50
P4
LANCASTER MEETING 1996
Plant Cuticles - an Integrated Functional Approach
Organised by G. Kerstiens for the Environmental Physiology Group
Biosynthetic pathways of cutin and waxes and their
sensitivity to environmental stresses. [P4.3]
P.E. Kolattukudy, Neurobiotechnology Center, Ohio State University,
Columbus, USAPlant cuticle Is composed of a structural polymer, cutin, that is
embedded In a complex mixture of highly hydrophoblc soluble
materials called waxes. Cutin is a polyester derived from hydroxy and
hydroxyepoxy fatty acids generated from cellular fatty acids. This
process Includes co-hydroxylation and mid-chain hydroxylatlon at 9 or
10 positions In palmitic acid and co-hydroxylatlon and epoxldatlon of
the olefln at C-9 In olelc acid and linolelc acid followed by hydration of
the epoxide. The monomers are esterlfled to the free hydroxyl groups
In the growing polymer at the extracellular location. Phenolic acids may
also be esterlfied. Cuticular waxes are generated by elongation of
cellular fatty acids to C^ to C32 level, reduction of the very-long-chain
acyl-CoA to aldehyde followed by decarbonylatlon to yield hydrocarbons. Acyl-CoA can be reduced to alcohol followed by esterification
to give wax esters. Environmental factors such as light Intensity and
temperature can differentially affect the synthesis of the cuticular
components. Cuticle synthesis Is a specialized function of epidermal
cells. Injury such as wounding leads to suberizatlon of cell walls that
leads to cell death. Highly suberized cell layer protects stems, tubers
and roots. Suberin Is composed of aliphatic and aromatic domains and
associated waxes. Cuticle can remove hydrophoblc sell Inhibitors in
fungal conidia and thus promote germination. Fungi use specific
cuticular components as signals to initiate developmental changes
needed to penetrate into the host. Fungal gene expression triggered by
the cuticular components will be discussed.
Mutants affected in the biosynthesis of cuticular
waxes (cer) and male fertility in Arabidopsis thaliana.
[P4.4]
R.P. Hodge, R. Hearn and R.J. Scott, Botany, University of Leicester;
M. Aarts and A. Pereira Molecular Biology, CPRO-DLO, Wagenlngen.
The ms2 mutant of Arabidopsis thaliana was generated by
transposon tagging and the gene subsequently cloned (Aarts et al.,
1993). We believe that this gene encodes a fatty acyl reductase which Is
essential for pollen exine formation In wlldtype plants. The cerl mutant
has also be tagged and the gene cloned (Aarts etal., 1995). This mutant
exhibits conditional male sterility due to alteration of the tryphlne layer
covering the pollen grain. The effect of these two genes both Involved
In wax biosynthesis and both affecting pollen wall development and
male fertility will be discussed.
1
Aarts et al., (1993) Nature 363 No. 6431 pp 715-717.
2
Aarts etal., (1995) Plant Cell 7 pp 2115-2127.
Diffusion through cuticles: principles and models.
[P4.5]
L Schrelber and M. Riederer, Julius-von-Sachs-lnstltut fur
Blowissenschaften, Universitat WQrzburg, Germany.
The most Important function of plant cuticles is to restrict the loss
of water and solutes from Interior tissues. Thus, the analysis of the
diffusion of matter across cuticles is essential for the understanding of
numerous cuticular functions. The paper will treat the principles of
diffusive transport across cuticular membranes under steady state
conditions and the factors Influencing cuticular penetration will be
identified. The sensitivity of the transport rates to the driving force, the
solubility and mobility of the solute In the cuticle, the tortuosity of the
diffusion pathway and the heterogeneity of cuticular membranes will
be analysed. Some characteristic results from studies on cuticular
transport properties will be presented. The focus will be on the
experimental methods used for investigating cuticular permeability on
the levels of the intact leaf, the Isolated cuticle and reconstituted
cuticular wax. The available knowledge will be used to formulate a
model of the cuticular transport barrier from a functional point of view.
In this context, the dependence of cuticular transport on the physicochemical properties of the permeant will be analysed. In addition the
molecular structure of this barrier will be discussed and parallels drawn
to similar technical membranes.
Parameters describing permeability of plant cuticles
follow a lognormal distribution. [P4.6]
P. Baur, Phytodermatology Group, University of Hannover.
Water permeability and organic solute mobility in plant cuticles
were found to follow a lognormal distribution. This result was first
obtained from the analysis of a number of 750 joined values for
published (Schdnherr, 1993, Pestic. Sci. 38, 155-164; 39, 213-223)
mobilities of 2,4-D in Citrus aurantium (L.) cuticles coming from one
population of leaves and isolated at one time. A histogram of mobilities
showed a skew distribution with a strong tailing to higher values.
Cuticular membranes with high values did not deviate from others by
visual appearance and were not leaky. After log-transformation of the
original data an almost perfect normal distribution was obtained. The
same distribution was found also for the mobility of NAA in 102 cuticles
of Pyrus communis (L.) and other organic solutes, and also for water
permeances of Isolated cuticles of both leaves and fruits of other
species. A lognormal distribution was found for cuticles of plants from
growth chambers, glasshouse and from natural habitats as well as for
water permeability of intact leaves of Hedera helix(L.). In all these cases
plotting the log-transformed values resulted In straight lines in a
probability plot while the original values where curvilinear. Other
statistical tests like the Kolmogorov-Smirnow-Lltlefors-test also suggested that a normal distribution of original data has to be rejected (p =•
0.05). A normal distribution of original values was acceptable only when
variability was low, but in such cases a lognormal distribution was also
acceptable. Assuming a normal distribution for the original values
overestimated the mean In these large samples by 6 to 22% If the
extreme values which are no outliers are included.
Cuticular permeability of three tree species:
comparison between intact leaves and isolated
cuticles. [P4.7]
T. Klrsch and L Schreiber, Julius-von-Sachs-lnstltut fur
Biowissenschaften, Universitat Wflrzburg, Germany.
Cuticular permeabilities of the three 14C-labelIed acids benzole acid,
salicylic acid and 2,4-dichlorophenoxyacetic acid (2,4-D) were
measured with the three tree species Prunus laurocerasus L, Ginkgo
biloba L and Juglans regia L. It was the aim to test the hypothesis,
whether transport properties of Isolated cuticles are similar to cuticular
transport properties of Intact leave surfaces. Permeances measured
with all three compounds and all three species ranged from 9.6xl0-10
m-s-1 (benzole acid, P. laurocerasus) to 4.4xlO-9 m-s-1 (2,4-0 and J. regia).
In every case permeances obtained with Isolated cuticles were equal to
ABSTRACTS - PLANT AND CELL TOPICS
permeances measured with Intact leaves. Thus, cuticle structure was
not damaged during Isolation and It can be concluded that the
investigation of isolated cuticles reflected qualitative as well as
quantitative transport properties of intact leaf surfaces. Permeances
measured with all three compounds were always lowest with P.
laurocerasus, Intermediate with G. biloba and highest with J. regia. This
is interpreted as an ecological adaptation of the respective species. The
evergreen species P. laurocerasus having the lowest cutlcular permeability must be more adapted to drought and frost Injury compared
to the two deciduous species C biloba and J. regia having significantly
higher cutlcular permeabilities.
Cuticular and whole-leaf exchange of water and gases:
artefacts and interactions. [P4.8]
G. Kerstlens, Biology, Lancaster University.
It appears unlikely that the literature contains more than a couple of
good estimates of strictly cutlcular water and gas exchange rates as
they occur under field conditions. Whole-leaf or canopy measurements
suffer from the uncertainty over the completeness of stomatal closure.
Therefore, they can only provide reliable information about minimum
conductance but not cutlcular permeance for water or air pollutants.
Most measurements with astomatous leaf surfaces or isolated cuticles,
on the other hand, were conducted under highly artificial conditions
not mimicking the natural content and distribution of moisture in the
cuticle, which is known to affect cutlcular permeability. Literature
surveys for both types of experiments, and for water and various air
pollutants, will be presented.
Although cutlcular water permeances are small compared with
(maximum) stomatal conductances, It has been suggested that cutlcular
transpiration may have physiological Importance with respect to (1)
the stomatal response to leaf-to-air water vapour pressure difference,
(2) drought resistance of crops, and (3) winter survival of conifer
needles near the tree line (frost-drought). The evidence for these
hypothetical interactions will be reviewed.
Effects of temperature, surfactants and other
adjuvants on rates of uptake of organics. [P4.9]
J. Schdnherr and P. Baur, Phytodermatology Group, University of
Hannover, Germany.
Rates of foliar uptake of organics depend on permeability of cuticles
and on driving forces. Cutlcular waxes associated with cutln are
responsible for low permeability and this is caused by both low
solubilities and low mobilities of organics In cuticular waxes. Permeabilities of cuticles greatly increase with temperature. Arrhenlus
activation energies from 120 to 170 kJ/mol have been measured between
15 and 35'C, which corresponds to Qio-values ranging from 5 to 10.
Activation energies Increase with molar volumes of organics and they
are higher with cuticles having low permeabilities. Certain adjuvants
called accelerators also increase permeabilities of cuticles by
increasing solute mobility In cutlcular waxes. Examples are fatty
alcohols, ethoxylated alcohols, dialkylesters of dicarboxyllc acids and
trlalkylesters of phosphoric acid. These compounds are sorbed In
cuticular waxes and Increase their fluidity. Accelerators can Increase
mobilities of organics by more than 100-fold and their effects are
proportional to the volume fraction of accelerators. In the presence of
accelerators activation energies of diffusion are greatly reduced. It will
be argued that temperature and accelerators have the same mode of
action, they both Increase fluidity of cutlcular waxes and these effects
are additive. If adjuvants form a spray residue on the surface of cuticles
this always affects equilibrium distribution of organics between cuticles
and residues. This phenomenon Is best treated as an effect on driving
forces.
