Transcript Louisiana-Pacific Sagola, MI

Abscission
Jennie Lund
Bioinformatics
May 1, 2003
Abscission
Abscission: the shedding of leaves,
flowers, & fruit from living plants,
separates senescent organs
http://homepage.ntlworld.com/tir
ain/galleries/autumn/vert.htm
Senescence: normal, energy-dependent
developmental process controlled by
genes (compare necrosis-death from physical
damage)
-photoperiod, temperature, shading
from other leaves
-salvages resources in leaves
-> back into plant through phloem
*enzymes break down proteins,
carbohydrates, nucleic acids into
sugars, nucleosides, amino acids
*minerals
Abscission
Abscission layer: distinct layer of cells--located in
the abscission zone--with weakened cell walls that
permit abscission
Abscission zone: region that contains the
abscission layer and is located near the petiole of leaves
Ethylene: gaseous hormone synthesized from methionine;
increases abscission (also involved with plant growth & development,
including stimulating or inhibiting elongation of stems, roots, depending on
conditions & species; enhances fruit development; suppresses flowering in
most species; increases RNA transcription of many genes)
Auxin: plant hormone that delays abscission (also involved in
inducing cell elongation in isolated coleoptile or stem sections, cell division in
callus tissue in presence of cytokinin, lateral root formation at cut stem
surfaces, parthenocarpic fruit growth, ethylene formation)
Abscission
More about ethylene:
-used commercially for fruit ripening, inducing abscission for
fruit thinning or drop in cotton, cherry, & walnut
-induces cucumber femaleness, also maple trees
-prevent self pollination, increase yield, inhibit terminal growth &
increases lateral growth
-store fruits at low & low temperature to inhibit ethylene
biosynthesis, CO2 also prevents ethylene action in ripening
-Transgenic tomato: insert antisense
of ethylene precursor = delayed
softening, can delay
tomato & petunia wilt &
senescence for weeks
www.hylineorchards.com/cherryharvest.html
www.gardeners.com
Abscission
Ethylene regulates abscission, auxin suppresses ethylene effect
(large amounts of auxin can cause ethylene production, resulting in abscission,
auxin analogs in Agent Orange)
Effects of auxin & ethylene:
-target cells in abscission zone “protected” by auxin from being
sensitized by ethylene
-auxin produced in the leaf travels down through petiole
-if auxin is removed by cutting off leaf blade, the petiole soon
falls off, too
-if auxin is put in place of the leaf blade, petiole abscission &
ethylene-induced accumulation of cellulase mRNA & protein are
inhibited
-if auxin is put on the tree side of cut petiole, abscission
speeds up
Therefore, auxin gradient determines ethylene sensitivity (more on
tree side increases it, more on blade side decreases it)
Abscission
What triggers sensitivity, & what
happens when target cells become
sensitive?
Leaf senescence reduces auxin
transport from leaf blade, so target
cells lose the “auxin protection” &
become sensitive; produces
ethylene
Low concentrations of ethylene cause target cells to synthesize &
secrete cellulase & other cell-wall degrading enzymes, & mature
parenchyma cells begin dividing into cells with weaker cell walls
-when cell walls break down, protoplasts are freed
-form stable spherical shapes, increase in volume, & push leaf apart
Cells in abscission layer produce suberin which begins to block water
& raw material flow to leaf
-chlorophyll cannot form -> fading leaves
-further decrease of auxin flow
Abscission
Auxin
Auxin
Ethylene
Separation layer
digested
Yellowing
Abscission
Original plan:
Find & compare abscission cellulases in
different tree species, especially
comparing those with different leaf drop
times
However……..no Quercus AND abscission AND cellulase
results on NCBI
search for abscission AND cellulase
-BLASTp with result for Sambucus nigra, European elder
-chose 15 proteins; 10 had E-values of 0.0; 5 had E-values
between -139 & -131
-to increase variety of species
Abscission
Species:
-Sambucus nigra, European elder
-Capsicum annuum, red pepper
-Lycopersicon esculentum, tomato
-Phaseolus vulgaris, common bean
-Arabidopsis thaliana
-Populus alba, (European) white poplar
-Pinus radiata, Monterey pine
-Citrus sinensis, sweet orange
-Fragaria X ananassa, strawberry
Abscission
Looked at paper: “The gene promoter for a bean
abscission cellulase is ethylene-induced in transgenic
tomato & shows high sequence conservation with a
soybean abscission cellulase” by Susan Koehler, et al. 1996.
