Plant Physiol. (1998) 116: 901-911
Epicuticular Wax Accumulation and Fatty Acid Elongation
Activities Are Induced during Leaf Development of Leeks1
Yoon Rhee,
Alenka Hlousek-Radojcic2,
Jayakumar Ponsamuel3,
Dehua Liu4, and
Dusty Post-Beittenmiller*
Plant Biology Division, The Samuel Roberts Noble Foundation, P.O.
Box 2180, Ardmore, Oklahoma 73402
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ABSTRACT |
Epicuticular
wax production was evaluated along the length of expanding leek
(Allium porrum L.) leaves to gain insight into the
regulation of wax production. Leaf segments from the bottom to the top
were analyzed for (a) wax composition and load; (b) microsomal fatty
acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier
protein (ACP) thioesterase activities; and (c) tissue and cellular
morphological changes. The level of total wax, which was low at the
bottom, increased 23-fold along the length of the leaf, whereas
accumulation of the hentriacontan-16-one increased more than 1000-fold.
The onset of wax accumulation was not linked to cell elongation but,
rather, occurred several centimeters above the leaf base. Peak
microsomal fatty acid elongation activity preceded the onset of wax
accumulation, and the maximum fatty acid synthase activity was
coincident with the onset. The C16:0- and
C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among
cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax
crystals. These studies demonstrated that wax production and microsomal
fatty acid elongation activities were induced within a defined and
identifiable region of the expanding leek leaf and provide the
foundation for future molecular studies.
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INTRODUCTION |
The ubiquitous presence of surface waxes among terrestrial plant
species is a strong testament that they are essential for life in an
aerial environment (Gülz, 1994
). Cuticular waxes provide the
hydrophobic barrier of the plant surface, and as such they function
primarily to shed water and prevent nonstomatal water loss. In
addition, they provide a first line of defense against bacterial and
fungal pathogens and against abiotic stresses such as drought and UV
damage (Kolattukudy, 1980). Epicuticular waxes also play a role in
plant-insect communication, by either attracting or deterring insects
(Eigenbrode and Espelie, 1995). Waxes differ widely among plant species
and among the organs and tissues of a single plant, attesting to the
genetic diversity and developmental influences (for recent reviews,
see von Wettstein-Knowles, 1995; Lemieux, 1996;
Post-Beittenmiller, 1996). In addition, wax content and composition are
affected by environmental conditions. Relatively high-humidity
conditions, such as in tissue culture, suppress wax production (Sutter
and Langhans, 1979, 1982), and the photoperiod affects the chain length
of wax components (von Wettstein-Knowles et al., 1980). Despite their
vital importance to plant survival and protection, and extensive
studies of wax composition, very little is known about the initiation
of epicuticular wax production and how production may be influenced by
developmental and environmental factors.
Plant waxes are a complex heterogeneous mixture of very-long-chain
(C20-C34) fatty acids and their derivatives. The fatty acid primers
used for elongation are derived from plastidial de novo fatty acid
biosynthesis and are exported to the cytoplasm after hydrolysis by
acyl-ACP thioesterases. These fatty acids are then partitioned among
membrane glycerolipid, cutin, and wax biosynthetic pathways. Elongation
of long-chain fatty acids occurs in the microsomal membranes of
epidermal cells (Kolattukudy and Buckner, 1972; Cassagne and Lessire,
1978) and proceeds in a series of enzymatic reactions similar to the
reactions of de novo fatty acid biosynthesis (Fehling and Mukherjee,
1991). The enzymes that catalyze these reactions are collectively
referred to as elongases. During wax biosynthesis very-long-chain fatty
acids are further modified to aldehydes, alkanes, and ketones, which
are the major components of mature leek (Allium porrum L.)
leaf epicuticular waxes. Thus, elongases are important for wax
production and as such would be expected to be the target for
environmental and developmental controls. In addition, because plant
epicuticular waxes predominantly contain components that are derived
from saturated fatty acids, it follows that C16:0-ACP, C18:0-ACP, and
C18:1-ACP thioesterase activities may be differentially regulated to
provide the required increase in the pool of saturated fatty
acids.
In leek epidermal cells that are actively synthesizing wax, the demand
for fatty acids can be quite high compared with the other tissues of
the leaf. For example, in leek leaf, hentriacontan-16-one, a single
component of epicuticular wax, makes up more than 15% of the total
leaf lipid, but is synthesized only by epidermal cells that constitute
less than 4% of the leaf fresh weight (calculated from Liu and
Post-Beittenmiller, 1995). Therefore, to provide sufficient pools of
fatty acid precursors for wax production the expression of plastidial
FAS, acyl-ACP thioesterases and microsomal fatty acid elongases may be
coordinately regulated. To begin to elucidate the controls on
epicuticular wax production, we have examined the relationships between
wax production, FAS, microsomal fatty acid elongase, and acyl-ACP
thioesterase activities and the changes in the epidermal cellular
morphology in the region where wax is being actively deposited.
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MATERIALS AND METHODS |
Plant Material and Wax Analyses
Leeks (Allium porrum L.) were regrown for 14 d
from commercially produced plants as described previously (Evenson and
Post-Beittenmiller, 1995; Liu and Post-Beittenmiller, 1995). The third
interior leaf, counting from the outermost leaf that expanded after
cutting and replanting, was used for all studies. The third leaf was
cut in transverse segments serially 3, 5, 7, 9, 14, 19, and 24 cm from the leaf base and segments were designated I to VII. The surface areas
of each segment were measured (surface area calculations included both
adaxial [inner] and abaxial [outer] surfaces), and then the
epicuticular wax from each segment was extracted by immersion in
CHCl3 for 60 s with gentle agitation.
