Whats relatively new in plant cell walls

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Whats (relatively) new in plant cell walls?
A postharvest biology perspective focusing on in
formation relevant to ongoing studies designed to
understand and then manipulate texture changes in
ripening fruits This talk will discuss (1) pr
oteins that appear to play important roles in the
metabolism of specific cell wall polysaccharides
and (2) possibilities for altering important ele
ments of the structures of pectin polysaccharides
that are the presumed targets for known enzymes.
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Pectin Lyase has been considered to be a
microbial pectin- degrading enzyme, not a plant
enzyme. Things are different now!
Marin-Rodriguez et al. (2002) Pectate lyases,
cell wall degradation and fruit softening. J.
Exp. Bot. 532115-2119. Jimenez-Barbudez et al. (
2002) Manipulation of strawberry fruit ripening
by antisense expression of a pectate lyase gene.
Plant Physiol. 128751-759. Pectin lyase is an
enzyme that breaks polymer backbone glycosidic
linkages in a-1,4-linked galacturonans. The
location of cleavage is like that for PG, but the
reaction mechanism for the cleavage is trans- or
b-elimination. The reaction creates a
4,5-unsaturated galacturonosyl residue at the
non-reducing end of one of the shorter
galacturonan products generated in the reaction.
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Earlier work had shown a sequence in ripening
strawberry that encoded a PL. Jimenez-Barbudez
et al. developed a construct of the antisense
orientation of this PL sequence driven by the 35S
CaMV promoter to transform strawberries. The
result was a number of Apel lines that showed
altered PL gene expression.
While the data show some variability, fully ripe
fruit have higher external and internal firmness
than controls (i.e., those not transformed or
transformed only with the GUS-encoding sequence).
Internal firmness readings are based on
measurements at points where epidermal tissue was
peeled away. The greater the fruit size, the
greater the disparity in external firmness
between Apel and control fruit (below).
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The different Apel lines showed differing degrees
of suppression of PL gene expression, altered
pectin metabolism, and ripe fruit showed less
leaky membranes (based on electrolyte leakage
from excised fruit cylinders, data not shown).
The left panels show Northern analysis (A B)
looking at strawberry PL gene expression and (C)
a Western blot using anti-strawberry PL serum.
The table (above) shows the extent of swelling of
isolated cell walls and the amount of cell wall
pectin isolated from cell walls prepared from
Apel and control lines.
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The firmness values of the Apel fruit diverge
from values for controls as ripening proceeds
(left figure) and Apel fruit show improved
firmness retention after ripe fruit are held for
4 days at 25C (right figure).
There are PL genes in many species that yield
edible fruits. The list includes grapes, bananas
and tomatoes (in ripening fruit).
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Studies of the effects of manipulation of genes
encoding presumptive wall-modifying enzymes in
ripening tomato fruits have been extensive (see
review by Brummell and Harpster (2001) Cell wall
metabolism in fruit softening and quality and its
manipulation in transgenic fruits. Plant Mol.
Biol. 47311-340). 1. Antisense suppression of
PG expression during ripening retards softening,
but only once fruit have reached the red ripe
stage. 2. Sense construct-based suppression of
fruit expansin (Exp) gene expression delays
softening in its early stages. Models of Exp
action have led researchers to conclude that Exp
plays a role in the breakdown of xyloglucan
polymers, but the effect of Exp gene suppression
in tomato fruit is accompanied by a somewhat
confusing effect on pectin metabolism during
ripening. Does the examination of presently acc
epted cell wall models help us to understand what
is going on in tissues as cell wall metabolism is
modified?
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The cell wall has two coextensive
polysaccharide networks they are the cellulose -
xyloglucan network and the pectin network.
The cell wall also has porosity. That is, the
gaps not filled by wall polymers or the water
shells that surround them define the sizes of
polymers that can diffuse through the wall.
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We should also be aware of the structures of the
molecules that might (1) contribute to fruit
texture and (2) be metabolized to result in fruit
softening during ripening.
HGA is a simple pectin. Its backbone bears no
branches. Its building-block, backbone sugars
are all galacturonosyl (GalUA) residues that are
linked a-1,4- to neighbors. The polymer will be
digested by PG or PL, but the presence of methyl
esters on GalUA carboxyl groups can influence
this. HGAs are thought to be held in the wall
via bridges through Ca2 ions to other HGAs.
