1. Ben Merritt 11/18/2015
Plant Biotechnology
Plant Cell Wall Modification
In our world, energy is constantly recycled through living organisms. For
example, a plant absorbs inorganic nutrients from the soil and energy from the sunlight to
grow. After it dies, unicellular organisms in the soil digest the remaining, energy-rich
body, allowing they themselves to grow and reproduce. In this way, excess waste does
not accumulate. Humans, however, currently make little use of this natural cycle for our
own benefit. Instead, we have been relying on fossil fuels and natural gases to bolster
and nurture the formation of our complex industrial societies. In fact, most of our energy
is obtained through the combustion of hydrocarbon molecules, and the waste is lost,
forever, into the air. Currently, we have no efficient means of recycling the gaseous
waste products formed as a result of our constant energy usage. However, plant biomass
provides a large reservoir of unused carbohydrates that can be enzymatically converted
into glucose and eventually ethanol, an efficient, and in this case, sustainable fuel. It is
estimated that in the US, annually, 1.5 billion tons of unused plant matter are discarded,
which equivalates to about 442 billion liters of ethanol (Abramson et al. 2013).
According to another source, this amount is “roughly equivalent to the energy content of
all imported petroleum” (1.4 billion tons) (Somerville and Milne, 2005). Despite the fact
that ethanol is a combustion fuel, its production is infinite if obtained from cellulose. It is
possible to take any unwanted, non-nutritional cellulose material and break it down into
glucose and ultimately ethanol. While the science of carbohydrate energy extraction is
still highly experimental, great potential exists within plant cell walls for use by humans.

2. In plants, the cell wall generally consists of three layers (from extra-cellular to
intra): middle lamella, primary cell wall, secondary cell wall – then the plasma
membrane. Not all cells have a secondary wall; it is created only in mature cells and
contains lignin, a compound used to bolster the wall’s strength (xylem). The middle
lamella is mostly pectin, and this layer holds adjacent cells together. Both the primary
and secondary cell walls contain pectin, cellulose, and hemicellulose. However, the
secondary cell wall is distinct because of its high lignin content. Pectin is known as a
structural heteropolysaccharide. Cellulose is formed from repeating units of cellobiose,
which itself is the disaccharide of D-glucose (1,4-ß-glyclosidic linkages, polymer called
1,4-ß-glucan). This creates a very straight chain, which can form microfibrils, generally
composed 36 cellulose strands (Delmer and Amor, 1995). These can also wrap together
again and form larger bundles. Tightly aligned (parallel) strands hydrogen bond strongly,
forming a highly ordered, crystalline structure. Amorphous cellulose also exists, which is
less resistant to degradation (Delmer and Amor, 1995). Hemicellulose interconnects
cellulose strands together with lignin, which further increases the strength of the wall.
The lignin is essentially a sheet of stable polyphenols stuck together in a tight latticework
to provide rigidity to the system. 1,4-ß-glucan is the most common polysaccharide on
earth, with lignin being the second-most common biopolymer (Abramson et al., 2013).
In order for the cells to grow, however, the cell wall must be loosened to allow for
expansion. In plant cells, this occurs via the action of ß-glucanases (endo/exo), which
break apart cellulose fibrils at their glycosidic bonds. The plants then utilize non-
enzymatic proteins called expansins to help tease the fibers apart. This reduction in
tension allows water to push out at the loosened membrane, facilitating expansion and

3. cell elongation. These events generally occur in actively growing tissues, during the
ripening of fruits, and abscission (Levy et al., 2002). Cell elongation can be mediated by
auxin, which is thought to decrease the pH near the cell wall and activate loosening
proteins in the cell wall matrix. Another word for this is “acid growth”, because it was
observed that plant cells would elongate after acute exposure to acidic solutions (Rayle
and Cleland, 1992). Therefore, it is conceivably possible to alter the plant cell wall in a
way that is analogous to what is found in nature. In terms of biomass production,
understanding the underlying mechanisms of cell wall formation/degradation will help to
design plant systems that can benefit humans. There are many challenges, however,
because the cell wall is very stable and resistant to degradation.
In order to maximize glucose isolation for further processing into ethanol (via
yeast fermentation), the glucose monomers must be extracted from the other relatively
impervious cell wall constituents. Cellulose is the best choice on which to focus for
glucose extraction, as it is a uniform polymer of glucose (compared to hemicellulose
which has many unique, branching sugars). Therefore, hemicelluloses and lignins are the
main obstacles in the way of isolating/purifying significant amounts of cellulose/glucose.
Because all three components are so intimately intertwined in-vivo, it is difficult to
introduce cellulases and efficiently break up the latticework post-harvest. Most
procedures involve processing the material for glucose extraction after the plant is
already uprooted. Ideally, the plant used should be high in cellulose, low in lignin, and
low in ash (zero energy inorganics) (Furtado et al., 2014). This way, less processing
would be necessary to remove lignin and hemicelluloses from the crude cell wall mixture.
There are so many different kinds of plants that choosing the right one can be difficult.