Triacontane was added as an internal standard and used to calculate the
amount of individual components. Total wax load was reported as the sum
of the identified components only and was therefore a conservative
measure of the total wax load. Complete wax composition and content
analyses were carried out on segments from six individual leaves.
Aliquots of some samples were also methylated as previously described
(Liu and Post-Beittenmiller, 1995).
Samples were analyzed using a gas chromatograph (model 5890) and a mass
spectrometer (model 5971, Hewlett-Packard) and separated using a
30-m × 250-µm capillary column (model 5MS, Hewlett-Packard). The injector temperature was 250°C and the detector temperature was
280°C. The oven was held at 150°C for 10 min and then increased to
300°C at 10°C/min and held for 10 min. Peaks were identified by
retention times and by comparison of mass spectra to standards and to
the NBS75K library (Hewlett-Packard). Where indicated, only the level
of hentriacontan-16-one was monitored. The surface area was measured as
described above, and the epidermis was peeled from the segments, frozen
in liquid nitrogen, and ground to a powder in a chilled mortar and
pestle. Microscopic examination of these peels indicated that they were
free of underlying parenchymal cells over most of the length of the
leaf. However, epidermal peels from the lower portion (segments I and
II) of the leaf had variable but small amounts of contamination from
underlying tissue. Contaminating tissue would not contribute to the
hentriacontan-16-one levels or to microsomal fatty acid elongase
activities (K.J. Evenson and D. Post-Beittenmiller, unpublished
data) but might contribute to FAS and thioesterase activities. An
aliquot (2-10 mg) of frozen, powdered tissue was added to
CHCl3 (triacontane was added as an internal
standard), vortexed briefly, and filtered through
CHCl3-washed glass wool to remove cell debris.
The collected CHCl3 extract was reduced under
nitrogen, and the residue was resuspended in 100 µL of
CHCl3 and analyzed by GC-MS as described
above. The hentriacontan-16-one levels were determined for samples used
for all microscopy by measuring the levels from adjacent leaf segments (1 cm in length).
Enzyme Assays
Specific enzyme activities were expressed per unit of surface area
to relate them to the accumulation of surface waxes and to each other.
This was necessary because each enzyme activity was assayed from a
different fraction (see below) and therefore the protein contents
differed for each enzyme assay. Furthermore, the surface area is a more
appropriate unit for comparing wax loads than either protein
concentration or fresh weight, which may vary considerably with the
developmental stage of the leek leaf segment. We found substantial
changes in protein and fresh weight along the length of the leaf that
did not correlate with the production of surface waxes.
Segments for enzyme assays were prepared as described above with one
exception. The first segment that we used for analysis of enzyme
activities represented the first 5 cm above the base of the stem,
combining segments I and II, as shown in Figure 1. Epidermis peeled
from individual segments was frozen in liquid nitrogen, ground to
powder, and stored at 
75°C until used. Enzyme assays were carried
out using fractions prepared from segments of four to seven individual
leaves. Each segment was analyzed for hentriacontan-16-one levels.
Samples were added to chilled homogenization buffer (80 mm
Hepes, pH 7.2, 2 mm EDTA, 320 mm Suc, 2 mm DTT, and 0.3 mm PMSF) at a ratio of 1:6
(w/v) and homogenized with a micropestle in a chilled microfuge tube.
Samples were centrifuged (15 min, 5000 rpm, 4°C) and supernatants
were transferred to airfuge tubes. Microsomal membranes were collected
by centrifugation in an airfuge (15 min, 22 psi, 4°C, Beckman) and
pellets were resuspended in buffer (80 mm Hepes, pH 7.2, with KOH, 15% glycerol, and 2 mm DTT). Membranes were
frozen in liquid nitrogen stored at 75°C until used for elongase
assays. Elongase assays and product analyses were conducted according
to the method of Evenson and Post-Beittenmiller (1995).
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| Figure 1.
Accumulation of total wax (gray bars) and
hentriacontan-16-one (blue bars) from segments (I-VII) of a single
leaf. The leaf segments are indicated by brackets along the top of the
leaf pictured below the graph. The brackets are proportional to the
segment distance as described in ``Materials and Methods''. Data from
a representative sample are shown, and the segment with  2 nmol/cm2 of hentriacontan-16-one was segment III. This
experiment was carried out six times. In different leaf samples the
maximum levels of total wax and hentriacontan-16-one ranged from 50 to
68 and 27 to 60 nmol/cm2, respectively.
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Supernatants from microsomal membrane preparations were used for the
FAS and acyl-ACP thioesterase assays. Supernatants were collected,
glycerol was added to 5% (v/v), and they were stored at 75°C until
used. Thioesterase assays were performed using the supernatant without
further preparation, according to the method of Ohlrogge et al. (1978).
FAS assays, however, required a concentration of supernatant proteins
before the activity could be assayed under initial velocity conditions.
