Variability in waxiness of
Eucalyptus gunnii
foliage for floriculture
Michelle G. Wirthensohn, Graham Collins,
Graham P. Jones, Margaret Sedgley
*Department of Horticulture, Viticulture and Oenology, Waite Campus, University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia
Accepted 16 March 1999
Abstract
Eucalyptus gunnii leaves can appear as green or glaucous phenotypes with the latter more desirable for floriculture. The epicuticular wax from these two types were compared morphologically using environmental scanning electron microscopy, chemically using gas chromatography, and molecular markers were found to distinguish the two types using RAPDs and bulked segregant analysis. Both phenotypes had wax tubes, which in the glaucous type were significantly longer and thicker and the surface area was covered more densely than on the green type. When compared chemically, the glaucous wax seemed to contain a higher percentage of alkanes, alcohols and free fatty acids, but lower percentages ofb-diketones and esters than the wax from the green type. The glaucous type had a greater yield of wax per unit area of leaf than the green. Seven molecular markers were found which would distinguish the green and glaucous bulks. No individual marker could totally distinguish all green individuals from all glaucous individuals but this could be achieved using combinations of markers.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Eucalyptus gunnii; Epicuticular wax; Bulked segregant analysis; RAPDs; Floriculture; Scanning electron microscopy; TLC; GC
1. Introduction
Stems ofEucalyptus gunniiare one of the most popular of the foliages because of their attractive small, round leaves which are normally very glaucous. The
* Corresponding author. Tel.: +61-8-8303-7242; fax: +61-8-8303-7116
E-mail address:msedgley@waite.adelaide.edu.au (M. Sedgley)
species is cultivated for floriculture in Italy, France, USA and Australia, and the glaucousness or waxiness gives the leaves a grey green bloom (Jones and Sedgley, 1993). In a seedling population of cultivatedE. gunniithere appear up to 10% of trees whose leaves have a green phenotype which is not as desirable in the cut foliage market. This phenomenon is well known in several Tasmanian species ofEucalyptusincluding E. gunniiand is due to clinal variation within a species where the green phenotypes are found in the more sheltered environments and the glaucous phenotypes are more frequent at exposed, higher altitudes (Potts and Reid, 1985a, b). There has been little information published, however, on how the wax structure or composition varies between the phenotypes.
Bulked segregant analysis (Michelmore et al., 1991) examines specific genomic regions against a background of random genetic background of unlinked loci. It detects polymorphisms generated by RAPD markers between two bulk DNA samples derived from a population segregating for a gene of interest. Comparison of bulks of extreme individuals with green or glaucous leaf colour may identify markers linked to this qualitative trait. The objective of this study was to correlate genetic markers, chemical composition and ultrastructure of epicuticular wax with glaucousness ofEucalyptus gunnii.
2. Materials and methods
2.1. Plant material
Fresh leaf material of Eucalyptus gunnii was collected from seedling derived trees in a commercial plantation at Forest Range, South Australia, 348540 S,
1388470 E. Trees were chosen on the basis of extremes of leaf colour, with 15
glaucous trees and 13 green trees chosen. Young leaves (nodes 1±5) were sealed in plastic bags and kept cold until returned to the laboratory. The samples were processed immediately for chemical analysis and frozen at ÿ208C for DNA extraction. Fresh leaves were also used for electron microscopy.
2.2. Scanning electron microscopy
Rasband at the US National Institute of Health and available from the Internet by anonymous ftp from zippy.nimh.nih.gov). Measurements were taken of tube length and tube diameter at 10 randomly selected areas on the image. Measurement of the percentage of the adaxial leaf surface covered by wax utilised the whole image. Observations were made on the gross morphology of the wax. Data were analysed by ANOVA using Genstat 5.1.
2.3. Wax extraction
Epicuticular wax from nodes three to five of 13 green and 15 grey trees, was extracted by immersing leaves in 40 ml chloroform for 30 s. The extracts were filtered, evaporated to dryness at room temperature and the wax yield calculated per unit surface area of leaf (mg cmÿ2
). Total surface area of leaves was calculated by photocopying the leaves, carefully cutting them out and weighing them and calculating the area from a standard curve constructed from paper cut to known surface areas.