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The typical cell wall also contains more complex,
RG-type pectins. RG-I is shown. Its backbone
has alternating residues of GalUA and the neutral
sugar Rhamnose.
RG-I also has a large array of possible side
chains. They contain the 6 carbon neutral sugar
Galactose (Gal) the 5 carbon neutral sugar
Arabinose (Ara). Breaking the RG-I backbone wo
uld require a Rhamno-galacturonase (RGase) but
the RGase could only act if it has access to the
backbone. Access might be provided if
a-arabinosidase and/or b-galactosidase were to
remove some side chains. These enzymes could also
act to increase wall porosity
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RG-II is even more complex. Its backbone might
be cleaved by PG or PL, but many enzymes would be
required to provide access to that backbone.
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In the past several years researchers have begun
to examine the roles of enzymes that cooperate
in the digestion of pectins to see if greater
understanding and control of ripening-related
softening would result. Smith et al. (2002) Down-
regulation of tomato b-galactosidase 4 results in
decreased fruit softening. Plant Physiol.
1291755-1762. Sozzi et al. (2002) Gibberellic ac
id, synthetic auxins, and ethylene differentially
modulate a-arabinofuranosidase activities in
antisense 1-aminocyclopropane-1-carboxylic acid
synthase tomato pericarp disks. Plant Physiol.
129930-940. These papers recognize that many g
lycosidase enzymes are present in isoforms
encoded by gene families. Smith, Gross et al.
have already characterized the tomato
b-galactosidase gene family. The work of Sozzi in
our lab has just recently made clear that there
is a family of a-arabinosidases in tomato fruits
and we (Powell, Lurie, Brummell, Greve and
Labavitch) are developing a research proposal to
characterize them.
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In Smith et al. (2002), expression of the
ripening fruit b-galactosidase (b-Gal II) was
suppressed with an antisense construct. This is
the only tomato b-galactosidase that will act on
a naturally-occurring tomato galactan polymer
that is, most likely,a polymer that is attached
as a side chain on an RG-I pectin.
The Northern analysis (above) shows that b-Gal II
comes up in normal fruits (A P) but the mRNA
level is relatively reduced in some of the
transgenic lines 3 days after breaker stage (B3),
but often not reduced 7 days after breaker (B7).
The activity against a model substrate (right,
panel A)is often greater in transgenics than
controls, but, at B3, the activity against the
side chain mimic substrate (panel B) is
substantially reduced not, however, at B7.
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The overall reduction in cell wall Gal content
that is seen in normally ripening tomatoes is
seen in the transgenic lines also. The
manipulations clearly do not alter the general
metabolism of Gal-containing wall molecules in
ripening fruit. Nevertheless, a few of the
transgenic lines did have firmer fruit, based on
compression tests.
Panel A shows data from a flat plate compression
test, panel B shows data from a spherical
indenter compression test. If polymer cross
links affect cell wall strength and wall strength
relates to fruit firm-ness, how can integrity of
wall Gal-containing polymers affect firmness?
After all, the polymers with Gal are present as
side chains that are not cross linking. Could
the galactans influence wall porosity or the
access of pectin backbones to enzymes that might
otherwise alter the backbones and cross-links,
and, hence, wall integrity? We (Powell, Brummell,
Greve and Labavitch with Smith and Gross) are
currently developing a plan to suppress the
expression of both b-gal II and PG in tomatoes to
see if the effect reported in Smith et al. can be
enhanced.
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Gabriel Sozzi, a Ph.D. student at the University
of Buenos Aires, came to our lab with an interest
in tomato fruit a-arabinosidases (a-Afs). a-Afs
cleave terminal Ara residues from polysaccharides
and glycoproteins therefore they might act to
trim side chains of Ara residues from backbones
of polymers like RG-I.
Gabriel showed that a-Af activity (fresh weight
basis) declined over the course of fruit growth
(left figure) and then rose again as the fruit
ripened (right figure, note y-axis scale
difference). He studied both wild-type tomatoes
and those with antisense suppression of ACC
synthase (ACC-S). Activity of a-Af did not
increase in ACC-S fruit unless they were treated
with ethylene.
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He was able to separate, based on their size,
three distinct a-Af activities from extracts of
early ripening fruits. The three represented
only a small amount of the extracted protein
(upper left panel).