Aliquots (200-500 µL) of each supernatant fraction were adjusted to
70% ammonium sulfate (percentage saturation) using saturated ammonium
sulfate. The proteins were allowed to precipitate on ice for 30 min and were then collected by centrifugation at 18,000g for 30 min
at 4°C. The pellet was resuspended in one-tenth volume of 10 mm Tris, pH 8.0, and desalted using a Sephadex G-50 spun
column (9:1, bed volume/sample volume) that had been equilibrated with
10 mm Tris, pH 8.0. The sample was collected by
centrifugation at 3,000g for 4 min at 4°C. The samples
were adjusted to 5% glycerol (v/v) and used directly in FAS assays, as
described by Clough et al. (1992) except that the concentration of
[14C]malonyl-CoA was 60 µm, and
assays were run for 30 min. All enzyme assays were performed under
initial velocity conditions. Proteins were measured using a Bio-Rad
protein assay reagent according to the manufacturer's recommendation.
Microscopic Examinations
For SEM examination, 1- × 0.5-cm leaf segments were prepared from
the appropriate region of leek leaf as indicated. The samples were air
dried for 72 h at room temperature, then mounted onto a copper
holder with double-adhesive carbon tape, sputter coated with gold for 3 min, and examined under an electron microscope (model JSM 880, JEOL).
Secondary electron images were collected at 15 kV of accelerating
voltage.
Leaf segments (5 × 0.5 cm) were cut starting from 5 cm above the
base of the leaf for TEM examination. The tissue was fixed with 3%
(v/v) paraformaldehyde and 1% glutaraldehyde (v/v) in 0.1 m sodium cacodylate buffer, pH 7.4, at room temperature for approximately 1 h. During the initial fixation a vacuum was
applied for approximately 15 min to facilitate fixative penetration.
Fixation continued overnight at 4°C. The tissue was rinsed three
times in 0.1 m sodium cacodylate buffer, pH 7.4, and then
fixed in 2% (w/v) osmium tetroxide in 0.1 m sodium
cacodylate buffer, pH 7.4, at room temperature for 2 h. The
samples were washed three times in 0.1 m sodium cacodylate
buffer and then dehydrated in a graded ethanol series of 10, 30, 50, 70, 95, 100, 100, and 100% for 30 min for each step. The dehydrated
leaf segments were infiltrated with Embed 812 (EM Sciences, Fort
Washington, PA) at room temperature under the following schedule: Embed
812:ethanol (1:2, v/v) for 4 h; Embed 812:ethanol (2:1, v/v)
overnight; and Embed 812 without ethanol for 8 to 12 h three
times. Finally, the samples were polymerized overnight in an oven at
70°C. Ultrathin sections were viewed using TEM (with either a model
10C microscope [Zeiss] at 80 kV or a model JEM2000FX microscope
[JEOL] at 100 kV).
Epidermal peelings were made from 0.5-cm segments and the unstained
peels were examined using bright-field microscopy (Nikon FX).
Cell-length measurements were made using a calibrated ocular micrometer. In the bottommost tissue, including the first three tiers
of cells, the tier organization was not distinct; therefore, 10 cells
were measured randomly through this region without regard to tier
association. In tiers 4 through 10, 10 cells were measured for each
tier. In the segments from 0.5 to 17 cm, 10 cells were counted in the
intact tier closest to the end most distal to the base of the leaf. To
ascertain whether distortion was created while preparing the epidermal
peels that might lead to erroneous measurements, negative cellulose
acetate replicas were also made from contiguous segments. The cellular
dimensions measured from the replicas showed similar cell dimensions as
the epidermal peels. Data are reported as tiers (below 0.5 cm from the
leaf base), single segments (at 0.5-1 cm above the leaf base), or two
combined segments, where there were no significant differences between two segments (above 1 cm from the leaf base).
| |
RESULTS |
Total Wax Accumulation and Composition
As a monocot, leek leaves expand in a simple, linear fashion from
the base of the leaf, in contrast to dicots, which expand in all
directions of the leaf plane. To establish whether epicuticular waxes
are deposited uniformly on the expanding leaf surface, or in a manner
consistent with a developmental influence, the total wax load and
composition were determined along the length of individual rapidly
expanding leek leaves. The wax load from the bottom to the top of one
of six individual leaves examined is shown in Figure 1. Very little
CHCl3-extractable lipid was detected from the
base of the leaf to approximately 7 cm above the leaf base. At 7 to 9 cm the onset of a dramatic increase (more than 30-fold in most samples)
in the wax load was observed (Fig. 1). The increase continued for most
of the length of the leaf, and then in segments VI and VII the
amount of wax per surface area was similar. The accumulation of
hentriacontan-16-one reflected that of the total wax. Although comparable trends were observed for all six leaves examined, the onset
(segment III or IV) and duration (through segment VI or VII) of wax
accumulation, as well as the absolute levels of wax, varied among the
leaves of different plants. Therefore, it was essential for later
biochemical studies to be able to identify the onset of wax
accumulation.
The wax composition of each segment was also determined from the six
leaves and the averages with ses are shown in Table
I. Leek leaf waxes consisted primarily of
fatty acids (C16-22) and longer-chain (C26-31) derivatives
(aldehydes, alkanes, and ketones). In this study samples were not
derivatized with silylating reagents and therefore alcohols, which are
a minor component (D. Huhman and D. Post-Beittenmiller, unpublished
data), were not analyzed. Other unidentified but minor components were
also not included in these analyses.
