Download 109 GENES OF ARABIDOPSIS THALIANA INVOLVED IN WAX

Analele ştiinţifice ale Universităţii “Al. I. Cuza” Iaşi
Tomul LIV, fasc. 2, s.II a. Biologie vegetală, 2008
GENES OF ARABIDOPSIS THALIANA INVOLVED IN WAX METABOLISM
ROXANA-IULIANA TEODOR*, AL. YEPHREMOV*
Abstract: The aerial surfaces of land plants are covered with cuticle that acts as a barrier providing
protection against water loss, pathogen invasion and other environmental aggressions. Besides physical
and chemical barriers, such as a waxy cuticle, plant defense mechanisms also involve a coordinated
activation of cellular responses to limit damage.
The eceriferum (cer) mutants of Arabidopsis define multiple genes required for various steps in cuticular
wax biosynthesis, transport and regulation of lipid-related pathways. Although the basic biochemistry of
wax production has been elucidated, very little is known about its regulation and its contribution to the
natural immunity. This review presents recently cloned wax biosynthetic genes and discusses regulatory
aspects of wax biosynthesis.
Key words: wax, Arabidopsis mutants
Role of cuticle for plants
A distinctive characteristic of all epidermal cell types is the presence of cuticle
covering their outer surface as a continuous lipophilic layer, which forms a barrier over the
aerial organs of land plants during their primary stages of development [7; 14].
Plant cuticle is formed mainly of cutin, a polyester of hydroxy and hydroxy-epoxy
fatty acid derivatives generated from cellular fatty acids [11], embedded in and covered
with a complex mixture of highly hydrophobic soluble materials (aliphatic compounds
comprised mainly of C24-C34 alkanes, alcohols and ketones) called cuticular waxes, as
well as other minor compounds, of an extremely diverse nature [30]. As they are physically
very closely associated, it is difficult to distinguish between the relative contribution of the
cutin matrix and that of cuticular waxes to the physical properties and the biological roles
of the cuticle.
However, in biochemical experiments, the cutin and cuticular waxes are usually
considered and analyzed separately, due to the soluble properties of the waxes versus the
cutin polymer, which remains insoluble. Such a structure gives the cuticle a set of highly
protective features and these were studied initially as to limiting nonstomatal water loss and
gaseous exchanges, controlling the absorption of lipophilic compounds, and providing
mechanical strength and viscoelastic properties [2; 25].
Additionally, the cuticle also functions in normal plant developmental processes,
including the prevention of postgenital organ fusion and pollen–pistil interactions [17; 28],
as well as protecting the plant from biotic and non-biotic environmental stress factors [27].
Pruitt et al. [24] and Sieber et al. [28] have suggested that cuticle permeability also
influences cell-to-cell communication by enhancing or attenuating the passage of signal
molecules. For example, such signals could be required for organ adhesion, when they
*
Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, Cologne,
Germany
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would be moving across the cuticle, or for mediating signaling between trichomes and
stomata, when moving within the developing epidermis [18; 13].
Arabidopsis mutants offer information on the regulation of cuticle development
Mutants with deficient or altered wax coatings have been identified due to their
nonglaucous or glossy phenotype. Thus, there are no reports of mutants that lack wax
completely, indicating the vital function that waxes play for the normal development of
plants.
On the other hand, changes in a minor wax component are less likely to lead to a
clearly discernable phenotype and therefor have not been reported. Similarly, plants that
overproduce waxes are not easily detected by visual screenings.
In Arabidopsis, as well as in other species, the mutants that have been identified to
be defective in wax and/or cutin formation facilitated the identification of enzymes
associated with the cutin and wax pathways. Some of the enzymes catalyzing various steps
in the wax pathway have been characterized or their function has been proposed based on
the phenotype of the corresponding mutants.
The identification of eceriferum (cer) mutant lines described mainly by Koornneef
et al. [12] and by McNevin et al. [20] has led to the isolation and characterization of various
genes associated with cuticular wax metabolism in Arabidopsis. CER1 intended to encode
an aldehyde decarbonylase [1]. Several genes playing a role in the fatty acid elongation
pathway that generates very long chain fatty acid (VLCFA) wax precursors have also been
characterized. They include the FATTY ACID ELONGATION1 homologs (FAE1) [9],
FIDDLEHEAD (FDH) [38; 24], 3-KETOACYL-CoA SYNTHASE [33], CUT1/CER6, and
CER60 [21; 5]. CER6 has been suggested to be the key condensing enzyme for wax
biosynthesis in Arabidopsis, due to its expression throughout all stages of stem and leaf
development, as well as in the inflorescence [8]. CER2 encodes a CoA-dependent
acyltransferase, a component of the fatty acid elongase complex, apparently located in the
nucleus [37; 14]. The cer2 mutant shows reduced levels of the decarbonylation pathway
products and it accumulates C26 and C28 acyl groups, primary alcohols, and wax esters but
the precise function of the gene is still unknown. Furthermore, its nuclear localization is
very intriguing for a protein of the fatty acid elongase complex. Many of the cer mutants
remain still to be characterized and the isolation of their corresponding genes might bring
valuable information on the mechanisms of wax metabolism.