51
In vivo surfactant uptake into plant leaves mechanism and structure interactions. [P4.I0]
J A Zabkiewicz, R.E. Gaskin, WA Forster and Z.Q. Uu, Plant
Protection Chemistry, NZ Forest Research Institute, Rotorua, New
Zealand.
Surfactants play an important role in enhancing uptake of pesticides
Into plant foliage. In the last decade, a series of organosllicone
surfactants have come to prominence for this purpose with a wide range
of pesticides and plant species. Extensive studies of the performance of
these surfactants In in vivo systems has determined that their ability to
enhance pesticide uptake into leaves is dependent on both rapid
stomatal Infiltration of solution, as well as slower cuticular penetration.
Non-radiotracer analytical methods have been developed recently,
which allow extensive study of surfactant behaviour on and In plant
foliage, either in conjunction with radlolabelled pesticides, or on their
own.
Surfactant movement into intact leaves has been studied quantitatively using high performance liquid chromatography, and qualitatively using confocal microscopy. These methods have considerable
potential to elucidate the Interactions of plant cuticles with solute and
water movement.
Rates of surfactant uptake varied considerably; between conventional and organosllicone types, with surfactant concentrations, and
with plant species. The causes of such variability are poorly understood
at present, although some common denominators have been identified.
It Is possible that In the future, surfactant enhanced solution movement
through cuticles, could be used as a probe for further characterisation
of plant cuticle structure.
Sorption of monodisperse and polydisperse nonionic
surfactants in isolated plant cuticles and reconstituted
cuticular wax. [P4.I I ]
M. Burghardt and M. Rlederer, JulIus-von-Sachs-lnstltut fur
Blowissenschaften, Universitat Wurzburg, Germany.
Nonionic surfactants are used In the majority of agrochemlcal
formulations to enhance the effectiveness of active ingredients.
Technical surfactants are polydisperse mixtures containing numerous
homologous compounds. In order to understand the mechanism of the
interaction of ethoxylated nonionic surfactants with plant cuticles and
to assess which homologues mostly contribute to an Increase in
cutlcular permeability, the sorption of monodisperse alcohol ethoxylates in cuticles Isolated from leaves of bitter orange (Citrus aumntium
L.) and reconstituted cutlcular waxes from barley leaves (Hordeum
vulgare L) were investigated. Below the critical micelle concentration
(CMC) sorption was linear related to the surfactant concentration In
the external aqueous solution. Above the CMC the surfactant concentration In the cutlcular phase and the wax phase remained constant at
a maximum level. The maximum concentration in the cuticle was gtven
by the product of the CMC and the cuticle/water partition coefficient.
Partition coefficients ranged from 25 (C8E4) to 148000 (C16E8). Wax/
water partition coefficients were about one order of magnitude lower
than the cuticle/water partition coefficients and a linear free energy
relationship between the partitioning systems cuticle/water and wax/
water was found. CMCs, partition coefficients and maximum concentrations could be predicted using the alkyl chain length and the
degree of ethoxylation as molecular descriptors. A correlation between
the effects of the alcohol ethoxylates on the mobility of the pesticide
bitertanol In reconstituted cutlcular wax and their maximum concentrations In the wax Indicated that unspeciflc Interactions are
responsible for the modifications of the cuticular barrier properties.
52
Optical properties of epicuticular waxes and cuticular
membranes. [P4.12]
J.D. Barnes, Agricultural and Environmental Science, Newcastle
University.
The leaf surface, like any other dielectric boundary, scatters and
transmits electromagnetic radiation. This paper will highlight the role
played by the cuticle and external wax deposits In attenuating the
amount and quality of the radiation received by the mesophyll mainly
through their affects on leaf reflectance. The ecophyslologlcal significance of the development of a thicker cuticle and extensive wax
deposits In species naturally exposed to high radiation environments
will be discussed, along with Impact of enhanced UV-B radiation on
plant leaf surfaces and vice versa. Finally, the Importance of Information
on bidirectional scattering properties of leaves/plant canopies will be
discussed in relation to the interpretation of data gathered using remote
sensing methods.
Fungal penetration. [P4.I3]
K. Mendgen, University of Konstanz, Germany.
Fungal penetration of the plant cuticle and the wall layers beneath is
a multlstep process with several requirements. The spore and the
infection structures need proper adhesion under more or less humid
conditions. Even if the cuticle is covered with water, the fungus needs
good anchoring to the wax crystals. During differentiation of the
penetration hypha, turgor pressure, cytoskeletal elements and cell wall
degrading enzymes contribute to the forward movement of the tip of
the penetration hypha. The importance of each factor varies depending
on the host-parasite system. The host plant reacts to fungal penetration
by reinforcement of the cell wall and the addition of new wall layers.
The secret of success of the pathogenic fungus may be a balance of
enzyme secretion, application of pressure, and the ability to keep the
host defence responses as low as possible.
Cuticular fracturing in the cherry fruit - causes and
consequences. [P4.14]
L. Sekse, The Norwegian Crop Research Institute, UUensvang
Research Centre, Norway.
Fruits are covered by a cuticle layer of which main functions are to
protect the underlaying tissue from Intrusions by water and pathogens.
Sweet cherries are susceptible to fruit cracking caused by rain on the
fruit surface. Moreover, they are easily attacked by spoilage fungi both
prior to harvest and during postharvest handling. An Intact cuticle layer
covering the sweet cherry fruit Is therefore crucial for protecting the
fruit from cracking and rot development.
Sweet cherry fruits develop cuticular fractures during the last part
of their growth period. The fractures occur in concentric patterns
around the apical cavity and can be studied with a good magnifier. A
method for quantifying them has been suggested. In orchards in
western Norway cuticular fructures in sweet cherry fruits are common.
Their amounts differ between cultivars.
Irregular water supply to cherry trees Increased the amount of
cuticular fractures. This indicates that the fruit volume expansion
caused by water uptake through the fruit pedicel may occur faster than
the fruit cuticle can correspondingly stretch. Consequently fractures
develop. Their patterns seem to be a function of the fruit shape The
cherry fruit has a double slgmoid growth pattern. This may contribute
to the fracture development In the last part of the growth period of the
fruit
Fractures in the fruit cuticle expose the fleshy fruit tissue to water
and spoilage fungi spores. This increases fruit cracking when the fruit
surface is wetted and makes the fruit less resistant to rot Infections.
LANCASTER MEETING 1996
Structure and chemistry of cuticular waxes. [P4.I5]
M. Riederer, Julius-von-Sachs-lnstitut Kir Biowissenschaften,
Universitat Wurzburg, Germany.
In terms of function, the waxes are the most Important constituents
of plant cuticles. This paper will give an overview of the experimental
and analytical methods employed for the qualitative and quantitative
analysis of cuticular waxes and will point at some factors critically
influencing wax composition and analytical results. In contrast to the
functional importance of cuticular waxes only fairly limited information
on their chemical composition Is available. The question will be
discussed whether we really know what cuticular waxes are and how
our knowledge may be biased by the selection of species and
developmental stages Investigated so far, by sample preparation and
by analytical shortcomings.
In comparison to wax chemistry even less is known about the
physical structure of plant cuticular waxes. The main results obtained
so far from x-ray diffraction, DSC, NMR, ESR, photobleachlng, FTIR and
crystallisation studies on cuticular waxes will be assessed and a
preliminary hypothesis of the physical structure of cuticular waxes will
be deduced. This hypothesis will serve as the background for a
discussion of the relationships between chemical composition and
physical structure and between physical structure and functional
properties of plant cuticles like permeability and wettabillty.
Seasonal development of cuticular waxes of beech
leaves: kinetic analysis of wax biosynthesis. [P4.16]
C. Markstadter and M. Riederer, Julius-von-Sachs-lnstitut fur
Biowissenschaften, Universitat WQrzburg.
The amount and composition of cuticular waxes of beech leaves
(Fagus sylvatica L.) were analyzed over the whole vegetation period,
during three years. Rates of biosynthesis of the waxes were closely
correlated to the development of the leaf areas. The velocity of leaf
growth and the rate of wax biosynthesis were strongly dependent on
the climatic factors prevailing during leaf development, whereas the
sequence of the synthesis of the individual wax components was not
affected. Different stages during the leaf development were characterised by specific wax compositions and chain-length distributions.
Slgmoid functions were fitted by non-linear regression procedures to
the kinetics of the leaf-area growth and the biosynthesis of cuticular
waxes. The velocity of leaf growth and the net production rates of
cuticular waxes were obtained by the calculation of the first derivative
of the slgmoid functions. This, finally, allowed to describe the kinetics
of the biosynthesis of all acyl-precursors of cuticular waxes, having
chain-lengths between C20 and C30, Including the different alcohol and
add components of Isomeric n-alkyl esters. On the basis of these results
It Is suggested that three different elongases are Involved in the
synthesis of acyl-precursors. Furthermore during the synthesis of
different substance classes the chain-length speclflty seems to be
caused by spatial separation of elongases Into single compartments and
by a specific association with enzymes catalysing further functlonallsatlons of acyl-precursors.
Structure and composition of plant surface lipids
affecting insect behavior. [P4.17]
S.D. Eigenbrode, Plant, Soil and Entomological Sciences, University
of Idaho, USA.