4. However, cell wall modification procedures are as diverse as the molecules that make up
the wall. Currently, the idea is to get as much cellulose return as possible per weight of
plant material while spending comparatively as little time and money. Therefore, this
problem has been approached from many angles.
Each method of modification involves optimizing the ratios of cell wall
components. First, simply increasing cell wall concentration in cells is possible. By
overexpressing miR156 (micro RNA) in switch grass plants, total biomass was increased
by 58-101% (Fu et al. 2012). Sahoo et al. (2013) attempted to increase cellulose
concentrations by expressing a mutant cellulose synthase gene (CES3A) from
Arabidopsis in tobacco, which resulted in more soluble sugars forming from cellulose.
However, Joshi et al. (2011) tried to upregulate the cellulose synthase gene (CesA) and
this actually resulted in its silencing and poor plant growth. However, it is also possible
to eliminate the undesired components. For example, hemicellulose may be targeted for
removal. Lee et al. (2009) used RNA interference to disrupt hemicellulose formation via
inhibition of a glycosyltransferase transcript, the protein product of which is used to make
hemicellulose. This was done in poplar. Xylose (hemicellulose) decreased 43-77% in
extracts and 54-79% in whole cells, compared to wild type. However, glucose levels
were lower than the wild type. Many of these experiments are faced with downstream
pleiotropic effects that were not predicted during original rationalization of the
experiments. This gives many of these modification experiments highly unpredictable
consequences. The opposite of removing hemicellulose, which would be adding soluble
cellulose units, has also been theorized. The rationale here is that adding soluble modules
to the wholly insoluble cellulose matrix will make it more penetrable to hydrolyzation for

5. downstream glucose extraction. Algal and viral soluble polysaccharides would be
introduced into growing cells, which should theoretically incorporate normally. During
processing, however, the soluble polysaccharides would make it easier to degrade the
surrounding cellulose matrices, as they would not be as tightly bound together
(Abramson et al., 2013). The final major component conveying structure to the cell wall
is lignin. It is the toughest material to remove or degrade, as well as the most costly.
Most attempts at removing lignin have been geared toward either degrading intra-lignin
or lignin-cellulose bonds. Attempts at inhibiting lignin formation via upstream
modulation have also been attempted. An experiment involving bond breaking was done
where ferulic acid esterase was expressed in F. arundinacea (grass), which resulted in
increased digestibility of the cellulose and later enzymatic release of monomeric sugars
(Buanafina et al., 2010). This worked because the esterase broke the bonds holding
lignins to cellulose, effectively freeing up the matrix. In another example, RNA
interference was performed to down-regulate COMT, a methyltransferase enzyme
necessary for the synthesis of lignins. Disruptions of this protein are not known to affect
development and growth of plants, and the RNAi was able to reduce lignin content by
3.9-13.7% (Jung et al., 2012). Finally, the most effective lignin-disruption experiment
involved antisense down regulation of 4CL in aspen, an enzyme upstream in the synthesis
of a lignin monomer. This prevents proper lignin formation, and indeed lignin content
was decreased by 45% and cellulose actually increased by 15% (Hu et al., 1999).
Another method of “loosening” the cell wall involves direct introduction of cellulases
into living plant cells. Of course, the biggest problem in this case involves preventing
constitutive in-vivo degradation of the growing plant cell walls. Expression of

6. endoglucanases to loosen the cell wall has been shown to promote cellular growth. Park
et al. (2004) introduced a fungal xyloglucanase into poplar transformants and observed
xylem tissue with increased cellulose content. In Brazil, a eucalyptus tree was
transformed with Agrobacterium to inherit cel1, which codes for a cellulase that can
degrade crystalline cellulose. It is possible that wall loosening allows for more cellulose
synthesis, which results in increased growth. The eucalyptus tree is reported to produce
20% more wood than conventional trees (FuturaGene, 2014). A more controlled method
of this procedure, in the future, may involve regulating exactly when the cellulose
synthase protein is upregulated, or perhaps confining the proteins to certain
compartmentalized areas in the cell for release later during processing. A relatively
recent work done by Verma et al. (2010) utilizes compartmentalization of proteins,
whereby they inserted various non-phytogenic cellulases and other cell wall degrading
enzymes into the chloroplasts of tobacco. They inserted it with the flanking sequence
16S trnI/trnA, and this resulted in the chloroplasts producing a veritable cocktail of
cellulases. Their rationale was that fermentation systems for producing proteins are
costly and low yielding, so these chloroplast-derived cocktails provide a means around
this. They tested their cocktail on a variety of substrates, and deemed that purification of
proteins was unnecessary.
As it stands, plant cell wall modification is in its infancy. More work needs to be
done to fully understand the mechanisms involved with cellulose biosynthesis and its
regulation of incorporation into the cell wall. So many factors influence this, such as the
presence of expansins, endoglucanases, and cytoskeletal elements. However, so far there

7. are promising results, especially in lignin reduction. Plants contain so much carbon that
is simply thrown away, where it could be used to our benefit, again and again.

8. Works Cited
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Buanafina, M.M., T. Langdon, B. Hauck, S. Dalton, E. Timms-Taravella, and P. Morris.
“Targeting Expression of a Fungal Ferulic Acid Esterase to the Apoplast,
Endoplasmic Reticulum or Golgi can Disrupt Feruloylation of the Growing Cell
Wall and Increase the Biodegradability of Tall Fescue (Festuca arundinacea).
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virgatum L. Results in Various Morphological Alterations and Leads to Improved
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