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Abscission Jennie Lund Bioinformatics May 1, 2003.

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Presentation on theme: "Abscission Jennie Lund Bioinformatics May 1, 2003."— Presentation transcript:

1 Abscission Jennie Lund Bioinformatics May 1, 2003

2 Abscission Abscission: the shedding of leaves, flowers, & fruit from living plants, separates senescent organs 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 ain/galleries/autumn/vert.htm

3 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)

4 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, CO 2 also prevents ethylene action in ripening -Transgenic tomato: insert antisense of ethylene precursor = delayed softening, can delay tomato & petunia wilt & senescence for weeks

5 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)

6 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

7 Abscission Auxin Auxin Ethylene Separation layer digested Yellowing

8 Abscission Original plan: Find & compare abscission cellulases in different tree species, especially comparing those with different leaf drop times However…… 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

9 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

10 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 (endo- 1,4-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

11 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

12 Abscission An ethylene responsive element (ERE) for pathogenesis- related (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

13 Abscission I used pileup & pretty to get a consensus I also obtained a consensus from clustalw, & I looked at JalView:

14 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

15 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 Tomato Red pepper Bean Bean Bean Arabidopsis Arabidopsis Tomato White poplar Sweet orange Monterey Pine Strawberry

16 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

17 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

18 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. Taiz, Lincoln; and Eduardo Zeiger. Plant Physiology, Third Edition Sinnauer Associates, Inc.: Sunderland, MA. 2002: 370-531.

19 Works Cited Pictures Red maple, paper birch, sugar maple, red oak Chemical of the week. The chemistry of autumn colors yellow maple leaves cherry shaker tomato =CNB883 Phaseolus vulgaris, common bean, string and bush bean strawberry white poplar fallen leaves copyright © 2001-2002, Morey

20 Thank you! Questions?

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