CHCl3-extractable lipids from the lower leaf segments were primarily hexadecanoic (C16) and octadecanoic (C18) acids. Smaller amounts of eicosanoic (C20) and docosanoic (C22) acids
were also detected. The level of hexadecanoic and octadecanoic acids
increased almost 4- and 2-fold, respectively, from the bottom (segment
I) to the top of the leaf (segment VII). Even though C16 and C18 fatty
acids were a major component (78-92%) of the CHCl3-extractable surface lipids in the bottom 5 cm, they were minor components (12-13%) in the top 5 cm. Undetectable
or very low levels (< 5% of the total wax load) of
very-long-chain alkanes, aldehydes, and ketones were also observed in
the bottom segments. These components, however, increased dramatically
in the middle region of the leaf to more than 75% in the top 5 cm.
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Table I.
Composition of epicuticular wax on segments of leek
leaves
Values are presented as averages with ses in parentheses
(n = 3 for fatty acid; n = 6 for other
components). Distance measurements are from the leaf base.
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Hentriacontan-16-one represented 58 to 72% of the total wax load in
the middle of the leaf (segments III-V). By comparison, aldehydes and alkanes represented substantially smaller proportions (5-9%) of the total wax content in the same segments. They
appeared similarly with hentriacontan-16-one, but their rapid
accumulation (beginning in segment V) lagged behind the
accumulation of hentriacontan-16-one (beginning in segment III),
becoming relatively constant in the topmost leaf segments (compare
segments VI and VII). Because the appearance of hentriacontan-16-one
began essentially with the rapid increase in total wax load,
hentriacontan-16-one levels were monitored and used as a marker to
identify the onset of the wax accumulation in all subsequent studies.
We defined the segment in which the level of hentriacontan-16-one was 2 nmol/cm2 as the onset of wax
accumulation for the ease of discussion in this paper. Whether this was
segment III (5-7 cm above the leaf base), as shown in Figure 1 and
Table I, or segment V, as shown in Figure 3 (see below), it was
variable among the plants used in these studies. Identification of the
onset was of particular interest, as the region preceding the onset of
accumulation is presumably where genes and enzymes involved in wax
production would be induced.
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| Figure 3.
Assay of enzyme activities in each segment of leek
epidermis compared with hentriacontan-16-one accumulation. Segments
indicated along x axis are as described in Figure 1. A,
Fatty acid elongase activity. B, FAS activity. C, Acyl-ACP-hydrolyzing
activity. Solid bars, 16:0-ACP hydrolysis; open bars, 18:0-ACP
hydrolysis; and hatched bars, 18:1-ACP hydrolysis. Hentriacontan-16-one
( ) was used as a marker for wax load in each experiment. The segment containing 2 nmol/cm2 hentriacontan-16-one was
segment V. Mean values with ses are presented. In A and B,
n = 7, and in C, n = 2 to 4.
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Wax Accumulation Began after Cell Division and Elongation Ceased
To determine whether wax biosynthesis was concomitant with cell
division and elongation in expanding leek leaf, we measured the length
of cells from near the base of the leaf to 17 cm above. As indicated in
the previous section, the level of hentriacontan-16-one was used to
identify the onset of wax accumulation. The results are shown in Figure
2. Substantial increases in the length of the epidermal cells were observed up to the 10th cell layer. Above the
10th cell layer increases in the cell length slowed, and above 1 cm,
further increases in the cell length were less than 8%. Analyses of
hentriacontan-16-one levels indicated that the onset of wax
accumulation (at 9-10 cm from the leaf base in this sample, data not
shown) was well above the region of epidermal cell elongation.
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| Figure 2.
Measurement of cell length to determine cessation
of cell elongation in the abaxial epidermal cells of leek leaves. The
numbers above the bars indicate the mean cell lengths. se
bars are indicated (n = 10). For each segment 10 fields were counted. Cell layers were noted below 0.5 cm. Cell lengths
were measured in 0.5-cm segments and data are shown as the lengths in a
1-cm segment; the two 0.5-cm segments did not vary significantly.
Hentriacontan-16-one was monitored in 1-cm segments at the same
position from the leaf base as the cell-length segments. The onset of
wax accumulation began 9 to 10 cm from the leaf base on this leaf. Cell
lengths did not change substantially above 7 cm and therefore data are not shown for segments above 7 cm.
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Enzyme Activities Associated with Fatty Acid Synthesis and
Elongation
The increasing wax accumulation on the leaf surface suggested that
there may be a corresponding increase in the enzyme activities associated with the biosynthesis of wax components. One of the key
activities leading to the production of hentriacontan-16-one is
microsomal elongation of fatty acyl primers. Therefore, we examined the
activities of microsomal elongases and FAS in each leaf segment. These
experiments were conducted on seven individual leaves. Each leaf was
segmented, the epidermis was peeled, and microsomal and supernatant
fractions from each segment were assayed separately for elongase and
FAS activities, respectively, and hentriacontan-16-one levels were
monitored. All individual leaves showed similar trends, although
maximum levels of in vitro activities varied. The averages of seven
experiments are shown in Figure 3. The
maximal elongase (Fig. 3A) and FAS (Fig. 3B) activities were 5- and
6-fold greater, respectively, compared with the activities in the lower
segments. Surprisingly, peak elongase and FAS activities occurred in
different segments. The peak of fatty acid elongase activity (segment
IV) preceded the onset of hentriacontan-16-one accumulation (segment
V), as was predicted. However, FAS activity peaked later, in segment V,
which was above the segment with peak elongase activity and which
coincided with the onset of hentriacontan-16-one accumulation. This
relationship between fatty acid elongase and FAS activities was seen in
all leaves assayed with the exception of one. In the exceptional case
the peak elongase activity was observed in the later segment (V)
simultaneously with peak FAS activity.
