Physic Meets Biology Lkdin

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


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


MOULD

Positive feed-back:“one rotten apple spoils the whole

bushel”

Flower Development

Fertilisation


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

AND FUNGAL SPREAD ON FRUITS

TOBACCO FLOWERS TOMATO (BERRY) FRUIT

Pollination in tobacco

C2H4 (nl/hour/flower)

0 20 40 60 80 1000

10

20

0 20 40 60 80 1000

10

20

0 20 40 60 80 1000

10

20

TIME (hours)

Not pollinatedHeated N.tabacum pollen

AVG + N.tabacum pollenN.tabacum pollen

N.tabacum 16h lightN.tabacum 8h darkN.tabacum continuous light

A

B

C



pollen

0-3h germination, Penetration into the stigma

3-30 hPenetration 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


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


A

B

C



0 20 40 60 800

10

20


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


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.


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


A

B

C



0 20 40 60 800

10

20


0 20 40 60 80 1000

10

20

0 20 40 60 80 1000

10

20

0 20 40 60 80 1000

10

20

TIME (hours)

Not pollinatedHeated N.tabacum pollen

AVG + N.tabacum pollenN.tabacum pollen

N.tabacum 16h lightN.tabacum 8h darkN.tabacum continuous light

A

B

C



•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 )


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


•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

http://en.wikipedia.org/wiki/Image:Resveratrol_trans_isomer.png

Post-harvest resistanceMOULD

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

http://en.wikipedia.org/wiki/Image:Resveratrol_trans_isomer.png

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 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.


MOULD

Flower Development

Fertilisation


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

Physic Meets Biology Lkdin

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