Several reports have also provided insights into the biosynthesis of cutin monomers
in plants. Chen et al. [3] reported the isolation of the WAX2 gene and showed that the
protein it encodes for has 32% similarity to CER1 and contains certain regions with
homology to sterol desaturases and short-chain dehydrogenases/reductases. It was
suggested therefore that WAX2 plays a metabolic role in both wax and cutin synthesis, thus
pointing to a link between wax and cutin metabolism. ADHESION OF CALYX
EDGES/HOTHEAD (ACE/HTH) is proposed to be an oxidase catalyzing the formation of
dioic acids from ω-hydroxy acyl-CoAs [13; 16]. The Arabidopsis LACERATE (LCR) gene
[36] encodes a cytochrome P450; enzyme activity assays using the recombinant LCR
protein showed that it could efficiently catalyze the formation of ω-hydroxy fatty acids
(ranging from C12 to C18:1). Expression of LCR gene is predominant in inflorescence and
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siliques, as well as in roots and young seedling tissue and it is the first cytochrome P450 ωhydroxylase for which a mutant has been isolated. Results of microarray analysis conducted
in our group (Yephremov et al., unpublished data) on three independent cuticular mutants
has revealed that a palmytoil protein thyoesterase (PPT) is almost ten fold up-regulated in
three mutants, as compared to wild type. In humans, PPT is a lysosomal long-chain fatty
acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins,
and the defective enzyme causes infantile neuronal ceroid lipofuscinosis, a recessive
hereditary neuro-degenerative disorder [34]. In plants, acyl-acyl carrier protein (ACP)
thioesterases play an essential role in chain termination during de novo fatty acid synthesis
and in the channeling of carbon flux between the lipid biosynthesis pathways [10].
Epidermal differentiation and implicitly cuticle formation is essential for the general
development of the whole plant, starting from the very early embryo stage. This fact is
supported by the characterization of the abnormal leaf shape 1 (ale1) mutant of
Arabidopsis, which shows impaired cuticle formation, adhesion of endosperm and embryo,
as well as fusion of cotyledons and leaves. The corresponding ALE1 gene encodes a
member of the subtilisin-like serine protease family and it is preferentially expressed during
seed development, showing a weak transcript expression in young embryo and a strong one
within the endosperm cells closely surrounding the developing embryo [32]. Three
aminoacid residues (aspartic acid, histidine and serine) are consistently conserved in the
catalytic regions of subtilisin-like serin proteases.
In animals, such proteases activate precursors of hormones, growth factors, or
receptors involved in the control of various developmental processes, including embryonic
patterning and proper epidermal differentiation. Although many members of this family of
proteases were reported in plants [29; 26], little is known, with few exceptions, about their
precise role. In addition to the developmental factors controling the synthesis of cuticular
lipids, environmental signals such as light intensity, photoperiod [19; 35], humidity [31],
chilling [22; 23] and seasonal variation [6; 4] have also been shown to ifluence wax
biosynthesis. In 1984, Sutter [31] described the dramatic response of wax production to
environmental cues, during tissue culture, observing that when the relative humidity is
high, wax production is low. When tissue-culture-grown plants are transfered to an
environment with less humidity (growth chabinet or greenhouse), production of wax is
stimulated and within a rather short period of time of a few days only, the plant synthesizes
a complete protective layer of wax.
Conclusion
The fact that cuticular wax is ubiquitously present is testimony to its essential role in
the adaptation of plants to the aerial environment, with all its implications. On the other
hand, the fact that environmental cues have an influence upon wax composition and
quantity is evidence that wax production is an actively regulated process.
An active regulatory netword is indicated also by the high diversity of proteins that
have been shown, through the Arabidopsis mutants, to be involved in the process of wax
biosynthesis. Although the biosynthesis of plant cuticular components has been studied for
over four decades, we still know little about the factors regulating the partitioning of fatty
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acid precursors and the synthesis of waxes with the synthesis of cutin and with other
cuticular compounds. However, the cloning of wax biosynthetic genes and the further
characterization of the respective proteins promises to bring valuable insights into the very
deep regulatory mechanisms of wax and cuticle development.
REFERENCES
1.
2.
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5.
6.
7.
8.
9.
10.
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12.
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14.
15.
16.
17.
18.
AARTS, M.G.M., KEIJZER, C.J., STIEKEMA, W.J., and PEREIRA, A., 1995 - Molecular
Characterization of the CER1 Gene of Arabidopsis Involved in Epicuticular Wax Biosynthesis and Pollen
Fertility. The Plant Cell Online, 7: 2115-2127.
