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Characterization of the leaf cuticle properties
of willow and poplar clones with potential as bioenergy
crops
Kim Cameron
In this study, we hypothesize that the characteristics of the leaf surface and the properties of the epicuticular wax directly influence biomass production. Our long-term goal is to identify characteristics in the epicuticular wax that can be used as early selectable traits in our breeding program.
We have compared the characteristics of the leaf cuticle of three willow species and two hybrid poplar clones currently growing in high density stands in Tully, NY three times over the growing season. Three samples were collected from duplicate plots for each clone at each sampling period.
To prove our hypothesis we need to:
1. Identify the characteristics of the leaf surface and the epicuticular
wax.
2. Determine the diversity in epicuticular wax characteristics among the different clones.
3. Correlate this data to biomass production.
4. Correlate biomass production to cuticle composition and deposition under different environmental conditions.
To date, we have focused our attention on the first two aspects of this study.
Properties of the epicuticular wax
The epicuticular
wax, a waxy layer on the surface of a leaf, acts as a barrier to water
loss. The topography of the leaf surface and the characteristics of the
cuticle affect biomass production. But, the characteristics of the epicuticular
wax vary with each species and under different environmental conditions.
The goal of our research is to determine which characteristics of the
wax influence biomass production and whether there are specific characteristics
that we can use to identify clones that will perform well under a wide
variety of environmental conditions.
First we
examined the surface of the different species of willow and poplar using
scanning electron microscopy. This type of microscopy allows us to examine
the surface of the leaf without melting or distorting the wax. The wax
coats the surface of the leaf. Note the difference in the surface of the
three willow clones, Salix purpurea, S. dasyclados, and S. eriocephala.
Crystals form on the surface of S. purpurea leaves whereas the other two
clones have smooth surfaces. Both S. dasyclados and S. eriocephala have
trichomes, unlike S. purpurea. ). But this diversity is not evident with
the two hybrid poplar clones. Both of these 
clones
have smooth surfaces and no trichomes.
Next we extracted the wax from the leaf surface. Leaf discs were cut out of the leaves with a cork borer and placed in a test tube with dimethyl chloride for 30 secs. The waxy components on the surface of the leaf dissolve in the dimethyl chloride. Then the solution evaporates leaving a residue of waxes.
The residue was resolubilized in iso-octane and analyzed by gas chromatography. In this procedure the components of the wax were separated based on their chemical properties.
Each one
of these peaks represents a single component of the wax. The wax is a
complex mixture of components that can be grouped into specific organic
classes - the most common ones are alkanes, alcohols, fatty acids, wax
esters, and sterols. We added an internal standard so that we could actually
quantify the amount of wax we extracted. Although most of the components
are the same in all three of the willow clones, some components are unique
to only one clone (arrow) and sometimes the concentration of a particular
component or group of components is very different between the clones
(example circled).
These pie
charts represent the concentration of each group of organic components
(alkanes, alcohols, fatty acids, and other) early in the growing season
(samples were taken late May). The top three pie charts represent the
willow clones and the bottom two represent the hybrid poplars. Large differences
are obvious between the willow clones. For example, S. dasylcados has
a much higher percentage of alkanes compared to S. purpurea or S. eriocephala
whereas S. eriocephala has a large amount of alcohols. Also note how similar
the hybrid poplars are. There is almost no difference in their wax composition.
In this graph
we are looking at the concentration of only one group of organic compounds,
the alkanes, over the three sampling periods. Notice how differently each
willow clone behaves over the growing season. S. purpurea and S. eriocephala
decrease their alkane concentration in July, whereas S. dasyclados increases
its alkane concentration over the same period. Interestingly, the two
hybrid poplars have similar, but different, patterns from the willow clones.
They both steadily decrease their alkane concentration over time.
In this graph
we can compare the total amount of wax for all five clones over the three
sampling periods. Once again we see a large difference between the willow
clones, but almost no difference between the hybrid poplar clones. In
particular, notice that S. eriocephala dramatically increases its wax
load in September compared to the July sampling. But, the opposite pattern
was observed with the S. dasyclados clone. It had a high wax load at the
end of May, then the wax load declined over time.
Every three
years the trees are cut back to stumps, the wood is dried in an oven,
and then weighed to determine the biomass. In the intervening years the
amount of biomass is estimated by taking a series of measurements (average
width of stem, number of stems, etc.). The samples collected for this
research were obtained during the summer of 2000, the third year in our
three-year rotation. Since these samples were taken, the trees have been
cut back to small stumps and will resprout in the spring of 2001. Here
we present an estimate of biomass for each clone for each year of the
rotation. In another graph we have provided data on the seasonal rainfall
collected from a weather station near the field plots. S. eriocephala
and S. dasyclados do not show any response to the environmental conditions
(note how the biomass continues to increase a similar amount each year
even though the precipitation data is dramatically different). But, the
other three clones appear to be very sensitive to environmental conditions.
They had almost no increase in biomass in 1999, a very dry year, whereas
they had a dramatic increase in 2000, a very wet year.
Conclusions
This is only a preliminary study and covers only one growing season so
we cannot assume that the trends we see will continue over multiple seasons.
However, there are strong indications that the three willow clones show
a large amount of diversity, both in their leaf topography and in the
wax deposition, compared to the two hybrid poplar clones. This suggests
that we may be able to use characteristics of the wax as selectable traits
in our breeding program. We have characterized the waxes, but we have
not addressed whether the characteristics of the wax affect biomass production.
We will begin to address that question in a study designed to examine
the wax of clones grown next to each other in irrigated and non-irrigated
plots.
Acknowledgements
Many thanks to all the people involved in this project. Special thanks
to Pradeep Tharakan who established all the plots I collect samples from
and provided the biomass and precipitation data. Also special thanks to
Dr. Larry Smart, my major professor and mentor, Dr. Mark Teece for his
advice and knowledge, as well as use of his GC and Dr. Edward Bevilacqua
for all of his help with the statistics. Dr. Robert Hanna and Dr. Sue
Anagnost very kindly performed the scanning electron microscopy work and
Tim Volk provided wonderful pictures of the harvesting procedure. Also
thanks to Dr. Larry Abrahamson in charge of the willow breeding project.
For more information Contact: Kim
Cameron
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