Molecular clues on eco-physiological response of lichens to nitrogen

Molecular clues on eco-physiological response of lichens to nitrogen

S. Munzi

1

_{, C. Cruz}

1

_{, L.J. Sheppard}

2

_{, I.D. Leith}

2

_{, L. Bini}

3

_{, A. Gagliardi}

3

_{, G. Cai}

3

_{, L. Parrotta}

3

_{, C. Gouveia}

1

_{, A. Varela Coelho}

4

_{, C. Branquinho}

1

1

_{Center for Ecology, Evolution and Environmental Changes, Universidade de Lisboa,}

2

_{Centre for Ecology & Hydrology, Edinburgh,}

3

_{Dipartimento di Scienze della}

Vita, Università degli Studi di Siena ,

4

_{Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa}

At Whim, an unmanaged

ombrothrophic bog …

•

2 forms: NH

_{and NO}

-•

3 doses: 16, 32 and 64 kg N ha

_yr

•

Cumulative exposure of 6 months (short-term) and 11

years (long-term)

•

Addition/no addition of phosphorous and potassium:

added as K

HPO

at a 1:14 PN ratio to give 16 kg N +

0.57 kg P ha

_yr

_{and 64 kg N + 4 kg P ha}

_yr

… where N manipulation experiments

have been carried on since 2001,…

… thalli of Cladonia portentosa were transplanted

and exposed to nitrogen treatments for 6 months…

…under local climatic

conditions.

p

r

o

t

e

o

m

e

Acknowledgements to the European Union 7

_{Framework Programme under grant agreement n° [301785] and under the ExpeER project; Fundaçâo para Ciência e Tecnologia Investigador grant (IF/00964/2013/CP1159/CT0021)}

M&M

The proteome of C. portentosa was extracted and

analyzed following Nicolardi et al., 2012 (Environmental

Pollution 160, 1-10).

Significantly different spots between treatments were

selected when comparing: 6-months treatments with

different doses of NH

₄

+

_{and with and without PK;}

6-months treatments with different doses of NO

₃

-

_{and with}

and without PK.

Results

MOLECULAR RESPONSE

ECO-PHYSIOLOGICAL OBSERVATIONS

?

Master (virtual) gel obtained from the PDQuest-based

comparison of 2-D electrophoresis gels of a) NO

treated

samples and b) NH

_{treated samples. Spots analysed by}

MALDI-TOF are circled and numbered.

Summary of changes in protein expression caused by addition of nitrogen

Summary of changes in protein expression (black=upregulation,

brown=downregulation, violet=no defined pattern). Abbreviations:

aa=aminoacids,

GS/GOGAT=glutamate

synthase/glutamine

oxoglutarate aminotransferase; see table for proteins.

Response to PK downregulated downregulated downregulated unclear downregulated downregulated upregulated at 64 kg unclear upregulated at t64 kg downregulated at 64 kg downregulated downregulated at 64 kg upregulated at 64 kg downregulated unclear downregulated unclear unclear upregulated downregulated unclear downregulated at 64 kg upregulated at 64 kg unclear Threshold yes yes yes yes no no no yes no yes yes yes no no yes yes yes unclear no yes unclear yes yes unclear Response to N dose upregulated downregulated at 64 upregulated unclear unclear upregulated no upregulated unclear downregulated slightly upregulated upregulated downregulated upregulated unclear unclear upregulated unclear no upregulated unclear no upregulated unclear N form NO3 -NO3 -NO3 -NH4+ NH4+ NH4+ NO3 -NH4+ NH4+ NO3 -NO3 -NO3 -NH4+ NH4+ NH4+ NH4+ NH4+ NH4+ NO3 -NO3 -NO3 -NO3 -NH4+ NH4+ Organism algae algae algae algae algae bacteria fungus fungus fungus fungus fungus fungus fungus fungus fungus amoeba fungus fungus fungus bacteria algae, fungus fungus algae, fungus fungus Function Photosynthesis Photosynthesis Photosynthesis Photosynthesis Photosynthesis Protein synthesis Folding Folding Folding Regulation Regulation Regulation Regulation Secretion Secretion Secretion Protein degradation Stress response Stress response Stress response Respiration Respiration Respiration Respiration Protein (abbreviation)

ATP synthase subunit beta (ATPase)

Oxygen-evolving enhancer protein 1 (OEP1) Fructose-bisphosphate aldolase (FBA)

ATP synthase subunit beta (ATPase)

Oxygen-evolving enhancer protein 1 (OEP1) Prolyl-tRNA synthetase

Protein disulfide-isomerase (PDI) Protein disulfide-isomerase (PDI)

Peptidyl-prolyl cis-trans isomerase (PPIases) Ran-specific GTPase-activating protein 1 (RGTPase)

Hypothetical protein AN4402.2

Peptidyl-prolyl cis-trans isomerase (PPIases) Putative prohibitin

Clathrin light chain (CLA)

Ran-specific GTPase-activating protein 1 (RGTPase)

Outer membrane autotransporter putative Cell division control protein 48 (CDP48) Woronin body major protein (WB)

Thiol-specific antioxidant protein 3 Hypothetical protein DSC_01120 Malate dehydrogenase (MDH) ATP synthase subunit beta (ATPase) Malate dehydrogenase (MDH)

NADH-quinone oxidoreductase (NADHox) Spot n. 3 2 5 19 15 22 12 14-17 24 4-10 6 7 27 13 16 20 18 23 11 8 1 9 25-26 21

In progress

?

X

a

b

The majority of the under/over expressed

proteins belong to the fungus and are involved

in protein transportation and regulation

(specific tolerance mechanisms?)

The photobiont showed upregulation of

energetic metabolism and photosynthesis

Analysis of chlorophyll a fluorescence parameters

showed no impairment of algal functionality

Lichen species with the same photobiont can have

different tolerance degrees to N

Several protein showed a bell-shaped curve

with highest expression at the intermediate

concentration (existence of a threshold for N)

_{Repeated exposures can exacerbate the toxic}

effects of N

Fertilizing effect of a small amount of traffic

related N

NH

₄

+

_{is more harmful to lichens than NO}

3

-Upregulation of enzymes involved in protein

degradation in case of NH

₄

+

_{but not NO}

3