BAKER, C.J., MCCORMICK, S.L., and BATEMAN, D.F., 1982 - Effects of Purified Cutin Esterase Upon
the Permeability and Mechanical Strength of Cutin Membranes. Phytopathology ,72: 420-423.
CHEN, X., GOODWIN, S.M., BOROFF, V.L., LIU, X., and JENKS, M.A., 2003 - Cloning and
Characterization of the WAX2 Gene of Arabidopsis Involved in Cuticle Membrane and Wax Production
Article, publication date, and citation information can be found at www. plantcell. org/cgi/doi/10.1105/tpc.
010926. The Plant Cell Online, 15: 1170-1185.
FAINI, F., LABBÉ, C., AND COLL, J., 1999 - Seasonal changes in chemical composition of epicuticular
waxes from the leaves of Baccharis linearis. Biochemical Systematics and Ecology, 27: 673-679.
FIEBIG, A., MAYFIELD, J.A., MILEY, N.L., CHAU, S., FISCHER, R.L., AND PREUSS, D., 2000 Alterations in CER6, a Gene Identical to CUT1, Differentially Affect Long-Chain Lipid Content on the
Surface of Pollen and Stems. The Plant Cell Online, 12: 2001-2008.
GÜLZ, P.G., AND MÜLLER, E., 1992 - Seasonal variation in the composition of epicuticular waxes of
Quercus robur leaves. Zeitschrift für Naturforschung. C. A journal of biosciences, 47: 800-806.
HOLLOWAY, P.J. (1982). Structure and histochemistry of plant cuticular membranes: an overview.
HOOKER, T.S., MILLAR, A.A., AND KUNST, L., 2002 - Significance of the Expression of the CER6
Condensing Enzyme for Cuticular Wax Production in Arabidopsis. Plant Physiology, 129: 1568-1580.
JAMES, D.W.J., LIM, E., KELLER, J., PLOOY, I., RALSTON, E., AND DOONER, H.K., 1995 - Directed
Tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) Gene with the Maize Transposon
Activator. The Plant Cell Online 7, 309-319.
JONES, A., DAVIES, H.M., AND VOELKER, T.A., 1995 - Palmitoyl-Acyl Carrier Protein (ACP)
Thioesterase and the Evolutionary Origin of Plant Acyl-ACP Thioesterases. The Plant Cell Online, 7: 359371.
KOLATTUKUDY, P.E., 2001 - Polyesters in higher plants. Advances in biochemical engineering,
biotechnology, 71, 1-49.
KOORNNEEF, M., HANHART, C.J., AND THIEL, F., 1989 - A Genetic and Phenotypic Description of
Eceriferum (cer) Mutants in Arabidopsis thaliana. Journal of Heredity, 80: 118.
KROLIKOWSKI, K.A., VICTOR, J.L., WAGLER, T.N., LOLLE, S.J., AND PRUITT, R.E., 2003 Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD. The Plant Journal, 35: 501511.
KUNST, L., and SAMUELS, A.L., 2003 - Biosynthesis and secretion of plant cuticular wax. Prog. Lipid
Res, 42: 51-80.
KUNST, L., SAMUELS, A.L., AND JETTER, R., 2005 - The plant cuticle: formation and structure of
epidermal surfaces. (Oxford,UK: Blackwell).
KURDYUKOV, S., FAUST, A., TRENKAMP, S., BÄR, S., FRANKE, R., EFREMOVA, N., TIETJEN,
K., SCHREIBER, L., SAEDLER, H., AND YEPHREMOV, A., 2006 - Genetic and biochemical evidence
for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and
formation of extracellular matrix. Planta, 224: 315 - 329.
LOLLE, S.J., HSU, W., AND PRUITT, R.E., 1998 - Genetic Analysis of Organ Fusion in Arabidopsis
thaliana. Genetics, 149: 607-619.
LOLLE, S.J., BERLYN, G.P., ENGSTROM, E.M., KROLIKOWSKI, K.A., REITER, W.D., AND PRUITT,
R.E., 1997 - Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for
the epidermal cell wall and cuticle. Dev. Biol, 189: 311–321.
112
19. MACEY, M.J.K., 1970 - The effect of light on wax synthesis in leaves of Brassica oleracea. Phytochemistry
9: 757-761.
20. MCNEVIN, J.P., WOODWARD, W., HANNOUFA, A., FELDMANN, K.A., AND LEMIEUX, B., 1993Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis
thaliana. Genome, 36: 610-618.
21. MILLAR, A.A., CLEMENS, S., ZACHGO, S., GIBLIN, E.M., TAYLOR, D.C., AND KUNST, L., 1999 CUT1, an Arabidopsis Gene Required for Cuticular Wax Biosynthesis and Pollen Fertility, Encodes a VeryLong-Chain Fatty Acid Condensing Enzyme. The Plant Cell Online, 11: 825-838.