Plant epicuticular waxes are predominant components of the
physical Interface between Insects and plants. They potentially
Influence the behaviours of both phytophagous and entomophagous
insects In search of host plant or prey. Both chemical and morphological attributes of plant waxes demonstrabry modify Insect behaviors.
Crystalline surface waxes can reduce the effectiveness of Insect
ABSTRACTS - PLANT AND CELL TOPICS
adhestve structures by sloughing off on contact and through direct
chemical adhesive Interactions with the Insect cuticle. This mechanism
not only contributes to Insect trapping by carnivorous plants but also
may provide a defense against herblvory In autotrophlc plants such as
Eucalyptus and Brassica spp. In crops, however, reduced wax blooms
are often associated with less rather than more susceptibility to insects.
Evidence Is presented that reduced-bloom plants obtain protection
from herblvory because attachment, and thus the agility and efficiency,
of active predators and parasitolds Is enhanced on them. In addition,
wax morphology may Influence insect-plant Interactions indirectly
through their affects on water relations that can in turn alter plant
secondary chemistry. Waxes also influence host plant acceptability by
shifting the spectrum of light reflected from the plant surface.
Both major aliphatic components and less-typical minor wax
components can function as allelochemicals affecting Insects. These
may act directly or in synergy with polar components at the plant
surface and may either deter or enhance feeding and opposition. The
evidence supports the notion that plant surface waxes contribute In
important ways to plant-insect and multltrophlc Interactions.
Effects of environment on the composition of
epicuticular wax from kale and swede. [P4.18]
T. Shepherd, G.W. Robertson and D.W. Griffiths, Chemistry; A.N.E.
Birch, Soft Fruit and Perennial Crops; G. Duncan, Virology, Scottish
Crop Research Institute, Dundee.
The leaf surface Is thought to be a major factor In host selection by
the turnip root fly, Delia floralis, a major pest of brassicas in Northern
Europe and Fennoscandla. As part of our investigation of the role of leaf
surface chemicals In insect host selection, the epicuticular wax
compositions of a series kale and swede genotypes which exhibit
varying degrees of antixenotlc resistance to Delia floralis, were
determined by gas chromatography-mass spectrometry. Plants, were
grown indoors (I) and outdoors (0) at SCRI, Scotland and outdoors at
Wadenswil In Switzerland (S). Epicuticular waxes from outdoor-grown
plants (OfS) had higher proportions of n- alkanes, octacosanolc acid,
primary alcohols and long-chain esters but lower proportions of
aldehydes, ketones, ketols and secondary alcohols than waxes from
indoor-grown plants (I). Outdoor-grown plants also had proportionally
more shorter chain-length compounds and indoor-grown plants
proportionally more compounds of longer chain-length.
The patterns of antixenotic resistance exhibited by the brassica
genotypes to Delia floralis were similar to their distribution patterns for
overall wax yields and for certain wax components, particularly the
alkanes, octacosanoic acid and the long-chain esters. Levels of the latter
compounds were more than 50% greater In wax from the most resistant
genotype compared to the most susceptible genotype.
Differences in leaf surface wax ultrastructure, between species, and
between different growth conditions were detected using scanning
electron microscopy.
Shepherd, T., Robertson, G. W., Griffiths, D. W., Birch, A. N. E. and
Duncan, G (1995). Phytochemistry 40, 407-417.
The role of polar and non-polar leaf surface chemicals
in host recognition by cabbage and turnip root flies
(Delia radicum, D. floralis). [P4.I9]
A.N.E. Birch and D.W. Griffiths, Scottish Crop Research Institute,
Dundee; R.H. Hopkins, Swedish University of Agricultural Sciences,
Uppsala; R. Baur and E. Stadler, Swiss Federal Research Station,
Wadenswil; M.S.J. Simmonds, Royal Botanic Gardens, Kew.
Four Brassica genotypes were studied, representing a range of
susceptibility/resistance to oviposltion by both root fly species.
Behavioural studies showed that exploration of the leaf surface was the
key step in host selection for oviposition. Non-polar (dichloromethane)
53
and polar (methanol) leaf surface extracts were sprayed onto artificial
leaves and tested In ovlposition-choice assays. Coating artificial leaves
In paraffin wax greatly enhanced the stimulatory activity of polar leaf
surface extracts In bioassays, indicating that structural or chemical
characteristics of waxes may Interact with polar stimulatory compounds. Polar extracts of the four Brassicas sprayed onto wax-coated
artificial leaves gave the same oviposition preference ranking as real
plants, whilst non-polar surface extracts contained little stimulatory
activity. Electrophysiological responses from specific tarsal chemoreceptors (gustatory) were used to track stimulatory activity in
fractions produced from polar surface extracts. For both fry species
leaf surface glucosinolates were active as oviposition stimuli, but less
active than a recently discovered non-glucosinolate stimulant, called
CIF. Together the glucoslnolate and CD7 contents of the leaf surface polar
extracts largely accounted for the observed oviposition preference
amongst Brassica genotypes. Structure-activity studies using purified
compounds on tarsal receptors Indicated that chemical characteristics
(e.g. overall length and type of side-chain) Influenced activity. Previous
studies showed that the most active glucosinolates (e.g. glucobrassicin)
can be detected by tarsal receptors of D. radicum at 10-8 M, whilst CIF
can be detected at 10-12 M.
1
Baur, R., A.N.E. Birch, RJ. Hopkins, D.W. Griffiths, M.SJ.
Simmonds and E. StSdler (1996). Entomologia experimentalis et
applicata (In press).
Integrated case study: tobacco leaf surface
components and their effects on insect pests and
diseases. [P4.21]
D.M. Jackson, ARS-USDA, U.S. Vegetable Laboratory, Charleston,
USA; DA. Danehower, Crop Science Department, North Carolina
State University, USA.
Most of the 66 species in the genus Nicotiana have glandular
trichomes that produce complex chemical exudates. These cuticular
components consist primarily of diterpenes (duvanes and/or labdanes)
and/or sugar esters (sucrose esters and glucose esters). These
nonvolatile cuticular components have a variety of biological activities,
Including allelochemlcal effects on plants, microorganisms, and Insects.
Duvane diterpenes (especially (a- and (P-4,8,13-duvatriene-l,3-dIols)
and sucrose esters (6-O-aceryl-3,3,4-tri-O-acylsucrose with C3 to C7 acyl
groups) from tobacco, N. tabacum, stimulate the ovlposltlonal behavior
of the lepldopteran pests, Heliothis uirescens (F.) and Manduca sexta
(L). Volatile components produced by Nicotiana species also affect
orientation and alighting behavior of insect pests and beneficials.
Duvane diterpenes (especially, (a- and (fJ-4,8,13-duvatrien-l-ols) and
sucrose esters from several Nicotiana species are highly toxic when
applied topically to the tobacco aphid, Myzus nicotianae Blackman, the
greenhouse whitefly, Trialeurodes vaporariorum (Westwood), the
sweetpotato whltefly, Bemisia tabaci (Gennadius), and the silverleaf
whitefly, Bemisia argentifolii Bellows and Perring. Because pests come
In contact with exudates on the leaf surface of Nicotiana species,
breeding lines with high levels of these components are being used In
tobacco breeding programs. Recent studies have concentrated on
developing sugar esters as natural insecticides for control of softbodied insects such as whlteflies and aphids. Nicotiana trigonophylla,
N. gossei, N. palmeri, and N. glutinosa are good candidates for the
production of natural sugar esters for use as biolnsectlcides.
Primary metabolites and cations on the leaf surface:
their role in insect pest host plant selection. [P4.22]
S. Derrid) and B.R. Wu, Phytopharmacy and Chemical Mediators,
INRA, Versailles, France.
Primary metabolites and cations are substances which are
perceived by Insects and which have an Influence on their behavior.
54
They were neglected In Insect ovipositing behavior partly because they
were not supposed to act on moths during the host plant selection and
their presence on the leaf surface was Ignored. We collected on the leaf
surface of several plant species, soluble carbohydrates, 21 free amlno
acids, organic acids, wax liplds, cations (Na, K, Mg, Ca). Monosaccharldes and cations were vlzualized by S.E.M. and mlcroanalyse on
the leaf surface of different plant species. By labelling 13CO2 we
demonstrated that carbohydrates at the leaf surface could have a
photosynthetic origin and appear as early as 0.5 h after pulse. Free
amino acids composition are plant species specific. According to Insect
behavioral events, substances which are detected on the leaf surface
may be localized in different cutlcular layers. Collecting method Is thus
Important to Interpret substance insect relationships. An activity of the
primary metabolites and cations present at the very above surface, was
observed on the ovlposltion site selection and sensorial perception of
polyphagous lepidoptera moths Os'rinia nubilalis Hbn.. The Insect
showed a very precise selectivity and a sensitivity adapted to the
quantities of substances (ng.cm-2) collected on the leaf surfaces.
Influences of host surface features on development of
Er/siphe graminis and Erysiphe pisl. [P4.23]
T.LW. Carver, S.M. Ingerson and B.J. Thomas, IGER, Aberystwyth.
The Ascomycete fungi Erysiphe graminis DC and Erysiphe pisi DC
cause powdery mildew disease of cereals and peas respectively. An
essential pre-requislte to infection by both fungi is the formation of a
specialised Infection structure, the appressorlum, from which a
penetration peg develops to penetrate host epidermal cells directly.
Appressorlum formation by both fungi represents the culmination of
fungal growth and differentiation processes which occur in an ordered
and predictable sequence driven by the perception of, and response to,
characteristics of the host leaf surface (substratum). We have
attempted to Identify important characteristics by comparing fungal
development on leaves and artificial substrata. When held in a simulated airborne state, spores germinate but appressoria are not formed.