Again using WT and ACC-S fruit, he showed that
a-Af-1(triangles) was low through development,
a-Af-2 fell steadily as fruit grew to full size
(squares), and that (lower right panel) a-Af-3
appeared early in ripening and rose steadily in
WT fruit (filled diamonds), but not in ACC-S
fruit (open diamonds) unless they were treated
with ethylene ().
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Gabriel adapted the excised pericarp disc fruit
ripening system that we have used for several
years for use with the ACC-S fruit and then
tested the effects of synthetic auxins, GA3, and
ethylene on the a-Af isoforms. The disks of
ACC-S fruit and discs did not change color
(ripen) unless they were treated with ethylene
(lower left panel). Changes in the activities of
a-Af -1, 2 and 3 in MG disks treated with the
hormones are shown in panels A, B C,
respectively. The different responses to hormones
and different patterns of change in growing and
ripening fruits convince us that there are at
least 3 a-Afs in tomato and that a-Af-3 is
ripening-specific.
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Manipulation of changes in a-Af gene expression
could affect softening in ripening tomato fruit,
just as manipulation of the expression of
b-galactosidase gene expression does. The main
roles of these enzymes may be in cooperating with
pectolytic enzymes in more effective digestion of
crucial bonds in pectin polymers.
One reason that attempts to genetically modify
the extent of ripening-related fruit softening
have not been more successful could be that both
wall polymer systems (cellulose-XG and pectins)
are involved in conferring wall strength and so
the metabolism of both must be considered when
trying to modify wall weakening.
A group of researchers in Vegetable Crops
(Gurrieri, Bennett, Powell), Food Science
(Kalamaki), and Pomology (Brummell, Greve,
Struijs, Van linden and Labavitch) has been
examining the effects of simultaneous
down-regulation of PG and Exp in tomato fruit on
fruit texture characteristics. The effect on
fruit softening is not clear (relative to the
lines suppressed in PG or Exp gene expression),
but processed tomato products from the doubly
suppressed fruits have improved consistency.
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Rather than discuss additional research that has
looked at the effects of the expression of genes
encoding other putative cell wall-digesting
enzymes in ripening fruit, I would like to end
this seminar with a bit of speculation about
other ways to alter the strength of cell walls
and, perhaps, the ways that development-related
changes in cell wall metabolism and strength
might be engineered. Most work done to date has
attempted to take advantage of what was known
about fruit enzymes that acted on cell wall
polysaccharides and test whether that knowledge
could lead to altered fruit softening. Well,
maybe we really didnt know enough, but the
various lines of fruit have proven to be valuable
experimental material, but not necessarily
commercially useful lines. Studies of these
engineered lines have taught us a lot more. But,
why not engineer the cell wall substrates, rather
than the enzymes? If we could change the polysacc
harides that are components of the cell wall
fabric we could probably determine which polymers
are most important in influencing various
physical properties (extensibility,
compressibility, juice consistency) of fruits and
their products. We might even make the fruit
walls more responsive to our manipulations of
wall-degrading enzyme production.
20
An international group of researchers has
collaborated on some work done in Denmark and
England that has demonstrated the feasibility of
engineering changes in pectin polymers in potato
tubers. The work is based on the expression of fu
ngal genes encoding pectin polymer- digesting
enzymes in tubers at the time when tubers are
growing. They do this by driving expression of
the fungal genes with a potato promoter that
normally regulates expression of a tuber starch
synthesizing enzyme. The fungal enzymes that are
expressed in tubers all have as their targets
aspects of the structure of the RG-I
polysaccharide. 1. Sorensen et al. (2000) PNAS US
A 977639-7644 examines the effect of expressing
a galactan side-chain-degrading enzyme that is
active in the apoplast. 2. Oomen et al. (2002) Pl
ant J. 30403-413 examines the effect of
expressing an RG-I backbone-cleaving enzyme,
RG-Lyase, that is active in the apoplast.
3. Skjot et al. (2002) Plant Physiol. 12995-102
examines the effect of expressing an arabinan
side chain-cleaving enzyme in either the Golgi
system, where RG-I is synthesized, or in the
apoplast.
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Here, again, is RG-I, the wall target of these
manipulations.
