When physic meets biology:  high-resolution laser-based techniques  to study plant-microbe interactions <ul><li>Domenico D...
The life cycle of a flowering plant OVARY Ovule SEPALS PETALS Anthers Stigma Style pollen pollen tube UPON FERTILISATION E...
The life cycle of a flowering plant MOULD Flower Development Fertilisation Seed/fruit development Fruit ripening Overipe, ...
the ethylene biosynthetic pathway Yang (Methionine) Cycle methionine S-adenosyl- methionine (SAM) Methylthioadenosine MTA ...
The life cycle of a flowering plant MOULD Positive feed-back: “ one rotten apple spoils the whole bushel ” Flower Developm...
ACO gene expression GENE CLONING ENABLED TO SELECT A OVULE SPECIFIC ACC-OXIDASE  (the “ethylene-forming enzyme”)  GENE
ACO gene silencing Silencing Gene Expression  of the Ethylene-Forming Enzyme  Resulted  in a Reversible Inhibition  of Ovu...
Pollen tube  vs . Fungal hyphae Images above Downloaded from Internet POLLEN TUBES AND FUNGAL HYPHAE PRESENT SOME SIMILARI...
Pollination  vs . infection A LASER SYSTEM THAT ENABLES TO MEASURE GAS RELEASE FROM PLANT TISSUES ONLINE WAS USED TO STUDY...
Pollination in tobacco pollen 0-3h germination,  Penetration into the stigma 3-30 h Penetration into the TT 60 h  Most of ...
Pollination in tobacco N. Repanda  pollen In this cross most pollen tubes stopped are arrested in the stigma, and no furth...
Pollination in tobacco pollen Petunia pollen <ul><li>pollen tube growth slower than that of self-pollinated tobacco  </li>...
Pollination in tobacco pollen <ul><li>ethylene release in the tobacco flower is a direct consequence of pollination and do...
Pollination in tobacco <ul><li>ethylene release in the tobacco flower is a direct consequence of pollination and does not ...
Grey mould  Botrytis cinerea Botrytis can produce ethylene Via  the “KMBA pathway”  (K-keto Q-methylthiobutyric acid )
Grey mould  Botrytis cinerea Dinamic of  in vitro  ethylene production resembles greatly production during fungus-fruit in...
Grey mould  Botrytis cinerea <ul><li>B. cinerea  is able to produce ethylene  in vitro , and the emission of ethylene foll...
Post-harvest resistance MOULD Resveratrol is a substance that is produced by several plants and that is sold as a nutritio...
Post-harvest resistance MOULD Plant Physiol, January 2003, Vol. 131, pp. 129-138  trans-Resveratrol and Grape Disease Resi...
Post-harvest resistance Reduced microbial bloom Conserved water content Unaltered nutritional contents
Post-harvest resistance Resveratrol  treatment resulted effective on fruit that normally does not accumulate such metaboli...
The life cycle of a flowering plant MOULD Flower Development Fertilisation Seed/fruit development Fruit ripening Overipe, ...
“ Raccolta e conservazione della frutta fresca:nuovi metodi per problemi antichi”.  J.B. Jiménez, J.M. Orea, C. Montero, A...
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Physic Meets Biology Lkdin

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THIS PRESENTATION REPRESENTS IN A NUTSHELL THE CORE OF RESEARCH MADE BETWEEN 1995 AND 2001 AT THE UNIVERSITY OF NIJMEGEN (NL) AND AT ENEA CASACCIA RESEARCH CENTRE (IT).