2.4. Wax analysis
Single epicuticular wax samples from each tree were separated into compound classes by thin layer chromatography (TLC) on Kieselgel 60 F254 (Merck,
Darmstadt) using a solvent system of n-hexane-diethyl ether-acetic acid (80 : 20 : 1) (Misra and Ghosh, 1992). Spots were visualised by exposing the TLC plate to UV light 254 nm and to iodine vapour. Compound classes of hydrocarbons, fatty alcohols, fatty esters and b-diketones were identified by comparing theirRf-values with those ofn-nonadecane, nonadecanol, and methyl
heptadecanoate and with those in the literature (Misra and Ghosh, 1992). The spots were removed from the plates and eluted with chloroform and analysed by GC after addition of the internal standards n-nonadecane for nonpolar compounds, nonadecanol for polar compounds and methyl heptadecanoate for the ester fractions. Quantification was carried out by comparison to the corresponding standards by peak area integration of the flame ionisation detector (FID) signal and expressed as percentage of whole wax. The analyses were performed using a gas chromatograph (GC) (Shimadzu 14A, Kyoto) equipped with an AT-35 capillary column (30 m, film thickness 0.25mm, 0.32 mm i.d., Alltech, Baulkham Hills) with an on column injection system (Shimadzu AOC-17) and autosampler (Shimadzu AOC-1400). GC was carried out with pro-grammed injection at 2508C, over 2 min at 1608C, 68C minÿ1
to 3208C, then held at 3208C for 20 min, detector at 3008C, N2 carrier gas head pressure of
1.25 kg cmÿ3
2.5. DNA isolation
DNA was extracted from all green and grey trees using the CTAB isolation method (Doyle and Doyle, 1990), was subjected to gel electrophoresis on 1% agarose gels in TBE buffer (Sambrook et al., 1989), and stained with ethidium bromide. DNA concentration was estimated by visual assessment of band intensities, compared to known genomic DNA standards. The concentration of DNA extracted varied from 5 to 100 ng/ml and the concentration used for RAPD analysis was adjusted to 5 ng/ml prior to bulking. Aliquots (50 ng of DNA) of each individual with green or glaucous leaves were bulked together to form a green and glaucous bulk.
2.6. PCR amplification
The optimised PCR reactions of all 28 plants were carried out in 25ml volumes containing 1Taqbuffer (Gibco-BRL, Gaithersburg), 3 mM MgCl2, 200mM of
dGTP, dATP, dCTP, dTTP (Promega, Madison), 1mM 10-mer primer (Operon Technologies, Alameda), 1 unit Taq polymerase (Gibco-BRL, Gaithersburg), 0.5ml Gene 32 Protein (Pharmacia Biotech, Uppsala), and 20 ng of genomic DNA for both bulked and individual tree reactions (Michelmore et al., 1991). Each reaction mix was overlaid with PCR-grade paraffin oil. Amplification was performed in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown) programmed for an initial denaturation step at 948C for 2 min, followed by 41 cycles of 948C for 1 min, 368C for 1 min, 728C for 2 min and terminated with a final extension step at 728C for 5 min. DNA amplification fragments were electrophoresed in 1.75% agarose gels using SeaKem GTG, (FMC BioProducts, Rockland) in 1TBE buffer. A negative control was added in each run to test for contamination. A 100 bp ladder molecular-weight marker (Gibco-BRL, Gaithersburg) was used on each gel to aid interpretation of band identity between gels. Polymorphisms between bulks was confirmed by repeating the amplification three times. DNA fragments were visualised by ethidium bromide staining.
2.7. Screening of RAPD markers
3. Results
3.1. Scanning electron microscopy
Both green and glaucous E. gunnii leaves had tube waxes and there were significant differences between the two types in tube length and diameter and in percentage surface area covered by wax (Table 1). Glaucous types had 2.1mm longer and 0.08mm wider tubes than green types and 37% larger surface area covered by wax, all of which were highly significant.