Topographical features of epicuticular waxes do not stimulate
appressorlum formation, but leaf surface hydrophobicity appears to be
Important. Additional stimulation to appressorium formation may be
provided if enzymes released by the fungi are able to degrade leaf waxes
and cutin allowing fungal contact with the underlying host cell wall.
Cuticle and cell wall components released by this activity may In turn
be perceived by the fungi and further stimulate development.
Identification of plant surface characteristics which Impair recognition
of the host by the fungus, and thus impede appressorlum formation,
should facilitate breeding for disease resistance.
Influence of epiphylllc microflora on leaf surface
wetting of Juglans regla L. [P4.24]
D. Knoll and L. Schrelber, Jullus-von-Sachs-lnstitut fur
Biowissenschaften, Universltat WOrzburg, Germany.
The phylloplane of field-grown plants Is often colonised by mlcroblal
populations, which increase with leaf age. One of the most Important
physico-chemical properties of the waxy leaf surface Is Its low
wettabillty by water. Therefore, the Influence of epiphylllc microorganisms on leaf surface wetting was examined measuring contact
angles of aqueous solutions of different pH-values on the upper leaf
surface of Juglans regia L during one vegetation period. Contact angles
decreased from initially 73.0° In June to 49.4" In October. At the end of
the vegetation period wetting was significant lower using basic aqueous
solutions compared to acidic solutions. Contact angle titratlon curves
showed Inflection points around 7.5 Indicating the existence of ionlzable
carboxylic groups In the Interface of the phylloplane. These lnterfaclal
carboxyllc groups might be constituents of the epicuticular wax.
However wax analysis revealed no significant Increase in the amount of
LANCASTER MEETING 1996
alkanoic acids during the vegetation period. However, scanning
electron micrographs of the leaf surface clearly demonstrated an
increasing colonisation of the phylloplane by epiphylllc microorganisms towards the end of the vegetation period. Thus, It was
concluded that age-dependent increase and pH-dependency of wetting
detected at the end of the vegetation period Is caused by epiphylllc
microorganisms. This conclusion could be confirmed measuring
wetting properties of artificial surfaces covered with epiphyllic bacteria.
From these studies It must be concluded that leaf surface wetting
properties can be affected significantly by the presence of epiphyllic
microorganisms on the phylloplane.
Surface wetness and pollutant deposition. [P4.26]
J.N. Cape, Institute of Terrestrial Ecology, Peniculk, Scotland.
Rain and dew are obvious sources of liquid water on leaf surfaces,
but "wetness1 can also extend to molecular films of water molecules in
apparently dry conditions. The structure of a leaf determines how wet
It becomes, and how quickly water evaporates. Both physical and
chemical factors are Involved In the capture and retention of cloud and
rain drops, and in the establishment of continuous aqueous films across
a leaf. Previous exposure to pollutants can also play a large role In
determining how leaf surfaces are wetted. Surface water mobilises
solutes which may have been deposited In dry conditions, allowing
access to Ion exchange sites, and also acts as a sink for the removal of
water-soluble trace gases from the atmosphere. For most trace gases
this Is a reversible process as surface water evaporates, unless chemical
reaction can occur on or with the leaf surface, to render the pollutant
involatlle. The effect of wetness on pollutant deposition is examined at
several scales: at the molecular level, where pollutants Interact directly
with the leaf surface; for the whole-leaf, where surface composition and
structure determine the way leaf surfaces Interact with cloud and rain
drops; and at the canopy level, where surface wetness determines rates
of pollutant removal from the atmosphere, and deposition to the earth's
surface. A simple model is presented to show how leaves are different
from Inert surfaces In modifying pollutant deposition when wet.
Interactions between microbes and deposited
compounds. [P4.27]
S. Huttunen, Botany, University of Oulu, Finland.
Foliar associated micro-organisms can be classified as epiphytes,
endophytes Inhabiting internal plant tissues without causing apparent
Injury to their host, and plant pathogens,causing visible Injury to the
host plants. Aerial algae react positively to Increased atmospheric N
deposition.Epiphytic microfungl on leaves and needles are usually
reduced by acid treatment, and needle endophytes are negatively
affected by acid treatment. A flux of ozone to plant surfaces results in
Its decomposition at the surface and Its uptake through the cuticle.
Ozone deposition velocity increases with the Increase of moisture. A
range of reponses Is true of the Interactions of pathogens and ozone.
Effects on the host plant, on the pathogen or on both may lead to
stimulations or inhibitions. The critical importance of the coincidence
of the timing of exposure and the Infective period to any effect of the
pollutants on the establishment of disease must be emphasized.
Atmospheric deposition occurs by three processes: wet deposition, dry
deposition, both particles and gases, and cloud water deposition.
Surface chemistry and roughness affect leaf wettabillty. Organic acids
may play a significant role on wetted surfaces. Water is the only solvent
naturally available in the phyllosphere.The role of surface water In
modifying pollutant flux rates to the leaf surface Is crucial. Plant
surfaces are somewhat acid, and, in conifers pH varies somewhat less
than in broadleaved plants .Plant surfaces are also negatively charged.
Anionic surfactants, sulphates, and nitrates decay the surfaces. Plant
leaves accumulate llpophlllc compounds to a considerable extent
ABSTRACTS - PLANT A N D CELL TOPICS
55
The design of unwettable plant surfaces: efforts
towards reduced contamination. [P4.28]
Responses of Scots pine needle cuticles to acid
deposition and heavy metals. [P4.30]
W. Barthlott and C. Nelnhuls, Botanlsches Instltut, Universitat Bonn.
M. Turunen, Arctic Centre, University of Lapland; S. Huttunen,
Department of Biology, University of Oulu; K.E. Percy, Canadian
Forest Service.
Cutlcular surfaces represent multifunctional interfaces between
plants and their environments. The cuticle, first of all, serves as a
protection against uncontrolled water loss. Eplcuticular waxes,
however, Influence the spectral properties of plant surfaces, they may
cause microturbulences and they reduce the adhesion of water and
contaminating particles.
Water repellency, as a macroscopic effect, is well known and
documented. While some papers indicate the connection between the
wettability of surfaces and their degree of contamination, they are not
substantiated by experimental data. Recently, we were able to verify
this Interdependence and called It "Lotus-Effect". Water droplets, rolling
off the leaves, quantitatively remove particles (dust, fungal spores etc.),
leading to an almost complete purification of the surfaces.
These anti-adhesive surfaces are characterised by convex to
papillose epidermal cells with a very dense layer of epicutlcular waxes.
In addition, the plants maintain the ability to regenerate waxes for a
considerable period of time. Based on the relief of the surfaces, the selfcleaning properties are hardly affected by natural environmental
influences, whereas they are by certain artificial substances. Tensldes,
in particular, important Ingredients in pesticides, cause a considerable
alteration of wax micromorphology, resulting in enhanced wettability
and increased contamination.
In our opinion, the Lotus-Effect represents the first and very effective
barrier against pathogens. Spores and conidia are removed from the
leaves with every precipitation and are deprived of the free water
essential for germination. In addition, anti-adhesive biological surfaces
serve as a model for several technical applications.
Reasons for the formation of micro-wetness on leaf
surfaces. [P4.29]
J. Burkhardt, R. Elden, University of Bayreuth, Germany.
The presence of an Invisible micro-wetness seems to be of general
importance on plant surfaces as could be shown on a variety of different
plant leaves under field and laboratory conditions. Electrical
conductance measurements indicated continuous water connections
even on very hydrophobic leaf surfaces, which could be attributed at
least partially to a superficial water film.
This indicates that the macroscopically noted contact angles might
not be of primary importance for micro-wetness, compared to other
influencing factors like micro-roughness or the presence of salt particles
on the surface. The micro-roughness of the surfaces Influences wetness
formation directly, leading to capillary condensation, as well as
Indirectly by affecting the dry deposition of sub-micron aerosols. This
latter observation was made during experiments In a wind tunnel using
fluorescent particles.
Salts on the leaf surface may either come from the atmosphere by
deposition or out of the foliage by leaching. The salts will dissolve at
relative humidities (r.h.) superseding their deliquescence point which
in most cases Is substantially lower than 100% r.h.. The sources of water
vapour condensing on the leaves are the stomata and the atmosphere.
Atmospheric trace gases may dissolve In superficial water films,
depending on chemical composition and on Ionic strength of the
solution. Trace gas deposition may be reversible or irreversible, in the
latter case possibly leading to the formation of new hygroscopic salts.
To gain an understanding of the response of the needle surfaces of
Scots pines (Pinus sylvestris L.) to acid deposition and heavy metals, the
physlcochemlcal characteristics of these surfaces were investigated in
multlseason experiments using acid rain (sulphuric and nitric acid pH
3-4) and heavy metals (nickel and copper sulphate, pH 5.7) and in a field
Investigation including a Cu-Ni smelter environment. A harsh subarctic
climate during the needle's first winter had stronger effects on the
structure and chemical composition of the epicuticular wax than the
pollutant treatments. Increased wettability was most evident in the
oldest needles after 2-3 growing seasons with pH 3 acid rain treatment
(1,2,3). With decreasing distance towards the smelter, the eplstomatal
wax tube distribution of overwintered needles decreased and needle
wettability Increased, and a significant site effect was found in the case
of diterpene acids, secondary alcohols and hydroxy fatty acids, but not
for the other wax classes (4,5). The results indicate that pollutantinduced, nonspecific structural and wettability changes were most
evident In the eplcuticular wax of the needles that had overwintered, as
a result of accelerated natural ageing, whereas changes In Its
composition or quantity were mainly caused by metabolic disturbances
in the young developing needles. Despite the prominent protective role
of epicutlcular wax, the exposure of pine needles to the combined
effects of harsh subarctic climate and ambient or applied pollutants led
- to measurable, irreversible deterioration in this layer, which could have
serious ecophyslologlcal consequences in the long term.