The termination of FAS and the initiation of microsomal fatty acid
elongation is a point where partitioning between extraplastidial glycerolipid and wax biosynthetic pathways might be controlled. The
enzymes responsible in part for FAS termination and export of fatty
acids from the plastid are the acyl-ACP thioesterases. These enzymes
have three substrates available in epidermal leucoplasts: C16:0-ACP, C18:0-ACP, and C18:1-ACP. Extracts from the
epidermal peels of segmented leaves were assayed for hydrolyzing
activity with each acyl-ACP substrate. The averages of two to four of
the same seven leaves used for the FAS and elongase assays are shown in
Figure 3C. Essentially, the levels of C16:0-ACP- and
C18:0-ACP-hydrolyzing activities did not change along the length of the
developing leaf. Except for a small (20%) but consistent increase in
C18:1-ACP-hydrolyzing activity in the segment preceding the increased
fatty acid elongase activity, the level of C18:1-ACP-hydrolyzing
activity gradually decreased to 42% (from 430 to 182 pmol
h
1 cm2) along the
length of the leaf.
Cellular Morphology Changed prior to the Appearance of Wax Crystals
We examined the leaf surfaces by SEM and the cellular morphology
of leaf epidermal cells by TEM to identify changes that may correlate
with the onset of wax production. Figure
4 is a composite of scanning electron
micrographs taken from various sections along the length of two
different leek leaves. Figure 4e is a picture of the abaxial (outer)
side of a leek leaf with circles drawn to indicate the areas of the
leaf that were examined by SEM. No crystals were observed on either
side close to the base of the leaf, well below the onset of wax
accumulation (Fig. 4a). On one leaf crystals began to appear in the
region that was identified by GC-MS as the onset of wax accumulation
(> 2 nmol/cm2, Fig. 4b). It was evident
that the formation of crystals (i.e. wax deposition) did not occur
uniformly across a given latitude of the leaf, because individual cells
within the region differed in the abundance of surface wax crystals.
The rectangle marked on Figure 4e is a region of the second leaf that
was below the onset of wax accumulation (< 2 nmol/cm2) in which a survey of the surface
crystals was conducted along a single latitudinal line, indicated by
the unidirectional arrow. The four SEM fields shown in Figure 4, f to
i, were taken from the survey and represented a progression from the
top to the bottom of the rectangle. The crystalline deposits were found
to be sparse (Fig. 4, f), moderate (Fig. 4, g), dense (Fig. 4, h), and
then sparse again (Fig. 4, i) as scanning proceeded latitudinally
across the leek leaf. Approximately 1 cm above the region shown in
Figure 4, f to i, but in a region where the levels of
hentriacontan-16-one were still < 2 nmol/cm2, the abaxial leaf surface was densely
covered with wax crystals (Fig. 4c). Solid plates were seen near the
leaf surface, with branched rods projecting out from the plates,
creating waffle-shaped crystals. Wax crystals were not observed on the
adaxial (inner) surface in this same region. Crystals, primarily
branched rods, were heavily distributed on the abaxial surface in a
region well above the onset of wax accumulation by GC-MS analysis (Fig.
4d). Densely packed crystals were also seen on the adaxial surface in
this region (data not shown). Wax crystals were more abundant around the stomata in all regions (data not shown), where they presumably provided extra protection against water loss.
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| Figure 4.
Scanning electron micrographs of the abaxial
surface of a leek leaf. a, Region below the onset of wax accumulation,
where no hentriacontan-16-one was detected by GC-MS and no wax crystals were observed. b, Region within the segment showing the onset of wax
accumulation as determined by the presence of hentriacontan-16-one 2 nmol/cm2. c, Waffle-like structures of wax
crystals were observed below the onset of wax accumulation. d, Region
well above the onset of wax accumulation showing numerous branched rods
and plates. e, Photograph of a leek leaf showing the abaxial surface.
All electron micrographs are shown in the same orientation as the pictured leaf. Circles indicate areas examined for a through d, and the
rectangle indicates the area examined for f through i. The
unidirectional arrow within the rectangle indicates the latitudinal survey covered between 10.5 and 11.5 cm above the leaf base, which was
identified as below the onset of wax accumulation by GC-MS analysis on
this leaf. Areas shown in f through i were taken along the same lateral
line as illustrated in the rectangle, showing the uneven deposition of
crystals in adjacent regions below the segment identified as the onset
of wax accumulation. a and b are from the third leaf of one plant, and
c through i are from the third leaf of a second plant.
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Morphological changes in the epidermis were also observed by TEM of
transverse sections. The most conspicuous changes occurred in the OTWs.
In the epidermal layer, approximately 5 cm above the base of the leaf,
the OTWs (Fig. 5a) of the abaxial
epidermal cells were smooth and similar in thickness (1 µm) to the
inner tangential cell walls (not shown), and the cuticle was a thin line. Farther up the leaf, at approximately 8 cm, the OTW had thickened
approximately 4-fold, and in each epidermal cell, a single central
ridge was apparent (Fig. 5b). The cuticle had also thickened, but
because it was quite thin at 5 cm above the leaf base, the increase at
8 cm could not be determined. The changes in the cuticular surface of
the OTW progressed rapidly and at 9 cm had become distinctly irregular
(Fig. 5c). The appearance of wax crystals observed by SEM (Fig. 4b) was
accompanied by the increased OTW thickness on the abaxial surface, as
observed by TEM (Fig. 5b). GC-MS analysis of the region represented by
Figure 5b detected 3.5 nmol/cm2
hentriacontan-16-one. The changes observed in the OTW of the abaxial
surface apparently occurred developmentally earlier than the cell wall
changes of the adaxial surface. For example, the thickening of the OTW
(from 1 to 4 µm) the increased cuticle thickness, and the appearance
of the central ridge on the cells of the abaxial surface were observed
at 8 cm, whereas at 10 cm, the OTW on the adaxial surface was only 2 µm, the cuticular surface was smooth, and the cuticle was thin (Fig.