3.2. Wax analysis
Wax yield was 23% greater in the glaucous leaves at 25.2mg cmÿ2
compared to the green leaves at 19.4mg cmÿ2. This was due in part to the greater amount of wax extracted from the glaucous leaves and also the smaller surface area of the glaucous leaves at 75 cm2compared to the green leaves at 80.5 cm2. The harvest of wax indicates that glaucousness is related to wax yield.
GC of all wax samples showed that the major classes of compounds of leaf waxes of E. gunnii were long chain hydrocarbons, free primary alcohols, long chain b-diketones, free fatty acids and long chain esters (Figs. 1 and 2). The
b-diketones were the major class of compounds identified in both the glaucous and green epicuticular leaf wax, amounting to 53% and 65% of the wax, respectively (Fig. 1). The homologue range was from C31 to C37, however, C35
and C36were not detected in either type and C32, C34and C37were not detected in
the glaucous type.n-Hentriacontan-14,16-dione (C31) was significantly greater in
green than glaucous types at 51% and 49%, respectively, as was by n
-tritriacontan-16,18-dione (C33) at 13% and 4%, respectively.
The next largest compound class detected was the long chain esters which ranged from C26to C38and accounted for 19.7% and 23.5% of the whole wax of
Table 1
Epicuticular wax measurements on green and glaucousE. gunniileaves
Leaf type Wax tube length
aStandard error of the mean. b
Number of measurements.
c
glaucous and green types, respectively (Fig. 2). The major esters identified in the glaucous types were methyl esters of odd carbon number ranging from C27to C31.
The green types showed a greater range of esters with the major ones being 1,2-benzenedicarboxylic acid decyl octyl ester (C26) and an alkyl ester (C38) which
were significantly greater in the green types.
Fig. 1. Percentage composition (%) and standard error ofb-diketones from leaf waxes of green and glaucousEucalyptus gunnii.X-axis legend: the numbers on thex-axis denote carbon number ofb-diketone.
Fig. 2. Percentage composition (%) and standard error of major compound classes from leaf waxes of green and glaucous Eucalyptus gunnii. X-axis legend: alk18±23C18±C23 alkane; alc18±
Alkanes comprised 12.7% of the glaucous wax, significantly greater than that of the green wax at 4.5%. Alkanes detected were C18, C22and C23, however, C22
was not detected in the glaucous wax. Primary alcohols made up 5.4% of the glaucous wax compared to 3.4% of the green wax. The alcohol homologues detected were C18, C20, C24, C28, and C30, with C20 and C24 not detected in
glaucous wax. Free fatty acids accounted for 8.9% of the glaucous wax and 3.4% of the green wax, with the range including C19, C25, C29 and C31, with C25 and
C29not detectable in the glaucous wax (Fig. 2).
3.3. Bulked segregant analysis
An average of 12 bands were amplified per primer with two primers giving no amplification products. The fragments ranged in size from 300 to 2100 bp. Primers OPA-7 (GAAACGGGTG), OPA-19 (CAAACGTCGG), OPB-3 (CATCCCCCTG), OPB-11 (GTAGACCCGT), and OPD-20 (ACCCGGTCAC)
Fig. 3. Segregation analysis with primer OPD-20 and DNA from 15 individuals of glaucousE. gunnii(lane 2±9, 11±17) and 13 individuals of greenE. gunnii(lane 18±30). The solid arrowheads indicate the glaucous-linked RAPD fragments OPD-20_850, OPD-20_1250, and OPD-20_1250.
provided reproducible polymorphic fragments between the green and glaucous bulks. Fragment OPD-20_850was present in all glaucous plants and absent in all
but two green plants (Fig. 3). Fragment OPD-20_1380was present in all glaucous
plants and absent in all but two green plants and fragment OPD-20_1250 was
present in all but two glaucous plants and absent in all but two green plants (Fig. 3). Fragment OPA-7_650was absent in all glaucous individuals and present
in all except four green individuals. Fragments OPA-19_1700 and OPB-11_720
were present in all but four glaucous plants and absent in all but one and two green plants, respectively; OPB-3_700 fragment was present in all but one
glaucous plant and absent in all but one green plant.