1. Turunen M., Huttunen S. 1991. Effect of simulated acid rain on
the epicuticular wax of Scots pine needles under northerly
conditions. Can. J. Bot. 69:412-419.
2. Turunen M., Huttunen S., Back J., Lamppu J. 1995. Acid raininduced changes in cuticles and Ca distribution in Scots pine and
Norway spruce seedlings. Can. J. For. Res. 25:1313-1325.
3. Turunen M., Huttunen S., Percy K.E., McLaughlin C.K. 1996.
Eplcuticular wax of subarctic Scots pine needles: response to
ageing, sulphuric acid and heavy metals (manuscript submitted
for publication).
4. Turunen M., Huttunen S. 1996. Scots pine needle surfaces on the
radial transects across the north-boreal area (manuscript
submitted for publication)
5. Turunen M., Huttunen S., Percy K.E., McLaughlin C.K., Lamppu J.
1996. Structure, chemical composition and wettability of Scots
pine needle eplcuticular wax in a northern smelter area
(manuscript submitted for publication)
The chemical and structural effects of dry air
pollutants on epicuticular wax tubules. [P4.31]
R. Jetter, Inst. Biological Chemistry, Washington State University;
M. Riederer, Botany, Universitat WQrzburg; K.J. Lendzian, Botany,
Technische Universitat Munchen.
The stomata of most conifer species are surrounded by protruding
subsidiary cells forming an eplstomatal chamber. A delicate network of
tubular eplcuticular crystals consisting of (S>nonacosan-10-ol fills this
antechamber as a means to reduce transpirational water loss and to
protect stomata from wetting. These functions may be hampered by
the degradation of tubules into planar masses clogging the antechamber. It has been hypothesised that this degradation Is due to the
direct action of Inorganic air pollutants.
Wax tubules were reconstituted in vitro by crystallising nonacosanlO-ol from the soluble cuticular llplds of Picea pungens on a glass
support and fumigated with dry O3, SO2 or NO2. The resulting crystal
shapes and compositions were monitored by SEM and GC, respectively.
56
Exposure to 1.8% O3 for 100 h, to 100% SO2 for 100 h or to 0.1% NO2 for
up to 1500 h had no effect on reconstituted nonacosan-10-ol tubules. In
contrast, the fumigation with 1% NO2 for 100 h or with 100% NO2 for
0.25-1 h yielded planar crystals of nonacosan-10-one. Further exposure
(1% NO2 for 264 h or 100% NO2 for 2 h) resulted in the complete
degradation of aggregates and the formation of nonadecanoic and
eicosanolc acids. Thus, the stepwise oxidation of nonacosan-10-ol
depended on the applied doses of NO2.
At ambient concentrations of air pollutants, equivalent doses would
accumulate only over thousands of years. Direct chemical Interactions
alone (e.g. oxidations) can therefore not account for the much faster in
vivo degradation of wax tubules on conifer needles.
Structural characteristics of a resistant biopolymer
Isolated from plant cuticles. [P4.35]
J.F. Vlllena, C.G. Casado, P. Luque and A. Heredia, Departamento de
Bioqulmica y Biologfa Molecular, Unlversldad de Malaga, Spain.
The cuticular matrix of plant cuticles Is mainly formed by the
biopolymer cutln, a high-molecular-welght polyester composed of
various interesterified C16 and Cis hydroxyalkanolc acids. After all the
wax and cutln components have been removed from an isolated cuticle
preparation there Is usually some residual material left. For many
species the bulk of this Is mainly cellulose. However, In some species
the residue is noticeable In weight and also contains other type of
aliphatic biopojymer named cutan. To date the molecular structure and
other physical properties of cutan remains unknown; cutans are
extremely resistant to chemical degradation and spectroscoplc and
pyrolitlc studies of cutan Isolated from Isolated leaf cuticles revealed
that, in addition to the celluloslc material, the aliphatic part Is
polymethyienlc In nature.
Data on the Isolation, monomerlc composition and structure
structure of the aliphatic and non-saponifiable fraction of cutan of the
Isolated leaf cuticles of Agave americana and Ciivia miniata are
presented In this work. It Is mainly composed by a mixture of C16 and
C)8 fatty acids In addition to a series of n-alkanes ranging from C19 to C«
with no predominance of odd over even number of carbon atoms.
Calorimetric behaviour of the samples agrees the picture that these
components form a heterogeneous mixture In a layered arrangement
which represents In fact an Insoluble, resistant, contlnous and
hydrophobic biopolymer. The high hydrophoblc character of this
biopolymer has been evaluated after physlcochemlcal studies of
sorption of fatty acids and water permeability through it
Bark and cuticle: a comparative approach. [P4.36]
J.P. Garrec, Pollution Atmospherique, INRA Nancy.
The role of cuticle Is to protect leaves from external blotlc and
abiotic agressors, and to prevent losses of water and elements (Ions,
organic compounds). A similar role is played by the bark for the trunk.
Despite similarities In their properties, bark and cuticle display
completely different structures. As a consequence, bark has to be about
100 times thicker than the cuticle to reach a similar efficiency.
Fixation and permeability coefficients of the cuticle are known for
many elements, but little is known for the bark, although the latter Is a
frequently used path for the penetration of phytosanitary products.
LANCASTER MEETING 1996
Chemical composition of lipophilic biopolymers
isolated from Agapanthus africanus (L.) Hoffmgg.:
comparison between leaf cuticles and endodermal
cell walls of roots. [P4.37]
J. Zeler and L. Schrelber, Julius-von-Sachs-lnstltut hlr
Biowissenschaften, Unlversitat WOrzburg, Germany.
The cuticle forming the Interface between leaf and atmosphere limits
the loss of water, whereas the root endodermis representing the barrier
between the stele and the cortex limits the passive apoplastic path of
diffusion from the soil Into the plant. The cuticle consists of the
biopolymer cutin and associated wax-like lipids, which Is well
established by modern analytical methods. The chemical nature of the
root endodermis Is reported to be cutln-, suberln- and/or llgnin-like, as
concluded essentially from indirect histochemical methods. In order to
obtain more precise analytical data on its chemical nature, endodermal
cell walls and cuticles of Agapanthus africanus (L.) Hoffmgg. were
Isolated enzymatlcally. Scanning electron microscopy Indicated that
endodermal Isolates were predominantly In the tertiary developmental
state. Endodermal cell walls as well as cuticles were subjected to
different chemical degradatlve reactions. The main cuticle monomers
recovered after depolymerlsation by transesterlficatlon were the
typical cutln acids 9,16/10,16-dihydroxyhexadecanolc acid, 9,10-epoxy18-hydroxyoctadecanolc acid and 9,10,18-trihydroxyoctadecanolc acid.
The endodermal Isolates released mainly 16-hydroxyhexadecanolc
acid, 18-hydroxy-9-octadecenoic acid, 9-octadecenolc-l,18-dlacld and In
smaller amounts oo-hydroxyaclds (C16-C2O. l.co-dlacids (C16-C24), fatty
acids (C16-C25), 1-alcohols (C22,C24) and various phenylpropane
monomers. Furthermore, thloacldolysis and cupric oxide oxidation
indicated significant amounts of lignln in the endodermal cell walls.
Therefore, the tertiary developmental state of the endodermal walls of
A. africanus roots is characterised by lignlfled and suberized cell walls.
A detailed knowledge of the chemical composition will finally help to
better understand the barrier properties of the root endodermis.
An FTIR-spectroscopic method for the physical
characterization of plant cuticular waxes. [P4.38]
S. Bach and M. Riederer, Jullus-von-Sachs-lnstltut fur
Biowissenschaften, Unlversitat WOrzburg, Germany;
A. Blume, Unlversitat Kalserslautern, Germany.
An FTIR-spectroscopic method has been developed for the
determination of melting regions and crystalllnlty of aliphatic
components of plant cuticular waxes. Temperature-dependent spectra
of waxes extracted from isolated cuticles can be measured. For this
purpose a thermostated sample holder for horlzontal-ATR was
constructed. The control of temperature, equilibration and scanning of
the sample Is automated. For our measurements the temperatures
range from 3 to 95°C at 2K Intervals. Selected absorption bands are
indicative for the physical state of aliphatic waxes: A shift In the
absorption maxima of asymmetrical and symmetrical CH? stretching
bands at 2920 and 2850 cm-< respectively can be used for the
determination of the melting region. The relative peak areas of the CH2
wagging band doublet at 720 cm-' Is taken as a measure for
orthorhomblc crystalllnlty.
More than 20 waxes have so far been investigated. In most cases, the
crystalllnity of aliphatic components at room temperature is higher
than 80%. Parallel measurements of water permeation through
astomatous Isolated cuticular membranes at different temperatures are
In process and the obtained data will be tested on correlation with the
respective crystalllnltles of the cuticular waxes.
57
ABSTRACTS - PLANT A N D CELL TOPICS
Determination of the composition of intact alkyl
esters in leaf epicuticular wax of swede by capillary
gas chromatography and electron-impact mass
spectrometry. [P4.39]
Sorption of volatile organic chemicals to plant
surfaces. [P4.42]
T. Shepherd, G.W. Robertson and D.W. Griffiths, Chemistry, Scottish
Crop Research Institute, Dundee.
