ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ ÊÊÊÊÊÊ 
ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ ÊÊÊÊÊÊÊÊÊÊÊÊÊ 
Enzyme production is relocated and
photosynthesis becomes more efficient as Nepenthes
tissues mature
Feinberg,
Instructed by Hardie, J. and Mooney, J.
LBS 145, laboratory section W2
Abstract
Controversy has always surrounded the methods of digestion by carnivorous plants, especially from the genus Nepenthes.Ê It has been established that the plant uses an endogenous enzyme, nepenthesin, in digestion; the effects of the enzyme on the plant are unclear.Ê The relation between maturation of plants and production of the enzyme is unclear.Ê This paper investigates changes within a maturing pitcher plant to elucidate these problems.Ê
ÊÊÊÊÊÊÊÊÊÊÊ To
test this hypothesis, we performed a series of tests comparing mature pitchers
to immature ones.Ê We compared protein
levels in walls, leaves, and fluid of the plants using the
The results of these tests showed that the closed pitcher had more protein below the fluid line, the opened pitcher more above.Ê Sugar tests showed the different parts of the plant contained varying amounts of sugar, and the fluid contained none.Ê The action spectrum showed that the leaves performed more photosynthesis than the pitchers.Ê Our pH and observational studies showed that the open pitcher had a higher pH and digested better.Ê Our conclusions from these results are that nepenthesin is still produced in the pitcher just at different locals at different stages of maturation.
Figure 1.Ê Suggested amount of the nepenthesin glycoprotein enzyme found in the closed and opened pitcher plants. The inside layers (segment a) of the opened pitcher plant have roughly the same suggested amounts of nepenthesin, as do those segments of the closed pitcher even though they are somewhat less. The outside layers (segment b) of the closed pitcher plant also have roughly the same suggested amounts of nepenthesin. The upper outside layer (segment 1b) has over three times the amount of suggested nepenthesin amount while the lower outside layer (segment 2b) has over five times the nepenthesin content. The fluid shows no nepenthesin, while the opened leaf shows the most out of all of the segments and the closed leaf has the most out of all of the closed segments.
These suggested nepenthesin levels were determined solely by the presence of a glucose/ protein correlation; no qualitative protein assays were used.
DiscussionÊÊÊ
Though many researchers have elucidated the means by which carnivorous plants digest their prey, there seems to be a gap in our knowledge of these plants, especially those in the genus Nepenthes. A major challenge facing researchers lies in understanding which mechanisms are in place to prevent the plantâs own proteins from being digested by nepenthesin, its digestive enzyme.Ê To try to fill these gaps, we hypothesized that some enzymatic and photosynthetic changes occur in tandem with the structural changes of a developing pitcher.Ê We predicted that this structural response would come in the form of a ãsealing offä of the enzymatic canals from the interior of the pitcher, and an inactivation of the cells that secrete nepenthesin. This would prevent the activated enzyme from washing back into the pores that secrete the inactive enzyme, which could destroy some of the plantâs other proteins.Ê Such protection against self- digestion requires a mechanism analogous to that of the human stomach. In this organ, the enzyme pepsinogen is produced by the chief cells, which are found in the lining of the stomach. The acidic content of the stomach activates pepsinogen into pepsin, which then digests proteins (Freeman 2000).Ê In drawing this parallel, we rely on the fact that nepenthesin is produced in the lining of the pitcher wall and activated by an acid within the mature pitcher fluid, specifically hydrochloric acid (Frazier, 2000).Ê
ÊÊÊÊÊÊÊÊÊÊÊ We also hypothesized that the pitcherâs maturation would affect the plantâs energy acquisition.Ê This idea was inspired by the fact that many nutrients are involved in the photosynthetic process; magnesium exists in chlorophyll, iron is incorporated into cytochromes, and ãpotassium is involved in stomatal openingä (David, 2003).Ê We predicted that the leaf of the open pitcher (O.L.) would store the most starch and perform the most efficient photosynthesis because its associated pitcher is a rich nutrient source (Gotelli, 2002).Ê Likewise, C.L. would store less starch and perform photosynthesis less efficiently because of its relative nutrient deprivation.Ê Finally, we predicted that the two pitchers (both open and closed) would contain far less starch than either of the two leaves, and would perform photosynthesis with the least efficiency.
