|
DISCUSSION
We chose
F.
benjamina
and
H. fortunei
as representative C3 plants because they were the plants with
relatively thick leaves. Since our hypothesis involves looking at
bundle-sheath cells and such cells are located on the underside of
the leaves, this necessitated scraping off the underside layer.
Thicker leaves would thus ensure that we would not get any mesophyll
cells included in our sample (which is the photosynthetic layer of
cells, usually more densely-colored green with chloroplasts, in C3
plants). We chose
K. daigremontiana
and
C. argentea
as representative CAM plants because their leaves were thick as
well, and since the bundle-sheath cells of CAM plants are located on
the innermost layers, thicker leaves would reduce the probabiliy of
our sample scrapings including non-bundle-sheath layer cells. The
purpose behind isolating bundle-sheath cells was to look at Rubisco
(large subunit) expression in these cells, reasons which are
detailed in the Introduction section.
Although we initially started out with four plant samples (Sample 1=
F. benjamina,
2=
H. fortunei,
3=
K. daigremontiana,
4=
C. argentea),
we could not use sample 2 because we found the protein concentration
in the sample to be too low. Therefore we used
F.
benjamina
(sample 1) as the representative C3 plant and
K. daigremontiana
(sample 3) and
C. argentea
(sample 4) as our representative CAM plants. After running real-time
PCR, we found that sample 3 revealed no significant amount of
Rubisco. This was probably due to a very low DNA concentration in
sample 3 to begin with. If sample 3 did not have such a low DNA
concentration, then we could alternatively conclude that there was
so low amount of Rubisco present that it would have taken more PCR
cycles to reveal substantial build-up of the product (However, this
was a standard PCR run with 32 cycles) or it could be that certain
plant samples had some Taq polymerase still present.
Since sample 1 (corresponding to the
C3 plant) had a higher C(t) value of an average of 11.05 than sample
4 (corresponding to the CAM plant) with a lower C(t) value of 15.04,
we could conclude that more DNA encoding for Rubisco was present in
the C3 plant. This is because the C(t) value essentially reveals the
cycle number at which enough product accumulated to be viewed above
threshold. For samples with less of the amplified product (in this
case, less DNA as revealed by PCR would correlate/imply less Rubisco
large subunit), it would take more cycles to show up above
threshold. This is the case with the CAM plant. For all samples, the
same primer (primer C) was used.
Our results from taking DNA fragments from the RT-PCR reaction and
running them on an agarose gel (Figure 1,
Results
second page) show a bright signal for
C.
argentea
in lanes 2 and 3, however this is not
representative of Rubisco and rather indicates the strong presence
of primer-dimers. We could have used another primer (rather than
just use Primer C) and this may have lessened this problem. We also
saw signal in lanes 7 and 8 for the other CAM plant (K.
daigremontiana),
yet this did not correspond to Rubisco as well. The only sample of
DNA which yielded signal at 200 bp (and therefore indicating
presence of primer bound to Rubisco DNA) was sample 1, which
represents the C3 plant
F.
benjamina.
After performing our Western blot on
one C3 plant (F.
benjamina)
and one CAM plant (C.
argentea)
with the most significant amount of protein present, we found that
there was more Rubisco protein present in the C3 plant as indicated
in Figure 3,
Results
second page). Therefore we started to conclude that the CAM plant
had less Rubisco, which is supported by literature (detailed in the
Introduction), but particularly important since now our results may
confirm the actual location of this lower Rubisco concentration (in
the bundle-sheath cells) in comparison to C3 plants. This
conclusion, although supportive of our hypothesis, is nevertheless
weak because we did not additionally test for presence of Rubisco in
other parts of the plant (such as in mesophyll cells) and we do not
have enough repeated trials and have only taken samples from one
species of CAM plant and one species of C3 plant.
In order to determine and compare levels of Rubisco large subunit
protein expression in our three samples, we ran a Western blot. We
were looking for dark "smudges" around 47,600 KDa which would be
indicative of the antibody binding to the large Rubisco subunit
protein. Since we found these dark smudges for our two repeated
lanes for sample 1, we concluded the large Rubisco subunit was
present in the C3 plant and not present (at least not significantly
visible) at all in the CAM plant since no dark smudges appeared in
the lanes corresponding to sample 3 and sample 4.
We could have improved our experiment by testing more CAM plants and
C3 plants, and by accumulating more grams of original leaf sample
and performing less dilution. This may yield us a higher
concentration of protein in the future. Due to high presence of
primer-dimers with using primer C, we could have used another kind
of primer (more than one primer) in the future. Also, we could take
further steps in the future to ensure our plant samples are all of
similar age since activity and amount of Rubisco decreases as leaves
mature (Luttge, 2004).
|