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Physic Meets Biology Lkdin

  1. 1. When physic meets biology: high-resolution laser-based techniques to study plant-microbe interactions <ul><li>Domenico De Martinis </li></ul>THIS PRESENTATION REPRESENTS IN A NUTSHELL THE CORE OF RESEARCH MADE BETWEEN 1995 AND 2001 AT THE UNIVERSITY OF NIJMEGEN (NL) AND AT ENEA CASACCIA RESEARCH CENTRE (IT). AN IMPORTANT COLLABORATION FOR THIS STUDIES WAS ALSO WITH THE INSTITUTO PLURIDISCIPLINAR OF THE COMPLUTENSE UNIVERSITY OF MADRID (ES)
  2. 2. The life cycle of a flowering plant OVARY Ovule SEPALS PETALS Anthers Stigma Style pollen pollen tube UPON FERTILISATION EACH (FERTILISED) OVULE will DEVELOP in a SEED and the OVARY in the FRUIT THE STUDIES FOCUSED ON THE DEVELOPMENT AND REPRODUCTION OF THE TOBACCO FLOWER AND MATURATION/RIPENING OF TOMATO BERRY FRUIT Stamen Tomato berry fruit Tobacco capsule (approx. 2000 seeds)
  3. 3. The life cycle of a flowering plant MOULD Flower Development Fertilisation Seed/fruit development Fruit ripening Overipe, Spoilage Ovary/ovule development megagametogenesis Pistil development megasporogenesis Pollen-Pistil interaction Fertilisation Flower Senescence Embryogenesis Seed development Ovary transition into fruit Pigmentation Placenta development Tissue softening Sugar accumuation Flavour HOW TOBACCO FLOWER AND TOMATO BERRY FRUIT ARE AFFECTED BY ETHYLENE?
  4. 4. the ethylene biosynthetic pathway Yang (Methionine) Cycle methionine S-adenosyl- methionine (SAM) Methylthioadenosine MTA MTR MTR1-P KBR C H 2 C H 2 C NH 3 + COO - H H H C=C H ACCsynthase ACCoxidase Ethylene 1/2O 2 CO 2 +HCN+H 2 O Perception/signal transduction ACC CH 3 -S-CH 2 -CH 2 -CH-COO - NH 3 + CH 3 -S- CH 2 -CH 2 -CH-COO - NH 3 + CH 2 + Adenine OH OH CH 3 -S CH 2 Adenine OH OH
  5. 5. The life cycle of a flowering plant MOULD Positive feed-back: “ one rotten apple spoils the whole bushel ” Flower Development Fertilisation Seed/fruit development Fruit ripening Overipe, Spoilage Pollination results in a burst of ethylene that relate with flower senescence Climateric phase, increase in respiration, ethylene production Pathogenesis-Related Ethylene burst Ovary/ovule development megagametogenesis Pistil development megasporogenesis Pollen-Pistil interaction Fertilisation Flower Senescence Embryogenesis Seed development Ovary transition into fruit Pigmentation Placenta development Tissue softening Sugar accumuation Flavour
  6. 6. ACO gene expression GENE CLONING ENABLED TO SELECT A OVULE SPECIFIC ACC-OXIDASE (the “ethylene-forming enzyme”) GENE
  7. 7. ACO gene silencing Silencing Gene Expression of the Ethylene-Forming Enzyme Resulted in a Reversible Inhibition of Ovule Development In Transgenic Tobacco Plants <ul><li>Arrested/retarded ovule development </li></ul><ul><li>Lack of fertilisation </li></ul><ul><li>Low or absent seed set </li></ul>
  8. 8. Pollen tube vs . Fungal hyphae Images above Downloaded from Internet POLLEN TUBES AND FUNGAL HYPHAE PRESENT SOME SIMILARITIES. WE TRIED TO STUDY THE EFFECT OF THEIR PENETRATION ON THE PLANT TISSUES WITH THE SAME APPROACH FROM MY IMAGE PORTFOLIO A B C D
  9. 9. Pollination vs . infection A LASER SYSTEM THAT ENABLES TO MEASURE GAS RELEASE FROM PLANT TISSUES ONLINE WAS USED TO STUDY FLOWER POLLINATION AND FUNGAL SPREAD ON FRUITS TOBACCO FLOWERS TOMATO (BERRY) FRUIT
  10. 10. Pollination in tobacco pollen 0-3h germination, Penetration into the stigma 3-30 h Penetration into the TT 60 h Most of the ovules fertilised neither senescence, nor mock-pollination with heat killed pollen does elicit ethylene production ethylene is de novo synthesized from the tobacco pistil C 2 H 4 (nl/hour/flower) TIME (hours) Not pollinated Heated N.tabacum pollen AVG + N.tabacum pollen N.tabacum pollen N.tabacum 16h light N.tabacum 8h dark N.tabacum continuous light A B C C 2 H 4 (nl/hour/flower) C 2 H 4 (nl/hour/flower)