To test our first hypothesis (regarding the pitchersâ enzymatic changes), we needed to determine where nepenthesin is present and when.Ê Since nepenthesin is a glycoprotein studded with glucose molecules (Athauda et al, 2004), we conducted macromolecule tests in search for glucose and protein.Ê Additionally, we conducted an observational test to further substantiate Frazierâs claim that nepenthesin is activated by hydrochloric acid.Ê To test our second hypothesis (regarding the effects of pitcher maturation on photosynthesis), we employed the Iodine test and the Hill Reaction.
The results of three macromolecules tests (Bradford, Barfoedâs, and Selivanoffâs) substantiated our hypothesis and predictions only to a certain degree.Ê Selivanoffâs test, inherently, was not suitable for our purposes.Ê We were hoping to gather information regarding the presence of aldehydes (because glucose is an aldehyde) in each tissue sample, but the presence of some ketoses in all of the samples ãmaskedä the possible presence of aldoses.Ê To help us determine if aldoses were present along with the ketoses, we would need to run tests on combinations of controls (i.e., glucose and fructose in the same test tube) to see if such combinations have characteristic color changing patterns that could be matched to our samplesâ results.Ê Until then, though, we cannot factor the results of Selivanoffâs test into our search for the presence and abundance of nepenthesin.Ê However, based upon the numbers derived by multiplying the results from Barfoedâs and Bradfordâs tests (which in essence gave us a numerical value which very likely correlates with the presence of nepenthesin) (Table 8), our prediction that nepenthesin would be more prevalent in the lower half (below the fluid and enzymatic glands) of the pitchers was confirmed.Ê However, our prediction that nepenthesin would be far more abundant in the tissues of the closed pitcher than in those of the open pitcher was not entirely supported by our data.Ê Our data showed that, in an immature pitcher, the most nepenthesin is found in both the inside and outside layers of the lower half.Ê We also found that, in the mature pitcher, high concentrations of nepenthesin are consistently found in the outside (section b) tissue of the plant.Ê Perhaps, then, the mature pitcher does not cease producing nepenthesin as we had once thought, but simply transfers the site of inactive enzyme production to the outer reaches of the tissue.Ê Perhaps this is the way that Nepenthes pitchers are able to protect their metabolic enzymes from the activated nepenthesin within the pitcher lumen.
Since the
reasoning for the above tests was grounded in the belief that nepenthesin is produced in an inactive form and activated
by HCl, our research team performed an observational
test to ensure the validity of said reasoning.Ê
This test was designed to determine the ability of open and closed
fluids to digest proteins, and the effects of HCl
upon this ability.Ê The results did align
with our prediction by showing that closed fluid had greater digestive
capabilities when mixed with HCl, and by showing that
open fluid had greater digestive capabilities than closed fluid in general.Ê
To test our second
hypothesis (regarding the role of pitcher maturation in photosynthesis), we
employed tests that compared the starch levels and photosynthesis rates between
tissue samples.Ê The Iodine test showed
that the pitchers store far less starch than the leaves, and that the leaf
associated with the closed pitcher contains more starch than that associated
with the opened pitcher.Ê This was in
direct contradiction with our previous predictions that the leaf of the opened
pitcher would be able to store more starch due to increased nutrient
uptake.Ê However, an equally plausible
explanation (inspired by Dr. Jim Smithâs discussion of the starch storage in
carrots) is that the leaf of the closed pitcher is ãpreparingä for an upcoming
energy crisis; it will soon have to provide the energy to increase the
pitcherâs size, open the pitcherâs lid, and prepare absorptive canals.Ê Thus, this starch storage may simply be a
preparatory mechanism (Anonymous-2.Ê Unknown).Ê The other
test aimed at discerning photosynthetic differences, the Hill Reaction,
produced results only marginally consistent with our original predictions.Ê As expected, the leaf associated with the
open pitcher was able to conduct photosynthesis more efficiently than that of
the closed pitcher.Ê However, our results
showed that, for both opened and closed samples, the leaves performed far less photosynthesis than the
pitchers did.Ê This seems very
counterintuitive, because a flat piece of tissue (namely, the leaf) would
probably absorb far more light than a tubular, upright piece of tissue (Angelo,
1999).Ê However, perhaps this very
concept is the reason for the increased photosynthetic capabilities of the
pitcher tissue; the pitchers must possess more light-capturing pigments to compensate for their geometry.Ê Since the Hill reaction took into account only the tissue composition, and not
pitcher shape, it makes sense that our results turned out as they did.
