Nanoindentation of mutated Arabidobsis cell walls
*
Winston ,
1
Bahr ,
2
Yang ,
Scott K.
Dr. D.F.
Dr. Hong
Dr.
2
2
Norman G. Lewis , Dr. Laurence B. Davin
(*)Department of Materials Science and Engineering; Clemson University; Clemson, SC 29631
(1) Department of Mechanical and Materials Engineering; Washington State University; Pullman, WA 99164
(2) Institute of Biological Chemistry; Washington State University; Pullman, WA 99164
IMAGING AND INDENTATION
Genetic manipulations of lignin production pathways in herbaceous plants allow
for nonlethal and highly visible phenotypic changes to be made. Until recently,
there has been no way to quantify exactly how these manipulations affect the
mechanical properties of the cell wall at the microscale and consequently
connect any changes to the specific genes that have been modified. It is
unknown if a replacement cell that provides mechanical support for a plant will
behave like the wild type or the donor plant the gene came from, in this case, a
poplar tree. Understanding exactly how specific genetic alterations affect these
properties will allow scientists and engineers to develop more effective biofuels
and bioproducts. Nanoindentation has been employed as a method to measure
these properties.
METHODS
ANALYSIS AND RESULTS
The experimental procedure to account for structurally-induced compliances in
non-ideal specimens presented by Jakes et al. focuses on correlating said
compliances with indentation distance from a free edge (such as a cell lumen).
Arabidobsis cell walls are approximately 1 to 3 μm in width; as such, an indent
made by a Berkovich tip takes up the majority of the cell wall width. Therefore,
it is not necessary to consider the distance the indent is from the cell lumen.
10
Figure 3. A diagram of the cell wall structure of typical
herbaceous plants blown out from a 40 μm x 40 μm SEM image
of an NAC1 mutant. The features are color coded, with white
being the primary cell wall, black the S1 layer, green the S2 layer,
and red the S3 layer.
The S2 layer of the cell wall is most responsible for providing structural support
ot the plant. As such, it is the most important layer to investigate when
considering mechanical properties.
8
E (GPa)
INTRODUCTION
4
2
Several varieties of Arabidobsis stems were examined:
0
8
Wild type—used as a control
1
2
3
4
5
6
7
8
9
7
•
SND1-NST1—a lignin-deficient mutant
•
NAC1—a mutant injected with poplar DNA responsible for activating lignin
production pathways
6
E (GPa)
•
6
5
4
3
2
1
0
1
2
3
4
5
6
7
E (GPa)
20
Figure 1. Comparison of the lignified areas (stained purple) of stem cross-sections of a wild type (left),
SND1-NST1 mutant (middle), and NAC1 mutant (right). Images show an area of 1000 μm (length) by 700
μm (height).
15
10
5
PROCEDURE
Step 1—Stem samples are cut from the basal section of the Arabidobsis plant or
mutant and cryotomed at -15°C then stored in an aqueous environment until
indentation.
Figure 3 (above) . A 10x10 μm image of a wild
type cell wall. Three indents from a Berkovich tip
are visible.
0
1
2
CONCLUSIONS
Step 3—Using an imaging technique similar to AFM, interfasicular cell walls are
located and indented with a partial unloading technique up to a depth of 200
nm.
• Wild Type: E = 6.36 GPa ± 1.18
2.7
Indents were performed with a Berkovich tip on samples five to seven hours
after removal from an aqueous environment. The lab was kept at room
temperature and approximately 40% relative humidity.
300
1000
2.5
• SND1-NST1: E = 3.46 GPa ± 1.88
• NAC1: E = 18.0 GPa ± 1.92
According to a Wilcoxon test, the p-value of the wild type and SND1-NST1
mutants is .03125; there is a very low chance that these two data sets are from
the same distribution. It is safe to say that these two specimens are statistically
different.
250
These data indicate that incorporating DNA responsible for lignin production
produces cell walls more closely resembling the mechanical properties of the
donor plant rather than the wild type. The NAC1 mutant has a cell wall modulus
coinciding with published values of the S2 cell wall layer of poplar tracheids
(16.9 GPa ± 1.9).
800
2.4
200
2.3
Load (µN)
600
Load (µN)
1/2
1/2
(nm/N )
y = 1.556 + 0.085398x R= 0.97379
Compliance * (Max Load)
5
Average Elastic modulus data:
LOAD-DISPLACEMENT DATA
2.6
4
Figure 6. Modulus data for wild type (top), SND1-NST1 (middle), and NAC1 (bottom) corrected for
structual compliances.
Figure 4 (right). A 20x20 μm image of an SND1NST1 lignin-deficient mutant. At this resolution,
the greater cellular array is more readily visible.
Step 2—Cryotomed stem samples are stood upright on a spiked platform and
placed in the indenter.
Step 4—Account for structurally-induced compliances using a SYS plot (Figure 2,
shown below) and calculate the elastic modulus.
3
150
400
100
2.2
𝐶t𝐿MAX1/2
= 𝐶m +
𝐶s 𝐿MAX1/2
+
𝐽01/2
Future work might consider the effects of moisture content on cell wall
modulus, different mutant varieties, and different cell types within the
Arabidobsis plant.
200
50
2.1
0
2
0
0
50
100
150
Depth (nm)
1.9
4
6
8
(Max Load)
10
1/2
12
14
1/2
(N )
Figure 2. A SYS plot, the slope of which indicates the extra, artificial compliances read by the nanoindenter
due to structural effects and machine compliances. The equation superimposed on the graph shows the
mathematical relationship described by the plot where Ct is the total compliance read by the machine, Cm
is the compliance of the machine, Cs is the structual compliance, and J0 is the Joslin-Oliver parameter.
200
250
0
50
100
Depth (nm)
150
200
Figure 5. A comparison of load-displacement curves. The SND1-NST1 mutant (left) requires
substantially less force to a chieve a depth of 200 nm than the NAC1 mutant (right).
Figure 5 above shows typical load-displacement data read by the machine.
These curves are obtained with a partial unloading technique—it took almost 4
times as much force to indent to a depth of 200nm in the NAC1 mutant as it did
for the SND1-NST1 mutant.
ACKNOWLEDGEMENTS: In addition to the other authors listed on this
poster, I would like to thank Dr. Katerina Bellou and Anqi Qiu for their
insights and guidance on this project.
This work was supported by the National Science Foundation’s REU
program under grant number DMR-0755055.