-abscission related to de novo accumulation of cellulase (endo1,4-B-D-glucanase)
-so unless the samples used for obtaining protein sequences
in NCBI were collected during abscission, probably shouldn’t
be able to find them--some must have been collected then
-de novo based on radioimmunoassays & fact that after
ethylene exposure, there is a 12-18 hour lag period then rapid
decrease in break strength to pull petioles off stem
-break strength decrease occurs with 40-fold increase of
cellulase activity
Abscission
Determined that block must be transcriptional, so there
must be some cis-acting intron somewhere
(-auxin inhibits abscission & blocks accumulation of
cellulase mRNA & protein (find mRNA if translational)
-ethylene increases RNA synthesis in abscission layer
-RNA synthesis inhibitor blocks synthesis & cellulase
mRNA & protein
-polysome-associated BAC mRNA occurred with
increased BAC activity in ethylene-induced
abscission zones
-ethylene required for sustaining accumulation of this
mRNA)
Now interested in promoters
Abscission
An ethylene responsive element (ERE) for pathogenesisrelated (PR) proteins & carnation are known. Koehler et al.
did not find either sequence in bean or soybean abscission cellulase.
-not all of my proteins started with M (ATG), so I searched for these
patterns in them
PR ERE: TAAGAGCCGCC
carnation ERE: ATTTCAAA
-I translated the ERE sequences & searched my proteins with them
-> 27 finds for PR ERE (0 for carnation ERE), but those 27 were
located at least 100 aa in, & target sequence only 2 aa long
-I backtranslated my proteins & searched for ERE in them-> no
matches for either until 7 mismatches, & target sequences only 11 &
8 bp long -> with 7 mismatch PR ERE = 1664, carnation ERE = 90
Expected, since Koehler et al. did not find any either, & my
sequences probably do not include promoter regions
Abscission
I used pileup & pretty to get a consensus
I also obtained a consensus from clustalw, & I looked at JalView:
Abscission
I did doublegap to see the % identity
-some were very high
-comparisons between abscission
cellulases in the same species were
98-100%
-comparison between red pepper & tomato was 89%
identity
-comparison between white poplar & sweet orange was
83% identity
-most were between mid-50s
to mid-60s % identities
Abscission
I used clustalw &
treeview to prepare this
phylogenetic tree:
-tomato is interesting, only
54% identity between 2
tomatoes from doublegap
-maybe one for leaf, one for
fruit
-79% identity for bottom
tomato & white poplar
-56% for white poplar & top
tomato
-60% identity for Monterey
pine & strawberry
-57% for strawberry & sweet
orange
European elder
European elder
Tomato
Red pepper
Red pepper
Bean
Bean
Bean
Arabidopsis
Arabidopsis
Tomato
White poplar
Sweet orange
Monterey Pine
Strawberry
Abscission
I used PSORT to see where the
sequence for Sambucus nigra is
located (this was my target sequence
for BLASTp)
PSORT results showed localization was most likely in
the microbody, but plasma membrane had a value only
0.005 lower
Conclusion
-Auxin “protects” target cells in the abscission zone from being
sensitive to ethylene
-When auxin is reduced or removed--such as by senescence,
target cells become sensitive to ethylene
-This causes them to produce cellulase & other cell wall
degrading enzymes, & newly produced cells have weak
walls
-“Released” protoplasts “round out” & help push the layer apart
-My sequences did not contain the published EREs
-My proteins were quite conserved
-Some of my proteins may have been for fruit abscission
-My first protein--used to get all the rest--is most likely
localized in the microbody or plasma membrane
-This makes sense--close to the cell wall
Works Cited
Koehler, Susan M.; Gali L. Matters; Pravendra Nath; Elizabeth C.
Kemmerer; and Mark L. Tucker. 1996. The gene promoter for a bean
abscission cellulase is ethylene-induced in transgenic tomato and shows
high sequence conservation with a soybean abscission cellulase. Plant
Molecular Biology 31: 595-606.
Nikon Microscopy. Confocal Microscopy Image Gallery: Abscission Layer.
http://www.microscopyu.com/galleries/confocal/abscission.html
Taiz, Lincoln; and Eduardo Zeiger. Plant Physiology, Third Edition
Sinnauer Associates, Inc.: Sunderland, MA. 2002: 370-531.
Works Cited
Pictures
Red maple, paper birch, sugar maple, red oak
Chemical of the week. The chemistry of autumn colors
http://scifun.chem.wisc.edu/chemweek/fallcolr/fallcolr.html
yellow maple leaves
http://homepage.ntlworld.com/tirain/galleries/autumn/vert.htm
cherry shaker
http://www.hylineorchards.com/cherryharvest.html
tomato
http://www.gardeners.com/department.asp?CMP=IL8892&DeptPGID=16979&lstCategory=17065&SC
=CNB883
Phaseolus vulgaris, common bean, string and bush bean
http://www.mpiz-koeln.mpg.de/~pr/garten/schau/PhaseolusvulgarisL/Common_bean.html
strawberry
http://www.noursefarms.com/catalog/strawberries.html
white poplar
http://www.nps.gov/plants/alien/fact/poal1.htm
fallen leaves
http://www.digitalphotography.tv/mff2001_01/2.html
copyright © 2001-2002, digitalphotography.tv/Charlie Morey
Thank you!
Questions?