22. NORDBY, H.E., AND MCDONALD, R.E., 1991 - Relationship of epicuticular wax composition of
grapefruit to chilling injury. Journal of Agricultural and Food Chemistry, 39: 957-962.
23. NORDBY, H.E., AND MCDONALD, R.E., 1995 - Variations in Chilling Injury and Epicuticular Wax
Composition of White Grapefruit with Canopy Position and Fruit Development during the Season. Journal of
Agricultural and Food Chemistry, 43: 1828-1833.
24. PRUITT, R.E., VIELLE-CALZADA, J.P., PLOENSE, S.E., GROSSNIKLAUS, U., AND LOLLE, S.J., 2000
- FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative
lipid biosynthetic enzyme, 97: 1311-1316.
25. RIEDERER, M., and SCHREIBER, L., 2001 -. Protecting against water loss: analysis of the barrier properties
of plant cuticles. Journal of Experimental Botany, 52: 2023.
26. SCHALLER, A., 2004 - A cut above the rest: the regulatory function of plant proteases. Planta 220, 183-197.
27. SCHWEIZER, P., FELIX, G., BUCHALA, A., MÜLLER, C., AND MÉTRAUX, J.P. (1996). Perception of
free cutin monomers by plant cells. The Plant Journal, 10: 331-341.
28. SIEBER, P., SCHORDERETA, M., RYSERA, U., BUCHALAA, A., KOLATTUKUDYB, P., MÉTRAUXA,
J.-P., AND NAWRATH, C., 2000 - Transgenic Arabidopsis Plants Expressing a Fungal Cutinase Show
Alterations in the Structure and Properties of the Cuticle and Postgenital Organ Fusions. Plant Cell, 12: 721738.
29. SIEZEN, R.J., 1997 - Subtilases: The superfamily of subtilisin-like serine proteases. Protein Science, 6: 501523.
30. SUH, M.C., SAMUELS, A.L., JETTER, R., KUNST, L., POLLARD, M., OHLROGGE, J., AND BEISSON,
F., 2005 - Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem
Epidermis 1 [W]. Plant Physiology, 139: 1649-1665.
31. SUTTER, E., 1984 - Chemical composition of epicuticular wax in cabbage plants grown in vitro. Canadian
Journal of Botany, 62: 74-77.
32. TANAKA, H., ONOUCHI, H., KONDO, M., HARA-NISHIMURA, I., NISHIMURA, M., MACHIDA, C.,
AND MACHIDA, Y., 2001 - A subtilisin-like serine protease is required for epidermal surface formation in
Arabidopsis embryos and juvenile plants. Development, 128: 4681-4689.
33. TODD, J., POST-BEITTENMILLER, D., AND JAWORSKI, J.G., 1999 - KCS1encodes a fatty acid elongase
3-ketoacyl-CoA synthase affecting wax biosynthesis inArabidopsis thaliana. The Plant Journal, 17: 119-130.
34. VESA, J., HELLSTEN, E., VERKRUYSE, L.A., CAMP, L.A., RAPOLA, J., SANTAVUORI, P.,
HOFMANN, S.L., AND PELTONEN, L., 2002 - Mutations in the palmitoyl protein thioesterase gene
causing infantile neuronal ceroid lipofuscinosis. Nature, 376: 584-587.
35. VON WETTSTEIN-KNOWLES, P., AVATO, P., AND MIKKELSEN, J.D., 1979 - Light promotes synthesis
of the very long chain fatty acyl chains in maize wax. Biogenesis and Function of Plant Lipids.
Elsevier/North Holland Biomedical Press, New York: 271–274.
36. WELLESEN, K., DURST, F., PINOT, F., BENVENISTE, I., NETTESHEIM, K., WISMAN, E., STEINERLANGE, S., SAEDLER, H., AND YEPHREMOV, A., 2001 - Functional analysis of the LACERATA gene
of Arabidopsis provides evidence for different roles of fatty acid omega-hydroxylation in development.
Proceedings of the National Academy of Sciences, 98: 9694.
37. XIA, Y., NIKOLAU, B.J., AND SCHNABLE, P.S., 1996 - Cloning and Characterization of CER2, an
Arabidopsis Gene That Affects Cuticular Wax Accumulation. The Plant Cell Online, 8: 1291-1304.
38. YEPHREMOV, A., WISMAN, E., HUIJSER, P., HUIJSER, C., WELLESEN, K., AND SAEDLER, H., 1999
- Characterization of the FIDDLEHEAD Gene of Arabidopsis Reveals a Link between Adhesion Response
and Cell Differentiation in the Epidermis. The Plant Cell Online, 11: 2187-2202.
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