The leaves of plants living under the conditions of a technical
civilization contain, In addition to the natural constituents, a complex
mixture of organic contaminants originating from Industrial activity,
combustion processes In traffic and homes and agriculture. Therefore,
the question arises how these chemicals may enter and accumulate in
plants. For organic compounds, there are in principle two different
routes of uptake: (1) the stomatal and (2) the cutlcular pathway. Playing
a central role during the foliar uptake the cuticle also functions as a
compartment of accumulation for lipophllic chemicals. Sorption
experiments were carried out with enzymaticalJy isolated tomato fruit
cuticles. Dewaxed cutlcular membranes (MX) were prepared by
exhaustive extraction in a Soxhlet-apparatus. Numerous volatile organic
compounds with varying physico-chemical properties, molecular
structures and functional groups were applied. Static headspace gas
chromatography was used to determine sorption Isotherms and cuticleair partition coefficients at 25°C. The Isotherms were linear within the
range of concentrations used. This corresponds to an ideal distribution
according to Henry's law. Boiling points and vapour pressures proved
to be applicable to coarse estimations of cuticle-air partition coefficients. In addition, air-water partition coefficients were obtained by
static headspace gas chromatography. They were used together with
cuticle-air partition coefficients for estimating cuticle-water partitioning.
Complex mixtures of more than 50 different Intact esters In
epicuticular wax of swede, Bmssica napus var. rapifera were separated
and Identified using combined gas chromatography-mass spectrometry
(GC-MS). The esters were separated at temperatures of up to 350'C by
automatic pressure programming (capillary GC) or by manually
increasing helium carrier gas pressure (GC-MS) to maintain flow at high
temperatures. Esters in the range C41-C50 were detected, with regular
repeating combinations of acid and alcohol moieties.
The identities of Individual acid and alcohol moieties were
determined from the molecular ions [M]* (RCO2R1) in the mass spectra
of intact esters and from fragment ions [RCO2H2]* derived from the acid
moieties by McLafferty rearrangement, together with the mass spectral
and chromatographic characteristics of the methyl esters of the acid
components (C14-C23) and trimethylsilyl derivatives of the alcohol
components (C23-C31) foliowing transesteriflcation and silylation.
The major components of the ester mixtures were doubly branched
C44 and C46 compounds consisting of anteiso Cn/anteiso- C27 (32.7%),
anteiso- Cn/anteiso- C29 (11.9%) and anteiso- Cy^anteiso- C27 (13.1%)
acid/alcohol combinations, respectively. Singly branched C43 and C45
esters, composed of n-C\danteiso- C27 (8.5%), anteiso- Cn/n-C26 (3.9%),
n- CisJanteiso- C27 (6.8%), anteiso- Cn/n- C28 (2.3%) and anteiso- C19/nC26 (1.6%) acid/alcohol combinations, were minor components.
Shepherd, T., Robertson, G.W. and Griffiths, D.W. (1995)
Phytochemical Analysis 6, 65-73.
Recrystallization of wax tubes on mechanically
injured surfaces of spruce needles. [P4.40]
E. Bermadlnger-Stabenthelner and D. Grill, Institute of Plant
Physiology, University of Graz; H. Waltinger, Centre of Electron
Microscopy, Graz.
The needles of several types of conifers are covered by wax forming
tube-like structures that are easily affected by physical (mechanical)
injury (Van Gardlngen et al. 1991, Plant, Cell and Environ. 14, 185-193;
Bermadinger-Stabentheiner 1995, New PhytoL 130, 67-74). To simulate
severe mechanical influence needles of Picea abies (L)Karst. and Picea
pungens Engelm. var. glauca BelBn. were wiped with a soft cotton cloth
and the needle surfaces were Investigated with a scanning electron
microscope. As a consequence of the experimental procedure the
epicuticular wax layer was smeared and the wax tubes were squashed
to an amorphous mass. As early as 48 hours later, however, a
considerable regrowth of wax tubes could be observed on needles of
both species. Regrown wax tubes were observed Independent of the
needle age class but the amount decreased with increasing needle age.
Corresponding to the originally present wax structures the diameters
of the regrown tubes increased with increasing needle age. A distinct
regrowth of wax tubes could also be observed on freeze-dried spruce
needles stored in a dry atmosphere for several months. With this dead
material a resynthesis of wax compounds can definitively be excluded.
Therefore the regrowth of wax tubes on mechanically injured needle
surfaces can be classified as re-crystalllzatlon. Epicuticular waxe Is
capable to organise Itself Into tubular structures (Jeffree et al. 1975,
New PhytoL 75,539-549). This capability can also be observed on natural
needle surfaces and obviously no solvents are necessary for this reorganisation.
B. Welke, M. Rlederer, JuIlus-von-Sachs-Instltut fur
Biowissenschaften, Unlversltat WOrzburg.
A leaf disc method for measuring cutlcular
conductance. [P4.43]
S.P. Hoad, University of Nottingham; J. Grace and C.E. Jeffree,
University of Edinburgh.
This paper describes a method for measuring cuticular conductance
(gj using a leaf disc sealed In a specially-designed envelope. Leaf discs
were punched out from attached Fagus syluatica leaves and placed
inside Individual envelopes that provided a water supply. Conductances
were determined from gravimetric measurements of water flux from an
exposed epidermal surface of the leaf disc. Conductances were
determined for astomatous (adaxlal) and stomatous (abaxial) surfaces.
All measurements were made under darkness. Conductance of the
adaxlal surface was referred to as &., whereas conductance of the
abaxial surface was considered as a minimum leaf surface conductance
(gtuMIN). The main advantages of this method are: (I) measurements
are made using intact leaf cuticles, (if) leaf relative water content is
maintained above 85% and (Hi) reduction In cutlcular hydratlon is
mfnlmised. Conductances determined from leaf discs in envelopes were
compared with those of whole leaves and leaf discs without envelopes.
Data In this study demonstrated a strong positive relationship between
conductance and RWC.
Hoad, S.P., Grace, J. and Jeffree, C.E. (1996) Journal of Experimental
Botany (in press)
Humidity response of cuticular conductance in beech
leaves. [P4.44]
S.P. Hoad, University of Nottingham; J. Grace and C.E. Jeffree,
University of Edinburgh.
Measurements of cuticular conductance (&) ' n leaves of beech
(Fagus syloatica L) were made using a leaf-disc envelope, as described
in Poster P4.43. Conductances were determined from gravimetric
measurements of water loss. Measurements were made for astomatous
(adaxlal) and stomatous (abaxial) surfaces. Conductance of the
astomatous surface was referred to as g^ whereas conductance of the
58
stomatous surface was considered as a minimum leaf surface
conductance (gfAfflV). In experiment I, leaf discs were placed under one
of a range vapour pressure deficits (D). Both gc and %<M1N decreased 2fold with an increase in D between 0.4 to 2.0 kPa. The decrease in g,. was
linear, but gjMWdeclined more steeply at D between 0.4 to 0.9 kPa than
at D between 0.9 to 2.0 kPa. In experiment II, leaf discs were exposed to
either a decrease or Increase In D. After a period of acclimation to D of
0.9 kPa, responses of &. and g<MlN to a stepwlse change in D were
recorded. The response times of ft. to increasing or decreasing D were
similar (< 80 min). By contrast, g<MIN responded more slowly to
increasing than to decreasing D. This study provided further evidence
for a significant response of cutlcular conductance to changes In D. The
hydratlon state of the cuticle may be the key factor in determining the
level of response of cutlcular conductance to an increase or decrease In
D.
Effects of elevated UV-B radiation on cuticular water
permeability of ivy grown under natural or artificial
light. [P4.45]
G. Kerstiens, SA. Moody and N.D. Paul, Biology, Lancaster
University.
Ivy (Hedera helix L var. Buttercup) was grown for 10 weeks in growth
cabinets (high pressure sodium lights, metal hallde lights and UV-A
black lights; PPFD: 0.8 mE m-2 s 1 ; 12h photoperlod plus "dawn" and
"dusk" periods of 2h each). Treated plants received 6.3 kj cH UV-B
(plant-weighted) over lOh (controls: none). This was approximately
4056 more than the dally ambient clear-sky UV-B dose during summer at
Lancaster. The treatment caused a significant Increase In cuticular
water permeance P of newly-grown leaves (+74%). Irrespective of UV-B
treatment, all plants grown in this experiment showed visible signs of
stress, such as slow growth and premature yellowing of leaves. Plants
from the same batch kept In a greenhouse did not show these
symptoms. Their P values were between those of the other two groups.
Ivy (H. helix ssp. hibemica) was grown from May to October in a
modulated field system in which plants received additional UV-B as a
percentage of incident UV-B (see PI. 18). UV-B additions were based on
a model assuming an average ozone reduction of 15% but with seasonal
variation, resulting in UV-B additions of 20-45% depending on season.
Control plants received no artificial light. A third group of plants was
grown in a greenhouse with no supplemental UV-B. No differences In P
between any treatment were found. Plants showed no signs of stress.
Attenuation of ultraviolet radiation by plant cuticles.
[P4.46]
P.S. Krauss, C. Markstadter and M. Riederer, Jullus-von-SachsInstltut fur Biowlssenschaften, Unlversitat WOrzburg.
As a consequence of the decline of stratospheric ozone concentrations enhanced levels of ultraviolet radiation reaching plant
communities are to be expected.
In primary plant organs, the cuticle is the first potential absorber for
Incidental radiation. The cutlcular membrane consists of the polymer
cutin and the soluble cuticular waxes and, in many species can be
separated from the cell wall by enzymatic hydrolysis of ester-links.