  11. 11. Pollination in tobacco N. Repanda pollen In this cross most pollen tubes stopped are arrested in the stigma, and no further growth could be observed 3h after pollination N. Rustica pollen For the first 12h after pollination, rustica pollen tubes grow into the style of tobacco slightly faster than that of self-pollinated tobacco. The growth rate eventually decreased and pollen tube arrested after approximately 24h at about 2/3 of the style N. trigonophylla pollen Pollen tubes of trigonophylla grow slowly into the tobacco style. 50h after pollination pollen tubes have reached less than 2/3 of the style. Sometimes the tobacco flowers were not able to sustain this inter-specific pollination up to 5 day and abscise AVG + N. repanda pollen N. repanda pollen AVG + N. rustica pollen N. rustica pollen 0 20 40 60 80 0 10 20 TIME (hours) 0 20 40 60 80 100 120 0 10 20 AVG + N. trigonophylla pollen N. trigonophylla pollen C 2 H 4 (nl/hour/flower) A B C C 2 H 4 (nl/hour/flower) C 2 H 4 (nl/hour/flower) 0 20 40 60 80 0 10 20
  12. 12. Pollination in tobacco pollen Petunia pollen <ul><li>pollen tube growth slower than that of self-pollinated tobacco </li></ul><ul><li>the pollen tubes reach halfway the style 24 h after pollination </li></ul><ul><li>petunia pollen can reach the ovary approximately 48 h after pollination </li></ul><ul><li>pollination results in a seed set comparable to that of self-pollinated tobacco </li></ul>In all cases the seeds obtained from pollination of tobacco pistils with petunia pollen were not viable, thus indicating that the inter-specific barriers between tobacco and petunia must be effective during post-fertilization development TIME (hours) C 2 H 4 (nl/hour /flower) 0 20 40 60 80 100 120 140 160 0 10 20 AVG + Petunia pollen Petunia pollen
  13. 13. Pollination in tobacco pollen <ul><li>ethylene release in the tobacco flower is a direct consequence of pollination and does not occur during flower senescence. </li></ul><ul><li>pollination-induced ethylene production represents a response of the flower to specific pollen recognition. </li></ul><ul><li>ethylene is de novo synthesized upon penetration of the pollen tubes into the style but its production does not correlate with the rate of pollen tube growth into the style but it depends on the type of pollen used. </li></ul><ul><li>Decreased ethylene production by AVG treatment of the stigma did not affect the process of fertilization self-pollination in tobacco, thus indicating that ethylene alone is not essential for pollen tube growth into the style. </li></ul>
  14. 14. Pollination in tobacco <ul><li>ethylene release in the tobacco flower is a direct consequence of pollination and does not occur during flower senescence. </li></ul><ul><li>pollination-induced ethylene production represents a response of the flower to specific pollen recognition. </li></ul><ul><li>ethylene is de novo synthesized upon penetration of the pollen tubes into the style but its production does not correlate with the rate of pollen tube growth into the style but it depends on the type of pollen used . </li></ul><ul><li>Decreased ethylene production by AVG treatment of the stigma did not affect the process of fertilization self-pollination in tobacco, thus indicating that ethylene alone is not essential for pollen tube growth into the style. </li></ul>AVG + N. repanda pollen N. repanda pollen AVG + N. rustica pollen N. rustica pollen 0 20 40 60 80 0 10 20 TIME (hours) 0 20 40 60 80 100 120 0 10 20 AVG + N. trigonophylla pollen N. trigonophylla pollen C 2 H 4 (nl/hour/flower) A B C C 2 H 4 (nl/hour/flower) C 2 H 4 (nl/hour/flower) 0 20 40 60 80 0 10 20 C 2 H 4 (nl/hour/flower) TIME (hours) Not pollinated Heated N.tabacum pollen AVG + N.tabacum pollen N.tabacum pollen N.tabacum 16h light N.tabacum 8h dark N.tabacum continuous light A B C C 2 H 4 (nl/hour/flower) C 2 H 4 (nl/hour/flower)
  15. 15. Grey mould Botrytis cinerea Botrytis can produce ethylene Via the “KMBA pathway” (K-keto Q-methylthiobutyric acid )
  16. 16. Grey mould Botrytis cinerea Dinamic of in vitro ethylene production resembles greatly production during fungus-fruit interactions m.maker daniela
