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).
When physic meets biology: high-resolution laser-based techniques to study plant-microbe interactions Domenico De Martinis 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)
Transcript
When physic meets biology:
high-resolution laser-based techniques
to study plant-microbe interactions
Domenico De Martinis
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)
The life cycle of a flowering plant
OVARY
OvuleSEPALS
PETALS
Anthers
Stigma
Stamen
Style
pollen
pollen tube
Tomato berry fruit
Tobacco capsule(approx. 2000 seeds)
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
The life cycle of a flowering plant
MOULD
Flower Development
Fertilisation
Seed/fruit development
Fruit ripening
Overipe,Spoilage
Ovary/ovule development
megagametogenesis
Pistildevelopment
megasporogenesis
Pollen-Pistil interactionFertilisation
Flower SenescenceEmbryogenesis
Seed development
Ovary transition into fruit Pigmentation
Placenta development
Tissue softeningSugar accumuation
Flavour
HOW TOBACCO FLOWER AND TOMATO BERRY FRUIT
ARE AFFECTED BY ETHYLENE?
the ethylene biosynthetic pathway
Yang(Methionine)
Cycle
methionine
S-adenosyl-methionine
(SAM)
MethylthioadenosineMTA
CH3-S-CH2-CH2-CH-COO-
NH3+
MTR
MTR1-P
KBR
CH3-S-CH2-CH2-CH-COO-
NH3+
CH2
+
Adenine
OH OHCH3-S
CH2
Adenine
OH OHC
H2C
H2C
NH3+
COO-
H
H
HC=C
H
ACCsynthase ACCoxidase
Ethylene
1/2O2
CO2+HCN+H2O
Perception/signal transduction
ACC
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
Pistildevelopment
megasporogenesis
Pollen-Pistil interactionFertilisation
Flower SenescenceEmbryogenesis
Seed development
Ovary transition into fruit Pigmentation
Placenta development
Tissue softeningSugar accumuation
Flavour
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 Ovule Development In Transgenic Tobacco Plants
•Arrested/retarded ovule development
•Lack of fertilisation
•Low or absent seed set
Pollen tube vs. Fungal hyphae
A B
C D
Images aboveDownloaded 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
Pollination vs. infection
A LASER SYSTEM THAT ENABLES TO MEASURE GAS RELEASE FROM PLANT TISSUES ONLINE WAS USED TO STUDY FLOWER POLLINATION
neither senescence, nor mock-pollination with heat killed pollen does elicit ethylene production
ethylene is de novo synthesized from the tobacco pistil
Pollination in tobacco
N. Repanda pollenIn this cross most pollen tubes stopped are arrested in the stigma, and no further growth could be observed 3h after pollination
N. Rustica pollenFor 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 pollenPollen 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 pollenN. repanda pollen
AVG + N. rustica pollenN. rustica pollen
0 20 40 60 800
10
20
TIME (hours)
0 20 40 60 80 100 1200
10
20
AVG + N. trigonophylla pollenN. trigonophylla pollen
C2H4 (nl/hour/flower)
A
B
C
C2H4 (nl/hour/flower)
C2H4 (nl/hour/flower)
0 20 40 60 800
10
20
Pollination in tobacco
pollen
Petunia pollen
TIME (hours)
C2H
4 (nl
/hou
r /f
low
er)
0 20 40 60 80 100 120 140 160
0
10
20
AVG + Petunia pollenPetunia pollen
•pollen tube growth slower than that of self-pollinated tobacco •the pollen tubes reach halfway the style 24 h after pollination •petunia pollen can reach the ovary approximately 48 h after pollination•pollination results in a seed set comparable to that of self-pollinated tobacco
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
Pollination in tobacco
pollen•ethylene release in the tobacco flower is a direct consequence of pollination and does not occur during flower senescence.
•pollination-induced ethylene production represents a response of the flower to specific pollen recognition.
•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.
•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.
Pollination in tobacco
AVG + N. repanda pollenN. repanda pollen
AVG + N. rustica pollenN. rustica pollen
0 20 40 60 800
10
20
TIME (hours)
0 20 40 60 80 100 1200
10
20
AVG + N. trigonophylla pollenN. trigonophylla pollen
•ethylene release in the tobacco flower is a direct consequence of pollination and does not occur during flower senescence.
•pollination-induced ethylene production represents a response of the flower to specific pollen recognition.
•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.
•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.
Grey mould Botrytis cinerea
Botrytis can produce ethylene
Via the “KMBA pathway” (K-keto Q-methylthiobutyric acid )
Grey mould Botrytis cinerea
0 12 24 36 48 60 72 840
10
20
30
40conidia/mlFUNGI on agar with 35 mM L-methionine
bb02
Ethylene (nl h
-1)
Time (hours)
1.5x108
2x107
2x105
Dinamic of in vitro ethylene production resembles greatly production during fungus-fruit interactions
m.maker
daniela
Grey mould Botrytis cinerea
•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.
•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.
This finding can be related to fungal neighbourhood sensing.
•B. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato
•Upon infection, the emission is synchronized with the growth rate of the fungus inside the tomato
Post-harvest resistanceMOULD
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
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 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.
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
“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