The transmission spectra of cutlcular membranes enzymatlcally
isolated from a variety of leaves and fruits were measured. In the visible
region, the transmission was In the order between 70% and 90%. Toward
the ultraviolet region of the spectrum transmission was increasingly
reduced. At a wavelength of 300 nm, transmission ranged from 1% to
55%. In selected cases transmission measurements were repeated after
wax extraction. The waxes contributed from 8?J to 30?J to the total
attenuation effected by the cuticle. In several species, Isolated cutlcular
membranes could not be obtained due to their mechanical Instability.
In these cases, leaf wax extracts only were examined. From transmission
spectra of these extracts it was calculated that their contribution to the
attenuation of ultraviolet radiation was below lOJS.
LANCASTER MEETING 1996
Leaf surface features (Populus x euramericana and
Populus tremula) developed under lowered or ambient
UV-B radiation. [P4.47]
M.S. Gflnthardt-Goerg, Swiss Federal Institute for Forest, Snow and
Landscape Research, Birmensdorf, Switzerland.
Whether natural UV-B radiation may be Increased through a
depletion of stratospheric 03, or decreased through anthropogenic
emmlsslons (smoggy clouds reflect incoming radiation, tropospheric
O3 reduces it), even very small changes are supposed to Influence the
vegetation. To assess the risk, we have to understand the plant
reactions to small changes in ambient UV-B radiation over at least the
life span of the leaves.
We therefore exposed cuttings in the field to ambient conditions
before leaf formation and let them develop until autumn leaf colouring.
Exposure was either to ambient radiation (100%), or to ambient with
reduced UV-B (transmlttance = 0 < 250, 72% at 290,93% > 400 nm), or to
another reduced UV-B (t - 0 < 250, 42% at 290, 93% > 400 nm), or to
ambient radiation without UV-B (t = 0 < 340 nm, 97% > 380 nm). The UVB reduction was established by adjustable Plexiglas roofs.
Because no UV-B radiation reaches the under leaf surface, the upper
leaf epidermis is supposed to be the primary site of the UV-B perception.
We therefore selected two poplar species, an amphlstomatous (P.
euramericana) and a hypostomatous (P. tremuloides). Results concerning the cutlcular and epidermal structure and cytochemistry, epidermal
wax extracts (peak at 273 nm) chlorophyll fluorescence, projected leaf
area and total bioraass will be presented.
Effects of UV-B treatment on the epicuticular wax
structure of Scots pine (P/nus sylvestris L.) needles.
[P4.48]
H. Klnnunen and S. Huttunen, Biology/Botany, University of Oulu,
Finland; E. Laitat, Blologle Vegetale, Gembloux, Belgium.
Structural properties and certain biochemical changes protect
plants from ultraviolet-B radiation. Adaption to UV-B radiation may be
best developed In species that occur in high UV-B lrradiance
environments. The purpose of this work was to study the effects of UVB treatment on Scots pine (Pinus sylvestris L.) needles from southern
and northern provenances.
The experiment was conducted during 13 weeks in May-July 1994 in
greenhouses In Belgium, Gembloux. The plants were Irradiated with
UV-Jamps (Philips UV-B TL 12 40 W). Three UV-BBE doses were used:
22.64 kJ m-2d-i, 11.32 kJ m-2d-' and 0 kJ m-2d-i. The plant material
consisted of Scots pine seedlings originating from Poland (52'N), France
(45'N and 47'N) and Finnish Lapland (68*N). Current year needles were
collected and mlcrographed under an SEM (JEOL JSM-6400).
High UV-B treatment Increased slightly the distribution of tubular
waxes In the Finnish pine needles, while there were no differences
between the treatments in the Polish or French pine needles. There
were no significant differences In the wax structure between the
provenances in this short-term experiment. Finnish pines, which grow
naturally in low UV-B conditions, may react against UV-B more rapidly
by producing more waxes.
Effects of fungal disease on cuticular integrity in
wheat. [P4.49]
S.P. Hoad, University of Nottingham.
Winter wheat (Triricum aestivum L. var. Beaver) was grown under
field conditions. One part of the crop was treated with fungicide to
minimise disease infection. A second area was left untreated to allow a
natural development of yellow rust (Puccinia striiformis). Disease levels
were assessed as a percentage of leaf area affected. Cutlcular Integrity
was assessed by using a dye tracer to detect discrete cutlcular lesions.
ABSTRACTS - PLANT A N D CELL TOPICS
A minimum leaf surface conductance (gm/fflV) was determined from
gravimetric measurements of water loss from a 3 cm2 portion of whole
detached leaves. All measurements were made on the adaxial surface
of the two uppermost leaves. In the fungicide-treated plants, yellow rust
infection was less 0.556. In the infected plants, percentage disease had
increased to 12% by late July. The number of cutlcular lesions increased
with leaf age, but was significantly greatest (33 lesions per cm2) in the
infected plants. g^^MW was approximately 2-fold greater in the infected
plants compared to uninfected plants. Generally, the number of
cutlcular lesions was positively related to the level of infection and
gsurMIN increased with an increase In the number of lesions. However,
after mid July, g^MlN had significantly declined in leaves with the
highest levels of infection. This may be attributed to a greater loss of
functional leaf area and/or hydraulic isolation of tissue adjacent to
cutlcular lesions. These data suggest that fungal diseases may have
significant consequences for cuticular Integrity and leaf water relations.
Synergism between manganese leaching and SO2
oxidation in dew on spruce needles. [P4.50]
J. Burkhardt, University of Bayreuth, Germany.
Mn2* ions are known to catalyze the oxidation of SO2. On the other
hand, Increasing amounts of Mn2* Ions are leached from leaves with
decreasing pH of the wetting solution. This study was conducted to
determine whether both processes form a positive feedback
mechanism between SO2 oxidation and manganese leaching on the
surface of spruce needles.
Three groups of 4-year-old spruce seedlings were treated with
nutrient solutions of different manganese concentrations. After three
months, the trees were placed In a dew chamber and sprayed in the
evenings with a neutral salt solution (lmM KC1). A gas concentration of
about 50 to ISOjig nr 3 SO2 was established. Each morning, the drops
were collected from the spruce needles. S(TV) was stabilized and the
sample pH was determined along with concentrations of sulfate,
manganese, and other ions.
Manganese concentrations in the needles were within the range of
natural occurring values. Sulfate production on the needle surfaces
increased significantly with increasing manganese content of the
nutrient solution. The formation of sulfate on the needles of trees with
the highest manganese nutrition level was about twice the amount as
those with the lowest manganese nutrition level.
An extrapolation of these results makes a positive feedback
mechanism on a higher spatial level reasonable: SO2 deposition leads
to an acidification of the soil. This will enhance manganese availability
and manganese uptake, if soil pH Is higher than the manganese buffer
system. According to the results of this study, increased manganese
uptake would increase SO2 deposition to the ecosystem, thus producing
further soil acidification.
The influence of leave microroughness on dry
deposition of fine particles. [P4.5I]
J. Burkhardt, K. Peters, University of Bayreuth, Germany;
A. Crossley, ITE, Bush Estate.
Monodlsperse particles (particle diameter x 0.5 \aa) were produced
by a particle generator and tagged with a fluorescent dye. The particles
were Injected Into a small wind tunnel, Into which single needles or
small branches of Picea abies (L) Karst, Pinus sylvestris L, and Abies
alba Mill, had been introduced. The needles were examined after
treatment, using fluorescence microscopy, and the spatial patterns of
particle deposition determined.
The particles deposited preferentially In the stomatal regions of the
needles. In these areas the Incidence of micro-roughness due to
epicutlcular waxes Is highest, reducing the laminar boundary layer of
the needle. Very little deposition of particles took place In regions
without structural surface waxes.
59
Atmospheric particles of less than 1 nm diameter are mostly
hygroscopic and are likely to be dissolved in the humid surroundings of
open stomata. Therefore, the effect of structural leaf surface waxes In
an environment of high atmospheric particle loads is rather to enhance
surface wetting than to reduce it.
Cuticular characteristics in maize lines selected for
differences in leaf ABA content. [P4.52]
Lj. Proki, Z. Jovanovi, R. Stlkl and S. PeH, Faculty of Agriculture,
University of Belgrade.
The effect of drought on leaf cutlcular thickness was Investigated In
different maize lines. The lines chosen for this Investigation were from
an absclslc acid (ABA) selection programme and, according to the leaf
ABA contents, were classified into high-ABA and low-ABA lines.
Therefore, the aim of our work was to examine the consequences of
genetic variation in leaf ABA content on leaf anatomy and xeromorphic
characteristics, of which cuticular thickness is one.
Eight lines of maize (four hlgh-ABA and four low-ABA) were grown In
the field under ralnfed and irrigated conditions. Shortly after flowering,
the lamina of the third leaf from the top was sampled for anatomical
measurements. Leaf sections were cut with a hand microtome, bleached
in diluted Parazone and stained with 1% safranln and 1% alclan blue.
After dehydration and mounting In Canada balsam, samples were
analysed with a Vegacame Image processing system.
Our results showed that drought induced cuticular thickening In
leaves of all the high- and low-ABA selections, as well as in leaves of
their early-maturing parental lines (PolJ17 and F-2, Jovanovi et al. P4.53,
SEB meeting, 1996). Although the lines differed significantly In leaf ABA
contents at the time of sampling, both In irrigated (hlgh-ABA lines ca.
527 and low-ABA lines ca. 183 ng/gdw) and droughted conditions (hlghABA lines ca. 680 and low-ABA lines ca. 169 ng/gdw), differences In
cutlcular thickness were not significant either in the irrigated (highABA lines 3.76 urn and low-ABA lines 3.73 urn) or the droughted field
(hlgh-ABA lines 4.22 urn and low-ABA lines 4.10 urn). Therefore, the
genotyplc variation In leaf ABA content did not Induce differences in
cuticle thickness and therefore consequences of such variation should
be tested using other leaf xeromorphic features.