  17. 17. Grey mould Botrytis cinerea <ul><li>B. cinerea is able to produce ethylene in vitro , and the emission of ethylene follow the pattern that is associated with hyphal growth rather than the germination of conidia. </li></ul><ul><li>Higher levels are observed when the concentration of conidia is higher. Note that at higher concentrations of conidia the ethylene emission rate is also faster. </li></ul><ul><li>This finding can be related to fungal neighbourhood sensing. </li></ul><ul><li>B. cinerea , although it is strongly synchronized with the growth rate of the fungus inside the tomato </li></ul><ul><li>Upon infection, the emission is synchronized with the growth rate of the fungus inside the tomato </li></ul>
  18. 18. Post-harvest resistance MOULD Resveratrol is a substance that is produced by several plants and that is sold as a nutritional supplement. A number of beneficial health effects, such as anti-cancer, anti-viral, neuroprotective, anti-aging, anti-inflammatory and life-prolonging effects Resveratrol FURTHER RESEARCH FOCUSED ON THE USE OF RESVERATROL AS A NATURAL PESTICIDE TO INCREASE SHELF LIFE OF FRUIT
  19. 19. Post-harvest resistance MOULD Plant Physiol, January 2003, Vol. 131, pp. 129-138 trans-Resveratrol and Grape Disease Resistance. A Dynamical Study by High-Resolution Laser-Based Techniques C. Montero, S.M. Cristescu, J.B. Jiménez, J.M. Orea, S. te Lintel Hekkert, F.J.M. Harren, and A. González Ureña Unidad de Láseres y Haces Moleculares Instituto Pluridisciplinar, Universidad Complutense de Madrid P° Juan XXIII, 1. 28040 Madrid, Spain (C.M., J.B.J., J.M.O., A.G.U.); and Department of Molecular and Laser Physics University of Nijmegen Toernooiveld, 6525 ED Nijmegen, The Netherlands (S.M.C., S.t.L.H., F.J.M.H.) Resveratrol
  20. 20. Post-harvest resistance Reduced microbial bloom Conserved water content Unaltered nutritional contents
  21. 21. Post-harvest resistance Resveratrol treatment resulted effective on fruit that normally does not accumulate such metabolite as, for example, tomatoes, apples, avocado pears and peppers. As a result, all treated fruits maintained their post-harvest quality and health longer than the untreated ones . This study demonstrates the potential of the use of resveratrol as “natural pesticide” to reduce post-harvest fungi development on a broad spectrum of fruit types.
  22. 22. The life cycle of a flowering plant MOULD Flower Development Fertilisation Seed/fruit development Fruit ripening Overipe, Spoilage Pollination results in a burst of ethylene that relate with flower senescence Climateric phase, increase in respiration, ethylene production Pathogenesis-Related Ethylene burst Pollination-induced ethylene production represents a response of the flower to specific pollen recognition ethylene controls ovule development can B.Cinerea sense ethylene? does B.Cinerea trigger ethylene production? resveratrol as “natural pesticide”, and added value to post-harvested fruit TO SUMMARISE
  23. 23. “ Raccolta e conservazione della frutta fresca:nuovi metodi per problemi antichi”. J.B. Jiménez, J.M. Orea, C. Montero, A. González Ureña, E. Navas, K. Slowing, M.P. Gómez Serrranillos, E. Carretero and D. De Martinis. (2005) Use of trans -resveratrol (3, 5, 4’-thrydroxystilbene) to control microbial flora, prolong shelf-life and preserve nutritional quality of fruit. J. Agric Food Chem . Mar 9;53(5):1526-1530 D. DeMartinis. (2003) “When physics meets biology: high-resolution laser-based techniques to study plant-microbe interactions”. Mycological Research , 107 (8), 899-900. S.M. Cristescu, D. De Martinis, S. te Lintel Hekkert, D. H. Parker, F.J..M. Harren (2002) ”Ethylene production by Botrytis cinerea in vitro and in tomatoes”. Applied and Environmental Microbiology , 68 (11) 5342-50. D. Pashkoulov, I. Giannetti, E. Benvenuto, and D. De Martinis (2002) ”Biochemical Characterisation of Polygalacturonases from five different isolates of Botrytis cinerea ” Mycological Research , 106 (7), 827-831. D. De Martinis, G. Cotti, S. te Lintel Hekkert, F.J.M. Harren and C. Mariani. (2002) “Ethylene response to pollen tube growth in Nicotiana tabacum flower “. Planta , 214(5)806-12 D. De Martinis and C. Mariani. (1999) ”Silencing Gene Expression of the Ethylene-Forming Enzyme Results in a Reversible Inhibition of Ovule Development in Transgenic Tobacco Plants”. the Plant Cell 11:1047-1060

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