RG-I also has side chains that are rich in the
neutral sugars Gal and Ara. Breaking the RG-I
backbone would require an enzyme (hydrolase or
lyase) that broke the glycosidic bond between
GalUA and Rha residues. Such an enzyme could act
only if it has access to the backbone. Access
might be provided if a-arabinosidase and/or
b-galactosidase were to remove some side chains.
22
The tubers transformed to express galactanase had
reduced cell wall galactose, but showed no
altered phenotype, although cooking properties,
ease of starch extraction etc. were not tested.
One might also imagine that wall porosity is
altered because of the apparent absence of some
pectin polymer side chains.
Visualization of wall galactans using the LM5
monoclonal antibody that recognizes
b-1,4-galactans reveals the changes made in muro.
Reflection scanning confocal microscopy (panels A
B) shows antibody presence in green.
Transmission EM (panels C D) shows antibody
localization based on appearance of electron
dense black particles.
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In muro fragmentation of the RG-I backbone in
potato by expression of RG-lyase altered cell
wall content of the RG-I, including the sugars
expected to be found in the RG-I side chains.
The approach taken in this work followed the sam
e lines as the earlier work on potato pectin
engineering. The concept was a bit different in
that the enzyme expressed was expected to break
the RG-I backbone, rather than remove its side
chains. The enzyme used was a fungal endo-RG
lyase (eRGL) that catalyses the trans-elimination
of the backbone between adjacent Rha and GalUA
residues.
Tubers from the transgenic lines were smaller,
had more deep-set eyes, and had a wrinkled
surface. The plants grew more slowly and tuber
eyes were relatively slow to sprout. Transgenics
produced only a few flowers.
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Light microscopy of sections through tubers,
showing periderm (Pdm), cortex (Ctx) and
perimedullary tissue (Pmed) show that the
alteration of polysaccharide solubility and sugar
content is linked to altered tissue organization.
Panel a shows WT. The tissue organization is
clear. Panel b, the transgenic line, shows
disordered arrangement of cells and occasional
large air spaces in the tissue, suggesting that
cell division planes have been altered.
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Although monoclonal antibodies that recognize
RG-I polymer backbones are available, they did
not prove to be useful in analysis of the tuber
wall RG-I polymer backbones. LM 5 (recognizes
b-1,4-galactan) and LM6 (shows a-1,5-arabinan)
were used. These reflectance confocal laser
scanning micrograph views show binding of the two
monoclonals as bright, yellow-green areas.
The distribution of the epitopes is substantiall
y reduced in the trans-genics. This is
consistent with the direct sugar analysis of
walls and extracts.
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Direct interference with RG-I biosynthesis in
Golgi vesicles. Skjot et al. (2002) Plant
Physiology 12995-102. Again the potato was used
as a target for transformation. A fungal
a-1,5-arabinanase was inserted into the potato
genome and regeneration was tested.
1. If the arabinanase was targeted to the
apoplast then no successful plants were
recovered. Plants lacked side shoots, flowers,
stolons and tubers. 2. If the arabinanase was t
argeted for localization in the Golgi, by using a
sequence encoding a fusion protein of the
arabinanase and a rat sialyl transferase normally
involved in glycoprotein oligosaccharide
synthesis, then transformed plants bearing tubers
were generated. 3. Sugar analysis and antibody st
udies confirmed that wall arabinan levels were
reduced in transformants.
Panel a shows the transgenic with the
a-arabinanase targeted to the apoplast, panel b
shows the transformed plant with Golgi-targeted
arabinanase, and panel c shows the WT potato line
used for the transformation.
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There is now a growing list of mutations that
affect cell wall polymer synthesis and, in some
cases, affect not only the cell walls sugar
content (hence the characteristics of specific
polysaccharides) but also the plants phenotype.
These mutations have not yet been inserted into
the genomes of fruiting species to test
horticultural impacts. Thus the engineering o
f changes in a plants wall biosynthetic
capacity and the final assembly of the wall in
the apoplast (hence, the walls chemical and
physical character) can follow many interesting
avenues. We have experimented with modifying the
complement of wall-metabolizing enzymes for
several years now. Thus we now can work on both t
he anabolic and catabolic sides of the cell wall
steady state equation. Whether we learn how to
manipulate the textural properties of ripening
fruits will remain an open question for at least
several more years, but the new experimental
material that is generated will help us to learn
a great deal about the biochemistry of cell wall
metabolism.