Since enzyme production within pitcher tissue is relocated throughout pitcher maturation, we are led to yet another question: just how, exactly, do certain cells in the pitcherâs wall cease secreting this enzyme, and how do others (namely, in the outer wall) start?Ê It seems that the cells that secrete HCl should be able to communicate to the cells that secrete nepenthesin to stop; perhaps these HCl-producing cells secrete a protein that alters the nepenthesin-producing cell (Sievi, 2002).Ê This protein, then, could work in any number of ways.Ê It may alter the ribosomes that produce nepenthesin in the first place, or it may alter the ãaddress tagä on nepenthesin, which would prevent it from being sent to the endoplasmic reticulum (Freeman, 2002).Ê This second idea is particularly interesting, considering that the ER is known to be able to glycosylize (add sugars onto) proteins (Freeman, 2002), and that nepenthesin is a glycoprotein!Ê Perhaps omitting this step in the production of nepenthesin renders it entirely functionless, and it simply floats aimlessly in the cell until it is degraded by cellular enzymes or by cellular chaperones.Ê This new set of predictions sets the stage for further experiments; perhaps if we could ever be supplied with the means to differentially centrifuge the components of pitcher tissue cells, we could examine the enzymatic changes within organelles through the maturation of the pitcherâs cells (Freeman, 2002).
ÊÊÊÊÊÊÊÊÊÊÊ Most
of the weaknesses with our experimental design were rooted in the source of our
Nepenthes pitchers and time
constraints. We were not granted entire pitcher plants, but only specific
clippings as needed.Ê Thus, we were
unable to control factors such as the feeding and watering of the plants that
could have led to dilution of enzymatic fluids (which would then throw off our
protein assay results).Ê Also, we were
unable to determine the exact age of each pitcher we used; we could only
differentiate between open and closed pitchers.Ê
Thus, a sample that still had the physical characteristics of a closed,
immature pitcher but that was already undergoing the enzymatic changes leading
to adulthood could give us skewed results.Ê
Other errors arose due to imperfections in the tests, pitchers, and
experimenters; a problem that arose during the experiment itself stemmed from our
collective forgetfulness: we removed the fluid from the closed pitcher plant
before noting the fluid line. Such inaccuracy could result in a mixing of
enzyme-rich and enzyme-poor plant tissue, thereby distorting our results.
However, to compensate, we cut the tissue at the distinct line where the distinctly
visible enzyme glands (Anonymous-3, unknown) ended (because that was where we
found the fluid line of the opened pitcher). Also, after grinding, straining,
and vortexing the plant tissue, a precipitate
gathered at the bottom of the storage vial over time and may have caused
concentration discrepancies in our results, despite our devotion to vortexing the tissue each time before sampling. In
addition, there may not have been a high enough enzyme concentration in the
fluid or plant tissue to produce reliable results with our tests. Regarding the
tests themselves,
ÊÊÊÊÊÊÊÊÊÊÊ A problem with our observational study was that it was very difficult to quantify the digestion that had occurred within each tube.Ê Some of the tubes without acid developed a cloudy appearance due to a mold growth, which masked the amount of digestion that had occurred (because cloudiness was one of the factors we used to gauge digestion).Ê A more effective way to test for enzyme activity, perhaps, would be to mimic previous experiments involving black and white film.Ê Because ãdigestive enzymes dissolve the gelatin layer of exposed film,ä researchers have devised methods to quantitatively gauge the enzymatic activity of carnivorous plants (Hartmeyer, 1997).ÊÊÊÊ
ÊÊÊÊÊÊÊÊÊÊÊ Knowing experimental alternatives such as that above would allow for the design of an excellent follow-up experiment.Ê It would be beneficial, of course, to have more quantitatively accurate assays (such as the above enzyme test) so that more solid conclusions could be drawn.Ê Additionally, tests run upon many different pitchers at different stages of maturation would give us a better idea of the relationships between pitcher age and all of the factors weâve tested; the more data available for analysis, the fewer problems one anomalous point will cause.Ê Knowing these possible improvements, and keeping in mind our previous error, our conclusions can be used as a firm ground for further research.Ê
Created on 02 Mar 05 by Kate Leitch.
Email her at leitchka@msu.edu
if you think this paper is so fantastic/terrible that
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