerl--cer4 loci as well as a previously unknown wax biosynthesis locus [26]. As epicuticular waxes are believed to play a significant role in plant-insect [27] and insect herbivore-parasitoidpredator interactions [28], the molecular cloning of the cer genes may eventually lead to the development of genetically engineered plants with altered surface lipid composition which may have higher levels of resistance to insect pests or be amenable to integrated pest management approaches. EXPERIMENTAL

Plant material. Seeds of the Landsberg erecta ecotype of A. thaliana (L.) Heynh. were obtained from Dr Chris

State University) while seeds of Arabidopsis eceriferum [8] were obtained from Dr Koornneef (Wageningen Agricultural University). Most of these mutants are available from the Arabidopsis Genetic Stock Center at Ohio State University. Plants were grown in chambers at 25” under continuous illumination in Growers Choice artificial soil mix (14 : 3 : 3 of peatmoss:vermiculite:perlite) which was supplemented with Arabidopsis mineral nutrients as described in ref. [26]. Wax analysis. Epicuticular waxes were isolated from individual plants by immersing stems of 6-week-old

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