5, compare c and d).
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| Figure 5.
Transmission electron micrographs of epidermal
cells at various positions along the length of the leaf. a, Abaxial
epidermal cells at 5 cm above the base of the leaf showing the thin OTW and smooth cuticular surface. The cuticle is a thin line on the surface
of the OTW. b, Abaxial (outer) epidermal cells at 8 cm showing the
thickened OTW and a single central ridge as the beginning of cuticular
surface irregularities. The single ridge was observed on the cuticular
surface of all abaxial epidermal cells examined in this region. c,
Abaxial epidermal cells at 9 cm showing the increasing irregular
cuticular surface and the pair of developing ridges over the radial
cell walls (double arrowhead). d, Adaxial (inner) epidermal cells at 10 cm showing the thin cuticle and the OTW, which was thin and smooth at
the cuticular surface. The epidermal cells on the abaxial surface at 9 cm had already undergone more extensive morphological changes (shown in
c) and had a heavy deposit of wax crystals. The adaxial surface did not
have wax crystals in this region, although dense wax crystals were
evident in segments from higher up on the leaf. Scale bars are
indicated.
|
|
| |
DISCUSSION |
Leeks have been used to study epicuticular wax production for more
than 25 years, but the emphasis has been limited primarily to fatty
acid elongases and not to pathway regulation. In the present study we
sought to determine whether wax production in leeks was induced along
the length of the expanding leaf, and if so, whether the region could
be readily identified among commercially produced plants for studies of
induction of wax biosynthesis using biochemical and molecular
approaches. Commercially produced leeks are readily available
year-round and the plants are considerably larger (2-4 cm in diameter)
than leek seedlings grown in greenhouses or growth chambers, which
require more than 6 months to reach a diameter of 1 to 2 cm. Space
becomes limiting when growing even moderate numbers of plants to sizes
approaching commercially produced plants. A distinct advantage is that
commercially produced leeks can be regrown from the cut and replanted
false stem. In 10 to 14 d the inner leaves grow 20 to 25 cm and
are actively producing epicuticular wax. The rapidly expanding
epidermal tissue is easily separated from the underlying parenchyma and
is an excellent source of stable and active microsomal fatty acid
elongases (Evenson and Post-Beittenmiller, 1995) and acyl-ACP
thioesterases (Liu and Post-Beittenmiller, 1995). The epidermis from
such regrown plants has been used to construct a cDNA library from
which a 3-ketoacyl-ACP synthase III cDNA (Chen and Post-Beittenmiller, 1996) and several cDNAs encoding acyl-ACP thioesterases (D. Liu, D. Hawkins, L. Yuan, J. Kridl, and D. Post-Beittenmiller, unpublished data) have been isolated and characterized. The regrown plants are also
the source of our explant material (flower stalk) for leek regeneration
and transformation studies (C. Maier and D. Post-Beittenmiller,
unpublished data).
As a monocot, the leek leaf expands from the intercalary meristem
(basipetal development), representing a developmental progression from
the leaf base to the leaf tip (Hay and Brown, 1988). We have used the
developing leaf as a means to study the induction of wax production.
GC-MS analyses demonstrated that accumulation of epicuticular waxes
began in a region 5 to 9 cm above the base of the leaf (Table I), but
in some leaves the onset was as high as 9 to 14 cm from the leaf base.
Because of the variability of the onset of wax accumulation among
regrown leek plants, and because of the small amounts of epidermal
tissue available for analysis from segmented samples, it was necessary
to identify a reliable, sensitive, and easy to measure marker for wax
production that would allow us to compare results from different
leaves. In all of these studies the levels of hentriacontan-16-one were
used to establish the onset of wax accumulation along the length of the
leaf.
Wax Biosynthetic Pathway May Be under Global Regulation
Fatty acids (C16-C22) were the predominant components in the
lowest leaf segments, making up 95 to 98% of the total wax. Although the total CHCl3-extractable lipids from the leaf
surface in this region were relatively low, total wax accumulation
increased 23-fold from the bottom (segment I) to the leaf tip (segment
VII) during the regrowth of the leek leaf, and accumulation of
hentriacontan-16-one increased more than 1000-fold from segments II to
VII. As the total wax load increased, very-long-chain aldehydes
and alkanes were also detected, although the accumulation of aldehydes
and alkanes lagged behind the accumulation of
hentriacontan-16-one. The rate of accumulation of all components
frequently slowed in the topmost segments. Figure
6 is a simplified schematic of the biosynthetic pathway leading to the production of hentriacontan-16-one. A recent report has shown that in pea, aldehyde components are produced
by a fatty acid reductase that is different from the fatty acid
reductase leading to primary alcohol production (Vioque and
Kolattukudy, 1997). The alcohol-producing reductase catalyzes the
two-step reduction without the release of an aldehyde intermediate. If
this is also the case for leeks, all aldehydes in leek epicuticular wax
are derived via the decarbonylation pathway. The boxed components are
products that accumulated in leek leaf epicuticular wax. Although we
assume that the ketone was derived from the corresponding secondary alcohol, the lack of hentriacontan-16-ol accumulation suggested that
oxidation to the ketone was very rapid. Similarly, the lack of
accumulation of very-long-chain fatty acids (C26-C32) indicated that
reduction to the corresponding aldehydes was also very fast. Alternatively, the alcohol and fatty acid precursors for the ketone and
aldehydes, respectively, may not be available for transport to the
surface. The fact that n-hentriacontane (C31) accumulation lagged behind that of hentriacontan-16-one (Table I) suggested that
there may be a decline in the rate of alkane hydroxylation in the upper
segments that allowed the precursor alkane to accumulate. Similarly,
the increased levels of shorter-chain aldehydes (C26-C30) and alkanes
(C29) in the upper segments suggested that the rate of C30 to C32
elongation may decrease somewhat earlier than the other fatty acids.