4. Discussion
Comparison of green and glaucous Eucalyptus gunnii leaf wax showed
differences in morphology, chemical composition and genomic DNA. Morpho-logically, the glaucous types had significantly longer and thicker wax tubes which covered a greater surface area of leaf than green types. Both the green and glaucous leaves had tube waxes, with the visible difference in appearance attributable to a difference in size and density of tubes. SomeEucalyptusspecies have plate waxes, which diffract the light differently from tubes, to give a greener visible appearance (Hallam, 1970), but plate waxes were not observed on the greenE. gunniileaves.
The amount of wax on the glaucous leaves was significantly more than on the green leaves. This may be due to a down regulation of wax biosynthesis such as C16 and C18 fatty acid precursor synthesis in the green types. Li et al. (1997)
found that E. gunnii showed much variation in morphological components between localities, including an increase in wax yield along the cline as well as a change in wax composition. Wax yield tended to be lower in the green forms of the species in that study. Morphological variation in glaucousness may occur within and between the species with clinal variation in morphological and physiological differences correlated with changes in the physical environment (Pryor, 1976). Clines occur along environmental gradients such as altitude, latitude or other factors, such as water availability or frost sensitivity. The cline of
E. gunniiis associated with increasing exposure to alpine environment (Potts and Reid, 1985a), and has been shown to be genetically based (Potts and Reid, 1985b). Eucalyptus gunnii is one of the most frost tolerant eucalypt species (Barber, 1955; Pryor, 1957) and maximum glaucousness occurs on trees which border frost hollows and grow in high light intensities (Potts and Reid, 1985a).
E. archerihad lessb-diketones and more alkanes than the glaucous phenotypes. This would suggest that glaucousness is not entirely related tob-diketone content which is confirmed by the fact that green E. viminalis has a high b-diketone content and green, adult E. globulus leaf wax contains greater amounts of
b-diketone than glaucous juvenile leaves (Li et al., 1997). Rather, glaucousness is related to the interaction of wax yield, chemistry and morphology as evidenced in this study.
A total of seven markers using five primers were found that would distinguish some green and glaucous types of E. gunnii. This relatively large number of markers and the fact that none distinguished all green and glaucous trees may be due to the fact that the loci of interest may occupy a large region of the genome, therefore, the probability of tagging is large, and in species ofEucalyptus, which preferentially outcross, the high levels of heterozygosity in the majority of the loci result in large amounts of DNA polymorphism. This however makes it harder to detect a dominant marker gene linked to the target gene. The most useful markers produced by bulked segregant analysis in this study were OPA-7_650,
OPB-3_700, OPD-20_850and OPD-20_1380. A combination of the following markers
would distinguish all green individuals from glaucous individuals: OPA-7 with either OPA-19, OPB-3 or OPD-20; OPA-19 with OPB-11 or OPD-20; and OPD-20 with either OPB-3 or OPB-11. Most traits of interest in forest tree improvement such as yield are controlled by more than one gene (Byrne et al., 1997).
There are likely to be many genes regulating wax biosynthesis due to the diversity of wax components. Using wax deficient mutants such as theglossyor
eceriferum (cer) mutants many workers have begun to isolate the genes responsible for the production of epicuticular wax in barley (von Wettstein-Knowles, 1979), maize (Bianchi et al., 1985),Arabidopsis(McNevin et al., 1993) andBrassica (Baker, 1974). Many of these mutants display glossy green stems and caryopses/fruits as well as leaves. Wax biosynthesis is a series of enzymatic steps in a number of pathways, any one of which could be altered, thus changing the final composition and amount of the epicuticular wax.
This research has shown the value of bulked segregant analysis and investigation of wax morphology and composition to eucalypt improvement. Using one segregating population bulked segregant analysis has identified molecular markers linked to glaucousness inE. gunnii. Further investigation of wax biosynthetic pathways and the genes involved will assist in explaining differences in epicuticular wax, as will analysis of more segregating populations.
Acknowledgements
the ESEM. M.G. Wirthensohn was supported by a University of Adelaide Postgraduate Research Scholarship.
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