Cuticular characteristics in maize lines differing in
drought resistance and maturity grouping. [P4.53]
Z. Jovanovi, LJ. Proki, R. Stlkl and S. Pekl, Faculty of Agriculture,
University of Belgrade.
Previous Investigations with several maize lines supported the
hypothesis that lines with higher levels of leaf abscisic acid (ABA) and
drought resistance are more xeromorphic than lines with lower levels
of leaf ABA and drought resistance (Rlstl and Cass 1991a, b). Since
cutlcular thickness Is of particular Interest for plant drought reactions,
the aim of this presentation was to test this hypothesis for cuticle
thickness In the same lines grown under field conditions.
For this Investigation we chose four maize lines from two diverse
maturity groups that also differed in leaf ABA content and drought
resistance. Two lines were investigated from an early maturity group
(Pol) 17, hlgh-ABA and drought resistant, and F-2, low-ABA and drought
susceptible) and from a late maturity group (ZPBL1304, high-ABA and
drought resistant, and ZPL389, low-ABA and drought susceptible).
Plants were grown in the field under ralnfed and irrigated conditions.
Shortly after flowering, the lamina of the third leaf from the top was
removed and used for anatomical measurements. Leaf pieces were cut
with a microtome, bleached In diluted Parazone and stained with 196
safranin and 1% alclan blue. After dehydration and mounting in Canada
balsam, samples were analysed with a Vegacame image processing
system.
Our results showed that drought caused an increase In cutlcular
thickness In all the genotypes. Comparison of the lines showed that
60
under field conditions the drought resistant, hlgh-ABA lines Pol) 17 and
ZPBL1304 had thicker cuticles (4.53 and 5.13 tun respectively) than the
drought susceptible, low-ABA lines F-2 and ZPL389 (3.81 and 4.13 nm
respectively). These results were In agreement with results obtained
from the same genotypes under drought stressed conditions in a
controlled environment (Rlsti and Cass 1991a), but differed from those
for well-watered plants of the same genotypes (Rlstl and Cass 1991b).
These results highlight the complexity of the investigations of plant
morphological adaptation to drought and indicate a need to extend
investigations to plants growing under different conditions.
Ristic, Z. and D.D. Cass (1991a) Bot Gaz. 152: 173-185.
Ristic, Z. and D.D. Cass (1991b) Bot Gaz. 152: 439-445.
A new SEM method for scoring the wax tube
distribution of needles. [P4.54]
S. Manninen, R. Peura and S. Huttunen, University of Oulu.
A more objective scanning electron microscopic method for
estimating the coverage of wax tubes has been developed using Scots
pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.)
needles as research material. The quantitative area fraction analysis is
based on backscattered electron images (BEI) (1300x), from which the
proportion of wax tubes is scored as a percentage of the total needle
surface area with a phase-analysing program of the EDS analyser. Three
colour bands provided by the program using the grey level thresholds
within the BEI have been Included: green for the wax tubes (brightest
areas) and red for the fused epicuticular waxes. Because of the
topographic structure of the stomatal areas of especially Scots pine
needles, some parts of the eplstomatal areas appear black, although
they are covered by wax tubes, and the proportions of the green and
black areas are therefore counted together. Successive analysis runs
can be accumulated In a file, and the results can be expressed as means
of ten analyses per needle or tree, for example. The SEM method thus
yields more exact values for the wax tube distribution than the other
classification methods. BEls are used to Increase the contrast between
the areas covered by wax tubes and the eroded areas. The method Is,
however, not highly practicable If there are many particles or biotlc
Infection on the needle surface, because they are analysed Into the
brightest phase together with the wax tubes.
Presence of crystals of calcium oxalate on the
phylloplane of cassava Manlhot escu/ento Crantz.
[P4.55]
S. Renard and C. Gaspar, Faculte des Sciences Agronomlques, U.E.R.
de Zoologie Generate et Appllquee, Gembloux, Belglque; B. Le RO,
O.R.S.T.O.M., Laboratolre d'Entomologie, Polnte Noire, Republique
du Congo; J. Le Lanic, Unlversite de Rennes I, Centre de Microscople
Electronique a Balayage, Rennes, France; E. Laltat, Faculty des
Sciences Agronomiques, U.E.R. de Blologle Vegfetale, Gembloux,
Belglque.
A study of the potential factors that could affect the resistance of
cassava (Manihot esculenta Crantz) to the mealy bug Phenacoccus
manihoti Matile-Ferrero (Homoptera, Pseudococcidae) included a
comparison of the physical characteristics of the phylloplane for three
LANCASTER MEETING 1996
varieties of cassava. Scanning electron microscopy was used to Identify
any secretion or structures on the leaf surface (e.g. glands or hairs) that
might play a role in the host recognition behaviour of the mealy bug.
An analysis of both the abaxial and adaxial surfaces of the leaf blade
revealed crystalline structures enclosed within spherical cells
embedded in the epidermis. These cells were 5-15 nm in diameter,
dependent on variety, with the crystalline structure commonly
arranged in groups of two or three.
The crystals were proposed to consist of calcium oxalate and this
hypothesis was confirmed by RX microprobe analysis. Although It Is
the first time that such structures have been described for M. esculenta,
excess calcium is known to be stored in crystalline form in several plant
species.
Financial support was provided by the FRIA.
Morphophysiological evaluation of needles of Pinus
sylvestris trees near local pollution sources in
Lithuania. [P4.56]
E. Kupclnskiene, Botany, Lithuanian Academy of Agriculture,
Kaunas-Noreikiskes, Lithuania.
Investigation of pollutant sources and the state of pollution In the
forests of Lithuania revealed Mazelklai oil refinery and Jonava nitrogen
fertilizer plants among the main point source polluters. Up to now,
studies of diagnostic tests for conifers Identified leaf surface
characteristics as good indicators of tree vitality. The main objective in
setting up this survey was to evaluate Pinus syloestris trees under the
influence of ambient pollution using some tests proposed to detect
latent injury. Pinus sylvestris trees were sampled in 1993 summerautumn period in both Mazelklai (near an oil refinery) and Jonava region
(near a nitrogen fertilizer plant) sites located according to prevailing
wind at different distance from pollution sources. Wettabillty
assessment showed that contact angles on current year needles ranged
between 64T-67.2* and on one-year-old needles were between 38.4'48.8* with the lowest contact angles in the site situated near the nitrogen
fertilizer plant Contact angles on current year needles from various
sites In Mazelkiai region ranged between 66.4'-65.9\ while on one-yearold needles, angles were 43.8'-48.1'. In the present work modified
Tuomlsto (1988) classification was used to analyze pine needles
surfaces by scanning electron microscope, both approaches used
(division of waxes into total eroded and total tubular wax as well as
more precise classification into initial tubes, tubes slightly fused, tubes
fused, net lying on the surface, alone tubes lying on the surface, uneven
amorphous crust, even amorphous crust) were important indicating
extent of surface damage. Significant differences occurred between sites
In the structural types of needle wax covering. Data obtained shows
possibility of effects of low level of pollutants to induce detectable
structural damages during one and half year existence of needles.
Comparison of results revealed the similarities between SEM
observation and contact angle estimations: both methods showed the
greatest needle erosion extent in the sites located near both pollution
sources (nitrogen fertilizer plant and oil refinery). The higher wax
degradation we found In the needles from the sites nearest to the
pollution sources (nitrogen fertilizer plant and oil refinery), as well as
in previously completed assessments (element analysis) could be
explained by direct pollutant mixture synergetlc effects on needle
surface rather than Indirect Influence through the root system.
ABSTRACTS - PLANT AND CELL TOPICS
61
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EMBRYOGENESIS
The generation of a plant
Edited by T.L. Wang & A.C. Cvuning
respectively John Innes Centre, Norwich and
University of Leeds, UK
Embryogenesis is an increasingly important research
topic in plant development. The recent application of
genetics and molecular biology to the subject promises
major advances in our understanding of plant
development.
This book brings together recent research on plant
embryogenesis by key workers in the field. It covers
the major formative stages from the inception of the
ovule, through early organ development and pattern
formation to the final dry embryo, and considers
major crop species such as maize, as well as the model
system, Arabidopsis.
1-85996-065-0; 19%; Case vound, 240 pages;
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Flexibility and aetllaatlea
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Contents
Perspectives on the molecular cytology of
embryogenesis, VRaghaven; Genes involved in the
elaboration of the apical pattern and form in
Arabidopsis thaliana: generic and molecular analysis, R.Torres-Ruiz et al.; Embryo-defective mutants
of Arabidopsis: cellular functions of disrupted
genes and developmental significance of mutant
phenotypes, D.W.Meinke; Insertional mutagenesis
to dissect embryonic developments in Arabidopsis,
K.Lindsey et al.; Protein kinase genes expressed
during plant reproductive development J.Tregear
et al.; Maize embryogenesis mutants, J.Clark; Gene
expression and embryonic maturation in cereals,
A.C.Cuming et al.; Genetic analysis of seed maturation and germination pathways in maize,
D.McCarty; Novel embryo-specific barley genes:
are they involved in desiccation tolerance?
D.Bartels et al.; The reprogrammed embryo: the
endosperm as a quick route to understanding
embryogenesis? R.C. Brown et al.; Initiation of
somatic embryos from single cells, M.A.J.Toonen &
S.C.de Vries; The generation of the legume embryo: morphological and cellular defects in pea
mutants, T.Wang et al.
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