These variations in the accumulation patterns of the individual
components were minor, however, and probably do not reflect substantial
differences in the expression of any single enzyme activity. Rather,
the coincidental increased accumulation of all of the components during
wax accumulation, as well as the generally slowed accumulation of all
components in the upper segments, implies that the pathway is
globally regulated.
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| Figure 6.
Schematic of the terminal steps of wax
biosynthesis in leek leaves leading to the production of
hentriacontan-16-one. The boxed components were found in the surface
wax. The brackets around hentriacontan-16-ol indicate that although it
is assumed to be the immediate precursor to hentriacontan-16-one, this
aspect of the pathway has not been characterized in leeks. The
synthesis of C26 to C32 fatty acids is evident from in vitro assays of
fatty acid elongases. The synthesis of the C32 aldehyde is deduced from the presence of the C26 to C30 aldehydes and the C31 alkane. Similar deductions were not made for hentriacontan-16-ol, since there is only
indirect evidence of secondary alcohol synthesis in leeks.
|
|
Wax Accumulation Is Not Linked to Cell Elongation
During plant growth the epidermis and cuticle necessarily expand
to provide a continuous protective layer. Expansion of the epidermis
has been proposed to be a limiting factor to organ growth (Kutschera,
1989). The cuticle is composed of polymerized cutin and embedded
hydrophobic compounds, such as fatty acids and other wax
components (Walton, 1990). Cutin has been shown to be synthesized in
the rapidly expanding internodal region of deepwater rice
(Hoffmann-Benning and Kende, 1994) and the bottom third of Clivia
miniata leaves (Lendzian and Schönherr, 1983).
Similarly, the waterproofing CHCl3-extractable
lipids would be expected to be synthesized at a developmental stage
similar to cutin biosynthesis. Our studies showed that in the region of
cell elongation (< 1 cm above the leaf base) free fatty acids
were present, but the major production of epicuticular waxes occurred
later. Similarly, the cuticle thickened in segments well above the
cessation of cell elongation. Since cell division occurs in the
intercalary meristem, below the region of cell elongation, wax
accumulation and cuticle thickening were also not strictly coordinated
with cell division. This result is in contrast to studies of two genes
involved in cuticular wax production in Arabidopsis. CER2
expression is associated with elongating tissues such as developing
siliques (Xia et al., 1996) and CER3 expression is
associated with meristematic regions (Hannoufa et al., 1996). It is
possible that the somewhat delayed induction of wax biosynthesis in
leeks differs from that in dicots because of the nature of tissue
exposure and tissue expansion in leeks. The telescoped stem discs of
the young leek leaves are protected from the environment by the outer,
more mature leaves until the young leaf expands beyond the outer
leaves, and therefore the meristematic region of the young leaf is
protected from the drying effects of an aerial environment.
CHCl3-extractable fatty acids on the lowest leaf
segments may provide the minimal waterproofing necessary on or in the
cuticle to protect the young, developing leek leaves. In dicots the
young leaves are exposed to an aerial environment soon after they
emerge from buds, and therefore a heavier or more complex mixture of
wax components may be required to protect the young dicot tissues from
more arid conditions compared with the conditions surrounding the young
leek tissues.
Elongase Activity Was Induced before the Onset of Wax
Accumulation
The low level of the CHCl3-extractable
lipids in the bottom segments (I and II) of the leaf followed by an
increase in accumulation of total wax load implied that leaf
epicuticular wax production may be due to an induction of the wax
biosynthetic enzymes. The increase in elongase activity (segment IV)
prior to the onset of wax accumulation supports this hypothesis (Fig.
3A). FAS activity increased in segments developmentally older than
segments with peak fatty acid elongase activity. This was observed in
six of seven leaves examined. The relative developmental difference
between the activity peaks suggests that fatty acid elongation and de novo fatty acid synthesis may be under different developmental regulation. The reason for the delay in peak FAS activity is not known,
but the delay suggests that a precursor pool is available for fatty
acid elongation prior to the stimulation of FAS activity. The
constitutive expression and the comparatively high levels of
thioesterase activities suggests that acyl-ACP thioesterases were not
induced and may not be limiting. In addition to wax and glycerolipid
biosynthesis, fatty acids are precursors for cutin biosynthesis. The
increase in cuticle thickness suggests that cutin was being synthesized
in the same regions as wax production, and, therefore, the thioesterase
activities may reflect the combined needs of glycerolipid, wax, and
cutin biosynthesis.
Cellular and Morphological Changes
SEM is more sensitive than GC-MS for detecting the onset of wax
production, since SEM analyses can detect a few crystals on a single
cell, whereas considerably greater levels of wax are required for
detection by GC-MS. However, SEM is not practical for the rapid and
routine detection required for many biochemical and molecular analyses.
SEM analyses showed that wax crystals were first evident in the segment
prior to the onset of wax accumulation, as identified by GC-MS analyses
of CHCl3 extracts. In addition, within this
segment there were areas in which some cells had a dense layer of
crystals and adjacent cells were almost devoid of crystals, indicating
that wax production was not strictly synchronous in cells of the same
latitude.
TEM analyses provided some insights regarding the changes in cellular
morphology that occurred in the segment preceding the onset of wax
accumulation. First, no wax crystals were observed in the lower leaf
segment, where the OTW and cuticle were thin. Second, the OTW
thickening was observed only in the epidermal cells that synthesize and
transport wax and cutin, not in the underlying parenchymal cells, and
it was only in the OTW and not on the inner tangential or radial cell
walls. Third, surface wax crystals were observed by SEM where the
irregular cuticular surface of the OTW also occurred. Fourth, these
morphological changes and the presence of wax crystals both occurred
developmentally earlier in cells of the abaxial surface than in cells
of the adaxial surface. Together, these observations suggest that the
thickening and the irregular cuticular surface may be related to
the deposition of cuticular wax. Additional studies are in progress to
address this issue.
Although environmental factors such as light and humidity have been
shown to affect wax production in some plants (von Wettstein-Knowles, 1995; Post-Beittenmiller, 1996), we suggest that the induction of wax
accumulation we observed in leek leaves was more likely to be
controlled primarily by developmental factors, but was potentially influenced by environmental factors. In this context it should be noted
that the greening of the leaf lamina, which is indicative of
light-induced chloroplast biogenesis in the underlying parenchymal cells, did not correlate with the onset of wax accumulation. The presence of wax crystals, the induction of microsomal fatty acid elongation, and increased levels of hentriacontan-16-one (> 2 nmol/cm2) were found in the nongreen regions of the leaf
sheath, deep within the layered leaves of the false stem. These leaf
segments were relatively protected from strong light and the potential drying effects of an arid environment. In addition, if humidity and
light play a dominant role in inducing wax accumulation, then both the
adaxial and abaxial surfaces of the leek leaf would be expected to have
similar responses when observed by SEM and TEM. However, whereas wax
crystals were abundant on the abaxial epidermis 11.5 to 12.5 cm above
the leaf base, they were absent from the adaxial epidermis in the same
region. Furthermore, wax crystals did accumulate to high levels on the
adaxial surface of the upper segments, suggesting that wax production
on the adaxial surface lagged behind that on the abaxial surface.
Detailed comparisons of the wax composition of the two surfaces by
GC-MS is beyond the scope of these studies but may lead to further
insights into the developmental aspects of wax production. In addition
to the observed differences in wax crystal formation, the abaxial side had more stomata per surface area than the adaxial side, further evidence of different developmental timing for these two surfaces.
Although these results imply that light and humidity are not involved
in the induction of wax biosynthesis in leeks, light has been
implicated in influencing fatty acid chain length in maize (von
Wettstein-Knowles et al., 1980), and high RH has been implicated in
suppression of wax accumulation in in vitro-grown cabbage and carnation
leaves (Sutter and Langhans, 1979; Sutter, 1984). Similarly,
environmental cues such as light or humidity may modulate the
developmental programming of cuticular wax production in leeks.
In these studies we have identified a region in the developing leek
leaf where wax accumulation increases dramatically. Fatty acid-elongation activity was induced in the region preceding the onset
of wax accumulation. The appearance of wax crystals in this region is
associated with morphological changes suggestive of active wax
deposition. The onset of wax accumulation can be identified by
monitoring the levels of hentriacontan-16-one, thus facilitating the
cloning of differentially expressed genes involved in wax production
(Liang and Pardee, 1992; Liang et al., 1995). These studies are
currently in progress.
| |
FOOTNOTES |
1
This research was supported by the Samuel
Roberts Noble Foundation, Ardmore, OK 73402.
2
Present address: Department of Biology, Richard
Bland College, Petersburg, VA 23805.
3
Present address: Department of Crop Sciences,
University of Illinois, Urbana, IL 61801.
4
Present address: Cold Spring Harbor
Laboratories, Cold Spring Harbor, NY 11724.
*
Corresponding author; e-mail dpost{at}noble.org; fax
1-405-221-7380.
Received September 18, 1997;
accepted November 14, 1997.
| |
ABBREVIATIONS |
Abbreviations:
ACP, acyl carrier protein.
Cx:y, fatty acid notation where x = number of carbon
atoms and y = number of double bonds.
FAS, fatty acid synthase.
OTW, outer tangential cell wall.
SEM, scanning electron microscopy.
TEM, transmission electron microscopy.
| |
ACKNOWLEDGMENTS |
We thank Drs. Richard Dixon, John Hipskind, and Jan Jaworski for
critical reading of the manuscript and Drs. Grattan Roughan and Jan
Jaworski for helpful discussions. We are grateful to Ms. Yuling Sun for
harvesting leek epidermis and to Mr. David Huhman for his
assistance in GC-MS analyses. We wish to extend our thanks to Mr.
Bill Chissoe and Mr. Greg Strout at the University of Oklahoma for
their help in SEM and TEM analyses.
| |
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[Abstract]
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